Hematology Flashcards
(500 cards)
1
Q
RBC
A
They make up the majority of cellular components of blood. They are highly specializesd and have a few uneique features: 1) they lack a nucleus 2) and mitochondria (are anaerobic dependent and therefore need ways to prevent build of O radicals in the cell). They loose these organelles prior to being released from the bone marrow into the periphery.
2
Q
Hemostatsis
A
(the arrest of bleeding), which allows blood to clot in response to damage to a blood vessel. Hemostasis results from the complex interactions between the platelet, the endothelial lining of blood vessels, and the blood coagulation factors in response to disruption of the endothelium at sites of injury. This process is counterbalanced by inhibitory factors and the fibrinolytic system, which is responsible for breaking down formed clots. This system has to be finely tuned to allow clotting to take place when necessary while preventing uncontrolled propagation of clots when they do form or formation of pathologic clots (thromboses).
3
Q
Anemia
A
decrease in the amount of red blood cells (RBCs) or the amount of hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry oxygen.
4
Q
Erythropoiesis
A
: the process, which produces red blood cells (erythrocytes). It is stimulated by decreased O2 in circulation, which is detected by the kidneys, which then secrete the hormone erythropoietin. This hormone stimulates proliferation and differentiation of red cell precursors, which activates increased erythropoiesis in the hemopoietic tissues, ultimately producing red blood cells.
5
Q
Lymphocytes
A
the key players in the adaptive immune response, which involves the development of “memory” following exposure to an infectious agent, providing the ability to respond more vigorously to repeated exposure to the same agent. Diameter: 7–12µm. Mostly small, can be large if reactive. Nucleus round or slightly indented. Condensed chromatin. Usually scanty bluish cytoplasm, may contain a few azurophilic granules
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Q
neutrophils
A
(also known as polymorphonuclear cells or PMNs): the most abundant (40% to 75%) type of white blood cells in mammals and form an essential part of the innate immune system. They are formed from stem cells in the bone marrow. They are short-lived and highly motile. Neutrophils may be subdivided into segmented neutrophils (or segs) and banded neutrophils (or bands). They form part of the polymorphonuclear cell family (PMNs) together with basophils and eosinophils. Diameter: 9–15µm, Cytoplasm slightly acidophilic, Many very fine granules, 2-5 nuclear segments/lobes
7
Q
monocytes
A
a type of white blood cells (leukocytes). They are the largest of all leukocytes. They are part of the innate immune system. They are amoeboid in shape, having clear cytoplasm. Monocytes have bean-shaped nuclei that are unilobar. Monocytes constitute 2% to 10% of all leukocytes in the human body. They play multiple roles in immune function: (1) replenishing resident macrophages under normal states, and (2) in response to inflammation signals, monocytes can move quickly to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Half of them are stored in the spleen. Monocytes are usually identified in stained smears by their large kidney shaped or notched nucleus. These change into macrophages after entering into the tissue spaces, and in endothelium can transform into foam cells. Diameter: 15–30µm. Large and eccentric nucleus, round, kidney/horseshoe-shaped or lobulated. Chromatin, skein-like or lacy appearance. Abundant cytoplasm, grayish-blue, few to many fine azurophilic granules. May have intracytoplasmic vacuoles
8
Q
eosinophils
A
are white blood cells and one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Diameter: 12–17µm. Numerous large, round and red-orange granules. 1-4 nuclear lobes, mostly 2.
9
Q
Basophils
A
Basophils contain large cytoplasmic granules which obscure the cell nucleus under the microscope when stained. However, when unstained, the nucleus is visible and it usually has two lobes. Basophils appear in many specific kinds of inflammatory reactions, particularly those that cause allergic symptoms. Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. They also contain the vasodilator histamine, which promotes blood flow to tissues. They can be found in unusually high numbers at sites of ectoparasite infection, e.g., ticks. Like eosinophils, basophils play a role in both parasitic infections and allergies. Diameter: 12µm. Numerous large round purple-black cytoplasmic granules. Usually two nuclear lobes, but often covered by granules
10
Q
complete blood count (CBC),
A
a very commonly used clinical test, will also calculate the hematocrit for you, but it will provide you with much more information as well. It will tell you the hemoglobin concentration in the blood. Hemoglobin is the protein in red blood cells that binds to and carries oxygen, so measuring hemoglobin gives you important information about the oxygen carrying capacity of someone’s blood. If you don’t have enough hemoglobin in your blood (for whatever reason – there are lots of them) you have anemia. A CBC also gives you a precise measurement of the number and size of each of the different blood cell types as well as percentages of the different types of white blood cells (the “differential”).
11
Q
peripheral smear
A
A drop of blood can also be smeared on a glass slide, stained, and examined under the microscope to look for any abnormally shaped cells or cellular inclusions.
12
Q
sickle cell mutation
A
mutations can lead to a situation where the hemoglobin molecules in certain situations tend to polymerize into long chains or form crystals, leading to abnormally-shaped cells that are fragile and easily destroyed. The most common of these mutations (a substitution of valine for glutamic acid at the 6th position of the beta-globin chain) makes hemoglobin S
13
Q
thalassemia mutation
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mutations in the promoter regions of the globin genes can lead to an imbalance in the number of alpha-globin and beta-globin chains produced in the RBC
14
Q
porphyria mutation
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mutations in the enzymes involved in the synthesis of the heme prosthetic group, leading to a rare disease known as porphyria.
15
Q
Other mutations in red blood cells
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There can be mutations of the hemoglobin molecule that cause it to bind with greater or lesser affinity to the oxygen. Other mutations can make the hemoglobin molecule unstable, leading to premature breakdown and RBC destruction (termed “hemolysis”). Finally, because mature RBCs lack nuclei, they can’t make new RNA, so they have limited ability to respond to changes in the environment. Once they’re released in the periphery, they’re stuck with what they’ve got and, if damaged, have limited ability to repair themselves. Also, since they lack mitochondria, they are dependent on anaerobic metabolism for generation of ATP to maintain critical cellular processes. Thus, mutations in the glycolytic pathway, such as pyruvate kinase deficiency, can lead to another type of hemolytic anemia.
16
Q
glucose-6-phosphate dehydrogenase (G6PD) deficiency
A
The most common cause of hemolytic anemia. RBCs must also have the ability to reduce reactive oxygen species which can accumulate in the cell with time and cause cellular damage. Mutations of the genes that encode the enzymes responsible for this function can also be a cause for hemolytic anemia. an X-linked disorder seen in ~15% of the African male population. G6PD is the most common human enzyme defect, being present in more than 400 million people worldwide.
17
Q
iron deficiency
A
Anemia can also occur when the bone marrow isn’t making enough RBCs. To make RBCs, the bone marrow needs enough of the necessary substrates. There must be enough iron available to be incorporated into the hemoglobin molecule; one of the most common causes of anemia is iron deficiency. When someone is iron deficient, it is important to know why. It may be due to decreased dietary intake. Often, however, it can be due to blood loss of some form or another that the patient may not even know about, such as occult (not clinically detectable) bleeding from the gastrointestinal tract due to a cancer in the colon. Vitamin B12 and Folic acid are also necessary for the developing RBCs to be able to undergo normal cell division, so when deficiencies of these vitamins occur, anemia results.
18
Q
erythropoietin
A
a hormone produced by the kidney called erythropoietin (“red making”), is essential for stimulating the marrow to make red blood cells. Under certain clinical conditions, such as kidney failure, RBC production is decreased due to a lack of erythropoietin production, and anemia results.
19
Q
“myeloid” cell types
A
neutrophils (also known as polymorphonuclear cells or PMNs), monocytes, eosinophils, and basophils. are critical components of the innate immune system. Innate immunity provides protection against infection that relies on mechanisms that exist before infection, are capable of a rapid response to microbes, and react in essentially the same way to repeat infections.
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Q
Hematologic malignancies
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These are all clonal, neoplastic conditions, meaning that the malignant cells have undergone a series of genetic mutations that have altered their differentiation and/or proliferative capacity. Some malignancies have classic mutations associated with them, such as the t(9;22) translocation (also known as the Philadelphia chromosome) associated with chronic myelogenous leukemia (CML). Others are not associated with any characteristic cytogenetic abnormalities.
21
Q
myeloma
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arising from one of the other cell types in the marrow
22
Q
anemia
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is not a diagnosis in of itself. Many causes: nutritional deficiencies (iron defecient, Vit B12, folate) Kidney disease, can also be due to inflammation, hemolysis (red cell destruction).
23
Q
Hematopoiesis
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The processes of making blood. After birth, both white and red blood cells are produced in the bone marrow and released into the peripheral blood when they reach maturity. All blood cells arised from a hematopoietic stem cell, differentiating along different lines of development to produce all the different blood cell types. With RBCs, the nucleus continues to shrink as divisions occurs and becomes red as hemoglobin becomes incorporated. Hematopoiesis is the complex process, usually occurring predominantly in the marrow, which results in the formation of the mature, functional red blood cells, white blood cells, platelets and miscellaneous other cell types (osteoclasts, dendritic cells, etc).
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Q
aplastic anemia
A
a disease in which the bone marrow, and the blood stem cells that reside there, are damaged. This causes a deficiency of all three blood cell types (pancytopenia): red blood cells (anemia), white blood cells (leukopenia), and platelets (thrombocytopenia). Aplastic refers to inability of the stem cells to generate the mature blood cells.
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spherocytosis
an auto-hemolytic anemia (a disease of the blood) characterized by the production of red blood cells (RBCs), or erythrocytes, that are sphere-shaped, rather than bi-concave disk shaped. Spherocytes are found in hereditary spherocytosis and autoimmune hemolytic anemia. This occurs because vesicles break off of RBC decreasing the surface area to volume ratio. No cenral pallor due to decreased cell membrance. increased MCHC
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Autoimmune hemolytic anemia
occurs when antibodies directed against the person's own red blood cells (RBCs) cause them to burst (lyse), leading to insufficient plasma concentration. The lifetime of the RBCs is reduced from the normal 100–120 days to just a few days in serious cases. The intracellular components of the RBCs are released into the circulating blood and into tissues, leading to some of the characteristic symptoms of this condition. The antibodies are usually directed against high-incidence antigens, therefore they also commonly act on allogenic RBCs (RBCs originating from outside the person themselves, e.g. in the case of a blood transfusion)[
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What COMPLETE BLOOD COUNT (CBC) can tell you
One of the most commonly ordered tests in medicine. Provides information on: Red blood cells: Number, size, hemoglobin content White blood cells: Total count, number and percentage for each type. Platelets: Number, size
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MCV
mean corpuscular volume. Mean size of red blood cells. Determined directly using the Coulter principle or manually (MCV = HCT ¸ RBC). Abnormal values due to: Low MCV: Microcytosis, iron deficiency anemia or thalassemia, High MCV: Macrocytosis, megaloblastic anemia, orAnemia can be classified as: Microcytic (MCV100)
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RDW
RBC distribution width 11.7-14.2. Measure of the variability in size of red cells. The wider the red cell histogram, the higher the RDW. Increased in anemia and disease with RBC destruction (i.e. schistocytosis)
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Normal RBC Morphology
Circular biconcave disc-shaped, Size: 6.7–7.7 µm, mean 7.5 µm, Lack of nuclei, Eosinophilic cytoplasm, Central area of pallor, <1/3 of diameter, the diameter is about half of a neutrophil
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Red Blood Cell Count
(4-6) Obtained using the Coulter principle. Abnormal values due to: Anemia: Decreased due to blood loss, peripheral destruction, or insufficient erythropoiesis in the marrow or Erythrocytosis/ Polycythemia: Increased due to reactive changes (smoking, renal cell carcinoma), thalassemia, or primary marrow neoplasm (polycythemia vera)
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HGB
Hemoglobin concentration (14-18) Determined spectrophotometrically after conversion to cyanmethemoglobin. Abnormal values due to: Anemia: Decreased due to blood loss, peripheral destruction, or ineffective erythropoiesis in the bone marrow. Erythrocytosis/Polycythemia: Increased due to reactive changes (smoking, renal cell carcinoma), or primary marrow neoplasm (polycythemia vera)
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HCT
hematocrit (M= 39-50, F= 35-47). Volume of red blood cells in whole blood. Obtained directly or by calculation (HCT = RBC × MCV). Rule of thumb: HCT = ~ 3 × HGB. Abnormal values due to: Decreased due to anemia or fluid overload or Increased due to erythrocytosis/polycythemia or dehydration. It is the volume of red blood cells in the whole blood (about 40%)
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MCH
Mean cell hemoglobin. Mean quantity of hemoglobin in a single red cell. Parallels MCV: MCV goes up or down, MCH goes up or down. Calculated from directly determined HGB and RBC: MCH = HGB ¸ RBC. Abnormal values due to: Low MCH: Hypochromatic, iron deficiency anemia. High MCH: Hyperchromatic, megaloblastic anemia. Normal value is 27.5-35
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MCHC
Mean cell hemoglobin concentration. Average concentration of hemoglobin in a single red cell or “concentration of hemoglobin in packed red cells”. Calculated from the directly determined HGB and the indirectly determined HCT: MCHC = HGB ¸ HCT = MCH ¸ MCV. Abnormal values due to: Decreased in moderate to severe microcytic anemia or Increased in hereditary spherocytosis. Normal= 32-36
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Neutropenia
Decreased absolute neutrophil count (ANC), < 0.5×109/L. Causes: Infections: Gram-negative septicemia, typhoid and paratyphoid fevers, CMV, HIV, EBV, HCV, measles, and HIV; Drugs, medication, ionizing radiation; Marrow diseases: leukemia, myelodysplastic syndromes, aplastic anemia; Bone marrow infiltration by tumors; Autoimmune disease: immune neutropenia, SLE, rheumatoid arthritis; Congenital: cyclical neutropenia, familial benign chronic neutropenia, severe congenital neutropenia, congenital aleukia
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Neutrophilia
Increased absolute neutrophil count (ANC), >11.1 × 109/L. Causes: Physiologic: neonates, exercise, emotion, pregnancy, parturition, lactation; Acute inflammation caused by infections; Acute inflammation caused by surgery, infarcts, autoimmune, etc.; Endocrine/metabolic: Cushing's syndrome, thyrotoxicosis, uremia, etc.; Myeloproliferative neoplasms and myelodysplastic/myeloproliferative neoplasms; Malignant diseases: carcinoma, lymphoma, other solid tumors; Drugs: adrenaline, corticosteroids, lithium
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Eosinopenia
Decreased eosinophils, <0.01 × 109/L. Causes: Drug induced: administration of corticotropin, corticosteroids, epinephrine or histamine; Acute inflammation or infection. No pathological affect
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Eosinophilia
Increased eosinophils, >0.4 × 109/L. Primary stimulating cytokines: IL-5, IL-3 and GM-CSF. Causes: Infections: parasites, fungi; Allergic disorders; Löffler's syndrome, tropical pulmonary eosinophilia, idiopathic hypereosinophilic syndrome; Leukemias, myeloproliferative neoplasms, myeloid and lymphoid neoplasms with abnormalities of PDGFRA, PDGFRB or FGFR1; Other malignant diseases: Mycosis fungoides, Sézary syndrome, Hodgkin's disease, T-cell lymphomas metastatic carcinoma; Churg–Strauss syndrome, systemic sclerosis, rheumatoid arthritis
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IL-5
an interleukin produced by T helper-2 cells and mast cells. Through binding to the IL-5 receptor, IL-5 stimulates B cell growth and increases immunoglobulin secretion. It is also a key mediator in eosinophil activation.
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Basopenia
Decreased basophil count, s syndrome and pregnancy; Administration of progesterone, corticosteroids or corticotrophin
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Basophilia
Increased basophil count, >0.2 × 109/L. Causes: Mastocytosis, CML, polycythemia vera, essential thrombocythemia, myelofibrosis, basophilic leukemia, eosinophilic leukemia, and Ph-positive acute leukemia
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Monocytopenia
Decreased monocyte count, <0.2 × 109/L. Causes: aplastic anemia, Cyclic neutropenia, hemodialysis, severe thermal injuries, AIDS, hairy cell leukemia.
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Monocytosis
Increased monocyte count, >1.0 × 109/L. May be a compensatory event in association with neutropenia. Causes: Physiologic; Infants; Certain infections; Marrow disease: MDS, MDS/MPN, acute immunoblastic and myelomonocytic leukemia; Hodgkin's disease, carcinoma, multiple myeloma, malignant histiocytosis
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Lymphopenia
Decreased lymphocyte count, s disease, aplastic anemia, agranulocytosis, and MDS
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Lymphocytosis
Increased lymphocyte count, >5 × 109/L. Causes: Physiologic: infants and young children; Certain viral infections: EBV, CMC, infectious hepatitis, chickenpox, smallpox, measles, rubella, mumps, influenza, primary HIV infection; Pertussis, brucellosis, tuberculosis, secondary and congenital syphilis; Chronic lymphoproliferative disorders, lymphomas; Post-splenectomy
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Thrombocytopenia
Decreased platelet count, < 140 × 109/L. Causes: Peripheral destruction: idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS), disseminated intravascular coagulation (DIC); Sequestration in spleen; Inadequate production: primary marrow disorders or metastatic tumor in the marrow; Inherited: Wiskott–Aldrich syndrome, May–Hegglin anomaly, Bernard–Soulier syndrome
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Thrombocytosis
Increased platelet count, > 400 × 109/L . Causes: Primary marrow neoplasm: essential thrombocythemia, chronic myelogenous leukemia, polycythemia vera, myelofibrosis; Reactive: inflammation, surgery, hyposplenism, splenectomy, asplenia, iron deficiency anemia or hemorrhage
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Bite cells
Bite-like detect due removal of Heinz body in spleen. Associated with G6PD deficiency
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Schistocytes
Fragmented RBCs, helmet cells. HUS, TTP, DIC, burns, HELLP, mechanical heart valves
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Target cells
Central hemoglobin, target shape. Thalassemia, hemoglobin C, iron deficiency, liver disease
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Sickle cell
Bipolar spiculated shape. Banana shape. Sickle disease
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Basophilic stippling
Morphology: evenly dispersed fine blue granules. Content: aggregated ribosomes (rRNA). Causes: Lead poisoning, porphyria, pyrimidine 5’ nucleotidase deficiency; Hemoglobinopathies, thalassemia; Myelodysplasia, sideroblastic anemia; Infection
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Howell-Jolly bodies
Morphology: single, dense, blue dot. Content: nuclear DNA remnant. Causes: Post-splenectomy; Functional asplenia; Megaloblastic anemia; Myelodysplasia
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Heinz Body
Not visible on Wright-Giemsa stain. Need to stain with supravital dye (crystal violet). Denatured/oxidized hemoglobin attached to the inner cell membrane. Cause: G6PD deficiency. Associated with bite cells
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Dohle Body
Pale blue inclusion at the periphery of the cytoplasm. Infection, inflammation, burns or pregnancy. Contents: condensed RNA.
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Toxic Granulation (Hypergranularity)
Increased Numbers and prominence of 10 granules. Due to rapid cell division (not enough time to dilute). Often associated with Döhle bodies and toxic vacuolization. Causes: bacterial infection, marrow recovery, G(M)-CSF
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Hypersegmented Neutrophils
More than five lobes. Associated with megaloblastic anemia
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hematocrit
also known as packed cell volume (PCV) or erythrocyte volume fraction (EVF), is the volume percentage (%) of red blood cells in blood. It is normally 45% for men and 40% for women.
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reticuloyte
young RBC, the count is important for evaulating anemia.
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Reticuloendothelial System
The mononuclear phagocyte system (MPS) (also called Reticuloendothelial System or Macrophage System) is a part of the immune system that consists of the phagocytic cells located in reticular connective tissue. The cells are primarily monocytes and macrophages, and they accumulate in lymph nodes and the spleen. The Kupffer cells of the liver and tissue histiocytes are also part of the MPS.
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Hepcidin
25 amino acid peptide produced by hepatocyte. Antibacterial peptide. Synthesis is increased by inflammation/infection or iron overload. Negative regulator of iron absorption by the intestinal epithelial cells, transport by the placenta, and release from macrophages (RE system). Modified by a protease associated with turnover. Implications for hemochromatosis (deficiency of hepcidin). During inflammation/infection, inhibition of ferroportin causes increased iron retention in macrophages contributing to anemia. Implication for iron resistant iron deficiency anemia. Hepcidin critical for new approaches for hemochromatosis and anemia of inflammation/infection. This can look like anemia.
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Characteristics of Iron Deficiency
Decreased Hgb synthesis. Decreased Cell proliferation (Hemolytic component). Multiple systems besides hematopoietic system involved: Hematopoietic: anemia (see previous slide); Neuromuscular: mild defect muscle performance, neuropsych dysfunction; Epithelial: nails (ridges, koilonychia); tongue (papillary atrophy); Upper GI: dysphagia, esophageal webs, achlorhydria, gastritis; Lower GI: protein losing enteropathy; Immune dysfunction: innate (phagocyte), adaptive (lymphocyte); Pica
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Diagnosis of iron deficiency anemia
History and physical exam (epidemiologic and etiologic factors). Routine laboratory tests. Decreased O2 carrying capacity (Hgb, Hct). Decreased production (reticulocyte count and index). Microcytosis (Decreased MCV, smear findings). Hypochromia (Decreased MCHC, smear findings). Wide range in size of cell (increased RDW). Additional studies may be indicated in difficult cases: Decreased serum Fe, increased TIBC, Decreased ferritin, increased FEP
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Differential diagnoses of iron defiency anemia
Anemia of chronic inflammation/infection; Anemia of chronic disease; Thalassemia; Sideroblastic anemias
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Timeline of normalization of iron levels after supplement is given
Serum iron > Hgb/retic > ferritin > MCV/FEP/RDW
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Iron Overload
Etiology: increased iron in diet, increased absorption (abnormality HLA-H gene), Repeat transfusions (chronic anemia). HLA-H gene encodes for a protein in duodenal crypt cells and reticuloendothelial cells which acts as a co-factor for iron absorption resulting in an increase in absorption and accumulation.
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Consequences of Iron Overload
Concequences: increased serum iron (sat >50%), increased ferritin, increased liver iron. Organ damage: Cardiac (arrhythmia, failure); liver (dysfunction, failure): endocrine dysfunction (e.g., pancreatic endocrine: diabetes) Treatment: Hemochromatosis → therapeutic phlebotomy; Hemosiderosis → iron chelators: Desferal (IV or subcutaneous infusion) and Exjade (Newer oral preparation)
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extramedullary hematopoiesis
Hematopoiesis outside of the bone marrow after birth and is distinctly abnormal
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Myeloid
although the root "myel-" just refers to marrow, the term myeloid most commonly refers to all non-erythroid and non-lymphoid lineages (i.e. the granulocytes, monocytes, megakaryocytes/platelets), though sometimes the term refers only to the granulocytic lineages.
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Lymphoid
Refers to T cells, B cells, and NK cells, and their precursors
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Bone marrow vasculature.
Nutrient arteries course into and through the bone marrow and branch into capillary-venous sinuses. Capillary-venous sinuses are composed of an endothelial cell layer, basement membrane, and an adventitial layer. Capillary-venous sinus blood eventually flow into a central vein, and from there into the systemic circulation. The passage of blood cells through the capillary-venous sinus endothelial layer is selective: Normally only mature cells are allowed to pass from the marrow into the sinuses and subsequently into the peripheral circulation; immature cells are retained.
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stromal elements
The bone marrow environment plays a critical role in hematopoiesis. Stromal elements play important roles in hematopoiesis. These stromal elements include endothelial cells of the capillary-venous sinuses; reticular cells lining the adventitial surface of the capillary-venous sinuses; fibroblasts; lymphocytes; macrophages; adipocytes; and the extracellular matrix produced by stromal cells (e.g. collagen fibers, laminin, fibronectin, chondroitin sulfate, hyaluronic acid, heparan sulfate).
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Erythropoietin (EPO)
made by certain kidney cells in response to hypoxia,
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Thrombopoietin (TPO)
promotes megakaryopoiesis
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Granulocyte-monocyte colony stimulating factor (GM-CSF)
promotes granulopoiesis and monopoiesis
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Granulocyte colony stimulating factor (G-CSF)
promotes granulopoiesis
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Monocyte colony stimulating factor (M-CSF)
promotes monopoiesis
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Interleukin-5 (IL-5)
promotes production of eosinophils
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Interleukin-3 (IL-3)
promotes production of basophils
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Features characterizing granulopoiesis
1) Nuclear maturation with acquisition of condensed chromatin, loss of nucleoli and progressive nuclear indentation and, ultimately, segmentation. 2) Acquisition of cytoplasmic primary (azurophilic), and then secondary (specific) granules. 3) Loss of the ability to replicate by the metamyelocyte stage.
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Eosinophils
The mature eosinophil is around 13 mm in diameter. The cytoplasm is full of large orange-red (eosinophilic) granules. The nucleus contains heavily condensed chromatin and is segmented, usually into two round to oval lobes.
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Basophils
The mature basophil is 10 mm in diameter. It contains a lobular but non-segmented nucleus. The nucleus is usually obscure by numerous blue-purple (basophilic) granules.
