Haematology 1 Flashcards
(207 cards)
1
Q
What does myeloid mean?
A
Of the marrow!
Refers to red cells, platelets, granulocytes and their precursors
2
Q
Myeloid diseases
A
Arise clonally within the marrow
e.g acute myeloid leukaemia, chronic myeloproliferative neoplasms
3
Q
Myeloid cells found in the bone marrow
A
Myeloblast Promyelocyte Myelocyte Metamyelocyte (band cells)
4
Q
Myeloid cells found in the blood
A
(band cells)
Neutrophils
5
Q
Clonal defect which prevents myeloid maturation
A
Acute myeloid leukaemia
6
Q
Clonal defect which causes defective myeloid maturation
A
Myelodysplastic syndromes
7
Q
Clonal defect which causes excessive myeloid maturation down one limb of the maturation pathway
A
Chronic myeloproliferative neoplasms
- Polycythaemia (RBCs)
- Essential thrombocythaemia (Platelets)
- Chronic myeloid leukaemia
- Myelofibrosis
8
Q
Features of AML
A
No neutrophils (total development block)
9
Q
Features of myelodysplastic syndromes
A
Abnormal cells
e.g granulocytes with no granules
10
Q
Features of CML
A
loads of neutrophils
philadelphia chrosome
11
Q
Eosinophilia associated with
A
- Allergic states
e. g asthma, dermatitis, drug rashes and with parasitic infections - Vasculitic disorders
- Myeloproliferative disease
12
Q
Proportion of lymphocytes that are T cells
A
75%
13
Q
Ratio of CD4:CD8 cells
A
2:1
14
Q
Causes of splenic infarcts
A
Haematological disorders include:
-polycythaemia -hypercoaguable states (protein C or protein S deficiency, sickle cell disease)
-malignant conditions (lymphomas and leukaemias)
In infiltrative haematological diseases the splenic circulation gets congested by abnormal cells.
Embolic causes:
- atrial fibrillation
- ventricular mural thrombus following a myocardial infarction
- septic emboli from infective endocarditis.
15
Q
Reed Sternberg cells
A
Giant cells found in Hodgkin’s lymphoma.
Neoplastic germinal centre-derived B cells
16
Q
Subtypes of classical Hodgkin lymphoma
A
- nodular sclerosing (NS) most common
- mixed cellularity (MC)
- lymphocyte-rich (LR)
- lymphocyte-depleted (LD)
17
Q
Morphology of lymph nodes in Hodgkin lymphoma
A
Enlarged and sometimes matted together
18
Q
What are B symptoms?
A
fever
night sweats
weight loss
19
Q
Prognositc significance of B symptoms
A
If untreated, death generally occurs within 6 to 24 months, but with treatment there is an 85% cure rate.
20
Q
Symptoms of a ruptured spleen
A
Sudden onset left upper abdo pain.
Symptoms and signs of hypovolaemic shock.
21
Q
Predisposing factors to splenic rupture
A
Malaria
Infectious mononucleosis
Lymphomas/leukaemias
Typhoid fever
22
Q
Precursor of platelets
A
Megakaryocytes- sheds platelets from cytoplasm
23
Q
Causes of decreased numbers of platelets
A
Primary platelet defect Marrow suppression Autoimmune attack Consumption Sepsis
24
Q
Difference between immunohistochemistry and immunophenotyping
A
IHC- tend to stain tissue
Immunophenotyping- single cell suspension, generally look for markers on the outside
25
Reed-Sternberg cells
aka lacunar histiocytes/ owls eyes
seen in Classical Hodgkin lymphoma
Multi-nucleate or multilobate large cell with each nucleus or lobe containing a prominent eosinophilic nucleolus with a modest rim of amphiphilic cytoplasm
26
Use of FISH
Fluorescence in situ hybridization
Chromosomal abnormalities
FISH can be used to identify missing, duplicated or translocated genetic material
27
CD20 monoclonal antibody
Rituximab
28
What type of FISH would you use to detect common translocations in which both partners are known?
Dual colour fusion probe FISH
29
What type of FISH would be useful if you don’t know the translocation partner?
Dual colour breakapart probe FISH
30
BCR-ABL tyrosine kinase inhibitor
Imatinib
first-line therapy for most patients with chronic myelogenous leukemia (CML). More than 90% of CML cases are caused by a chromosomal abnormality that results in the formation of a so-called Philadelphia chromosome
31
What is the Philadelphia chromosome?
Fusion between the Abelson (Abl) tyrosine kinase gene at chromosome 9 and the break point cluster (Bcr) gene at chromosome 22, resulting in a chimeric oncogene (Bcr-Abl) and a constitutively active Bcr-Abl tyrosine kinase that has been implicated in the pathogenesis of CML.
32
Mutation associated with myeloproliferative disease
JAK2 V617F
33
Mutation associated with haemochromatosis
HFE C282Y
34
What's HFE1?
Hereditary haemochromatosis
Hereditary disease characterized by excessive intestinal absorption of dietary iron resulting in a pathological increase in total body iron stores. Humans, like most animals, have no means to excrete excess iron.
Excess iron accumulates in tissues and organs disrupting their normal function. The most susceptible organs include the liver, adrenal glands, heart, skin, gonads, joints, and the pancreas; patients can present with cirrhosis, polyarthropathy, adrenal insufficiency, heart failure or diabetes.
35
Function of HFE gene
A working model describes the defect in the HFE gene, where a mutation puts the intestinal absorption of iron into overdrive. Normally, HFE facilitates the binding of transferrin, which is iron's carrier protein in the blood. Transferrin levels are typically elevated at times of iron depletion (low ferritin stimulates the release of transferrin from the liver). When transferrin is high, HFE works to increase the intestinal release of iron into the blood. When HFE is mutated, the intestines perpetually interpret a strong transferrin signal as if the body were deficient in iron. This leads to maximal iron absorption from ingested foods and iron overload in the tissues.
36
Mutation associated with alpha-1 antitrypsin deficiency
A1AT G324L
37
Mutation associated with familial adenomatous polyposis coli
APC
MUTYH
MUTYH glycosylase, involved in oxidative DNA damage repair. Base excision repair
38
BRCA mutations linked to which cancers
breast
ovarian
prostate cancer
39
Next generation sequencing based on...
```
DNA extraction
Amplification / library preparation
Sequence detection
Alignment to a reference sequence
Bioinformatics analysis
```
40
In what disease dose a translocation between the genes PML and RARA occur? What does the fusion protein do?
Seen in one form of acute myeloid leukaemia (AML) produces a fusion protein that blocks granulocytic differentiation.
41
Where and when does erythropoiesis occur in the foetus?
- Starts at about 21 days gestation
- Initially in the yolk sac “blood islands”
- Then in the fetal liver
- Then moves to the bone marrow
42
Where does erythropoiesis occur in