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Sideroblastic anemias
a rare group of congenital or acquired disorders. Vitamin B12 and folate deficiency are also included in underproduction anemias, but they are better characterized as a group of disorders with ineffective erythropoiesis. Sideroblastic anemias are a heterogeneous group of disorders with deposits of iron in mitochondria of erythroid precursors. The sequence below summarizes the pathophysiology. Impaired production of protoporphyrin or incorporation of iron in heme -> Accumulation of iron in mitochondria forming a ring around the nucleus-> Ring sideroblasts (Prussian blue).
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causes of folate deficiency
In contrast to B12 deficiency, the most common cause of folate deficiency leading to megaloblastic anemia is inadequate dietary intake. Other causes include malabsorption due to such things as tropical sprue or parasitic infection, which can lead to rapid depletion of folate through interruption of enterohepatic circulation, inborn errors of folate metabolism (very rare), and increased demands (hemolysis, pregnancy/lactation, rapid growth, psoriasis, myeloproliferative disorders). Alcohol consumption also can lead to rapid onset of folate deficiency, not only through decreased dietary intake but also through disruption of cycling from liver stores to tissues.
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Clinical and Laboratory Features of folic acid and vitamin B12 deficiency
Both folic acid and vitamin B12 deficiency result in megaloblastic anemia. The onset of folate deficiency can occur quite rapidly (within weeks), particularly in the setting of malabsorption or alcoholism. In someone who is well-nourished, Vitamin B12 deficiency takes several months to develop because of its long half-life within the body and large hepatic stores. Vitamin B12 deficiency develops more slowly and is more likely associated with malabsorption. The symptoms and signs of anemia in both cases are not distinguishable from other causes.
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BONE MARROW STRUCTURE
Marrow space is encased by cortical bone, and interspersed by trabecular bone lined by osteoblasts and osteoclasts. Between trabecula is a network of vascular sinusoids with walls of ‘leaky’ endothelial cells. Marrow stromal cells are bound to the non-luminal side of the endothelial cells. These stromal cells: produce the protein framework of the marrow, especially type IV collagen (reticulin); produce regulatory factors and adhesion molecules needed to induce and maintain hematopoiesis. Marrow and blood are interconnected compartments
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Erythropoiesis
Rate of erythropoiesis determines the hemoglobin level of normal individuals. Initiated by erythropoietin, a hormone produced by the kidneys. Erythropoietin production stimulated by hypoxia. Erythropoietin acts to: Activate stem cells of bone marrow to differentiate into pronormoblasts; Increases rate of mitosis and maturation process; Increases rate of hemoglobin production; Causes increased rate of reticulocyte release into peripheral blood.
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Distinguishing types of granulopoiesis
Granulocyte types are distinguished from each other by the appearance of their secondary (specific) cytoplasmic granules: Neutrophils: pink to rose-violet granules. Eosinophils: reddish-orange granules Basophils: dark purple granules
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AUER RODS
ARE SPECIFIC FOR MYELOBLASTS, BUT ARE ONLY SEEN IN ABNORMAL CONDITIONS
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Neutrophil Granulocytes
Granules contain destructive enzymes, most famously myeloperoxidase, used to destroy infectious organisms, most commonly bacteria. Also have prominent phagocytic activity. Mature segmented neutrophil granulocytes are also known as PMNs, for ‘polymorphonuclear leukocytes’ (due to their highly and variably segmented nuclei), and also by the abbreviated terms ‘segs’ and ‘polys.’
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Eosinophil Granulocytes
Similar maturation stages to neutrophils. Characterized by large, eosinophilic secondary granules. Mature eosinophils usually have 2 nuclear lobes. Lifespan of around 8-12 days. Granules contain destructive enzymes, which are generally used to fight organisms too big to phagocytose (fungi, protozoans, parasites). Also involved in modulation of mast cell activity in hypersensitivity response/allergic disease. Minimal degree of phagocytic activity. Main cytokine initiating eosinophil production: Interleukin-5 (IL-5)
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Basophil Granulocytes / Mast Cells
These cell types are though to be related in some manner, though this is a point of contention. Both of these cell types are involved in hypersensitivity/allergic processes, and in innate defenses against microbes. Main cytokines initiating their production are: Interleukin-3 (IL-3) for basophils and Stem Cell Factor (SCF) for mast cells
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MATURE BASOPHIL
Prominent large dark blue (basophilic) cytoplasmic granules, which obscure the nucleus. Multilobular but non-segmented nucleus. Found in blood and marrow at low levels
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MAST CELLS
Many reddish-purple (metachromatic) cytoplasmic granules. Round nuclei, abundant cytoplasm. Found in marrow at low levels, and at varying levels in different solid tissues
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Lymphopoiesis
T LINEAGE: Early T-lymphoid progenitor cells migrate to the thymus, the site of T cell maturation. Some mature T cells migrate back to reside in the marrow. B LINEAGE: B cell maturation takes place in the marrow. A few mature B cells and a fair population of plasma cells reside in the marrow
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Normal B-Lymphoblasts (Hematogones)
Size: 10-18 μm in diameter. Nucleus: lacy and fine chromatin, but less fine than myeloblasts 1-2 nucleoli. Cytoplasm: scant, non-granular
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Monopoiesis
Initiated by M-CSF (monocyte-colony stimulating factor). Time for monocyte maturation in marrow not well understood. Mature monocytes circulate in peripheral blood an average of 20 days, before entering tissue to become macrophages. Some mature monocytes and macrophages reside in the marrow
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Investigation of Cytopenias
caused either by increased distruction or decreased production of marrow lineage. In cytopenias due to increased destruction, growth factors should signal to the marrow to make more of whatever is being destroyed….thus, examination of the marrow shows a compensatory marrow hyperplasia of one or more lineage. In cytopenias due to decreased production, the marrow will not show a compensatory hyperplasia; serum growth factor levels may be increased or decreased. If increased, they are not really getting a response.
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PETECHIAE
microhemorrhages within skin (indicative of thrombocytopenia)
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GIANT PLATELET
Not an entirely specific finding on blood smear, but usually indicates increased thrombopoiesis, in much the same manner as increased reticulocytes (large RBCs) usually indicates increased erythropoiesis
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causes of neutrophilia
acute: infections (bacterial), burns, infarcts, drugs (steriods), stress (pain, extreme temp). Chronic: persistent infections, persistent inflammatory disease (IBD), continued exposure to neutrophilia inducing drugs, tumors producing growth factors, clonal proliferation of neutrophils (CML)
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possible causes microcytic and hypochromic anemia
iron deficient anemia, anemia of chronic disease, colon cancer, hemoglobinopathy (thalassemia), primary marrow disease
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Specific Testing for folate and vitamin B12 deficiency
Direct measurement of serum cobalamin levels and serum or red cell folate levels is useful in diagnosing deficiencies, although there can be problems with these tests. Cobalamin deficiency in the tissues can exist with a normal serum cobalamin level. Serum folate levels may reflect recent intake and not tissue stores, while red cell folate is a better indicator of tissue folate status but will be low with B12 deficiency as well. Measurement of plasma homocysteine levels has been used as a more sensitive marker of deficiency of B12 and folate in the tissues. As indicated in the metabolic pathways described above, vitamin B12 and folic acid are both required for the synthesis of methionine from homocysteine. So, deficiency of either one of these vitamins should lead to elevated homocysteine levels. Another reaction involving vitamin B12 but not folate is the synthesis of succinyl CoA from methylmalonyl CoA. Thus, in B12 but not folate deficiency, methylmalonic acid levels are increased, making measurement of methylmalonic acid a good way to distinguish the two.
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The Schilling Test
Once B12 deficiency is diagnosed, it is important to know the cause. Measurement of serum autoantibodies against intrinsic factor, the cobalamin-intrinsic factor complex, and parietal cells is now commonly used to diagnose pernicious anemia, with positivity in more than 60% of adults with the disease. The Schilling test is an older test used to diagnose defects in B12 absorption that is less commonly used today but still important to know about. In this test, 1 μg of radiolabeled cobalamin is given orally to a fasting individual. The IF produced in the stomach combines with the radiolabeled cobalamin (Cbl) which is absorbed in the terminal ileum. The tagged cobalamin is then bound to transcobalamin II (TcII) and enters the bloodstream. A dose of cold (unlabeled) cobalamin is given intramuscularly 2 hours later, causing some of the labeled cobalamin to be excreted in the urine over the following 24 hours (5-35%). If the patient isn’t absorbing the cobalamin given orally, less radiolabeled cobalamin will be excreted into the urine. If the test is positive, it is then repeated with the patient receiving intrinsic factor in addition to the cobalamin, with correction establishing the diagnosis of pernicious anemia. A variation on this test is the food Schilling test where the cobalamin is incorporated with food, allowing the diagnosis of other malabsorptive problems.
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Use of Transfusion and EPO in Chronic Anemia
Only transfuse red cells when the severity of anemia has potential for cardiovascular decompensation. Use EPO to treat anemias for which there is (1) an absolute deficiency (renal disease) or (2) a decrease of this cytokine out of proportion to the hematocrit level and for which a response has been documented. EPO is linked with strokes.
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Clinical findings of sideroblastic anemia
Variable anemia; hypochromic, microcytic RBCs; Pappenheimer bodies (precipitated iron in mitochondria), particularly in marrow normoblasts; accumulation of stored iron. Some syndromes are congenital without specific inheritance; others show an X-linked or autosomal recessive pattern. Sporadic cases and secondary sideroblastic anemias (alcoholism, copper deficiency, drug related) are described. The treatment includes vitamin B6 (some respond), supportive care (including transfusions) or treatment of a reversible secondary cause. Iron overload is treated with chelation therapy (desferal or newer oral chelators).
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low affinity hemoglobin disease
Hemoglobin mutations with low oxygen affinity are associated with a right-shifted oxyhemoglobin dissociation curve, decreased oxygen affinity, normal tissue oxygen, and mild anemia because of improved oxygen delivery (in some) and/or hemolysis (depending on the specific mutation).
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protein/ calorie malnutrition
Protein/calorie malnutrition, to low to support erthropoeisis, is associated with a normochromic, normocytic anemia. There may be associated vitamin and mineral deficiencies which may also play a role in the anemia.
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methyltetrahydrofolate
methyl donor critical for synthesis of methionine from homocysteine
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tetrahydrofolate
a subratrate for purine an pyrimidine synthesis
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entrohepatic circulation (EHC)
Substances are said to undergo an enterohepatic circulation (EHC) when they are excreted into the bile, pass into the lumen of the intestine, are reabsorbed and return to the liver via the circulation
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Hemoglobin A1 (a2b2)
also called Hemoglobin A - is the predominant form of hemoglobin in the adult. Because there is no substitute for the α-chain beyond the early gestational period, a total lack of α-globin chains is incompatible with life.
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Fetal Hemoglobin
(Hb F, a2g2) is the predominant form in the fetus and newborn. Hemoglobin A2 (α2δ2) makes up a small amount of the total hemoglobin in the adult. Fetal hemoglobin remains elevated in premature babies and infants of mothers with diabetes. It also remains elevated in people with hemolytic anemias and with certain diseases affecting the bone marrow such as myelodysplasia and leukemia. Hemoglobin A2 (α2δ2) comprises about 2% of normal adult hemoglobin. It is evenly distributed in red cells and functions much like hemoglobin A. It has the same Bohr effect, cooperativity and response to 2,3-BPG but is more heat stable and has slightly higher oxygen affinity. As you will learn later in the course, hemoglobin A2 can be elevated in certain clinical situations, such as β-thalassemia, sickle cell trait and disease, hyperthyroidism, and megaloblastic anemias.
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oxygen dissociation curve for hemoglobin
Because of cooperativity, when the % saturation of hemoglobin by oxygen is plotted as a function of the partial pressure of oxygen, the resulting curve turns out to be sigmoidal, or S-shaped. hemoglobin is an excellent protein to use for oxygen transport, since oxygen is easily loaded onto the molecule in the lung where the partial pressure of oxygen is ~100 mmHg but then readily unloads in the tissues where the partial pressure of oxygen is ~40 mmHg.
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myoglobin dissociation curve
a protein which stores oxygen in muscle cells and is very similar to hemoglobin except that it is a monomer rather than a tetramer and therefore does not undergo allosteric regulation or cooperativity. The myoglobin curve is shaped more like a hyperbola, giving the myoglobin molecule very high oxygen affinity at very low oxygen concentrations. Functionally, myoglobin is a very poor protein to use to transport oxygen from the lungs to the tissues, since it would hold tightly to the oxygen and not release it until the oxygen concentration got very low. On the other hand, myoglobin is a very good protein to use for storage of oxygen in the intracellular environment where oxygen concentration is very low (1-5 mmHg) and where high oxygen affinity is needed to transfer the oxygen from hemoglobin to myoglobin.
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P50
A way to quantify this difference in oxygen affinity is by determining the P50, which is defined as the partial pressure of oxygen at which the oxygen carrying protein is 50% saturated. Under normal conditions for temperature (37 C) and pH (7.4), the P50 for hemoglobin is approximately 27 mmHg while the P50 for myoglobin is 2.75 mmHg.
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Fetal hemoglobin
Chromosome 16 contains the “a-like” genes, including two copies of the a-globin gene itself along with variants expressed early in embryonic development; therefore, the genome contains a total of 4 copies of the a-globin gene (2 paternal and 2 maternal). The “b-like” genes (genes for the g-, d-, and b-globin chains along with variants produced early in embryonic development) are products of a set of genes on chromosome 11; one copy of the gene set is inherited from each parent.
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fetal hemoglobin patterns
Embryos have 3 distinct hemoglobins that are present only between 4 and 14 weeks gestation: Hemoglobin Gower I (z2e2), Hemoglobin Gower II (a2e2) and Hemoglobin Portland (z2g2). Each of these has a higher affinity for oxygen than does hemoglobin A. After week 8 of gestation, fetal hemoglobin or hemoglobin F (a2g2) predominates. The g-chain differs from the b-globin chain by 39 amino acids. Fetal red cells have a higher oxygen affinity than adult red cells, primarily because hemoglobin F binds 2,3-BPG poorly, stabilizing the hemoglobin in the R state and shifting the oxygen dissociation curve to the left. The Bohr effect is also increased by 20% in fetal hemoglobin, so that as fetal blood passes through the intravillous spaces of the placenta, H+ ions are transferred to the maternal circulation and the pH rises, leading to increased oxygen affinity and a further shift of the curve to the left. These changes favor transfer of oxygen from the maternal circulation to the fetal circulation
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Adult hemoglobin patterns
At birth, there is 65-95% hemoglobin F and about 20% hemoglobin A. The normal adult level of fetal hemoglobin is approached by 1 year and achieved by 5 years of age. Under normal conditions, adults have 96-97% of their hemoglobin as hemoglobin A (a2b2). In adults, hemoglobin F makes up <1% of the total hemoglobin and is unevenly distributed in red cells.
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Cyanosis
is visually perceptible when reduced hemoglobin exceeds 3 g/dL, which generally corresponds to an oxygen saturation level below 85 percent in a patient with a hemoglobin concentration of 15 g/dL. Clinically, it would be very useful to know a patient’s oxygenation status before it reaches this critical level. Traditionally, arterial blood gas analysis was performed to measure oxygen status. Pulse oximetry, a noninvasive alternative, is the norm now.
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A pulse oximeter
probe is a photo detector and two light-emitting diodes, one emitting at 660 nm and the other at 940 nm. They are in the red band (660 nm) where deoxyhemoglobin absorbs light maximally and the 940 nm infrared wavelength where oxyhemoglobin absorbs most. The detector and emitter face each other through tissue, so the probe is usually placed on the finger. Only pulsatile flow, representing arterial blood flow, is measured. The photodiodes switch on and off several hundred times/second and light absorption is measured. The displayed value is based on an average of the previous 3-6 seconds.
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extravascular hemolysis
the red cell is ingested by macrophages of the RE system. The heme is separated from globin, iron removed and stored in ferritin, and the porphyrin ring converted to bilirubin which is released from the cell. Taken up by a specific transport system in the liver, the bilirubin (lipid soluble) is converted to a water soluble compound by addition (conjugation) of a glucuronic acid. This is completed by the cytochrome P-450 enzyme(s) in liver parenchymal cells. After secretion into the biliary tract and small bowel, the glucuronic acid is removed and bilirubin converted into urobilinogen and other water soluble pigments. Urobilinogen may cycle between the gut and liver (entero-hepatic circulation) or excreted by the kidney into the urine.
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Unstable hemoglobins
Tendency to spontaneously denature, >60 variants. Often due to mutations that disrupt the stability of the heme-globin linkage. May not be detected until adulthood. Can also have altered oxygen affinity (higher or lower). May lead to a hemolytic anemia, with jaundice and splenomegaly. Can be referred to as Heinz body anemia. Usually don’t need blood transfusions. Give folic acid regularly. Splenectomy not curative. May not see Heinz bodies until after splenectomy
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CYANOSIS
Too much deoxyhemoglobin. >3 g/dL (~1.5 g/dl methemoglobin (8-12% assuming normal hemoglobin level). Sulfhemoglobinemia >0.5 g/dl sulfhemoglobin
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Differential Diagnosis of Cyanosis
Inadequate Oxygenation of Hemoglobin (common): Pulmonary disorders, Cardiac right-to-left shunt, Congestive heart failure, Cardiovascular collapse (shock). Low O2 affinity Hb variant (rare). Methemoglobinemia (rare): Congenital-Cytochrome-b5 reductase deficiency, Cytochrome-b5 deficiency, M hemoglobins. Acquired- Drugs, Industrial environmental toxins, etc. Sulfhemoglobinemia (rare): Acquired: drugs, toxins, etc.
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Hereditary spherocytosis
Hereditary spherocytosis (HS) is a familial disorder characterized by anemia, intermittent jaundice, splenomegaly and responsiveness to splenectomy. The heterogeneity of clinical features is associated with multiple molecular abnormalities of which spectrin deficiency is the most common. The hallmark of this syndrome is loss of plasma membrane and formation of the microspherocyte. Spherocytes are more susceptible in vitro to osmotic stress, the basis for a common test for the disorder. The basic pathophysiology is that spectrin, ankyrin or band 3 defects weaken the cytoskeleton and destabilize the lipid bilayer. Loss of membrane and formation of the spherocyte leads to decreased deformability and entrapment in the spleen. Conditioning in the red pulp leads to further loss of red cell membrane and, ultimately, removal by the macrophage. Most inherit the condition as an autosomal dominant; 25% are autosomal recessive.
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Clinical presentation and treatment of hereditary spherocytosis
Clinically, patients present with a variable degree of anemia as well as jaundice and splenomegaly. One third has hyperbilirubinemia as neonates. Treatment includes supportive care for chronic anemia and intermittent complications and splenectomy which usually resolves the clinical manifestations. Incidence 1/5,000. Anemia (severe 5%, moderate 60-75%, mild 20%), jaundice, splenomegaly. Variable onset: neonatal hyperbilirubinemia (1/3), childhood or adulthood (as one of two major complications). 75% have autosomal dominant pattern, 25% autosomal recessive. Presenting complications: hyperhemolysis, aplastic crisis. Supportive care (including supplementation with folate). Splenectomy usually resolves clinical manifestations
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Clinical features of G6PD
this disorder presents as intermittent episodes of acute hemolytic anemia and hyperbilirubinemia associated with oxidant stress (infection, drugs or ingestion of specific foods including fava beans). Rarely it may be characterized as chronic hemolytic anemia punctuated by episodes of acute exacerbation of anemia. This condition may also be a cause for neonatal hyperbilirubinemia. No specific morphologic features are associated with G-6-PD deficiency (originally categorized as congenital non-spherocytic hemolytic anemia) but occasionally the smear will show microspherocytes and “blister” or “bite” cells. Management consists of avoiding oxidant drugs and foods for the most common variants seen in the U.S. For severe cases with chronic anemia, supportive care and folate are included.
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neonatal hyperbilirubinemia
Neonatal jaundice can sometimes make the newborn sleepy, and interfere with feeding. Extreme jaundice can cause permanent neurological damage known as kernicterus. Kernicterus is a bilirubin-induced brain dysfunction. Bilirubin is a highly neurotoxic substance that may become elevated in the serum.
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aplastic crisis
can occur in anyone with hemolysis. Happens when hemopoeisis drops down occasionaly. Often associated with sickle cell disease. RBC survival is much shorter therefore cannot compensate.
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Function of Spleen
Clearance of intravascular particles. Adaptive immune response: origin of IgM agglutinins, especially for encapsulated organisms.
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Management of Splenectomy
Problem with splenectomy: overwhelming sepsis (particularly to S. pneumoniae). Risk highest in children 38.5°C (lifelong).
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Chronic RBC Adhesion/Vascular Occlusion with sickle cell
Even when not sickled, the red blood cell in sickle cell disease is “sticky” due to membrane injury and retention of adhesion molecules on the its surface, which results in adhesion of sickle RBCs in the microvascular circulation. This adhesion results in transient vaso-occlusion (partial or total blockage of blood flow through the vessel), vessel wall injury and endothelial remodeling, resulting in narrowing of the vessels and chronic organ damage due to slow or absent blood flow through this microcirculation. The most severely affected organs include the spleen, central nervous system vasculature, lung, kidney, retina, and others.
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Spleen with sickle cell
The microcirculation of the spleen is especially susceptible to occlusion and injury by sickle RBCs. When large numbers of sickle RBCs become abruptly trapped in microcirculation of the spleen, severe anemia and circulatory shock can occur, a complication called splenic sequestration. Even if overt evidence of sequestration isn’t seen, nearly all patients with sickle cell anemia (HbSS) chronically occlude the spleen’s microcirculation, resulting in “autoinfarction” (destruction) of the spleen by the age of 5. This process begins during the first year of life, and slowly compromises the spleen’s ability to kill encapsulated organisms (e.g. pneumococcus, meningococus, hemophilis). Sepsis (overwhelming blood infection) with these organisms, a common cause of death for infants and young children with sickle cell disease, is significantly reduced by the use of prophylactic penicillin and prompt treatment of fever with additional antibiotic therapy.
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Central Nervous System (CNS) Vasculature with sickle cell
The large blood vessels of the central nervous system can be significantly damaged by sickle RBCs. Up to 10% of children with sickle cell anemia (Hb SS) experience an overt large vessel stroke due to this chronic injury, and a larger percentage experience learning disabilities and more subtle neurologic problems. An increase in the velocity of blood flow through the middle cerebral artery, as detected by transcranial Doppler, can predict for an increased risk of stroke in children. This risk can be reduced by prophylactic blood transfusions. Adults with sickle cell disease are more like to have hemorrhages from progressive weakening and rupture of these vessels.
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Lung with sickle cell
The microcirculation of the lung is vulnerable to damage from sickle RBCs. Damage to these vessels makes it harder for blood to flow through the lungs, resulting in an increased pressure in the pulmonary arteries, a condition called pulmonary arterial hypertension (PAH). PAH, which puts strain on the right side of the heart (cor pulmonale), may affect 30-40% of patients with sickle cell disease and is now one of the most common chronic causes of death in adults with sickle cell disease.
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Kidney with sickle cell
The tubules of the kidney are damaged by chronic vaso-occlusion, resulting in the inability to concentrate the urine to avoid dehydration. Hematuria (blood in the urine) may occur due to ischemia to the collecting system (papillary necrosis), which may also cause severe flank pain. The glomerulus may be affected, most commonly initially manifested by an enlargement of the glomerulus and protein in the urine. Ultimately, up to 10% of adult sickle cell patients will develop renal insufficiency due to permanent damage and scarring of the glomerulus (focal segmental glomerular sclerosis), and some with require dialysis and/or renal transplantation.
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Retina with sickle cell
The retinal vessels are susceptible to chronic injury, resulting in the propensity for abnormal vessel formation and hemorrhage, which can lead to retinal detachment and blindness.
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Other Areas affected with sickle cell
The femoral and humeral heads may develop avascular necrosis, a source of chronic pain and progressive joint deterioration, leading to hip and shoulder replacement. Skin ulcers, likely due to microvascular ischemia and poor healing, most often form around the ankles.
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Acute RBC Adhesion/Occlusion – Sickle Cell “Crisis”
In the setting of hypoxia, dehydration, inflammation, infection, or other stresses, not only are RBCs more likely to sickle, but blood vessels can become acutely damaged and constricted, which may promote significant, sudden vaso-occlusion. This results in a “pain crisis”, in which acute severe pain develops relatively rapidly in a pattern that is unique to each patient and may involve any part of the body, most commonly in the arms, legs, chest or abdomen. The pain is likely due to many factors, including reversible ischemia, and resolves as the inciting factors (e.g. hypoxia, dehydration) are improved. During some pain crises, acute severe vaso-occlusion may occur in critical organs, causing acute end-organ injury. One example is the splenic sequestration described above. Other significant acute vaso-occlusive complications include: hand foot syndrome, Acute Chest Syndrome, Acute Multi-Organ Failure Syndrome, Priapism, Bone Infaction
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Hand-Foot Syndrome
Usually seen in infants with sickle cell anemia and Sβothalassemia, self-limited acute severe swelling of the hands and feet can occur, and may be one of the earliest manifestations of sickle cell disease.
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Acute Chest Syndrome.
Sickle RBCs can become trapped in the lung circulation, which damages the vessel lining (endothelium), promoting fluid to leak into the lungs, compromising the ability to oxygenate the blood. The patient may develop chest pain, fever, low oxygen saturation, and experience a fall in hemoglobin due to the trapping of RBCs in the lung. This is one of the most common acute causes of death in sickle cell disease, and may be triggered by pneumonia or embolization of fat from the bone marrow.
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Priapism
Sickle RBCs can be trapped in the penis, with obstruction of outflow, resulting in sustained painful erections. Unless treated, these can result in impotence.
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Bone Infaction
Focal areas of bone may sustain enough ischemia as to become permanently damaged, or necrotic. These areas can remain painful for extended periods of time and may become a source for chronic pain or a site of osteomyelitis (bone infection).
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PROGNOSIS of sickle cell
Despite the multiple potential health impacts of sickle cell disease, most people with even the most severe forms of the disease should expect to live into their 50s and 60s, provided they receive comprehensive medical care and appropriate interventions for acute and chronic complications.
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Beta globin gene cluster
epsilon, gamma (for hbF), delta (In low capacity), and beta (for HbA)
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alpha globin gene cluster
chromosome 26, zeta and alpha (for HbF and HbA)
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Polychromasia
Polychromasia (also known as Polychromatophilia) is a disorder where there is an abnormally high number of red blood cells found in the bloodstream as a result of being prematurely released from the bone marrow during blood formation. These cells are often shades of grayish blue. Polychromasia is usually a sign of bone marrow stress as well as immature red blood cells. 3 types are recognized, with types (1) and (2) being referred to as 'young red blood cells' and type (3) as 'old red blood cells'.
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HbF
Some of the excess chains form relatively unstable tetramer hemoglobin molecules. The main prenatal hemoglobin is Hb F (α2γ2), so in the fetus/newborn with α-thalassemia, there is an excess of γ-globin chains which forms Hb Barts, made up of 4 γ-chains (γ4). This hemoglobin can be identified by hemoglobin electrophoresis.
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HbH
In the first six months of life there is a shift to the production of hemoglobin A1 (α2β2). As this occurs in a person with α-thalassemia, there is an accumulation of β-globin chains, some of which form hemoglobin H, a relatively unstable tetramer of β chains (β4). Hb H can be detected on fresh blood samples using special hemoglobin separation techniques.
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Chronic Hemolytic Anemia with thalassemia
The fragile RBC has a very short half-life and is destroyed in the marrow or culled by the spleen from the circulation. This results in characteristic changes: Anemia with some increase in reticulocyte count- The degree of anemia and reticulocytosis will vary with the severity of the thalassemia. For patients with Cooley’s anemia (βothalassemia), severe anemia with hemoglobin values of <7 g/dL develops within the first year of life, and regular transfusions of RBCs are necessary to sustain life beyond the first 2-3 years. In milder forms of thalassemia, transfusion support may become necessary later in life. Abnormal peripheral smear- with microcytosis (small RBCs), target cells, polychromasia (blue-colored cells representing reticulocytes), mild anisocytosis (variation in size of RBCs), though the cells generally appear relatively homogenous without a large variation in size and shape. Thus, the RBC distribution of width (RDW) is often normal or minimally elevated in thalassemia traits. Abnormal chemistry profile- with increased total/indirect bilirubin, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) as they are released from the lysed RBCs. Splenomegaly-The spleen chronically removes fragile, damaged RBCs from the circulation, and may be enlarged even in persons with thalassemia traits and Hb EE disease. In some cases, the spleen traps increasing numbers of RBCs, resulting in more anemia (fewer circulating RBCs) and an increase in the volume of transfusions needed (hypersplenism), and must be removed.
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Expanded Bone Marrow and Extramedullary Hematopoiesis with thalassemia
In an effort to produce an adequate RBC mass, the bone marrow expands and fills with RBC precursors even though they fragile and ultimately destroyed (ineffective erythropoiesis). This may result in characteristic skeletal deformities, including frontal bossing, although gross changes are generally prevented by early transfusion therapy. Osteopenia often develops despite transfusion support, and in some patients is very painful. The liver and spleen may also enlarge as they become sites for erythropoiesis (extramedullary hematopoiesis).
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Increased Iron Absorption with thalassemia
Persons with severe forms of thalassemia increase absorption of iron from the diet in response to anemia. These are the same patients who require RBC transfusions, which also add to the iron burden and pathologic accumulation of iron in the heart, liver, and pituitary. Unless iron chelation therapy is used, there is a significant risk of mortality due to heart failure during late childhood or young adulthood.
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Delayed Growth and Development with thalassemia
Anemia, increased metabolism associated with ineffective erythropoiesis, and endocrinopathies likely contribute to delayed growth and development, including short stature and delayed-onset of puberty. This can be improved with effective transfusion and iron chelation therapy.
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Endocrinopathies with thalassemia
Nearly 2/3 of patients with Cooley’s anemia (β-thalassemia major) have abnormal endocrine function. The pituitary gland is commonly affected, and may lead to hypogonadotrophic hypogonadism (inadequate gonadal/reproductive function). Hypothyroidism (primary or due to pituitary dysfunction) and/or impaired glucose tolerance may occur in 40-60% of patients with β-thalassemia major.
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Pulmonary Hypertension with thalassemia
Persons with chronic hemolytic anemia, including thalassemia, appear to be at risk for the development of pulmonary hypertension. This is more common in those who have had a splenectomy.
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Hb C
(b6 Glu ->Lys).structural variant with abnormal function.
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Hb E
(b26 Glu ->Lys). Structural variant with decreased synthesis.
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Normal Adult Hemoglobins
Hb F a2 g 2 (95.0%), Hb A2 a2d2 (<3.5%)
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ANTIBODY SPECIFICITY
This can be thought of in terms of the “goodness of fit” (affinity) between an antigenic determinant and a B cell receptor or free antibody. The better the fit, the more that cell or antibody seems to be specific for the determinant. It can be amazingly good: antibodies with association constants (Ka) in the range of 1015 liters/mole have been described. This enormous affinity/specificity makes antibodies not only great protection but unique tools. Such minor differences in antigens can be distinguished that antibodies are essential in hundreds of clinical and research assays, for example, measuring one steroid hormone in the blood in the presence of many others; in drug assays; for Western Blots, and screening libraries of genes in expression vectors; in diagnostic kits, including pregnancy tests; and hundreds of other applications.
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steps to activate a B cell to produce antibodies
First, binding of antigen to the B cell’s receptors (membrane-bound versions of the antibody it will eventually secrete) occurs with a particular Ka. If this binding is strong enough, the second step, activation of the B cell, can take place. So an antigen which binds with low affinity may never activate the cell; but if another antigen comes along which not only binds but activates, the product of the cell (the secreted antibody) may combine with the low affinity antigen well enough to be inconvenient.
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rheumatic heart disease
The heart valves contain an antigen, laminin, which cross-reacts with Group A streptococci. Obviously, the antigen in the valves does not normally activate the corresponding B cells, or we’d all have an autoimmune disease. When people get a streptococcal infection, the streptococcal antigens do activate these B cells because they bind to them with sufficient affinity. Then the released antibody can react with heart valves; with low affinity, it is true, but occasionally, in some people, with enough affinity to lead to a destructive, complement- mediated, inflammatory process
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original theory of antibody design
The original theories were of the instructive type, that is, they said that the antigen told the immune system in some way to make an antibody of appropriate conformation, the way a potter’s mold informs the pot. These theories were Lamarckian; they implied that the outside world could instruct a cell to change its genetic information in some specific way so that a new protein was made.
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ANTIBODY GENETICS
The combining site (into which the epitope fits) is made up of the V (variable from one antibody to another) domains of H (heavy) and L (light) chains. If H and L chains are under separate genetic control and any two can associate randomly, then with 1000 L chains and 1000 H chains we can make 1000 x 1000= 1,000,000 antibody combining sites; we could make a million antibodies with 2000 genes.
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Receptor editing
Although we just said that a B cell tries to rearrange each allele just once, that isn’t strictly true. In some cells, when a rearrangement is detected as faulty (say a stop codon is generated), or if an anti-self receptor has been displayed, as long as the recombinases (RAG genes) are still active it can “try again.” The process is called receptor editing.
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Storage Issues of blood
Each component of blood has a different storage condition which is determined to provide optimum survival (recovery and turnover) and normal function. At day of outdate (the last day the product can be used), recovery of the specific blood component is at least 70% of what would be expected from a freshly collected product, and turnover of the transfused cells in the recipient approximates what should be normal for that blood constituent. For red cells, 2,3-DPG and O2 delivery are adequate for up to 10 days of storage. For products stored longer, 2,3-DPG and O2 dissociation curve for red cells are abnormal, but will return to normal within 12 hours after transfusion. Platelet concentrates infused into patients will achieve maximal peripheral counts and hemostasis 1 hour after the infusion, and platelet turnover approximates a normal rate.
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Production and Kinetics of Neutrophils
The production and kinetics of the neutrophil are useful to understand the innate immune response and to provide a conceptual scheme for organizing neutropenic disorders. Under non-stressed conditions, it takes 10 days to two weeks to move from pluripotent stem cells to mature neutrophils. Under stress, this can be shortened to 5 days. Earliest recognized myeloid cells (myeloblast promyelocyte and myelocyte) form the mitotic pool. These cells both divide and mature (by developing azurophilic and specific granules and other cellular constituents). The storage compartment contains metamyelocytes, bands, and segmented neutrophils (segs) which no longer have the capacity to divide but complete the process of maturing granules and their contents as well as cytosolic proteins to provide the mature cells with complete functional activity. The storage pool, made up of mostly bands and segmented neutrophils, provides a reserve of first responding phagocytic cells, easily mobilized into peripheral blood to fight infection. Once released into the circulation, these cells move to tissues to dispatch microbial colonizers and invaders or die (apoptosis) trying.
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Myelopoiesis
Regulation of production is provided by the cytokines noted previously to develop progenitors and precursors in combination with cytokines for maturation of specific cell lines (G-CSF, M-CSF, IL-5). Production of these cells can be enhanced during infection, stress, and trauma which release microbial products, cytokines, interleukins and other biologic response modifiers to induce endothelial cells, epithelial cells and lymphocytes to produce SCF, IL-11, IL-3, and other cytokines, causing proliferation of pluripotent stem cells and expansion on maturation of the myeloid/monocyte cell lines.
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Innate Immunity Response
Infection or tissue damage results in activation of Toll-like receptors (TLRs) and release of inflammatory mediators including lipids (PAF, LTB4), cytokines and interleukins (IL-1, TNF, IL-6, γ-INF), chemokines (IL-8, GROγ, NAP-2, MCP-1, RANTES), complement, kinins, and coagulation factors which cause vascular dilatation, permeability and emigration of leukocytes into a focus. Movement of neutrophils and monocytes into the area result in release of innate immune responses and inflammation and subsequent emigration of lymphocytes to interact with monocytes to initiate an adaptive immune response.
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Biochemistry and cell biology of neutrophil function
Recent studies in patients with recurrent infections and congenital neutrophil dysfunction syndromes have identified the nature of the defects and expanded our understanding of specific aspects of phagocyte function. Included in these are adhesion molecules, β2 integrins (CD11b/18) and selectins, the oxidase enzyme system, and granules and their constituents. Selectins are a family of proteins with a lectin binding domain, an EGF domain, a variable number of short consensus repeats, a transmembrane domain and a cytoplasmic domain. The lectin site binds sialyted, fucosylated carbohydrates. L-selectin and sialy-LeX on neutrophils interacts to induce rolling, the first adhesion function.
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β2 integrins
a family of glycosylated heterodimers. The α chain has a cytoplasmic domain, a single membrane spanning segment, and an extracellular portion with an I domain and divalent cation binding sites. The β chain has similar intracellular and membrane domains and a different extracellular portion with an I domain and cysteine-rich repeats. Various combinations produce CR3 and C3bi (CD11b/CD18 and CD11c/CD18, respectively) on the surface of the cell which function as receptors for adherence, chemotaxis, and ingestion.
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The oxidase enzyme system
is composed of 6 or more proteins which are distributed in the plasma membrane or specific granule membrane (gp91phox, p22phox) or in the cytosol (p47phox, p67phox, p40phox, and Rac2). With a phagocytic stimulus, assembly of the cytosolic components with the membrane components assembles the system and results in activity with addition of an electron to oxygen to form superoxide anion from which H2O2 and the other reactive oxygen species (ROS) can rapidly be formed.
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granule protein classes
Granule proteins and other constituents are contained in the two main granule classes (azurophilic and specific granules) as well as tertiary granules and secretory vesicles. The contents of these organelles contain constituents which aid in the disruption and dissolution of microbes. Stores of receptors of various classes as well as proinflammatory compounds support continued phagocyte function and inflammation. Congenital neutrophil defects may be categorized according to main functional characteristics of the cells.
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COMPLEMENT DEVELOPMENT
Newborn C levels are usually around those of adults; preemies are often low. Complement components are mostly made in the liver, though white blood cells also contribute.
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the two compartments of the thymus
The lymphocytes (“thymocytes”) whose precursors came in from the marrow, and the supporting structure or stroma, which develops in the neck region and moves down into the fetal chest.
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Granulocyte-colony stimulating factor (G-CSF or GCSF)
a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. Functionally, it is a cytokine and hormone, a type of colony-stimulating factor, and is produced by a number of different tissues. The pharmaceutical analogs of naturally occurring G-CSF are called filgrastim and lenograstim. G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils.
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high grade pathogens
live out side of cells (eg staph, strep). Affectively delt with with antibodies.
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low grade pathogens
live within the cells. T cell system can deal with this.
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Cytokines
Short-range mediators made by any cell, that affect the behavior of the same or another cell. IL-1, TNFα, IL-12
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Lymphokines
Short-range mediators made by lymphocytes, which affect the behavior of the same or another cell. A subset of cytokines. IL-2, IFNγ, IL-4, IL-5, IL-10
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Chemokines
Small (6-14 kD) short-range mediators made by any cell, that primarily cause inflammation. MIP-1 to -4, RANTES, CCL28, CXCL16, Eotaxin, IL-8
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ONTOGENY AND MATURATION OF T CELLS
T cells originate in the thymus, coming out as CTL or Th0 (there is a subtype of Treg that develop in the thymus, too). The thymus consists of epithelial cells, most of which arise from the III and IV pharyngeal pouches in fetal life; macrophages, derived from the bone marrow; and thymocytes (developing thymic lymphocytes), also bone-marrow derived. There are also, of course, supporting cells, fibroblasts, blood vessels, even nerves. There is a dense cortex and a somewhat looser medulla.T cell precursor cells arrive from the bone marrow via the blood, and land in the outer thymic cortex. There they begin to divide rapidly, and can be distinguished from other cells by their large size. At this stage they are ‘double-negative,’ that is, CD4-/CD8-, and have activated Rag-1 and Rag-2 DNA recombinases so they are beginning to rearrange their TCR variable domain genes. These cells will eventually give rise to the mature phenotype ‘single-positives,’ CD8+/CD4- and CD4+/CD8-. The first step is to become double-positive (going from CD4-/CD8- to CD4+/CD8+), and then during selection to turn off one or the other gene. If so, this suggests why the bulk of the cells in the thymus are, in fact, double positive; having failed to be selected for further maturation, they remain ‘stuck’ at the double positive stage until they die. Single-positive T cells acquire other phenotypic refinements as they mature in the thymus, such as recirculation specification molecules and the various molecules with which they interact with APC. Then they are exported from the medulla. ►Fewer than 2% of thymocytes are exported; the rest will die in the thymus. Why? Because the demands on the T cell repertoire are very strict and not many randomly-generated TCR fill the bill.
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What are the specifications for a successful T cell?
A T cell must: Not recognize “self”, that is, not bind so firmly to a self structure (MHC alone, or MHC loaded with a “self” peptide) that the T cell becomes activated; this would be autoimmunity. Not recognize free antigen (which is antibody’s job). Recognize antigenic peptide plus self MHC.The repertoire is generated and then selected within the thymus. Imagine a thymocyte that has just rearranged the genes for the alpha (V,J) and beta (V,D,J) chains of its TCR. (Note, these V(D)J are a different set from the ones B cells use.) It puts the receptors on its surface, and begins percolating through the thymus cortex, during which it will brush against the surfaces of thousands of macrophages and epithelial cells.Let us say that the CDRs of TCR a and b genes have been selected during evolution to produce receptors that are roughly complementary to the average shape of an MHC molecule. MHC is very highly polymorphic; there are thousands of alleles in the human species. Since the TCR rearrangements are random, a brand-new thymocyte’s receptors will bind to the particular MHC molecules it encounters on macrophages and epithelial cells on its trip through the thymus with either high, low, or no affinity.
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negative selection of T cells
The first possibility is that the immature T cell’s receptor binds to MHC (which will have a “self” peptide in it, derived from a normal protein) with high affinity. By high we mean high enough to result in the activation of the T cell. This is clearly an undesirable cell as its activation would result in autoimmunity. The fate of this immature cell is clear: it dies by the process of apoptosis. The proportion of cells that do this is hard to estimate, but it must be rather small and the phenomenon cannot be directly studied in the normal thymus. But with the development of transgenic techniques, it has become possible to create mice all of whose T cells bear the same receptor. With the appropriate cross-breeding scheme, mice can be generated in which all the developing T cells have high affinity for the MHC they find in the thymus. These mice have double-negative (CD4-/CD8-) thymocytes, but no double- or single- positives, and no T cells; they were all deleted. So negative selection takes place at the transition to the double-positive stage. The process is functionally identical to B cell clonal deletion. This mechanism would delete T cells reactive against the sorts of peptides you’d expect to find expressed in the thymus; but what about liver or thyroid or adrenal-specific gene products? Amazingly, the AIRE (autoimmune regulator) gene, which encodes a transcription factor, causes thymic medullary stromal cells to express a wide variety of otherwise-inexplicable “out- of-place” peptides so that reactive T cells may be removed from the repertoire. In fact, Aire- deficient people develop multiple autoimmunities.
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non-selection of T cells
Since the repertoire of T cells is generated by random association of V, (D), and J gene segments, it is reasonable to assume that most of the resultant TCR will have essentially no affinity for the particular MHC molecules they find expressed in the thymus. The cell thus receives no stimulation through its TCR. Under these circumstances it will die in a day or two, again by apoptosis.
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positive selection of T cells
If there is low but real affinity of binding between the TCR and the MHC of the thymic stroma (with a “self” peptide in the cleft), the cell binds just enough, not to be deleted, but to be signaled to mature (positive selection). The idea here is that low affinity for self might turn out in the periphery to be high affinity for self + some foreign peptide. This model explains MHC restriction: the T cells that emerge from the thymus of an “A” animal or person see antigen plus “A” MHC, because they were positively selected on “A”. There is plenty of experimental evidence to support this: for example, mice that genetically lack MHC Class I develop normal Th cells but no CTL, because there was nothing in the thymus for their developing CTL to bind to. Although it’s very difficult to do, enough x-ray crystal structures of TCR-peptide-MHC complexes have now been solved that we can say positive selection takes place when the CDR 1s and CDR2s of the TCR α and β chains interact adequately with amino acid residues on the alpha-helical sides of the peptide-binding MHC groove. This is not enough binding energy to be activating, but enough for selection. In the periphery, if the peptide that loads into MHC makes appropriately strong contacts with the CDR3s of the α and β chains, the total binding energy is now sufficient, and the T cell will be stimulated.
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extrinsic pathway.
Macrophages phagocytose antigens, digest them in phagolysosomes, load peptides onto MHC Class II, and recycle them to the surface. Of course, like all cells, macrophages also express Class I. B cells use surface Ig to bind an epitope of an antigen, internalize it, digest it to peptides which are loaded onto MHC Class II and recycled to the surface for interaction with Tfh2 cells. Dendritic cells, the best APC, take up antigen and process it for MHC Class II as do macrophages or B cells; but there is also “cross-presentation” by the intrinsic pathway, so some peptides are presented on MHC Class I as well. The result is that DC stimulate both Th and CTL.
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intrinsic pathway.
When body cells like this virus-infected hepatocyte make proteins, they shuttle peptides derived from the nascent protein from the cytosol to the endoplasmic reticulum, and thence to the surface, presented in MHC Class I.
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MINOR HISTOCOMPATIBILITY ANTIGENS
There are about 30 minors, mismatch at any of which may cause slow (chronic) rejection. One is H-Y, coded for on the Y chromosome. It’s not a transmembrane surface molecule, but an internal protein whose peptides are displayed on MHC Class I (only on male cells, of course). Because of H-Y, male skin grafts will be slowly rejected, even by fully syngeneic inbred females, while males accept female grafts without fuss.
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ARCANE TERMINOLOGY
Grafts between genetically identical individuals (e.g., inbred mice, identical twins) are called syngeneic or isografts; between non-identical members of the same species (e.g., people) are allogeneic or allografts; and between members of different species (e.g., baboon hearts into babies) are xenogeneic or xenografts. Grafts from one individual to himself (e.g., hair transplants) are autografts.
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autoimmune regulator (AIRE)
a protein that in humans is encoded by the AIRE gene.[1] AIRE is a transcription factor expressed in the medulla of the thymus and controls the mechanism that prevents the immune system from attacking the body itself. In the thymus, the autoimmune regulator causes transcription of a wide selection of organ-specific genes that create proteins that are usually only expressed in peripheral tissues, creating an "immunological self-shadow" in the thymus. It is important that self-reactive T cells that bind strongly to self-antigen are eliminated in the thymus (via the process of negative selection), otherwise they can later bind to their corresponding self-proteins and create an autoimmune reaction. So the expression of non-local proteins by AIRE reduces the threat of the occurrence of autoimmunity later on by allowing for the elimination of auto-reactive T cells that bind antigens not traditionally found in the thymus. Furthermore, it has been found that AIRE is expressed in a population of stromal cells located in secondary lymphoid tissues, however these cells appear to express a distinct set of TSAs compared to mTECs
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affects of stress on RBC development
Development from pluripotential hematopoietic stem cells to the most mature red cell in the marrow, the reticulocyte, takes 10-14 days. Reticulocytes may be identified in the peripheral blood for 1 day after release. During stress, the time for red cell production may decrease to 5-7 days, production can increase by 6-8 fold above baseline, and reticulocytes are released early. These “stress” reticulocytes may be detected in the circulation for several days.
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RBC turnover
Although the specific changes which limit the lifespan of normal red blood cells is not completely understood, several processes contribute to normal red cell turnover. Age related decreases in red cell enzyme activity, oxidative damage to cell constituents, changes in calcium balance, alterations in membrane carbohydrates and expression of senescent antigens to which there are naturally occurring antibodies all play a role in the 120 day lifespan of the normal red cell. Most of the turnover (90%) occurs in the spleen; the red pulp presents a challenging metabolic environment and the splenic macrophages provide a barrier that culls out old cells.
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Etiology
the study of the cause of disease and illness
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etiology of hemolysis
Red cells circulate as biconcave discs which have a diameter slightly larger than that of the capillary. Movement through capillary beds requires red cell flexibility which depends on the red cell plasma membrane and associated cytoskeleton. The plasma membrane is a lipid sheath containing phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol fixed to an underlying protein network. Two integral proteins penetrate the lipid bilayer, glycophorin A and component a, and are important for the negative membrane potential of the cell and glucose transport, respectively. Attached to the plasma membrane through an interaction with these integral membrane proteins, the cytoskeleton has two major constituents, spectrin and actin. These proteins, along with other associated proteins, comprise the cytoskeleton critical to the red cell shape and other mechanical properties. Defects in the cytoskeleton play a role in specific hemolytic disorders.
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metabolism of RBC
Because the mature red cell lacks a nucleus and other organelles such as mitochondria, its metabolism is simpler than other cells. Energy is generated by the breakdown of glucose through the Embden-Meyerhof pathway. The metabolism of glucose to lactate and pyruvate provides ATP necessary to maintain the plasma membrane and cytoskeleton, and energize metabolic pumps to control intracellular sodium, potassium and calcium. ATP is critical for cell survival. Three associated pathways are also important for cell function. The Rapoport Luebering pathway produces 2,3-diphosphoglycerate which stabilizes the deoxy form of hemoglobin and maximizes transport of O2 to tissues. The hexose monophosphate shunt (phosphogluconate or pentose pathway) produces reduced pyridine nucleotide which reduces glutathione and provides protection from oxidant stress. Finally, the methemoglobin reductase pathway maintains the iron in hemoglobin in the ferrous state required for reversible oxygen binding by hemoglobin. Dysfunction in these pathways may lead to decreased survival and/or altered function.
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location of erythropoiesis
The location of red cell production varies in fetal life, childhood and the adult. In the first two months of fetal life, production of red cells occurs in the yolk sac. Beginning at approximately the second month of gestation, production of red cells is transferred to the liver and spleen, peaking at about five months gestation and disappearing from these organs by normal parturition. Beginning somewhere in the second trimester, hematopoiesis moves to the bone marrow in the axial skeleton and distal long bones. At birth, this is the primary site for red cell production. During childhood, active marrow in long bones recedes so that by the adult years most hemapoiesis occurs in the axial skeleton.
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Erythropoiesis
Pluripotent stem cells are the source of all immune and hematologic cells. A regenerating pool of pluripotent stem cells retains the marrow hematopoietic capacity while development of lineage specific progenitor cells provides the required leukocytes, erythrocytes and platelets. The earliest identifiable cell in the red cell series is the burst forming unit erythroid (BFUE) and is controlled by growth factors which include IL-3 and GM-CSF derived from stromal cells, lymphocytes and macrophages. The next step (differentiation) is the formation of the colony-forming unit, erythroid (CFUE). This is under the control of the hormone, erythropoietin (EPO). The CFUE progresses (matures) into a series of erythroid precursor cells, easily identified in the marrow and termed normoblasts. Normoblasts progress through distinct steps as basophilic, polychromatophilic, orthochromatic normoblasts. This process takes 7-8 days and is associated with the progressive formation of hemoglobin, change in size of the cell and loss of mitochondria, RNA and the cell’s nucleus. The first mature red cell is termed the marrow reticulocyte which contains some of the remnants of messenger RNA and remains in the marrow for 3-4 days before being released into the peripheral blood. Reticulocytes can be identified in the peripheral blood (stain for the RNA) for an additional day with the mature erythrocyte in the circulation surviving for more than 100 days.
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Hemoglobin Structure and Synthesis
The hemoglobin molecule is composed of four subunits: two alpha chains and two beta chains. Each of the globin chains contains a pocket for heme molecule, and therefore, has a capacity to bind oxygen through its interaction with the iron (ferrous form) molecule contained in the heme ring. A single molecule of hemoglobin can bind up to four molecules of oxygen. 2,3 DPG binds to the two beta chains and stabilizes the deoxy form of hemoglobin. The globin molecules are gene products of the globin genes found on chromosomes 16 and 11. The major polypeptide genes include γ, α, δ, and β and hemoglobins include α2β2 (or A, adult hemoglobin); α2γ2 (fetal hemoglobin); α2δ2 (A2 hemoglobin). During human development there is sequential suppression and activation of individual globin genes to provide the predominant hemoglobin during that stage. In fetal life, the major form is fetal hemoglobin with high affinity for O2 (γ chains have no 2,3-DPG binding site, produce right-shifted oxy-hemoglobin dissociation curve, and favor bound oxygen at any oxygen tension) which allows the fetus to develop at the venous O2 saturation levels seen in placental circulation. During the postnatal period, there is a switch from fetal hemoglobin to adult (A1) hemoglobin. The transition takes place somewhere in the first two months of life, and after six months of life A1 is the predominant hemoglobin with a small amount of A2 hemoglobin also produced. Hemoglobin is produced in the mitochondria in maturing normoblasts. Protoporphyrin is synthesized in mitochondria and iron, which is obtained from transferrin and placed temporarily into ferritin stores, is added to the porphyrin ring to make heme. This is bound to the predominant globin chain to make hemoglobin.
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RBC membrane
is highly elastic allowing RBC to undergo large reversible deformations while maintaining its structural integrity. It is able to do this because of an extensive 2-dimensional elastic network of cytoskeletal proteins which are tethered to sites on cytoplasmic domains of transmembrane proteins embedded in the plasma membrane. Mutations in these proteins can lead to clinical conditions such as spherocytosis, where RBCs are fragile and easily destroyed leading to a chronic hemolytic anemia. Other causes include the situation where the body produces antibodies that bind to and coat the RBCs, leading to their destruction by the immune system.
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Lymphoma
are “extramedullary” (outside of the bone marrow) collections of malignant lymphoid cells, usually involving lymph nodes or other lymph organs.
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Acute leukemia
the cells are immature in their degree of differentiation and that the clinical course is usually rapidly progressive without intervention
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Chronic leukemia
that the cells are more mature in their differentiation and that the disease follows a more indolent clinical course.
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Platelets
(thrombocytes) are the cellular component of the blood responsible for hemostasis. Platelets are actually small cell fragments produced from large, polyploid cells in the bone marrow called megakaryocytes. It is estimated that a single megakaryocyte can produce up to 5,000 platelets. Small fragments of megakaryocyte cytoplasm. Diameter, 2–3 µm. Average volume, 7–8 fl. Irregular outline, light blue cytoplasm. Many small azurophilic granules. Normal platelet count: 150-400.
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Flow cytrometry
Blood counting machines aspirate a very small amount of the specimen through narrow tubing followed by an aperture and a laser flow cell. Laser eye sensors count the number of cells passing through the aperture.
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Coulter principle
state that particles pulled through an orifice, concurrent with an electronic current, produce a change in impedance that is proportional to the column of the particle traversing through the orifice.
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calculating MCH from HGB and RBC
MCH = (HGB ¸ RBC) ×10- mean cell hemoglobin=hemoglobin concentration/red blood cell count* 10
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calculating HCT from RBC and MCV
HCT(%) = (RBC × MCV) ¸ 10- hematocrit= (red blood cell count*mean corpuscular volume)/10
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calculating MCHC from HGB and HCT
MCHC = (HGB ¸ HCT) × 100- mean cell hemoglobin concentration=(hemoglobin concentration/hematocrit)*100
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calculating MCV from HCT and RBC
MCV = (HCT ¸ RBC) X 10- mean corpuscular volume=hematocrit/ red blood cell count)*10
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five types of white blood cells
neutrophils, lymphocytes, monocytes, eosinophils, and basophils
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Normal peripheral blood smear
circular uniform size, 6-8um. Central pallor, <1/3 of diameter.
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Microcytic and hypochromatic RBCs
RBCs are smaller with larger central pale area, often seen in iron deficiency anemia
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bite cells
due to removal of Heinz body in spleen. Associated with G6PD deficiency
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Schistocytes
hereditary of immune hemolytic anemia. Smmal and spherical, no central pallor
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Target cells
central hemoglobin, target shape. Thalassemia, hemoglobin c, iron deficiency, liver disease
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Basophilic stippling
evenly dispersed find blue granules compose of aggregated rRNA. Caused by lead poisoning, porphyria, infection, hemoglobinpathies, thalassemia, myelodysplasia, siderblastic anemia
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Howell-Jolly bodies
single, dense blue dot composed of nuclear DNA remnant. Caused by post-splenctomy, functional asplenia, megaloblastic anemia, myelodysplasia.
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Heinz Body
crystal violet with supravital dye composed of denature hemoglobin attached to the inner cell membrane. Caused by G6PD deficiency. Associated with bite cells.
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Dohle Body
pale blue inclusion at the periphery of the cytoplasm composed of condensed RNA. Caused by inflammation, burns, or pregnancy.
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Toxic Granulation (Hypergranularity)
increased number of granules, due to rapid cell division. Often associated with Dohle bodies and toxic vacuolization. Caused by bacterial infection, marrow recovery, stimulation of GM-CSF.
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Hypersegmented Neutrophils
contain 6 or more nuclear lobes. Can be caused by megaloblastic anemias (anemias caused by failure of bone marrow blood-forming cells to make DNA, often caused by vitaminB12 or folate deficiencies.
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Define the platelet count and recognize platelets on a peripheral smear.
Platelet count (PLT)- analogous to RBC. Mean platelet volume (MPV)
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pattern-recognition receptors (PRR
Most cells have pattern-recognition receptors (PRR), on outer or inner membranes that recognizes PAMPS.
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Toll-like receptor
a type of PRR, called because of homology to the Toll gene in the fruit fly (which controls innate immunity in invertebrates). Each TLR can recognize a foreign molecular structure that we humans don’t have. When TLR are activated, when a foreign pattern binds causing a signaling cascades causing expression of factors that cause or increase inflammation. TLR have a switch like response because a complex must form to activate TLR, keeping us from responding when its trivial. All TLR except TLR3 use the IRAK pathway (TLR3 uses IRF). The net effect is to activate NF-κB.
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damage-associated molecular patterns (DAMP)
Stress or damage indicator molecules expressed by body cells. As cells get damaged and stressed, they release certain of their internal molecules (the DAMPs), and some TLRs bind them, too, increasing the local inflammation.
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TLR4
binds lipopolysaccharide (part of the cell wall of Gram-negative bacteria)
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TLR2
binds peptidoglycan (Gram-positive bacteria)
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TLR3
binds double-stranded RNA, which almost all viruses make.
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Identify the final transcription factor that is most commonly activated in inflammation
The mother of all inflammatory transcription factors, NF-κB.
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Define cytokine and chemokine
They are factors that cause or increase inflammation (increased blood vessel diameter, stickiness, and leakiness causing efflux of fluid and phagocytic white blood cells). These again are examples of gene duplication. They act at close range.
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Describe the function of the innate immune response.
It’s main function is to detect intruders in the body and then inactivate, destruct and remove. It needs to be able to determine innocent and evil invaders. It is quick and is shared among all animals. Innate immunity recognizes three sorts of things: PAMP; DAMP; The absence of certain normal cell surface molecules, which would certainly indicate a problem. This recognition is done by NK cells. It’s impossible to have a good adaptive response if there isn’t an innate response to prime the pump (some vaccines have innate immune stimulators added for this reason).
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dendritic cells
(DC)
Special phagocytic cells at the interface between body and world. Their membranes are highly branched. At a wound site, immature DC are activated by cytokines and chemokines and take up foreign molecules derived from invaders. DC than leave the periphery and travel in lymoatics to lymph node where they present the antigen (thus antigen presenting cells) to lymphocytes. In the lymph nodes there is the correct balance of B cells, T cells, and DC to get the adaptive immune system started.
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Discuss in principle the role T cells play in immunity.
T cells recognize antigens with surface receptors that are presented to them by DC. This activates the T cell and it proliferates allowing the daughter cells to travel throughout the body. At infection sites, they are restimulated by local APC and release lymphokines (cytokines made by a lymphocyte)- helper. Another type of T cell are specialized for killing any body cell they recognize as containing abnormal molecules, which may be the result of damaging mutations, or the products of intracellular pathogens like viruses. They mature in the thymus. Cytotoxic or killer T cells, CTL for short, destroy any body cell they identify as bearing a foreign or abnormal antigen on its surface.
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B Cells
arrange for the phagocytosis and destruction of foreign materials. They also recognize antigens via surface receptors; then they release soluble versions of their receptors, namely antibodies. B cell receptors see antigen alone, and do not require the simultaneous recognition of an associated MHC molecule, or presentation, the way T cells do. When a B cell binds antigen, it is activated to proliferate and differentiate (usually after help from a Tfh cell.) A fully differentiated B cell is a plasma cell, a protein-production factory.
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Describe some of the functions of antibodies.
Antibodies bind to the corresponding antigen, and this may be enough to neutralize a toxin, or prevent a microorganism from binding to its target cell. The first time an antigen enters the body at the mucous membranes, it will reach the nearby lymphoid tissues, where there are T and B cells; the environment there favors the production of IgA and, in certain people, IgE too. The IgA is secreted and local immunity is established. If the antigen penetrates further into the body, it reaches draining lymph nodes or the spleen, and there the environment favors first IgM production, and then IgG, which bind up pathogens as they circulate. When most antigens enter the body, there will be both T and B cell responses to them. Some will be more important than others for that particular antigen.
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Type I immunopathology
immediate hypersensitivity. This is seen in patiens with too much IgE to an environment antigen, due to pollen or food leading to allergic symptoms. It is partly genetic.
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Type II immunopathology
autoimmunity due to antibodies which react against itself. There are a number of ways this can come about: For example, if a foreign antigen happens to look like a self-molecule, the response to the antigen may accidentally “cross-react” with self.
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Type III immunopathology
can occur whenever someone makes antibody against a soluble antigen. Immune complexes of antigen and antibody are usually eaten by phagocytes, but if they are a bit too small for that, they may instead get trapped in the basement membranes of capillaries they circulate through. The trapped complexes activate complement and the usual inflammatory response occurs, with the tissue damaged as an innocent bystander. No matter what the antigen is, the symptoms tend to be the same: arthritis, glomerulonephritis, pleurisy, rash. Foreign antigens that cause Type III include drugs like penicillin when given in large doses, and foreign serum, such as horse antiserum to rattlesnake venom. More troublesome is when the antigen is internal, as part of an autoimmune process. Thus people with systemic lupus erythematosus, SLE, make antibody to their own DNA, some of which can always be found free in blood.
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ype IV immunopathology
t cell mediated and can be autoimmune or innocent bystander injury from another infection. For example, in tuberculosis most of the cavity formation in lungs is T cell-mediated, not bacterium-mediated.
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Chronic frustrated immune response
when the antigen is not self, but is something else you cant get rid of (gut bacteria). Inflammatory bowel disease, celiac disease
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HIV/AIDS
The AIDS virus, HIV-1, infects Th cells because its envelope glycoprotein, gp120, binds to the CD4 molecules they have on their surface. Inside, it uses its enzyme, reverse transcriptase, to copy its RNA into DNA which becomes inserted into the cell’s own DNA. It then is latent, and seems to become reactivated when the T cell is activated by antigen, leading to a progressive loss in Th cells that, as you can imagine, is a critical blow to all branches of immunity.
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Anemia
Insufficient red cell mass to adequately deliver oxygen to peripheral tissues.
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Measurements to define the existence of anemia
Hemoglobin concentration (Hgb), g/dl; Hematocrit (Hct), % volume of red cells in blood; Red blood cell count, cells x1012/L. Reference range provides values below which anemia is defined: variation based on age, gender, geography. 3% is higher and lower than reference range.
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Reticulocyte Count/Index
It is not a count; it is a percentage. Reticulocytes (3 days in marrow, 1 day in blood) contain stainable mRNA. Counted as % of 1,000 cells with stain (0.4-1.7%). Normal range defined by steady state with production ~1% RBC mass/day. Increased RBC production, usually see 3-5 fold increase. More precise measures: Absolute count = % x RBC (~ 50,000/μl);
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Retic index
(correct for effect of altered red cell concentration and stress reticulocytes) RI= Retic count * patient Hgb/normal Hgb*1/stress retic factor (1.5, 2, 2.5)
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Anemia Clinical Signs and Symptoms
Red cell is one component of mass transport of O2 to tissue (cardiovascular and pulmonary). Anemia stresses oxygen transport. Degree of stress dependent on extent of anemia, ability of other systems to compensate, and speed at which anemia develops. Symptoms: shortness of breath, fatigue, rapid heart rate, dizziness, claudication, angina, pallor. Signs: tachycardia, tachypnea, dyspnea, pallor
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Iron Metabolism
Fe exists in two valence states and activity may depend on a specific state. In aqueous solutions, Fe forms insoluble hydroxides unless bound (protein, heme, etc.). Fe more soluble at low pH. Fe balance: controlled by absorption; no active excretion mechanism. Losses each day are small: loss from exfoliation of skin and mucosal surfaces (GI, skin); in the urine or with menstruation.
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Iron Distributions
65% in hemoglobin; 6% in myoglobin; 0.1 in transferrin; 13% in ferritin (storage); and 12% in hemosiderin (storage). Iron also exists in many other proteins that are important but do not make up a large %.
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Iron absorption
Two types of iron presented in food: elemental and heme-bound iron. Most information available for elemental iron. Occurs in the duodenum. In the mucosal cell, ferritin binds to iron and transport iron through the basal side (through ferroportin). Intraluminal factors that increase iron absorption: Gastric factors (low pH, gastroferrin); Presence of protein, amino acids; Vitamin C; Phytates, oxalates; Amount of iron ingested. Extraluminal factors: Erythropoietic activity
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Transferrin
transport iron, 84 kDa plasma protein. The Erthyrotes precursor cells have receptors that are internalized. As
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How to diagnose iron deficiency anemia
A sufficiently low hemoglobin (Hb) by definition makes the diagnosis of anemia, and a low hematocrit value is also characteristic of anemia. Further studies will be undertaken to determine the anemia's cause. If the anemia is due to iron deficiency, one of the first abnormal values to be noted on a CBC, as the body's iron stores begin to be depleted, will be a high red blood cell distribution width (RDW), reflecting an increased variability in the size of red blood cells (RBCs). In the course of slowly depleted iron status, an increasing RDW normally appears even before anemia appears. A low mean corpuscular volume (MCV) often appears next during the course of body iron depletion. It corresponds to a high number of abnormally small red blood cells. A low MCV, a low mean corpuscular hemoglobin and/or mean corpuscular hemoglobin concentration, and the appearance of the RBCs on visual examination of a peripheral blood smear narrows the problem to a microcytic anemia (literally, a "small red blood cell" anemia). The numerical values for these measures are all calculated by modern laboratory equipment. The blood smear of a patient with iron deficiency shows many hypochromic (pale and relatively colorless) and rather small RBCs, and may also show poikilocytosis (variation in shape) and anisocytosis (variation in size). With more severe iron-deficiency anemia, the peripheral blood smear may show target cells, hypochromic pencil-shaped cells, and occasionally small numbers of nucleated red blood cells. Very commonly, the platelet count is slightly above the high limit of normal in iron deficiency anemia (this is mild thrombocytosis). This effect was classically postulated to be due to high erythropoietin levels in the body as a result of anemia, cross-reacting to activate thrombopoietin receptors in the precursor cells that make platelets; however, this process has not been corroborated. Body-store iron deficiency is diagnosed by diagnostic tests, such as a low serum ferritin, a low serum iron level, an elevated serum transferrin and a high total iron binding capacity. A low serum ferritin is the most sensitive lab test for iron deficiency anemia. However, serum ferritin can be elevated by any type of chronic inflammation and so is not always a reliable test of iron status if it is within normal limits (i.e., this test is meaningful if abnormally low, but less meaningful if normal).
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Causes of iron deficiency anemia
parasitosis, blood loss (menorrhagia, peptic ulcer, GI bleed), lack of iron in the diet, inability to absorb iron (celiac disease, inflammatory bowel disease), pregnancy
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Describe the symptoms, signs, and laboratory findings associated with iron deficiency anemia.
Breathlessness, anxiety, irritability, angina, constipation, sleepiness, tinnitus, mouth ulcers, palpitations, hair loss, fainting or feeling faint, depression, breathlessness, twitching, pale skin, tingling, missed menstrual cycle, angular cheilitis
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Describe the effects of over accumulation of iron in the body and describe two treatments for iron overload.
The most important causes are hereditary haemochromatosis (HHC), a genetic disorder, and transfusional iron overload, which can result from repeated blood transfusion. Routine treatment in an otherwise-healthy person consists of regularly scheduled phlebotomies (bloodletting). For those unable to tolerate routine blood draws, there is a chelating agent available for use
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Pre-natal hematopoiesis
In the embryological stage, primitive blood cells (primarily RBC) are produced in the yolk sac, until about the third month of gestation. In the fetal stage, the hematopoiesis occurs in the liver from 2nd to 7th month and to a lesser extent in the spleen. At time of birth, bone marrow is the site.
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Childhood hematopoiesis
Most of the marrow cavity is hemapoietically active. As the child ages, it moves to the axial skeleton.
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Adult hematopoiesis
at about 18 years old, 90% of active marrow is located in the vertebrae, pelvis, sternum, ribs, and skull. The liver and spleen retain their ability for hematopoiesis during certain illness.
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Hematopoietic stem cells (HSC)
mother of all blood cells and gives rise to lymphoid and myeloid elements. Are multipotent, may self renew or commit to becoming one the the pluripotential stem cells. Very rare in marrow. Not morphologically recognizable. Express CD34, CD117. Unique function of ASYMMERTRIC CELL DIVISION-> 1 HSC daughter and 1 multipotent progenitor cell
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Pluripotential stem cells
colony forming units (CFUs), GEMM = granulocyte/erythroid/monocyte/megakaryocyte). Includes CFU-GEMM , CFU-L. CFU-L is the mother of all lymphoid cells. CFU-GEMM is the mother of all non-lympoid cells. Pluripotential stem cells have xome ability to self-renew or they may commit to becoming progenitor cells
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Progenitor cells
the cells ability to self renew is severly limited and they are irreversibly committed to differentiate along one or at most two lineages. Progenitor cells give rise to precursor cells. Multipotent – capable of differentiating to all lymphoid and myeloid lineages. Lineage-restricted progenitor cells. Oligopotent – common myeloid progenitor cells and common lymphoid progenitor cells
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The myeloid progenitors
CFU-GM (Granulocyte/Macrophage); CFU-G (Granulocyte); CFU-M (Monocyte); CFU-E (Erythroid); CFU-Meg (Megakaryocyte); CFU-Eo (Eosinophil); CFU-Baso (Basophil).
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Burst Forming Unit-Erythroid (BFU-E)
the progenitor cell that gives rise to CFU-E; the name derives from the impressive, exuberant appearance of the colonies that these cells form.
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Precursor cells
these cells are the recognizable, maturing cells that are visible and counted when a marrow specimen is examined (myeloblasts, myelocytes, orthochromic normoblast). Precursors are capable (up to a point) of cell division but cannot self-renew. Precursor cells give rise to the mature, functional cells in the peripheral blood, lymphoid organs, and reticuloendothelial system.
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Amplification
the self renewal phenomenom creates a situation where there can be a relative low number of stem cell giving rise to billions and billions of blood cells every day.
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Hematopoietic growth factors
there are many the important ones are erythropoietin (EPO), thrombopoietin (TPO), granulocyte-monocyte colony stimulating factor (GM-CSF), monocyte colony stimulating factor (M-CSF), IL5 and IL3
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Erythropoietin (EPO)
made by certain kidney cells in response to hypoxia, promotes erythropoiesis
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Thrombopoietin (TPO)
promotes megakaryopoiesis
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Granulocyte-monocyte colony stimulating factor (GM-CSF)
promotes granulopoiesis and monopoiesis
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Granulocyte colony stimulating factor (G-CSF)
promotes granulopoiesis
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Monocyte colony stimulating factor (M-CSF)
promotes monopoiesis
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Interleukin-5 (IL-5)
promotes production of eosinophils
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Interleukin-3 (IL-3)
promotes production of basophils
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Erthrocyte maturation
pronormoblast -> basophilic normoblast -> polychromatophilic normoblast -> orthochromatic normoblast -> reticulocyte -> erythrocyte.
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Pronormoblast
the first erythroid precursor, is up to 18um in diameter. The large nucleus contains finely granular chromatin, and one to two fairly inconspicuous nucleoli. The cytoplasm contains a lot of RNA so it stains intensely blue with the Wright stain.
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Basophilic normoblast
the second erythoid precursor. The cytoplasm is still pretty basophilic although a little lighter in color. Coarse condensation of the nuclear chromatin has begun and the cell is 12-14 um in diameter.
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Polchromatophilic normoblast
the third erythoid precursor is 10-12 um in diameter. Hemoglobin is starting to accumulate in this cell. The combination of this hemoglobin and still plentiful RNA gives it a purphlish-ble color to the cytoplasm with a Wright stain. The chromatin has condensed to form chunks.
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Orthochromatic normoblast
the fourth erthoid precursor is 8-10 um in diameter. The cytoplasm of this cell has a distint red-orange hue due to accumulated hemoglobin. The small, shrunken pyknotic nucleus is soon to be extruded.
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Reticulocyte
The fifth erthoid precursor. This anucleate cell still contains ribosomes and mitochondria. A Wright-stained reticulocyte will have a bluish-purple tinge and is said to be polychromatophilic. Supravital staining (e.g. new methylene blue) of a reticulocyte causes the ribosomes and mitochondria to condense and form strands; reticulocytes can be identified and counted under the light microscope after supravital staining. First stage to have no nucleus. Some residual RNA (not very much).
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Erythrocyte
reticulocyte ribosomes continue to produce hemoglobin for two to three days. At the end of this time ribosomes and associated RNA are degraded and cell is now mature erythrocyte. The diameter is 7-8 um. The cell is a biconcave-shaped disc; the biconcavities create the central area of pallor that is seen in erythrocytes in blood smears. The cytoplasm of a Wright-stained erythrocyte is orange-red in color.
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Granulocyte maturation
Granulocytic precursors in ascending order of maturity are: myeloblast -> promyelocyte -> myelocyte -> metamyelocyte -> band -> segmented granulocyte (neutrophil, most common, eosinophil, basophil).
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Myeloblast
The first neutrophilic precursor is around 15mm across, has a high nuclear to cytoplasmic (N:C) ratio, fine nuclear chromatin, and 1 or more nucleoli. The cytoplasm is stained blue (basophilic) by the Wright stain because it has a lot of RNA in it. By definition, the myeloblast contains no cytoplasmic granules.
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Promyelocyte
This cell is around 20 mm across. The chromatin of the promyelocyte is more condensed and therefore appears coarser; nucleoli are often still present. The defining feature of a promyelocyte is the presence of a variable number of large (0.8 mm), purplish primary granules (aka azurophilic granules). The cytoplasm is still decidedly blue.
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Myelocyte
The myelocyte is around 15 mm in diameter. The defining feature of the myelocyte is the appearance of lavender secondary granules, smaller than the primary granules, and a prominent paranuclear Golgi apparatus. There are less primary granules in the myelocyte than the promyelocyte. The round to oval nucleus has more condensed, coarser chromatin, and nucleoli are less conspicuous. The myelocyte is the last neutrophilic precursor with the ability to divide. paranuclear ‘hof’ (Golgi app.)
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Metamyelocyte
The metamyelocyte is 14-16 mm in diameter. The far more abundant secondary granules that impart a pinkish purple hue to the cytoplasm obscure primary granules, and primary granules may be absent altogether by this stage. The nuclear chromatin is distinctly condensed and coarse. The nucleus is indented (kidney shaped); by definition the indentation is less than half of the diameter of the nucleus.
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Band
band has a diameter of around 13 mm. The horseshoe‑shaped nucleus has evenly dispersed clumps of chromatin. Secondary granules far outnumber primary granules, if primary granules are even seen. Cytoplasm: same as metamyelocyte Nucleus: rod or band shaped coarsely clumped chromatin
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Segmented neutrophil
The segmented neutrophil has the same size and cytoplasmic properties as the band. The nuclear chromatin is coarsely clumped. The nucleus is segmented into two to five (usually three) distinct lobes that are connected by thin chromatin strands.
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Megakaryocyte maturation and platelet production
The megakaryocyte maturation scheme is as follows: megakaryoblast -> promegakaryocyte -> megakaryocyte -> platelet.
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Characterization of megakaryocyte
DNA in developing megakaryocytes undergoes repeated doublings without cell division. This process is known as endoreduplication and results in a multilobulated nucleus containing 16, 32 or 64 sets of chromosomes (32 sets is the most common). As the megakaryocyte matures, the cytoplasm becomes filled with small reddish- purple granules and there is a network of membranes that allow platelets to be shed from the mature megakaryocyte. They are shed directly into the marrow vascular sinuses from the ends of megakaryocyte cytoplasmic arms that extend through the endothelium into the vascular lumens.
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Megakaryoblast
This cell is 20 to 30 mm in diameter. It contains a large round or indented nucleus with nucleoli and a thin rim of basophilic (i.e. RNA-filled) cytoplasm. Often cannot be differentiated from other types of blasts based on morphology alone.
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Promegakaryocyte
Endoreduplication has commenced, so the nucleus is lobulated and the chromatin is more condensed. The appearance of granules in the cytoplasm attenuates the intense blue color seen in the cytoplasm of the megakaryoblast.
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Megakaryocyte
The mature megakaryocyte has a lobulated, endoreduplicated nucleus, and copious cytoplasm that appears finely granular and purplish. A million platelets are produced every second (around 100 trillion a day). Products can be increased by up to 20-fold (around 2 gazillion a day) in response to stress. Initiated by action of the cytokine thrombopoietin (TPO) on an megakaryocyte/erythroid progenitor cell. Earliest lineage-committed cells, megakaryoblasts, are very rare, and require special studies for identification. Very large cells with highly folded, multilobular nuclei and abundant finely granular cytoplasm. Very easily identified in normal marrow, though they account for only 0.05% of nucleated marrow cells. Possess pseudopods, which they insert in bone marrow sinuses to allow direct shedding of platelets into the circulation
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Platelet
The mature megakaryocyte hangs out beside the marrow sinuses. There it extends strands of its membrane bound cytoplasm into the lumen of the sinus; platelets are formed when small chunks of this strand break off and float away. Platelets are anucleate, 2 to 4 mm in diameter, and appear granular and purplish.
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Monocytes/macrophages
Monocyte precursors in ascending order of maturity are: monoblast -> promonocyte -> monocyte. Monocyte maturation is characterized by the monocyte nucleus progresses from a round, indented to variably and irregularly shaped. The cytoplasmic maturation is marked by the appearance of peroxidase- positive lysosomal granules and vacuoles. Monocytes travel through the blood to the connective tissue of the body where they become macrophages and comprise the active cells of the mononuclear phagocyte system.
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Monoblast
This cell is 16 mm in diameter. The nucleus is slightly indented, and contains fine chromatin and nucleoli. The cytoplasm is very blue and lacks granules. This cell may be difficult to distinguish from a myeloblast.
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Promonocyte
This cell is 16 to 18 mm in diameter. The nucleus is indented or folded and the chromatin is more condensed than in the promonocyte, and one or more nucleoli may still be present. Red-purple granules are scattered though the blue cytoplasm.
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Monocyte
With a diameter of 15 to 18 mm, this cell is the largest in the peripheral blood. The nucleus assumes a variety of shapes including reniform and even band-like. The chromatin does not have the clumpy appearance such as in the band or segmented neutrophil. Nucleoli are absent. The cytoplasm is bluish with an opaque ground-glass appearance. Red-purple granules are scattered through the cytoplasm, but are nowhere near as abundant as in the neutrophil.
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Erythropoiesis
3 – 5 mitotic divisions between pronormoblast and polychromatophilic normoblast stage. 2 – 7 days for pronormoblast to mature into orthochromic normoblast. 1 more day to extrude the nucleus from the orthochromic normoblast. Reticulocyte further matures for 2 – 3 days in bone marrow before it is released into the peripheral blood. Red cell has life span of 120 days in peripheral blood
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Neutrophil Granulocytes –Marrow
Main cytokine initiating neutrophil production: Granulocyte-colony stimulating factor (G-CSF). Myeloblasts, promyelocytes, and myelocytes undergo cell division (mitotic pool): (4 – 5 cell divisions, 3-6 days spent in this pool). Metamyelocytes, bands, and segs do not divide (maturation and storage pools). (5-7 days in maturation and storage pools; 3 times as many cells as mitotic pool) Blood: Leave storage pool (in bone marrow) and enter peripheral blood. 50% circulate freely (circulating pool); 50% adhere to walls of blood vessels (marginal pool). Neutrophils continually move between circulating and marginal pools. Average time spent in peripheral blood is 10 hours
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Erythrocytes
120 days life span in the peripheral circulation; around 175 billion produced per day by the average 70kg man
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Platelets
7 to 10 days life span in the peripheral circulation; around 200 billion produced per day by the average 70kg man
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Neutrophils
7 hours half-life in the peripheral circulation; around 70 billion produced per day by the average 70kg man
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Describe the process of bone marrow biopsy and aspirate.
Typically, the aspirate is performed first. An aspirate needle is inserted through the skin using manual pressure and force until it abuts the bone. Then, with a twisting motion of clinician's hand and wrist, the needle is advanced through the bony cortex (the hard outer layer of the bone) and into the marrow cavity. Once the needle is in the marrow cavity, a syringe is attached and used to aspirate ("suck out") liquid bone marrow. A twisting motion is performed during the aspiration to avoid excess content of blood in the sample, which might be the case if an excessively large sample from one single point is taken. Subsequently, the biopsy is performed if indicated. A different, larger trephine needle is inserted and anchored in the bony cortex. The needle is then advanced with a twisting motion and rotated to obtain a solid piece of bone marrow. This piece is then removed along with the needle. The entire procedure, once preparation is complete, typically takes 10–15 minutes.
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Myeloid: erthroid ratio (M:E ratio)
the ratio of granulocytic to erythoid precursors should be around 3:1, although there is a wide normal range.
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Maturation of precursors
a normal bone marrow has heterogeneous appearance due to the presence of megakaryocytes, and granulocytic and erythroid precursors at all different stages of maturation. Lack of maturation of hematopoietic precursor cells will impart a homogenous appearance to the marrow that could be inductive of such things as acute leukemia.
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Megakaryocytes
a large bone marrow cell with a lobulated nucleus responsible for the production of blood thrombocytes (platelets), which are necessary for normal blood clotting. Megakaryocytes are large and weird looking. If they increase, decrease or present in normal numbers is somewhat subjective.
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Iron storage in macrophages
Iron storage in macrophages is also somewhat subjective but too little or too much is indicative of pathology. The Prussian blue stain is used to asses iron stores.
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bone lesions in bone marrow
Lesion are indicative of fibrosis, metastic tumor and granulomas.
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marrow cellularity
Marrow cellularity means the portion of the marrow that is hematopoietically active; non- hematopoietically active is occupied by stromal elements, which is usually mostly fat. The cellularity of the bone marrow decreases with age (approximately 100-age). Could be hyperplastic due to increased proliferation of one more lineages; usually due to increased signalling by HGFs. (e.g. secondary erythroid hyperplasia in smokers). Could be neoplastic due to a neoplasm of hematopoietic cells HYPOCELLULAR MARROW: Classified as: hypocellular (cellularity decreased but some marrow cells present) or aplastic (marrow cells essentially absent). Possible causes include: autoimmune attack on marrow cells; viral attack on marrow cells; hematopoietic neoplasms; malnourished state (rare)
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Cellular components of bone marrow
fibroblasts, macrophages (they deliver iron for hemoglobin production), adipocytes, osteoblasts, osteoclasts, endothelial cells.
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Explain the generally expected findings in peripheral blood and marrow for cytopenias resulting from increased consumption/ destruction versus cytopenias resulting from decreased production.
With increased consumption/ destruction you would find an increased retic count. If it was due to hemolysis you would find a high bilirubin. With cytopenias caused by reduced production you would see a normal or low retic count, microcytic anemia.
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List different types of pathologic processes that might be seen on a bone marrow biopsy in a patient with peripheral cytopenias.
Thrombocytopenia or leukopenia in patients with chronic liver disease is often attributed to functional overactivity of the spleen (hypersplenism). Despite being a fairly common phenomenon, there is a paucity of reports on the prevalence of this syndrome in stable chronic liver disease patients with or without severe fibrosis/cirrhosis. Lupus can also cause cytopenia
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For iron deficiency anemia(IDA), list the typical CBC findings of IDA and recognize the typical morphologic features seen on the peripheral blood smear in cases of IDA.
RBC count is low. Small RBC, target cells. HCT, MCH, MCHC, HGD decrease. RDW increase, microcytic anemia
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For megaloblastic anemia, list the typical CBC findings of megaloblastic anemia, recognize the classic morphologic features seen on the peripheral blood smear in cases of megaloblastic anemia, and explain the etiology and pathogenesis of megaloblastic anemia.
Megaloblastic anemia (or megaloblastic anaemia) is an anemia (of macrocytic classification) that results from inhibition of DNA synthesis during red blood cell production. Hypersegmented neutrophil- more than 6 lobes on nucli. Target cells. Decrease RBC, HGB, HCT. Increased MCV, MCH, RDW. No change in MCHC. Decreased retic count. Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs). Macrocytes, ovalocytes, and howell-jolly bodies. Can be caused by vitamin B12 deficincy (due to IF factors), folate deficiency, myelodysplastic syndrome, chemo related. Methlymalonic acid (MMA)- is increased in vitamin B12 deficiency. Neutrophil granulocyte may show multisegmented nuclei,
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Leukocytes
nucleated cells of the blood; white blood cells. They make up the buffy coat
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Mononuclear cells
Leukocytes whose nucleus has a smooth outline; monocytes (immature, becoming mature macrophages in the tissues), and lymphocytes. Its sometimes hard to tell the difference between macrophages and lymphocytes, hence this blanket term.
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Polymorphonuclear cells
Cells whose nucleus is lobulated, also called granulocytes because they have (usually) very prominent cytoplasmic granules including eosinophis, basophils (closely related to mast cells), and neutrophils.
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Serum
What is left over after blood clots. the component that is neither a blood cell (serum does not contain white or red blood cells) nor a clotting factor; it is the blood plasma not including the fibrinogens. Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances (e.g., drugs and microorganisms).
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plasma
the pale-yellow liquid component of blood that normally holds the blood cells in whole blood in suspension. It makes up about 55% of the body's total blood volume.
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normal white blood differential
Adults: Total white blood cells (WBC): 4,500-10,500 per mL of blood (4.5-10.5 x 109/L). Neutrophils 40-60% (in toddlers this N:L ratio is reserved). Eosinophils 1-4%. Basophils 0.5-1%. Monocytes 2-8%. Lymphocytes 20-40%
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Central lymphoid organs
where lymphocytes develop; the bone marrow (B cells) and thymus (travel from bone marrow than mature in the thymus are the T cells).
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Peripheral lymphoid organ
where mature cells are organized to trap and respond to foreign invaders; these organs include lymph nodes, spleen, Peyer’s patches and mesenteric lymph nodes of the gut (GALT= gut associated lymphoid tissue), tonsils, and adenoids. Most lymphocytes are found in peripheral lymphoid organs but are all over in the blood and lymph.
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Describe the recirculation of lymphocytes from blood to lymph and back
Arterioles enter at the hilum and split up into capillaries, which drain into venules, veins exist at the hilum. Lymph channels (afferent) enter at the periphery and lymph flows into the subscapular sinus, percolates through the substance of the node and leaves in efferent lymphatics via the hilum.
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Cortex
The node’s outer region, under the subcapsular sinus, and it is full of tightly packed (but highly motile) lymphocytes arranged in follicles. The cortex is primarily B cells.
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germinal centers
very crowded areas with many dividing cells in the cortex and represent visible evidence of an immune response. They appear paler because lymphocytes are actively dividing, therefor are larger with more cytoplasm. It is a sign of antibodies being made by B cells.
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deep or paracortex
Sits below the germinal centers and is a little less dense, but still has huge numbers of lymphocytes. Dendritic cells that arrive in the afferent lymph tend to gather at the interface between the cortex (mostly B cells, arising from the bone marrow) and the paracortex (mostly T cells, arising from the thymus). Follicular helper T cells (Tfh) migrate from the deep cortex into the follicles where they help B cells get activated. The paracortex primarily contains T cells.
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Lymphocyte recirculation
a lymphocyte in the blood encounters the cells lining certain postcapillary venules in the peripheral lymphoid tissues, especially lymph nodes. These endothelial cells are unusual¾not flat as is the usual case, but high and cuboidal. Recirculating lymphocytes may bind to and pass between the endothelial cells into the lymph node, where they may stay, or move eventually into the lymph which drains from that lymph node. Lymph drains into the largest lymph channels (e.g. thoracic duct); from there it is emptied into the venous blood and the circulatory loop starts over again. Thus there are two lymphocyte circulations, blood and lymphatic, in which lymphocytes cross from blood to lymph at the nodes, and from lymph back to blood at the heart. All tissues, leukocytes preferentially leave the blood via postcapillary venules; but the ones in the lymph nodes are specialized for high-turnover recirculation.
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Medulla
deeper in lymph node, largely filtering.
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Antigen
a substance that can be recognized by the immune system. Immunogen: a type of antigen that is in a form that can give rise to an immune response, that is, which can immunize.
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Antigenic determinant
The part of an antigen that fits into the lymphocyte receptor
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Epitope
The part of an antigen that fits into the lymphocyte receptor
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Tolerogen
a form of an antigen that does not trigger an immune response and causes tolerance.
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Discuss lymphocyte activation by antigen with respect to: receptor binding, proliferation, differentiation
Each lymphocyte has receptors for antigen; there are thousands on each cell, but all are identical so that each cell has just a single specificity, different from nearly all the others. T cell receptors are composed of alpha and beta chains; B cell receptors are samples of the antibodies that the cell will eventually secrete. The part of an antigen that fits into the receptor is the antigenic determinant or epitope. To activate the T or B cell several conditions must be met: the fit between receptor and the antigen it sees must be good (specific) enough, several nearby receptors must be simultaneously bound by antigen, and other cell surface molecules must be involved too (accessory interactions or costimulation). Once the cell is correctly activated it begins to proliferate. Lymphocytes can divide as fast as every 6 hours, so in just a few days you have thousands of cells specific for the antigen that got the process started. These cells also differentiate: into effectors that do the job (B cell blasts and plasma cells that release antibodies into the blood; helper T cells that pour out cytokines; killer T cells that induce their targets to die) and into memory cells that recirculate efficiently and are very easily triggered by another exposure to antigen.
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T Lymphoblast
a stimulated T cell that becomes large and differentiated.
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Plasma cell
A B cell also becomes a (B) lymphoblast and then goes beyond that to the incredibly specialized plasma cell, with an enormous protein-making rough endoplasmic reticulum (RER). They work so hard to pump out antibody that many of them will die in a few days; others back off a few notches and remain as long-term memory cells.
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Humoral immunity
also called the antibody-mediated beta cellularis immune system, is the aspect of immunity that is mediated by macromolecules (as opposed to cell-mediated immunity) found in extracellular fluids such as secreted antibodies, complement proteins and certain antimicrobial peptides. Humoral immunity is so named because it involves substances found in the humours, or body fluids.
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Cell-mediated immunity
an immune response that does not involve antibodies, but rather involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Historically, the immune system was separated into two branches: humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens. Cytotoxic T cells cause death by apoptosis without using cytokines, therefore in cell mediated immunity cytokines are not always present.
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Major causes of underproduction anemias
Along with iron deficiency anemia, anemia associated with chronic infection or inflammation (infection: e.g., osteomyelitis, pulmonary abscesses/infection, meningitis, chronic GU infection) inflammatory disease (e.g., rheumatoid arthritis, systemic lupus erythematosis, rheumatic fever), malignant disease (e.g., carcinoma, Hodgkin’s disease, etc), renal insufficiency, endocrine disorders, lead intoxication, and protein calorie malnutrition. These are also called anemias of chronic disease.
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Clinical features of underproduction anemia
many of these anemias include those associated with the specific underlying condition. Infection and inflammation may include fever, arthritis, arthralgia or symptoms involved with an infected or inflamed area. Lead intoxication can present with additional personality changes, irritability, and headache; abdominal pain and vomiting may signify the effect of ingested lead. Renal dysfunction and its anemia may have an insidious onset but symptoms, signs, and lab findings will be consistent with the diagnosis. Endocrine disorders, particularly hypo- or hyper-thyroidism present with distinct changes in skin, hair, nails, activity, tone of voice, and vital signs.
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Clinical Laboratory findings of underproduction anemia
The anemia of chronic infection, inflammation, or malignancy (non-hematologic) may vary in its severity (Hgb, 8-12 gm/dl) with the level proportional to the underlying condition. The indices and smear may be normocytic, normochromic, or microcytic with some hypochroma. All have reticulocytopenia. Characteristically, serum iron is decreased, TIBC normal to decreased and ferritin normal to increased. Erythropoietin (EPO) levels are often inappropriately low for the degree of anemia. Lead intoxication is associated with mild to moderate anemia, decreased reticulocyte count, microcytosis, basophilic stippling (smear), and an increase in zinc protoporphyrin. Be observant for concurrent iron deficiency; and, of course, there is an increase in lead level. For anemia due to renal insufficiency, the anemia is typically not seen until there is significant renal impairment (kidney function <40% normal). This anemia is a normochromic, normocytic and is associated primarily with EPO deficiency, since EPO is produced and released by the kidney. Other conditions may also contribute to the anemia (e.g., iron and other nutritional deficiencies). Anemia in thyroid disorders presents mainly as normochromic and normocytic anemia but microcytosis (both) or macrocytosis (hypothyroidism) can be seen. Anemia found with adrenal insufficiency is usually normochromic and normocytic.
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anemia caused by Chronic Inflammation or Infection
| Malignancy
clinical features are Dependent upon underlying disease associated: may include fever, arthralgias, arthritis and fatigue. For infection, symptoms and signs relate to the focus (e.g., pain, cough, swelling). Laboratory findings Mild-moderate anemia (Hgb 8 -12 gm/dl). Severity proportional to underlying disease; may be normochromic/normocytic or microcytic with some hypochromia. decreased serum Fe, decreased TIBC, nl to increased ferritin, decreased EPO for Hct, decreased retic count.
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anemia caused by lead intoxication
clinical features are Personality changes, irritability, headache, weakness, wt loss, abdominal pain and vomiting presenting with insidious nature. Laboratory features include Mild to moderate anemia. decreased retic count. Microcytosis and mild hypochromia. Basophilic stippling. increased zinc protoporphyrin. May see concurrent iron deficiency confounding the diagnosis. Lead levels increased.
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anemia caused by renal insufficiency
clinical features are Signs and symptoms may be interrelated with those of renal dysfunction: fatigue, pallor, decreased exercise tolerance, dyspnea, tachypnea. Anemia may have other contributing factors. Usually don’t see anemia until kidney function <40% of normal. Moderate to severe anemia. Hgb 5-9 mg/dl. Normochromic, normocytic. decreased retic, occasionally abnormal morphology. EPO deficiency, decreased production. Renal insufficiency usually leads to pretty severe anemia.
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anemia caused by endocrine disorders
clinical features are Hyper or hypoactivity, weight gain or loss, systemic skin, nail, hair changes in hyper or hypothyroidism help suggest etiology. Nausea, vomiting, dehydration, weakness and circulatory collapse suggest adrenal insufficiency. labortory findings include Hypothyroidism: mild anemia; most normochromic, normocytic. May be microcytic or macrocytic. Hyperthyroidism: usually normocytic, may be microcytic. Adrenal: mild anemia, normocytic, all have decreased reticulocyte count and index. Anemia caused by endocrine are underproduction anemias.
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anemia caused by malignancies and sepsis
In malignancies and sepsis, TNF decreases iron availability from stores and decreases production of EPO, and INF-β inhibits erythropoiesis. In chronic infection or inflammation, IL-1 diminishes iron mobilization and EPO production, and INF-γ inhibits proliferation of erythroid precursors. The results are primarily related to inability to use iron stores, diminished EPO production for the degree of anemia, and decreased erythropoiesis. With renal insufficiency, the lack of EPO causes anemia and mainly leads to normachromic and normacytic anemia. Finally, in lead intoxication, lead inhibits synthesis of protoporphyrin and the enzyme that ligates iron to the porphyrin ring.
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anemia caused by iron deficiency
With iron deficiency the transferrin level goes up; if the anemia is caused by inflammation it stays the same because iron stays in the stores so nothing is transporting iron to developing erythrocyte. Also EPO is only slightly depressed in inflammation as compared to iron deficiency where it is greatly reduced? You see free protoporphyrin with lead poisoning because lead inhibits combining iron with protoporphyrin.
348
Creatinine
is a measure of kidney function
349
treatment of underproduction
EPO is used in specific conditions where there is an absolute deficiency or where EPO levels are decreased out of proportion to the degree of anemia and administration is known to induce a response.
350
treatment of anemia of chronic disease
Treat underlying disease (infection, malignancy) to decrease cytokines and interleukins. Treat co-morbid conditions (e.g., iron deficiency). EPO has been shown to be effective in some cases.
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treatment of anemia due to renal deficiency
Administration of EPO. Treat co-morbid conditions.
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treatment of anemia due to endocrine disorders
hormone replacement
353
Explain the biochemical basis for B12 and folate deficiency leading to a macrocytic anemia.
Folic acid and vitamin B12 (cobalamin) are critical co-factors for normal hematopoiesis. Methyltetrahydrofolate (a metabolite of folic acid) is a methyl group donor and essential co-factor, along with vitamin B12, in the synthesis of methionine from homocysteine. This reaction generates tetrahydrofolate which is a substrate for purine and pyrimidine synthesis and the conversion of deoxyuridylate to thymidylate (required for normal DNA synthesis). Deficiencies of folic acid and vitamin B12 profoundly affect the maturation process of red cell precursors in the marrow. The cells increase in size, arrest in S phase of mitosis, and then undergo destruction, resulting in ineffective erythropoiesis and anemia. Although we classify this as underproduction this is really arrest of erythropoiesis.
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dietary sources of vitamin B12
Vitamin B12 is originally synthesized by bacteria and algae, eventually working its way up the food chain to humans through consumption of meat, eggs, and milk. It is required as a vitamin by animals but not by higher plants - hence, plants do not contribute Vitamin B12 to the diet, and a strict vegan diet can lead to deficiency. Once ingested, Vitamin B12 in food is released in the acid environment in the stomach. The protein carrier, intrinsic factor (IF), is secreted by gastric parietal cells and binds vitamin B12. In the terminal ileum, the B12 is absorbed and released from IF, bound to transcobalamin binding protein II (TcII) and transported to the liver for storage or to other tissues like the bone marrow for use.
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dietary sources of folate
Sources of folate: Folate is widespread in food. With a typical diet, about one third of the daily folate intake is provided by cereals and bread, another one third by fruits and vegetables, and the remaining one third by meats and fish. Human milk provides enough folate for infants. Goat’s milk, however, contains little folate, and children maintained on it alone can develop severe deficiency. Overcooking can also lead to loss of folates in food
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Absorption, transport and storage of folate
Dietary folate is absorbed in the jejunum. It is hydrolyzed, reduced and methylated before distribution to the tissues or liver for storage (as methyltetrahydrofolate). The liver stores undergo turnover, secretion in the bile and reabsorption (enterohepatic circulation) supporting a constant supply to tissues. Folate is absorb in the jejunum.
357
where is iron absorbed?
in the duodenum
358
clinical and laboratory features of folate and vitamin B12
Both folic acid and vitamin B12 deficiency result in megaloblastic anemia. The onset of folate deficiency can occur quite rapidly (within weeks), particularly in the setting of malabsorption or alcoholism. In someone who is well-nourished, Vitamin B12 deficiency takes several months to develop because of its long half-life within the body and large hepatic stores. Vitamin B12 deficiency develops more slowly and is more likely associated with malabsorption. The symptoms and signs of anemia in both cases are not distinguishable from other causes.
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hematologic changes due to folic acid and vitamin B12 deficiency
In the bone marrow, erythroid hyperplasia (because cells are bigger and there is stress increase erythropoiesis) leading to an alteration of the myeloid:erythroid (M:E) ratio from a myeloid to an erythroid predominance is observed. Megaloblastic changes are seen in both erythroid and myeloid series. Cytoplasmic maturation is normal but at any stage of the development, marrow precursors show large, immature nuclei (termed nuclear-cytoplasmic asynchrony). In peripheral blood, the anemia is variable. There is macrocytosis (MCV >97 fl in adults). The reticulocyte count is decreased, with a reticulocyte index <1.0. On the peripheral smear, macro-ovalocytes and hyper-segmented (≥4-5 lobes) neutrophils can be observed. As the anemia progresses, poikilocytes and fragmentation may be seen. In severe cases, neutropenia and thrombocytopenia can be documented, as well as increases in bilirubin and LDH levels due to intramedullary (within the bone marrow) destruction of red cells.
360
Vitamin B12 deficiency cause
It is unusual for Vitamin B12 deficiency to be caused by inadequate dietary intake. The most common cause of Vitamin B12 deficiency is pernicious anemia, due to autoimmune destruction of IF-producing gastric parietal cells. This condition is most common in the older age population. Other causes include failure to produce IF (gastritis, gastrectomy, congenital), malabsorption (multiple disorders), defective transport or storage (TcII deficiency) and metabolic defects in pathways which utilize B12 as a substrate.
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Cause of folate deficiency
In contrast to B12 deficiency, the most common cause of folate deficiency leading to megaloblastic anemia is inadequate dietary intake. Other causes include malabsorption due to such things as tropical sprue or parasitic infection, which can lead to rapid depletion of folate through interruption of enterohepatic circulation, inborn errors of folate metabolism (very rare), and increased demands (hemolysis, pregnancy/lactation, rapid growth, psoriasis, myeloproliferative disorders). Alcohol consumption also can lead to rapid onset of folate deficiency, not only through decreased dietary intake but also through disruption of cycling from liver stores to tissues.
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Neurologic Features of Vitamin B12 Deficiency
Neurologic involvement is classic in B12 deficiency, though it can infrequently be seen in folate deficiency as well. Sensory abnormalities (numbness, tingling, loss of fine sensation) occur first. Loss of proprioception (perception of movement and spacial orientation) may also be documented. As the deficiency progresses, ataxia (gross lack of coordination of muscle movements), spasticity, gait disturbances, and a positive Babinski reflex (in response to stroking of the sole of the foot, extension rather than flexion of the great toe) may follow. Sometimes cerebral signs are seen including cognitive dysfunction and emotional changes. Anemia may be absent in 28% of patients with neurologic problems. Neurologic defects may not be completely reversible after B12 administration. Importantly, if a patient with undiagnosed B12 deficiency is treated with large doses of folic acid, neurologic damage can be exacerbated, making it critical to rule out B12 deficiency before initiating treatment in someone with folate deficiency. Methylmolonic acid is only increased in vitamin B12
363
Describe the appropriate therapies for B12 deficiency and folate deficiency.
Cobalamin deficiency: Intramuscular or subcutaneous injections of B12, with a typical schedule being daily for 2 weeks, then weekly until the hematocrit is normal, then monthly for life. If absorption is not an issue, replacement can be oral. In some cases of pernicious anemia, large oral doses given daily can overcome the absorption defect, but correction of the deficiency needs to be documented. Folate deficiency: 1 mg/day orally or parenterally. The response to anemia is rapid for folate or B12 deficiency. Reticulocytosis occurs in 2-3 days which peaks at 7-10 days. Hgb increases beginning 7-14 days. WBC and platelet counts increase in the first week. The MCV decreases over weeks to a few months. Normal counts, 8 weeks. Neurologic dysfunction shows slow improvement over 6 months to a year depending on duration of symptoms before treatment but may not completely resolve. Progression of symptoms rules out cobalamin deficiency.
364
Hemoglobin structure
a ~68-kD tetramer comprised of 2 pairs of globin polypeptide chains: one pair of a-globin chains and one pair of non-a-globin (g-, d-, or b-) chains.
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A heme prosthetic group
consisting of a protoporphyrin ring bound to iron, is associated with each globin chain of the hemoglobin tetramer. It is the heme group that binds oxygen. a-globin chains have 141 amino acids and b-globin chains have 146 amino acids. The heme iron is covalently linked to a histidine, binding the 87 position of the a-chain and the 92 position of the g-, d-, and b-chains. Charged amino acids such as lysine, arginine and glutamic acid lie on the surface of the molecule and are hydrophilic, helping to keep hemoglobin soluble in the red cell and preventing precipitation. The four heme groups lie in clefts on the surface of the hemoglobin molecule.
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Allosteric regulation
when hemoglobin binds to one oxygen, occupying one of its binding sites, the hemoglobin molecule changes its configuration, altering its binding affinity for additional oxygen molecules. Under conditions where the oxygen concentration is low enough that none of the four sites are occupied, the binding affinity to oxygen is relatively low. In this situation, the hemoglobin is in a taut or T configuration, due to inter-and intra-salt bonds within the molecule.
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Cooperativity
If the allosteric changes lead to increased affinity for the substrate at the other binding sites, its is positive cooperativity. As oxygen becomes more available and one of the binding sites becomes occupied, the configuration of the molecule changes such that the other three sites have higher binding affinity and can more easily bind to additional oxygen molecules. As the number of occupied sites increases, the affinity for the remaining sites continues to increase. This occurs through sequential breaking of the salt bonds, converting the hemoglobin to the relaxed or R form.
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p50
A way to quantify this difference in oxygen affinity is by determining the P50, which is defined as the partial pressure of oxygen at which the oxygen carrying protein is 50% saturated. Under normal conditions for temperature (37 C) and pH (7.4), the P50 for hemoglobin is approximately 27 mmHg while the P50 for myoglobin is 2.75 mmHg. Functionally, myoglobin is a very poor protein to use to transport oxygen from the lungs to the tissues, since it would hold tightly to the oxygen and not release it until the oxygen concentration got very low. On the other hand, myoglobin is a very good protein to use for storage of oxygen in the intracellular environment where oxygen concentration is very low (1-5 mmHg) and where high oxygen affinity is needed to transfer the oxygen from hemoglobin to myoglobin. By contrast, hemoglobin is an excellent protein to use for oxygen transport, since oxygen is easily loaded onto the molecule in the lung where the partial pressure of oxygen is ~100 mmHg but then readily unloads in the tissues where the partial pressure of oxygen is ~40 mmHg.
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Bohr effect
The oxygen affinity of hemoglobin increases over a pH range of 6-8.5, with oxygen being more tightly held by hemoglobin in alkaline situations (with a shift of the curve to the left) and more easily released from hemoglobin to the tissues when there is a lower pH (with a shift of the curve to the right).
370
CO2 concentration effect on oxygen affinity
CO2 produced by the tissues as a product of metabolism contributes to the Bohr effect, because when it is released into the bloodstream, the enzyme carbonic anhydrase converts CO2 and water into carbonic acid which decomposes into bicarbonate and H+, leading to a drop in pH. The Bohr effect makes sense from a physiologic viewpoint, since tissues with a higher metabolic rate, which are utilizing more oxygen, are going to be producing more CO2 and lactic acid, leading to a drop in pH, which then shifts the hemoglobin oxygen dissociation curve to the right, allowing greater release of oxygen to the tissues. Conversely, with CO2 efflux in the lungs, the pH of blood passing through the pulmonary circulation rises, leading to increased oxygen affinity and easier loading of oxygen onto the hemoglobin molecule to be delivered to the tissues.
371
Temperatures affect on oxygen binding
Hemoglobin’s oxygen affinity varies inversely with temperature so that at higher temperatures more oxygen is unloaded to tissues and less is bound by hemoglobin. Again, this makes physiologic sense, since with exercise or fever, metabolic rates are higher, leading to increased need for oxygen. With a shift of the curve to the right, more oxygen is made available to the tissues to meet this increased demand.
372
2,3-bisphosphoglycerate (2,3-BPG)
also known as 2,3-diphosphoglycerate or 2,3-DPG, is a byproduct of the anaerobic glycolytic pathway and is normally present in red cells at a concentration of ~5 mmol/L. When the level is higher, such as occurs during states of increased oxygen utilization and glycolysis, chronic hypoxia, and chronic anemia, oxygen affinity of hemoglobin decreases and shifts the curve to the right, increasing delivery of oxygen to tissues. Formation of 2,3-BPG is related to glycolysis, so more glycolysis leads to more 2,3-BPG. 2,3-BPG alters oxygen affinity by binding to deoxyhemoglobin and stabilizing it in the T configuration, leading to decreased affinity of the hemoglobin for oxygen.
373
Compare oxygen dissociation curves for myoglobin and hemoglobin and explain the reason for the differences.
Hemoglobin is sigmoidal shape because of allosteric regulation and cooperative binding. Myoglobin binding to oxygen is a monomer and does not undergo this. At very low partial pressure of O2 there is very tight binding, which makes it a good storage protein but not a good transporter.
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Fetal hemoglobin
Chromosome 16 contains the “a-like” genes, including two copies of the a-globin gene itself along with variants expressed early in embryonic development; therefore, the genome contains a total of 4 copies of the a-globin gene (2 paternal and 2 maternal). The “b-like” genes (genes for the g-, d-, and b-globin chains along with variants produced early in embryonic development) are products of a set of genes on chromosome 11; one copy of the gene set is inherited from each parent.
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fetal hemoglobin patterns
Embryos have 3 distinct hemoglobins that are present only between 4 and 14 weeks gestation: Hemoglobin Gower I (z2e2), Hemoglobin Gower II (a2e2) and Hemoglobin Portland (z2g2). Each of these has a higher affinity for oxygen than does hemoglobin A. After week 8 of gestation, fetal hemoglobin or hemoglobin F (a2g2) predominates. The g-chain differs from the b-globin chain by 39 amino acids. Fetal red cells have a higher oxygen affinity than adult red cells, primarily because hemoglobin F binds 2,3-BPG poorly, stabilizing the hemoglobin in the R state and shifting the oxygen dissociation curve to the left. The Bohr effect is also increased by 20% in fetal hemoglobin, so that as fetal blood passes through the intravillous spaces of the placenta, H+ ions are transferred to the maternal circulation and the pH rises, leading to increased oxygen affinity and a further shift of the curve to the left. These changes favor transfer of oxygen from the maternal circulation to the fetal circulation
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Adult hemoglobin patterns
At birth, there is 65-95% hemoglobin F and about 20% hemoglobin A. The normal adult level of fetal hemoglobin is approached by 1 year and achieved by 5 years of age. Under normal conditions, adults have 96-97% of their hemoglobin as hemoglobin A (a2b2). In adults, hemoglobin F makes up <1% of the total hemoglobin and is unevenly distributed in red cells.
377
Describe how structural differences in hemoglobin affect oxygen affinity
Most common are Hb S, Hb C, and Hb E. can lead to unstable hemoglobin, hemoglobins with altered oxygen affinity, hemoglobins associated with cyanosis.
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Hemoglobin Chesapeake
high-affinity hemoglobin variant, The genetic defect was a single point mutation. Erythrocytosis (an elevated red blood cell count) is generally found in people with high-affinity hemoglobins. This occurs because oxygen delivery to the tissues is reduced, leading to increased release of erythropoietin from the kidney, which stimulates increased red cell production. Affected people are generally well. Alpha-globin chain with oxygen affinity. Hemoglobin is usually stable. Usually have an abnormal hemoglobin electrophoresis. Typically with no hemolysis . P50 is left shifted. Leads to relative tissue hypoxia. Erythropoietin production by the kidney is stimulated and the red blood cell count increases. Affected generally are well and don’t need treatment
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Low-affinity hemoglobin mutants
are associated with cyanosis (a gray or bluish tint to the skin and mucus membranes). There are fewer low-affinity than high-affinity hemoglobin variants. More oxygen is delivered to the tissues with low-affinity hemoglobins and patients may have a physiologic anemia. Diagnosis of the variants is done by measuring the P50. Less common than high-affinity Hb. P50 is right shifted. Oxygen is released to the tissues more easily. Often with mild anemia. Can have cyanosis (blue/grey color to skin and mucus membranes).
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Hemoglobin Zurich
There are also unstable hemoglobins that may or may not bind oxygen differently than hemoglobin A. Hemoglobin Zurich has a single point mutation that does not affect oxygen binding but does increase binding to carbon monoxide. The carboxyhemoglobin levels are like that of smokers.
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Hemoglobin Köln
the most commonly recognized unstable hgb (though it is still very rare). Affected people have 10-25% hemoglobin Köln and have mild anemia, reticulocytosis, and splenomegaly (an enlarged spleen).
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Methemoglobin
To bind oxygen, the iron contained within hemoglobin needs to be in the reduced or ferrous (+2) form. If the iron is in the ferric (+3) form, methemoglobin results. Normally we have about 1% methemoglobin in our RBCs at any given time. The iron contained within hemoglobin is maintained in the ferrous form within the erythrocyte by the NADPH methemoglobin reductase pathway.
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Methemoglobinemia
can occur because of too much methemoglobin production or because of decreased methemoglobin reduction. It may be an acquired or genetic process. In the presence of abnormally high amounts of methemoglobin, not only is the capacity of hemoglobin to carry oxygen reduced, the oxygen dissociation curve also shifts left, leading to even less availability of oxygen to the tissues. Most people with congenital, chronically elevated methemoglobin levels are asymptomatic, even with methemoglobin up to 40%. They are, however, cyanotic. Cyanosis is apparent when methemoglobin levels are at least 1.5 g/dL, or when ~8-12% of hemoglobin is methemoglobin.
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Acquired methemoglobinemia
Oxidation of the heme by reaction with free radicals, hydrogen peroxide, nitric oxide, or OH- can generate methemoglobin. Acquired methemoglobinemia can occur with exposure to a number of different drugs and chemicals. For instance, benzocaine (a topical anesthetic) is a potential cause. Well water contaminated with nitrates can also cause methemoglobinemia. Newborn infants, who normally have a lower activity of cytochrome b5 reductase, can be especially at risk. Hemoglobin F oxidized more readily to ferric state. May become cyanotic with well water (nitrates), raw spinach, disinfectants, benzocaine
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cytochrome b5 reductase
There are several different hereditary causes of methemoglobinemia with different inheritance patterns (autosomal dominant or autosomal recessive). Most commonly, it is due to homozygous deficiency of cytochrome b5 reductase. (Involved in transfer of electrons from NADH generated by glyceraldehydes-3-phosphate in the glycolytic pathway to cytochrome b5).
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Hemoglobin M
methemoglobinemia may also be due to a mutation in hemoglobin resulting in production of hemoglobin M. In this situation, a mutation occurs in either the a-or b-globin chain that leads to inhibition of reduction of iron to the ferrous form.
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Diagnosis of methemoglobinemia
It is suspected when a person looks cyanotic but the arterial partial pressure of oxygen is normal on an arterial blood gas. With methemoglobinemia, the blood looks dark-red, chocolate or brown-blue and with oxygen exposure does not change whereas if the cyanosis is due to increased deoxygenated hemoglobin, the blood will turn bright red with addition of oxygen.
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Treatment of methemoglobinemia
Treatment depends on the cause. No treatment is needed for hemoglobin M. Cytochrome b5 deficient patients are only treated for cosmetic reasons with methylene blue or ascorbic acid. For acquired methemoglobinemia, methemoglobin levels below 30% in a healthy person produce minimal symptoms (fatigue, lightheadedness, and headache) or none, whereas levels from 30% to 50% produce moderate depression of the cardiovascular and central nervous systems [weakness, headache, tachycardia (fast heart rate), tachypnea (rapid breathing), and mild shortness of breath]. Levels between 50% and 70% cause severe symptoms [stupor, bradycardia (low heart rate), respiratory depression, convulsions, dysrhythmias, and acidosis]. Levels above 60% can be lethal, and levels above 70% usually are not compatible with life. Acquired methemoglobinemia is treated by removing the inciting drug or chemical. Methylene blue given intravenously provides an artificial electron acceptor for the reduction of methemoglobin via the NADPH-dependent pathway. Response is within one hour.
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Carbon monoxide poisoning
Carbon monoxide (CO) is an odorless, tasteless, colorless, nonirritating gas formed by hydrocarbon combustion. CO binds the heme moiety of hemoglobin with an affinity 240 times that of oxygen, producing carboxyhemoglobin. CO poisoning kills 5-6,000 people per year, usually as a result of poorly functioning heating systems, gasoline-dependent generators, and motor vehicles in poorly ventilated areas. Sniffing paint remover, which is converted to CO, is another etiology. Normal people have up to 3% CO; smokers have up to 10-15%. When one heme binds CO, an allosteric change occurs so the other 3 hemes download oxygen less well, increasing the affinity of hemoglobin for oxygen and decreasing delivery of oxygen to tissues.
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Diagnosis and treatment of CO poisoning
People with carbon monoxide poisoning usually present complaining of a headache. They may also have malaise, nausea and dizziness. If levels are high enough, seizures, coma, and myocardial infarction may ensue. 40% of affected people will have late neurologic deficits such as loss of cognition, personality change, and movement disorders. Diagnosis is by co-oximetry (not pulse oximetry, which does not differentiate carboxyhemoglobin from oxyhemoglobin). Treatment is with 100% oxygen, which will compete with CO for the binding sites on the heme moiety. Hyperbaric oxygen can also be considered, which decreases the half-life of CO hemoglobin from 40-80 minutes to 15-30 minutes.
391
Explain in basic terms how a pulse oximeter works. Describe situations where a pulse oximeter reading may inaccurately reflect a patient's true oxygenation status.
Pulse oximetry may be inaccurate if the probe is not placed correctly, if only one of the 2 diodes is working, if there is too much motion due to shivering or seizing, if nail polish is present, and with deeply pigmented skin, anemia, shock, or abnormal hemoglobins. Carboxyhemoglobin absorbs at 660 nm at a similar level to oxyhemoglobin so will give a falsely high reading. Methemoglobin absorbs at 660 nm and 940 nm so it will also give inaccurate results. Co-oximetry, which measures absorbance at 4 or more wavelengths rather than the two wavelengths measured by pulse oximetry, is needed to accurately quantify carboxyhemoglobin or methemoglobin levels.
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H chain L chain
Antibodies are composed of two identical light (MW about 25,000) and two identical heavy (MW about 50,000) chains.
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kappa and lambda chains
hinge region
Fab, F(ab2)
Fab= antigen binding. Antibodies are made of four fragments attached by disulfide bonds. The two identical ones are each called Fab. If you adjust conditions during antibody digestion with proteolytic enzymes, you can get 2 Fab fragments (S—S bonds between the H chains fully reduced) or you can leave the 2 Fabs still joined; that’s called F(ab2). Fab is univalent (binds only 1 antigen); like IgG, F(ab2) is divalent. When an IgG or IgM antibody binds antigen with at least one of its (two or ten) binding sites, there is a change in the angle between the Fab parts and the Fc, so that the molecule may be more Y or T shaped than before (this explains why the region between Fabs and Fc is called the Hinge.) Kappa and Lambda are the two families of light chains, you don’t get a mixed antibody with one kappa and one lambda. They are located on two different chromosomes
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Fc
This is the constant region, and it is made up of 1 (in L chains) to 4 (in epsilon and mu) compact, structurally-similar domains called C domains. It is composed of folded beta sheets rigidly held together by disulfide bonds.
395
Fv
Each chain also has, at its N-terminal, a domain that is different in sequence between antibodies of different specificities: the variable domain or V. It is located at the tip of Fab (N-terminal end). The variable bits extend from the beta sheets.
396
Hypervariable regions
complementary determining regions, they are interspersed in the variable domains.
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VL and CL
Each chain is composed of domains, which are compact areas held together by intrachain S---S bonds. Light chains have one variable domain, VL, and one constant domain, CL.
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VH and CH
Heavy chains have one variable domain, VH, and 3-4 constant domains, CH1, CH2, CH3, (CH4). There is considerable structural homology between different domains, which suggests that there was once an ancestral gene for one domain, which has duplicated many times.
399
Diagram an electrophoretic separation of human serum
If serum is separated in an electrical field, the proteins segregate into an albumin and several globulin bands. Antibody activity was found mostly in the gamma (γ) globulin zone; antibodies were called, and still sometimes are, gamma globulin or immune globulin. Since some activity was also located in the beta region, it was decided to come up with a better generic term: immunoglobulin.
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IgG
2 light and 2 gamma (g) chains. The most abundant immunoglobulin in the blood. It is the only class that passes the human placenta from mother to fetus (active transport required). It comes up in the blood a little later than IgM after primary immunization, but the antibody levels go higher and last longer. The plasma half-life of IgG is about 3 weeks. If antigens (pathogens) get into the blood stream IgG antibodies are very important; phagocytic cells have receptors for the Fc of bound IgG, and so IgG is opsonizing; vital for clearance of most extracellular bacteria. It takes two IgGs close together to activate the first component of complement, so this will only happen if the density of epitopes on the antigen is high enough for that to occur (binding a single epitope should be enough to allow an IgM molecule to activate complement).
401
IgE
2 light and 2 epsilon (e) chains. Because its Fc end binds to a corresponding receptor on mast cells and basophils, forming a trigger for these histamine-loaded cells, this antibody is the cause of immediate hypersensitivity or allergy. It is important for resistance to parasites, where it triggers the mast cells to release not only histamine, but eosinophil chemotactic factor. Eosinophils are uniquely effective at killing parasites.
402
IgD
2 light and 2 delta (d) chains. Although there is some IgD in the plasma, it is believed that the only important role for IgD is as a B cell receptor.
403
IgA
4 light, 4 alpha (a), I J, and 1 S.C. chain. This antibody is preferentially made by plasma cells in lymphoid tissues near mucous membranes. Two IgA’s are assembled into a dimer by the addition of a J chain while in the plasma cell, and then secreted into the interstitial space. Adjacent epithelial cells have receptors for IgA which binds to them, is taken up and moved through the epithelial cell to the luminal (mucous membrane) side. There the IgA is exocytosed, still bound to the receptor, which is now called Secretory Component. Secretory Component protects the IgA from digestion in the gut, and makes it work well as our first line of immunological defense against invading organisms. There is some monomer and dimer IgA in the plasma, where it can bind pathogens and activate complement (by the alternative pathway).
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IgM
10 light, 10 mu (m), and 1 J chain. This is the oldest antibody phylogenetically. It is also the first seen in blood after immunization (sensitive tests reveal an increase by day 2). It is decavalent, but in practice its shape rarely allows more than two of its binding sites to interact with antigenic determinants. It has a great capacity to activate complement; two adjacent Fcs are needed to get the complement cascade started, and IgM always has five adjacent. It can be as much as 500 times more efficient than IgG at activating complement. IgM, being so large, is viscous in solution; if we had only IgM at the same molarity as we have IgG we would scarcely be able to pump our blood. And there are no useful IgM receptors on phagocytes. IgM is the only antibody to be made by the fetus. We make a little secretory IgM, which perhaps helps out people who lack secretory IgA (a common condition). During an infection, initially cells will make IgM, then switch to IgG.
405
Arrange IgG, IgM and IgA in terms of molecular size, and give their approximate normal concentrations in serum.
IgM (900,000, 100 mg/dL) > IgA (400,000, 200 mg/dL) > IgE (190,000, 0.02 mg/dL) > IgD (180,000, 5mg/dL) > IgG (150,000, 1000 mg/dL)
406
Describe the structure of antibody combining sites.
The antibody’s combining site, which binds antigen, is made up of parts of the V domains of both the H and L chain (VH and VL.)
407
Explain why complementarity-determining regions are also called hypervariable regions.
Amino acid sequence variability is not distributed uniformly along the V domain; most of the variability is in 3 areas called, therefore, hypervariable regions. It is more functionally significant to call them complementarity-determining regions, CDR, because the amino acids in the hypervariable regions comprise the actual antigen-binding site.
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Subclass
On the basis of small differences in the amino acid sequences of their H chain C regions, the 5 main classes of immunoglobulins are divided into subclasses. These are isotypes.
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Allotype
Minor allelic differences in the sequence of immunoglobulins between individuals. The allotypes you express are determined by the allotypes your parents had, in the usual Mendelian fashion. Allotypes are useful in genetics, for example in determining relatedness, and sometimes in forensic medicine. Occasionally, an immunodeficient patient getting immunoglobulin treatments will make antibodies to someone else’s allotype; this could be awkward. If certain allotypes function more efficiently than others, it could explain why some people are more susceptible to some infections than other people
410
Idiotype
Each antibody will have its unique combining region, made up of the sequence of CDR amino acids of its L and H chains; this unique structure is an idiotype (idio means self). Antibodies can be made (most easily in another species) which recognize the unique sequence of that combining site, and no other. Such an antibody is an anti- idiotype. In other words, it is almost completely correct to think of an idiotype as an antibody’s unique set of CDRs considered as an antigen.
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Epitope
Not all of a protein antigen binds specifically to an antibody; the part that actually interacts is usually 10 to 20 amino acids long. It is also called antigenic determinant. Typical proteins have several epitopes which elicit, and bind to, different antibodies; this single protein from human papilloma virus (HPV) has at least a dozen identified. The bindings are charged and hydrophobic based, not covalent. An epitope may be based on the primary structure or tertiary structure of the protein.
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Antibody valence
How many epitopes that an antibody can bind to.
413
Precipitation
if you mix multivalent antigens and divalent antibodies than there is a good chance of cross-linking two antigens and an immune complex begins to grow. The large immune complexes that are formed at or near equivalence (where ratios of antigen to antibody are optimal) tend to become insoluble and fall out of solution or suspension. When the antigen is a molecule, the phenomenon is called precipitation.
414
Agglutination
when it’s a cell or cell-sized particle, it is called agglutination. Agglutination is more readily detected than precipitation, and so, for the same amount of antibody, an agglutination test is more sensitive.
415
Describe a quantitative precipitin test where amount of antigen/tube is varied while antibody/tube is constant.
The size of the precipitin depends on the ratio of antibodies and antigen. The long chains are achezied when the ratios are about even.
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Immunodiffusion
precipitation in gels. If you take a layer of agar gel in a dish, cut two holes in it, and put antibody in one and antigen in the other, they will begin diffusing radially out of their wells. They diffuse towards eachother and in the middle somewhere you get equivalence growing large complexes, which forms a line that precipitates
417
Compare and contrast precipitation and agglutination in terms of the nature of the antigens involved, and sensitivity of the tests.
When the antigen is a molecule, the phenomenon is called precipitation; when it’s a cell or cell-sized particle, it is called agglutination. Agglutination is more readily detected than precipitation, and so, for the same amount of antibody, an agglutination test is more sensitive.
418
Discuss how complement plays roles in both innate and adaptive immunity.
a large number of proteins, similar to the blood clotting system in that each exists in an inactive form, and when the first is activated the rest follow in a sort of cascade. There are at least three ways to activate the C cascade; the one that is most familiar is the classical pathway. Later, an alternative and a lectin pathway were described. Each pathway gets started differently but all represent different ways to activate C3.
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The classical pathway
is activated by complexes of IgG or IgM antibody with antigen; it seems to be the main way IgG and IgM antibodies deal with bacterial invaders There is a change in the Fc portions of the antibodies after interaction with antigen, which allows the binding and activation of C1q. The C1q must interact with two Fcs simultaneously; it does so either by finding two IgGs close together, or a single IgM (this reinforces the point that IgM is a much more efficient a C activator than is IgG). C1 activates C4 and then C2, which together activate C3, which can then activate C5. Classical C counts: 1-4-2-3-5-6-7-8-9. certain complement components, activation means splitting into 2 parts; one usually stays attached to the nearest membrane (e.g., C3b), and the other may float away (C3a, C4a, C5a) and have biological activity.
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The alternative pathway
is activated by certain cell wall structures of microorganisms such as dextrans, levans, zymosan, and endotoxin; a bacterium can activate C this way even in the absence of antibody. Therefore this pathway is part of the innate immune system. C3 is always breaking down at a low rate to C3a and C3b, which usually are rapidly degraded. So if C3b could be stabilized, C5 could be activated. The cell wall structures provide a surface for the binding of properdin (P), which acts as an anchor for the assembly of C3b, factor B, and factor D; a stable C3bDbC3b complex (trimer of Db and two C3b units) forms which can activate C5 (and thus 6-7-8-9). Recently the alternative pathway been shown to play a significant role in autoimmunity
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mannose-binding protein, MBP
The lectin pathway is mediated by mannose-binding protein, MBP or MBL, a lectin. Lectins are proteins that bind (usually foreign6) carbohydrates. MBP binds certain mannose–containing structures found in carbohydrates of bacteria but not humans. MBP is functionally similar to C1q in the classical complement pathway, so the lectin pathway goes MBP-4-2-3-5-6-7-8-9. There are several alleles of MBP in humans, and Caucasians have an allele that results in low levels of serum MBP; about 8% have very low levels. These people have marginal immunity—they may be fine except when the immune system is stressed (in infancy, in old age, in the presence of anything that compromises the immune system). Associating with MBP when it binds mannose are some serine proteases, the MASPs, which activate C2 and C4 and get the cascade rolling.
422
Discuss the different ways in which complement is activated by IgG and IgM.
In the classical pathway, the C1q must interact with two Fcs simultaneously; it does so either by finding two IgGs close together, or a single IgM (this reinforces the point that IgM is a much more efficient a C activator than is IgG).
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Membrane attack (lytic) complex or MAC
C5, activated by any of the three pathways described, but very strongly by the classical pathway, activates C6, C7, C8, and C9. C8 and C9 form a lesion on the target cell membrane which, on electron microscopy, looks like a hole, which in fact it is; the cell loses its ability to regulate its osmotic pressure and lyses or pops. Neisseria (gonorrhea, meningitis) are by far the most susceptible family of bacteria to C lysis because if they are eaten by phagocytosis they are not killed (they have defensives against this).
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OPSONIZING
One split product of activated C3, namely C3b, adheres to membranes. Phagocytic cells (PMN, macrophages) have C3b receptors, and so can get a firm grip on an antigen if it is opsonized with C3b. As we said before, IgG is also opsonizing, because phagocytes have receptors for its Fc end called FcR (there are several different FcR). There are no FcR for IgM, but the complement it activates is, of course, opsonizing. PMN attach to multiple FcR and zippers it up into the cell.
425
CHEMOTACTIC
The C5 activation product, C5a, is chemotactic for phagocytes, especially neutrophils. This explains much of the inflammation in an antibody-mediated reaction, and why PMN are the hallmarks of such a reaction. C3a and C4a are also chemotactic. (these are the ones that split off).
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ANAPHYLATOXIC
C3a, C4a and C5a are all capable of releasing histamine non-specifically from mast cells or basophils. This means that there will be increased blood flow in the area of antigen deposition, and a better chance for inflammatory cells to get out of the blood and into the tissues. Sometime, a person with a lot of complement activation will break out in hives, and you can confuse what’s going on with an allergic reaction.
427
Discuss how complement is important in immunity to bacteria even if the bacteria are resistant to lysis by C9.
Hydrophobic sites on C8 and C9 molecules are exposed when they bind to the complex, so they can also insert into the bilayer. C8 and C9 form a lesion on the target cell membrane which, on electron microscopy, looks like a hole, which in fact it is; the cell loses its ability to regulate its osmotic pressure and lyses or pops.
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hemolysis
Hemolysis is a decrease in red cell survival or increase in turnover beyond standard norms. The pace or rate of hemolysis, in part, determines whether anemia presents acutely or over a more insidious, chronic course. The degree of anemia is affected by the extent to which marrow production is increased. Increased production may compensate for the increased turnover (no or mild anemia) or may not be able to keep up with red cell destruction (uncompensated, moderate to severe anemia). Marrow production can increase by 6-8 fold and no more (normally production includes 1% red cell mass/day). During stress, maturation may ↓ 5-7 days and reticulocytes released early. Increased bilirubin leads to gallstones and some patients must have cholelithiasis.
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Two mechanisms for red cell destruction exist
1) turnover within the vascular space (intravascular) or 2) through ingestion and clearance by macrophages of the reticuloendothelial (RE) system (extravascular). This is where most of the turnover occurs, in the spleen. RBC pass by mononuclear macrophages internalize them based on changes: a) ↓ red cell enzyme activity with age, b) Oxidative injury over time, c) Changes in calcium balance, d) Changes in membrane carbohydrates and surface constituents, e) Antibodies to membrane constituents. One or more of these may be aggravated in pathologic conditions. The breakdown of hemoglobin in these two processes is distinct and may aid in the identification of hemolysis.
430
Intravascular hemolysis
Red cells undergoing intravascular hemolysis release hemoglobin into the circulation. The tetramer form of hemoglobin is unstable and dissociates into αβ dimers which may be immediately bind to haptoglobin. This complex is removed from the circulation by the liver. Although haptoglobin has a very high affinity and specificity for hemoglobin, its capacity may be easily overwhelmed by significant intravascular hemolysis leaving the released hemoglobin to be broken down by other pathways. The dimer form can pass through the kidney will get filtered and will not get reabsorbed, showing up in the urine. The iron in hemoglobin can be oxidized to form methemoglobin. Dissociation of globin releases metheme which may bind to albumin or hemopexin. These latter compounds may be taken up by hepatic parenchymal cells and the heme is converted to bilirubin. Alternately, the dimeric forms of methemoglobin or hemoglobin may be filtered and not reabsorbed by the kidney and appear in the urine.
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Extravascular hemolysis
the mononuclear phagocyte ingests the RBC and breaks down hemoglobin. Iron is taken up by transferren. Heme is converted into bilirubin which is taken up to the liver and a glucuronide group is added. This then moves to GI system where bacteria breaks it down into different products one of them being fecal urobilnogen. Some of these are reabsorbed and some are excreted.
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detection of hemolysis
is based on an understanding of the classification scheme of anemia and the pathways described above. The CBC will define whether anemia is present or not, the mean size of the red cells, and whether there is a significant variation of that size. Review of the red cell morphology on the blood smear allows characterization of the predominant shape of the circulating red cells, providing a hint of the etiology and basic disorder. In most, but not all cases, a shortened lifespan of the red cells will result in an increased reticulocyte count and index. Bilirubin is increased if hemolysis is brisk enough to overcome the bilirubin processing system of the liver; this leads to an increase in the unconjugated fraction which accounts for most of the elevated total bilirubin. Hemolysis is linked with unconjugated bilirubin. A decrease in serum haptoglobin levels, detection of hemoglobin in the urine or plasma and increase in metheme or methemalbumin all suggest intravascular hemolysis. Release of housekeeping cellular enzymes (SGOT, LDH) from damaged red cells resulting in elevated serum levels may also provide evidence for increased red cell destruction. However, this is not a specific finding for hemolysis and the enzymes may be elevated with increased turnover or damage to other cell types. Haptoglobin is a protein that is produced at a continuous level and cannot be decreased. With hemolysis, hepatoglobin will decrease with intravascular hemolysis.
433
Describe the major constituents of the RBC membrane and cytoskeleton. Identify the major defects in hereditary spherocytosis.
The basic pathophysiology is that spectrin, ankyrin or band 3 defects weaken the cytoskeleton and destabilize the lipid bilayer. Just below the phospholipid membrane, ankrin spectrin and band 3 provides support to the cytoskeleton and destabilizing plasma membrane. Loss of membrane and formation of the spherocyte leads to decreased deformability and entrapment in the spleen. Conditioning in the red pulp leads to further loss of red cell membrane and, ultimately, removal by the macrophage (extravascular hemolysis). There are abnormal response to hypotonic stress (osmotic fragility)
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Laboratory features of hereditary spherocytosis
include variable Hct and Hgb (a small group may have no or mild anemia), increased reticulocyte count and index, decreased MCV, spherocytes on smear, unconjugated hyperbilirubinemia and an abnormal osmotic fragility test. Two important clinical compli-cations include aplastic crises and bilirubin stones. Shortened red cell survival in the context of viral suppression of marrow production may lead to the rapid onset of severe, life-threatening anemia. Because of the large amount of bilirubin traversing the biliary tree, bilirubin stones affecting the gall bladder are a common cause of obstruction and cholecystitis requiring cholecystectomy. Milder syndromes are associated with different defects in the cytoskeleton and include hereditary elliptocytosis, stomatocytosis, acantho-cytosis and echinocytosis.
435
Explain when splenectomy is indicated for treatment of hereditary spherocytosis.
Processing and intrapment in the spleen can exerterbate hereditary spherocytosis. By removing the spleen, they have less hemolysis.
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glucose-6-phosphate dehydrogenase (G-6-PD) deficiency
Inherited as a sex-linked recessive (female carriers, males affected), many defects have been described which lead to decreased activity of the enzyme. These disorders have a world-wide distribution with the highest incidence in tropical and sub-tropical areas of the Eastern Hemisphere (Southern Europe, Africa, South China, India, Southeast Asia). The existence of G-6-PD deficiency may be associated with a selective resistance to plasmodium vivax (malaria).
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G-6-PD enzyme
an important enzyme in a pathway which provides protection against oxidant stress. Early loss of enzyme activity in the red cell results in inability to restore reduced glutathione. With oxidant stress, denatured hemoglobin attaches to the membrane and spectrin may be damaged. The resultant decrease in deformability and presence of abnormal membrane results in splenic trapping and extravascular hemolysis.
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Pyruvate Kinase (PK) Deficiency
PK deficiency is the second most common enzyme deficiency and most common glycolytic enzyme defect. Because of a decrease in converting phosphoenolpyruvate to pyruvate results in decreased ATP, increased 2,3-DPG, loss of membrane plasticity and increase in rigidity, and destruction in the spleen. Patients present with variable chronic anemia, hemolysis, splenomegaly, gallstones and aplastic crises. Laboratory features include mild to severe anemia, increased reticulocytes, and no specific morphology. Management consists of supportive care, folate, and transfusions if severe anemia occurs. Splenectomy may partially ameliorate the disorder. Other glycolytic enzyme defects are very rare and may affect other organs and tissues (e.g., muscle, CNS).
439
List some of the major foods, drugs, or other chemicals which can induce hemolytic anemia in patients with G6PD deficiency.
Associated with oxidant stress: infection, drug, ingestion of specific foods (e.g., fava beans)
440
Describe the pathophysiology and site of RBC destruction of immune-mediated hemlysis.
Antibodies to universal RBC antigens can cause hemolysis. Can cause either intravascular or extravascular hemolysis. Two general types : cold and warm.
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Direct Coombs test
Blood sample from a patient with immune mediated haemolytic anemia: antibodies are shown attached to antigens on the RBC surface. The patients washed RBC’s are incubated with antihuman antibodies (coombs reagent). RBCs aggluinate: antihuman antibodies form links between RNCs by binding to the human antibodies and RBCs.
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Indirect coombs
The indirect Coombs test (also known as the indirect antiglobulin test or IAT) is used to detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of antibodies present in a patient's plasma/serum prior to a blood transfusion. In antenatal care, the IAT is used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. The IAT can also be used for compatibility testing, antibody identification, RBC phenotyping, and titration studies.
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Warm Antibody Autoimmune Hemolytic Anemia (WAIHA)
is the most common of the autoimmune hemolytic diseases.[1] About half of the cases are idiopathic, with the other half attributable to a predisposing condition or medications being taken.The most common antibody involved in warm antibody AIHA is IgG, though sometimes IgA is found. The IgG antibodies attach to a red blood cell, leaving their FC portion exposed with maximal reactivity at 37°C (versus cold antibody induced hemolytic anemia whose antibodies only bind red blood cells at low body temperatures, typically 28-31°C). The FC region is recognized and grabbed onto by FC receptors found on monocytes and macrophages in the spleen. These cells will pick off portions of the red cell membrane, almost like they are taking a bite. The loss of membrane causes the red blood cells to become spherocytes. Spherocytes are not as flexible as normal RBCs, and will be singled-out for destruction in the red pulp of the spleen as well as other portions of the reticuloendothelial system. The red blood cells trapped in the spleen cause the spleen to enlarge, leading to the splenomegaly often seen in these patients.
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Cold agglutinin disease
an autoimmune disease characterized by the presence of high concentrations of circulating antibodies, usually IgM, directed against red blood cells. At body temperatures of 28-31 °C, such as those encountered during winter months, and occasionally at body temperatures of 37 °C, antibodies (generally IgM) bind to the polysaccharide region of glycoproteins on the surface of red blood cells (typically the I antigen, i antigen, and Pr antigens). Binding of antibodies to red blood cells activates the classical pathway of the complement system. If the complement response is sufficient, red blood cells are damaged by the membrane attack complex, an effector of the complement cascade. In the formation of the membrane attack complex, several complement proteins are inserted into the red blood cell membrane, forming pores that lead to membrane instability and intravascular hemolysis (destruction of the red blood cell within the blood vessels). If the complement response is insufficient to form membrane attack complexes, then extravascular lysis will be favored over intravascular red blood cell lysis.
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Indications for splenectomy
When it becomes very large such that it becomes destructive to platelets/red blood cells. For diagnosing certain lymphomas. Certain cases of wandering spleen. When platelets are destroyed in the spleen as a result of an auto-immune condition, such as idiopathic thrombocytopenic purpura. When the spleen bleeds following physical trauma. Following spontaneous rupture. For long-term treatment of congenital erythropoietic porphyria (CEP) if severe hemolytic anemia develops. The spread of gastric cancer to splenic tissue. When using the splenic artery for kidney revascularisation in renovascular hypertension. For long-term treatment of congenital pyruvate kinase (PK) deficiency
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Risks of splenectomy
As splenectomy causes an increased risk of sepsis due to encapsulated organisms (such as S. pneumoniae and Haemophilus influenzae) the patient should receive the pneumococcal conjugate vaccine (Prevnar), Hib vaccine, and the meningococcal vaccine; see asplenia. These bacteria often cause a sore throat under normal circumstances but after splenectomy, when infecting bacteria cannot be adequately opsonized, the infection becomes more severe. An increase in blood leukocytes can occur following a splenectomy. The post-splenectomy platelet count may rise to abnormally high levels (thrombocytosis), leading to an increased risk of potentially fatal clot formation. There also is some conjecture that post-splenectomy patients may be at elevated risk of subsequently developing diabetes. Splenectomy may also lead to chronic neutrophilia. Splenectomy patients typically have Howell-Jolly bodies and less commonly Heinz bodies in their blood smears. Heinz bodies are usually found in cases of G6PD (Glucose-6-Phosphate Dehydrogenase) and chronic liver disease
447
Explain when prophylactic antibiotics are indicated post-splenectomy and the role of vaccination.
These are recommended in patients at high risk of pneumococcal infections and the antibiotics of choice are oral penicillin V or macrolides. Patients developing infection, despite measures, must be given systemic antibiotics and admitted urgently to hospital.
448
Sickle cell disease
a autosomal recessive genetic disorder of hemoglobin, in which both β-globin genes are mutated, at least one with the characteristic single amino acid substitution (β6(glu®val)).
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Chronic Hemolytic Anemia
The sickle RBC is rigid and fragile, resulting in chronic RBC destruction (hemolytic anemia). Sickle RBCs in sickle cell anemia (HbSS) survive approximately 20 days in the circulation, as compared to 120 days for normal RBCs. Growth retardation/delay, which may be multifactorial and related to anemia, increased metabolic rate due to increased RBC production and vitamin deficiencies. Bilirubin (“pigmented”) gallstones, due to the chronic elevation in bilirubin, present in most patients with sickle cell disease, often by the second decade of life.
450
Describe the geographic distribution of sickle cell disease and a situation where people heterozygous for sickle cell disease may have a survival advantage.
Sickle cell probably arose in india content and three diff places in africa due to malaria. Malaria has to live in RBC for a certain amount of time. Sickle cell cant support malaria parasite so the infection is less sever. Its not just surviving but also fertility. Severe malaria can affect fertility. HbD punjab- middle east. HbE- south east asia.
451
Describe the findings on the CBC and peripheral blood smear in patients with sickle cell disease.
Anemia with compensatory increase in reticulocyte count. The severity of the anemia varies with type of sickle cell disease. Increased baseline white blood cell count (WBC) and platelet count in some patients due to exuberant bone marrow response to hemolytic anemia and other factors. An increased baseline WBC has been associated with increased mortality and morbidity in sickle cell anemia. Increased Red Cell Distribution of Width (RDW). Because sickle RBCs transition from sickled to unsickled, changing shape, and because reticulocytes are very young, large RBCs, there is significant variation in the size of RBCs as analyzed by automated cell counters. Abnormal peripheral smear with sickle forms, schistocytes (“broken”, irregular cells), polychromasia (blue-colored cells representing reticulocytes), anisocytosis (variation in size of RBCs), poikilocytosis (variation in shape of RBCs). Howell-Jolly bodies (small purple dots within RBCs) are seen in patients without a functional spleen. Target cells and hemoglobin C crystals (red “rods” within RBCs) are seen in hemoglobin SC disease (due to hemoglobin C). Microcytosis (with a low mean corpuscular volume or MCV) and target cells are found in Sβothalassemia and Sβ+thalassemia. Abnormal chemistry profile with increased total/indirect bilirubin, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) as they are released from the lysed RBCs.
452
Describe what “sickle trait” is and the consequences of having sickle cell trait.
When carried as a genetic trait in the heterozygous state (one gene normal, one gene mutated), the presence of these abnormal hemoglobins likely reduces the morbidity and mortality of malaria, providing the carriers with a survival advantage. Sickle cell trait, by contrast, occurs in a person with one sickle cell gene and one NORMAL gene. This normal gene, producing normal β globin chains in normal quantities, protects against the development of sickle cell disease. Persons with sickle cell trait do NOT develop sickle cell disease. RARE splenic infarct in white males at high altitude, hematuria, renal medullary carcinoma (VERY rare), ?death in athletes
453
Describe major variants of sickle cell disease, including sickle beta-thalassemia and SC disease.
β-thalassemia also occurs in these areas, as do other β-globin variants including hemoglobin C (β6(glu®lys)), DPunjab (β121(glu®gln)), E β26(glu®lys)and OArab (β121(glu®lys). Sickle cell disease occurs in persons who have two abnormal β-globin genes, one from each parent. By definition, at least one of these genes has the sickle mutation. The other β-globin gene must also be abnormal; if the other gene also has the sickle cell mutation, the disease is called “sickle cell anemia” (HbSS). Sickle cell disease also occurs if the other gene has a different mutation (e.g. for hemoglobin C), or there is underproduction of normal β globin chain (e.g. β-thalassemia)
454
Describe the precipitating factors and pathophysiologic process by which hemoglobin S causes sickling
When deoxygenated, sickle hemoglobin polymerizes into 14-strand helical fibers which distort the shape of the RBC into a “sickle form” or other irregular shapes and damages the RBC membrane. When reoxygenated, the polymers dissolve, and the RBC returns to its normal shape. After several deoxygenation-reoxygenation cycles, the cell becomes irreversibly sickled and is lysed (destroyed). The presence of other hemoglobins in the RBC, such as hemoglobin C (as in Hb SC disease) or some normal hemoglobin A1 (as in Hb Sβ+thalassemia), interfere with polymerization and lessen the tendency for RBC sickling and membrane injury, which may attenuate the severity of the clinical manifestations. Misformed and non-misformed RBCs are sticky and can interact with vessels, especialy post capillary venules (after releasing O2). This leads to Abnormal cellular adhesion to endothelium (sickle RBC and WBCs); Direct damage to endothelium: upregulation of adhesion molecules, exuberant repair mechanisms, apoptosis; and Abnormal vasoregulation. Abnormal cellular adhesion to endothelium (sickle RBC and WBCs) Retention of adhesion molecules on damaged RBC membranes, especially on reticulocytes. Adhesion of less dense cell (retics) noted at shear stresses seen in arterioles and post-capillary venules. Adhesion of dense cells in static systems
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Direct damage to endothelium from hemoglobin S
Difficult to distinguish effect of RBC adhesion itself from elements that enhance RBC adhesion e.g. hypoxia, cytokines. Effects of sickle RBCs: With they get stuck leads to sickle and lysis leading to oxidant injury. Infarction with tissue is hypoxic. WBCs get signals to clean up but there are innocent bystanders laeding to more injury.
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Abnormal vasoregulation with hemoglobin S
Less capacity to make NO can occur acutely. NO vasoregulate. When rbc lyse they release arginase, reducing bioavailability.
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Aplastic crisis
Since sickle cell disease patients rely on an increased reticulocyte count to compensate for increased RBC destruction, anything that compromises the bone marrow’s ability to rapidly produce RBCs can result a sudden drop in hemoglobin, called an aplastic crisis. The characteristic finding is a low reticulocyte count. In children, an important cause of aplastic crisis is Parvovirus B19, which causes “fifth disease” and infects RBC precursors, arresting their development into mature RBCs. This infection is usually transient, but patients may require transfusion of RBCs if the hemoglobin falls significantly. Other severe infections, medications or vitamin (e.g. folic acid) deficiencies can also result in an aplastic crisis.
458
Bone Marrow Transplantation
Allogenic bone marrow transplantation offers a potential cure for a patient with an HLA-matched full sibling unaffected by sickle cell disease, with >90% overall and disease-free survival. However, <20% of eligible patients have such a donor available. Mini-allogenic transplantation (less harsh preparative chemotherapy) has been attempted to lessen the morbidity and mortality of the procedure, but thus far has resulted in graft rejection and relapse of the sickle cell disease in most cases. The use of unrelated donors remains to be explored.
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Hydroxyurea Therapy
Hydroxyurea, an oral chemotherapy agent, is able to induce the production of fetal hemoglobin, which is otherwise “shut off” in most people within the first 6-12 months of life. Fetal hemoglobin significantly interferes with sickle hemoglobin polymerization; administration of hydroxyurea is associated with increased fetal hemoglobin levels, improvement in anemia, reduced frequency of acute pain crises and reduced mortality in adults with sickle cell anemia (Hb SS) and Hb Sβothalassemia. Hydroxyurea appears to be safe in children and may have benefits similar to those seen in adults. To date there are no data that chronic complications of sickle cell disease (splenic injury, stroke, lung disease, kidney disease) are reduced by hydroxyurea, but studies are ongoing.
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Transfusion Therapy
Most people with sickle cell disease do not require regular transfusions of RBCs, despite their anemia. In fact, transfusion of RBCs to above the patient’s usual hemoglobin level and/or above a hemoglobin of 10 g/dl in sickle cell anemia (HbSS) or Sβothalassemia may actually increase blood viscosity and cause harm. However, if there is acute worsening of anemia (e.g. splenic sequestration, aplastic crisis) or acute end-organ injury (e.g. acute chest syndrome), transfusion of RBCs may rapidly reverse the life-threatening process. Transfusion can be given by simply administering RBCs (simple transfusion) or by simultaneously removing the patient’s RBCs as normal RBCs are being given (exchange transfusion). In patients with recurrent or severe acute events, chronic transfusions may be given to sustain 50-70% normal RBCs in the circulation, reducing the sickle RBCs to less than 30-50%. This can reduce the chance of recurrent severe events, including splenic sequestration, stroke and acute chest syndrome. Unfortunately, transfusion may be associated with transmission of infectious agents and antibody formation which makes future transfusion difficult. If multiple simple transfusions are given, iron overload develops, which can cause organ damage (especially of the liver and heart) unless the iron is removed by a chelation agent.
461
Explain iron chelation therapy, its indications, and its drawbacks.
Iron overload is a common clinical problem, arising from disorders of increased iron absorption such as hereditary haemochromatosis or thalassaemia intermedia syndromes or as a consequence of chronic blood transfusions for various blood disorders. Can cause growth retardation, bone abnormalities, gastrointestinal problems
462
Explain how newborn screens can be used to diagnose sickle cell disease.
The type of sickle cell disease is usually determined by preparing a lysate of the RBCs to release the hemoglobins. These hemoglobins are then separated and identified by their characteristic locations on an electrophoretic gel or off a liquid chromatography (HPLC) column. The relative percentages of each hemoglobin are quantified
463
Review the normal structure of hemoglobin and indicate the globin chains that typically make it up. Describe how the composition of globin chains in hemoglobin changes during fetal development and after birth
The hemoglobin molecule is a heterodimer that contains 2 α-globin chains and another pair of different globin chains, normally β and δ; γ chains are produced by the fetus and newborn. The major hemoglobin in human red blood cells (RBCs) after 4-6 months of age is hemoglobin A1, which consists of 2 α chains and 2 β chains (α2β2). Production of α-globin chains is controlled by 4 genes, 2 on each chromosome 16. Production of other globin chains is controlled by one gene cluster on each chromosome 11.
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α-Thalassemia
In α-thalassemia, the α-globin chain is underproduced, most often due to an absence of one or more of the four genes which control production. α-thalassemia is most common in persons of southeast Asian, African and Mediterranean descent. The types and characteristics of α-thalassemia are described:
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β-Thalassemia
In β-thalassemia, the β-globin chain is underproduced, most often due to point mutations which result in dysfunctional genes. Hemoglobin E (Hb E) is a structurally abnormal hemoglobin, due to a point mutation in the β-globin gene (β26(glu®lys)), which is unstable. The amount of Hb E in the RBC is lower due to this instability, causing some of the same RBC changes as in classical thalassesmias.
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pathophysiology of thalassmia
Thalassemia defects result in the underproduction of either α- or β-globin chains, or from the production of an unstable hemoglobin like hemoglobin E. The consequences of this include: Low Concentration of Hemoglobin in RBCs. RBCs in thalassemia and hemoglobin E are smaller (low MCV) and have a low mean corpuscular hemoglobin concentration (MCHC). Despite the smaller size, there is an excess of RBC membrane, which results in a shape like a Mexican hat. When viewed in cross-section, the cell appears as a “target” cell, a characteristic feature of thalassemias and Hb E diseases. Imbalance in Chain Production: Underproduction of one globin chain results in unmatched excess of the other globin chain. For example, in β-thalassemia, there is a relative excess of α-globin chains. Excessive, unused globin chains can precipitate, undergo denaturation and oxidation, resulting in membrane damage and increased red cell fragility. This results in increased RBC apoptosis (death) and ineffective erythropoiesis (inability to make enough mature RBCs). Relative Increase in Other Hemoglobins: In β-thalassemia, there is an underproduction of β-globin chains. However, production of the other globins in the same gene cluster continue, so there is a relative increase in the percentage of Hb A2 (α2δ2) and persistent/increased production of Hb F (α2γ2).
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Describe the geographic distribution of thalassemia. Describe a situation where people heterozygous for thalassemia may have a survival advantage.
In contrast, for those of African descent, each chromosome usually has one intact α gene and one deleted (- α / - α), so offspring inherit at least one α gene. As long as there is one α gene, the fetus can survive. Thus, persons of African descent with α-thalassemia trait are less likely than those of Asian descent to have a pregnancy with hydrops fetalis. β-thalassemia occurs most commonly in persons of Mediterranean, African and southeast Asian descent; Hb E is most common in those of southeast Asian descent.
468
Explain why Southeast Asians with alpha-thalassemia are more likely than Africans with alpha-thalassemia to have a child with hydrops fetalis.
In those of southeast Asian descent, both genes on the same chromosome are usually missing, which means the person has one normal chromosome (with 2 α genes) and one without any α genes (- - / α α). This person can pass on a chromosome with no functional α genes, and if the other parent contributes a similar chromosome, the offspring will inherit no α genes. The fetus will be unable to make any normal form of hemoglobin, since α chains are necessary for all normal hemoglobin molecules, which leads to death in utero (called “hydrops fetalis”) or at birth. Bone marrow transplantation has been successfully performed in utero to prevent hydrops fetalis in affected pregnancies.
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Transfusion Support for thalassemia
In severe thalassemias (β-thalassemia major, HbEβothalassemia), RBC transfusions are started within the first 2 years of life to maintain hemoglobin values generally between 8 and 10 g/dL, which avoids excessive bone marrow expansion and extramedullary hematopoiesis, and permits adequate growth and development. However, within 1-2 years of the initiation of transfusion, significant iron overload occurs, and within 10 years, significant cardiac dysfunction develops unless chelation therapy is started. Splenectomy may reduce the amount of transfusion needed in patients with a very large spleen. Chelation therapy: involves the administration of medications with bind excess iron stores and remove them from the body. The most common chelation agent, deferoxamine, is infused subcutaneously over 8-12 hours, usually in the abdominal area, 5 to 7 times a week. Compliance with this therapy is challenging for some patients. Deferiprone (L1) is an oral chelator which is used in Europe, often in conjunction with deferoxamine. Deferasirox (ICL670), another oral chelator, has been preliminarily approved for use in the United States, and appears to be as effective as deferoxamine.
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Increase Fetal Hemoglobin (α2γ2) Production for thalassemia
: In β-thalassemia, there is an excess of α chains. In the absence of β chains, increasing the production of γ chains provides a pool of chains to combine with the excess α chains, reducing their harmful effects in the RBC. Hydroxyurea, butyrate, and decitabine have variable effects on γ-chain and Hb F production, and may benefit some patients with β-thalassemia major.
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Bone Marrow Transplantation for thalassemia
Thalassemia can be cured by successful bone marrow transplantation. Thalassemia-free survival at 20 years is about 70% in those who receive bone marrow from HLA-identical unaffected siblings. Unfortunately, only about 30% of patients have a matched sibling; ongoing work suggests that the use of HLA-matched unrelated donors may be an alternative. Sibling cord blood transplantation, as a source of stem cells, has not generally been successful to date due to graft rejection.
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DIAGNOSIS OF THALASSEMIAS
Hemoglobin separation techniques, including electrophoresis or HPLC, are commonly used to make the diagnosis of thalassemias. Genetic testing can also be performed in specialized labs, and for purposes of prenatal diagnosis. The diagnosis of β-thalassemia is based on the recognition of increased Hb A2 (α2δ2) and Hb F (α2γ2), relative to the underproduced Hb A1 (α2β2). The presence of hemoglobin E is detected by its characteristic mobility on hemoglobin electrophoresis, which is indistinguishable from Hb A2, as shown. However, patients with α-thalassemia underproduce all types of hemoglobin in proportion to each other, so the hemoglobin electrophoresis is normal.
473
Distinguishing iron deficiency anemia from thalassemia
Iron deficiency anemia, characterized by microcytosis and target cells, can be confused with thalassemia trait. If hemoglobin electrophoresis shows an elevated Hb A2, then β-thalassemia is diagnosed. However, in iron deficiency, Hb A2 may be reduced. So, if a person with β-thalassemia becomes iron deficient, the diagnosis of β-thalassemia may be missed because the Hb A2 value will appear normal rather than high because of the iron deficiency. It is important to be sure a person is not iron deficient before relying on hemoglobin electrophoresis to evaluate for thalassemia.
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Toxoid
toxin like. It retains its immunogenicity; if you’re immunized with toxoid, your antibodies cross-react with and neutralize toxin.
475
Primary RNA transcript
Heavy chain’s variable domain is coded by V, D, and J segments. The developing B cell first brings one random D segment close to one J; the DNA is cut, the intervening DNA is discarded and
476
RAG recombinases
The enzymes that do the recombination of antibody and T cell receptor DNA are called RAG-1 and RAG-2 recombinases. The recombinases first bind splice signals to the right of a D segment and the left of a J segment, pull them together, and then cut and splice. Then they look for a splice sequence to the right of a V segment and do it again. If RAGs are knocked out, mice make neither B nor T cells. It happens in humans, too—very rarely (Omenn Syndrome). The RAG gene system appears in evolution with the jawed vertebrates; lampreys don’t have it.
477
Somatic mutations
The V-D and D-J joins are “sloppy.” The cell uses randomizing mechanisms: First, exonucleases for chewing away a few nucleotides after the DNA is cut but before two gene segments (D to J, V to DJ) are joined. Second, for adding a few nucleotides as well, an enzyme called terminal deoxynucleotidyl transferase, TdT, which doesn’t use a template so its additions are random. Thus you can’t predict the sequence at the joining area (which is called an “N” region); it might be obvious that V7 has joined to D2, let’s say, but in this cell there’s an extra alanine and tyrosine there, and in that one there is a leucine missing. This produces a lot more completely random diversity. There is a price for it: two times out of three the N region, being of random length, will create a frame-shift mutation, that is, a nonsense codon which terminates transcription.
478
CROSS-REACTIVITY
This refers to the tendency of one antibody to react with more than one antigen. Again, it has to do with goodness of fit. An antibody is “against” mumps virus if it was obtained from an donor immunized with mumps, or if it reacts with mumps with a high association constant Ka (high affinity); but we must remember that all the antibody has is a combining site made up of six CDRs whose position, charge, and hydrophobicity distribution is such that an antigenic determinant of the mumps virus binds it with observable affinity. Other antigenic determinants might also fit it; if they did so detectably, the antibody cross-reacted with those determinants. T cell mediated immunity has similar specificity and cross-reactivity. The two cross-reacting epitopes may have very similar amino acid sequences. Sometimes they don’t: but then the charge/hydrophobicity distribution will be similar.
479
CLONAL SELECTION THEORY
each cell of the immune system is programmed to make only one antibody (T cells weren’t known yet, but the theory covers them, too); that the choice of which antibody the cell will make is random, not dependent on outside information; and that the entire population of potential antibody-making cells preexists in a normal individual, even before any contact with antigens. When a new antigen is introduced into the body, it comes into contact with a huge number of lymphocytes, and when it encounters one to whose receptors it binds with sufficient affinity, it activates it, resulting in expansion of that clone and production of that antibody. The best- fitting clones are selected by antigen. Clonal selection is Darwinian; survival of the fittingest, as it were.
480
Define allotypic exclusion and state the number of chromosomes in a cell which bear H or L genes, including the number that actually contribute to a single B cell's antibody product.
The lambda, kappa, and H chain gene families are all on different chromosomes. A potential problem arises because, since we’re diploid, each cell has two copies of each gene, maternal and paternal. Shouldn’t that one cell make two different H chains and four different L chains, and therefore, by random combination, many different antibodies? It doesn’t happen; only one H chain (maternal or paternal in origin) and one L chain (either kappa or lambda, either maternal or paternal) are synthesized in any one B cell. All the other genes are silenced. Though the person can make two H-chain allotypes, each individual B cell makes only one. Sloppy recombination often ends up with a frame-shift mutation; when one examines a particular B cell, one often finds H and L genes that have been abortively rearranged, that is, in such a way as to produce nonsense codons. When this happens the cell tries again with the other allele; if things work, it goes on to become a B cell, if not, complete antibody cannot be made and the cell dies. If it gets lucky on the first try, though, it doesn’t try the other allele. So although any one cell is theoretically capable of making 2 H chains (by rearranging both maternal and paternal loci,) and 4 light chains (maternal and paternal, κ and λ,) that doesn’t happen; it makes only one of each—all other alleles are excluded.
481
Explain why we commonly write V(D)J instead of VDJ.
the DNA which codes for the variable domain of an L or H chain the ‘V domain gene region’ rather than the V locus. This is because it turns out that at the DNA level, the information to code for a variable domain is actually broken up into segments or ‘minigenes.’ ->The variable domain region of heavy chain genes is composed of multiple V, multiple D, and multiple J gene segments; the V region of light chains into multiple V and J segments, so generically when talking about H and L genes we say ‘V(D)J.’ The cell will choose one of its V’s, one D, and one J to make a VH domain gene region.
482
CLASS SWITCHING
A single mature B cell starts by making both IgM and IgD, which it puts into its membrane as receptors. Later it may switch to making IgG, IgE, or IgA. In all cases, the V domain stays the same but the C region of the H chain changes. As may be anticipated by now, what happens is that the cell which has put its particular H-chain VDJ combination together with its mu and delta genes (as shown in the diagrams) goes back to its DNA, does a loop-out of mu and delta, and puts VDJ next to the C-region gene of gamma or epsilon or alpha, while excising and discarding the intervening DNA. The new mRNA, then, may be VDJα or VDJγ or VDJε. Thus a cell which is making IgM can go on to make IgG, but a cell making IgG cannot go back to making IgM; the mu information is physically gone. “M to G” or “M to A” or “M to E” class switching is common in antibody responses, and requires T cell help; without it, only IgM responses are possible. Remember that the cell switches heavy chain class, but doesn’t switch light chains; they remain the same throughout the B cell’s life.
483
GERM LINE VERSUS SOMATIC MUTATION
There used to be two schools of thought about antibody diversity: one said that all the V genes were in the germ line; if you looked at a fertilized ovum you could predict all potential antibodies that potential individual could potentially make. The other said that only a few were there. It postulated that during embryonic lymphoid development these genes underwent repeated (somatic) mutation until a full complement of antibodies was produced. Both theories, it turns out, were right. A lot of our diversity is in the germ line (that is, in the individual V, D, and J segments you’re born with). Even more diversity is also generated by variable (“sloppy”) V/J and V/D joining.
484
SOMATIC HYPERMUTATION
Another source of receptor diversity: the recombined V(D)J unit is “hypermutable”; each time a B cell divides after antigenic stimulation there is a good chance that one of the daughters will make a slightly different antibody. Selection by antigen of the best-fitting mutants after antigenic stimulation allows a gradual increase of affinity during an immune response—an exceptionally nice design feature called affinity maturation. (For T cells, somatic mutation after contact with antigen does not seem to take place.)
485
How hypermutation works
Activation-Induced [Cytidine] Deaminase (AID) converts a random cytosine in the CDR gene regions to uracil. So a cytosine: guanine pair becomes a uracil: guanine mismatch. The uracil bases are removed by the repair enzyme uracil-DNA glycosylase. Error- prone DNA polymerases then fill in the gap, creating mostly single-base substitution mutations, so at the end of cell division one daughter may be making a different (worse? better?) antibody.
486
N-region diversity
Random nucleotides added or subtracted at CD and DJ joints. Estimated to produce 100 times more diversity than the germ line
487
Describe the basic process of donor qualification and blood collection relating specific steps to blood safety.
Volunteer blood donation, without significant monetary incentives, provides the first level of safety for blood recipients. At the time of phlebotomy, the donor completes a questionnaire and provides multiple answers to questions about current or past illnesses or surgery, travel, vaccination, and high-risk behavior. In addition, the individual is urged to call back the phlebotomy site with the onset of any symptoms of viral syndrome or other infection. The abbreviated physical exam (vital signs, general appearance, skin, upper extremities) reaffirms the general health of the donor. Screening tests including Hematocrit (Hct) and, in the case of an apheresis platelet donor, platelet count certify the safety for both the recipient and the donor. Skin preparation for the phlebotomy reduces the risk of bacterial contamination of blood products, production of components. Blood products may be obtained by phlebotomy of whole blood or collection of specific components by apheresis. The latter is a technique where automated machines take anticoagulated blood from the donor, separate out one or more components by centrifugation and buoyant density, and return the unused components to the donor. From whole blood phlebotomy, 450-500 ml of whole blood is mixed with ~67 ml of preservative/anticoagulant solution. This may be stored as whole blood or separated into packed red blood cells (PRBCs) and fresh frozen plasma (FFP). Alternatively, the whole blood may be separated into FFP, PRBCs, and, if a two-step centrifugation is performed, random donor platelet concentrate (1 aliquot). Apheresis machines may collect platelet concentrate (by apheresis), FFP, or red blood cells individually or in combination. Usually, one or two (e.g., platelets and plasma or PRBCs, or one apheresis platelet concentrate) components are collected. The separation of products from whole blood must be completed within 8 hours of the blood draw. Each component has a separate set of storage conditions to assure optimal function of the specific blood component for the entire storage time.
488
Infectious Disease Screening
In addition to screening interview strategies noted previously, screening laboratory tests are completed on all donations and must be negative before the blood products are released for transfusion. This has added dramatically to the safety of homologous transfusions in the past 20 years. The standard tests include testing for antibody against and/or antigen for each infectious agent. In the last 6-8 years, the addition of nucleic acid amplification tests (NAT) for Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), and West Nile Virus (WNV) have been included in the testing scheme, detect viral genome in blood samples from donors, and have decreased the risk of transmission to very low levels. Donors found screen positive and confirmed for an agent are deferred from further donation. CMV transmission is infrequent with seronegative or leukoreduced products. Screening for Chagas disease is completed depending on region of donation in the U.S. Currently, family, social, travel, and exposure history are the basis for questionnaire screening for Creutzfeldt Jakob Disease (CJD) or variant CJD. Vaccination against Vaccinia results in a temporary deferral. There is concern about parvovirus, Epstein-Barr Virus (EBV), dengue and malaria, and screening tests may be implemented in the future. Deferral for travel to a malaria area or defined malaria disease is currently based on travel and personal medical history.
489
Whole blood
is kept at 4-6ºC for 35 days to optimize red cell recovery and survival. Platelet and neutrophil function degenerate by 24-48 hours of storage and clotting factors turn over more slowly but well before the outdate. Whole blood may be used in massive transfusions to replace oxygen carrying capacity and blood volume when 1 blood volume has been shed and replaced with crystalloid or colloid and packed red cells. This component must be crossmatched (anything with significant numbers of RBCs must be crossmatched) and infused through a microaggregate filter (routine infusion set) over 2-4 hrs or more rapidly with acute blood loss.
490
PRBCs
are also stored at 4-6ºC for 35 days or longer (42 days) with addition of special solutions to support higher ATP levels. With the plasma removed, the Hct is 70% in 200-250 ml volume and these products may be leukoreduced with high efficiency filters (<25-30. Age and disease related changes to level of Hct may apply to indication. PRBCs must be crossmatched and administered like whole blood.
491
FFP
is an acellular product which is kept at -18ºC for one year and contains >80% of all plasma procoagulant and anticoagulant proteins as well as complement factors. FFP may be used to treat coagulopathy related to procoagulant deficiency (DIC, liver failure, vitamin K deficiency, etc.). Specific clotting factor deficiencies (VIII, IX, VII) are treated with factor specific concentrates. FFP may support anticoagulants in general, but AT-III and protein C concentrates are available. FFP must be type specific and administered as tolerated over 1-3 hrs but not >4 hrs.
492
Cryoprecipitate
is made from fresh plasma frozen quickly at -80ºC and allowed to sit for 18 hours at 4ºC. Cryoprecipitable proteins are isolated after centrifugation and removal of cryopoor plasma. The resultant prep is frozen at -18ºC for up to one year. Cryoprecipitate contains 80-100 U factor VIII/bag, a biologically equivalent amount of von Willebrand’s factor, 200-250 mg of fibrinogen, and increased levels of factor XIII than is found in plasma. (Caution! IgM isoagglutinins for ABO antigens may also be concentrated and can cause hemolysis if incompatible with patient’s ABO type). Cryoprecipitate. The indications for use include low or absent fibrinogen associated with acquired or congenital/inherited disorders and replacement for factor XIII deficiency. Concentrates of factor VIII are used for hemophilia/factor VIII deficiencies. Two commercially available VIII concentrates also contain Von Willebrand’s Factor (VWF). If these are not available, cryoprecipitate may be used for bleeding in patients with von Willebrand’s disease. Consider giving ABO type specific or compatible; infuse over 30-45 minutes.
493
Platelet concentrates come in two types
Random donor unit (RDUs) concentrates (5x1010 platelets in 50 ml) or apheresis platelet concentrates (3x1011 platelets in 200-300 ml). These may be leukoreduced (by filtration after production or elutriation techniques applied to apheresis) for patients receiving many transfusions. Concentrates are stored at 22-24ºC for 5-7 days in gas permeable bags. During storage, reasonable platelet function is maintained but concentrates are a poor source of clotting factors. Apheresis platelets avoid exposure to multiple donors. Platelet concentrates. Indication is for bleeding associated with thrombocytopenia and/or platelet dysfunction. Consider ABO type specific/compatible. Infuse over 30-45 minutes. For pediatric patients, 10 ml/kg or for adults, 1 apheresis unit or a pool of 6-10 random donor units raise platelet count by 50-100,000/μl.
494
Granulocyte (white blood cell) concentrates
may be collected by apheresis procedures. No storage is allowed. Granulocytes are kept at room temperature and transfused within 8-12 hours. May collect 2x1010 – 2x1011 neutrophils depending on whether donors are given a single dose of G-CSF or not. Hc, 3-5%. Volume, 200-300 ml. Many platelets contaminate granulocyte products. Granulocyte concentrates. Reserved for severe bacterial or fungal infection in patients with ANC <4 hrs.
495
Discuss the basic blood groups (ABO and Rh) and contrast the different compatibility requirements of basic blood components.
For all transfusions, donor cells are typed for ABO and Rh(D), and the serum is screened for antibodies other than ABO. The transfusion recipient is tested the same way. Finally, the major crossmatch is completed: recipient’s serum and donor cells are mixed and evaluation for agglutination after centrifugation at room temperature is completed (immediate spin). Classically, the Coombs reagent is added to determine if the recipient’s serum sensitizes donor’s cells with IgG or complement; however, this is no longer required. The minor match (donor’s serum and recipient’s cells) was eliminated several years ago. The type and crossmatch takes ~ 45 minutes and markedly reduces the possibility of immediate hemolytic transfusion reactions. Urgent situations require an abbreviated procedure. (1) No time (e.g., exsanguinating hemorrhage, massive trauma). Give O, Rh(D) negative; or for males and non-childbearing females, use O, Rh(D). (2) Abbreviated testing: if the donor and recipient ABO and Rh(D) types are identical and the antibody screen is negative in both, the unit may be infused without completing the crossmatch. This should only be used in urgent situations where transfusion cannot be delayed for 45 min to complete the crossmatch. The risk of an adverse event is 1/17,000. When an antibody is detected in the recipient (antibody screen positive), the antibody must be identified by standard serologic techniques before transfusing the patient. If specific alloantibodies are detected, then units negative for the antigen(s) may be administered to avoid an adverse event. If autoantibodies are detected and no alloantibodies co-exist, the indication for the transfusion is re-evaluated. If transfusion is required, give least incompatible units and transfuse with in vivo crossmatch. If alloantibodies co-exist with autoantibodies, units which are negative for specific alloantigens are identified and are transfused with an in vivo crossmatch.
496
Explain the infectious risks of blood transfusion and describe testing strategies to reduce risks of specific agents.
The risks of transfusion for transmission of specific agents are as follows: Syphilis <1/300,000.
497
Febrile non-hemolytic transfusion reactions
(≥ 1°C rise in temperature from pre-transfusion level) and mild allergic reactions (hives, transient skin rashes) are the most common adverse events (1/200 and 1/400, respectively). After confirming that other types of reactions are not occurring in the patient, treatment with antipyretics (e.g., Tylenol) and antihistamines (e.g., Benadryl) are required before re-instating the infusion. Future transfusions may require pre-medication with antipyretics and/or antihistamines or leukoreduced cell containing blood products.
498
Immediate hemolytic transfusion reactions
as a result of infusing incompatible blood products (usually ABO) present an infrequent (<1/30,000) but severe and possibly life-threatening event. The cause is usually misidentifying or mislabeling samples from the patient or donor. Activation of complement and intravascular hemolysis may lead to shock, acute renal failure and disseminated intravascular coagulation. Vigorous supportive care, diuretics and heparin may be required.
499
Delayed hemolytic reactions
represent the process of alloantibody production (1/2,500 transfusions) and slow destruction of the sensitizing red cells with very few symptoms and signs. Documentation of these alloantibodies should become part of the medical record since their existence may increase the risk of immediate hemolytic transfusion reactions with future transfusions.
500
Anaphylactic reactions
are fortunately rare (1/150,000 transfusions) and usually occur without identifying the specific reagent. The bronchospasm and/or large airway response is treated with epinephrine, benadryl and steroids. May be seen in IgA deficient individuals (the most common humoral immunodeficiency syndrome).
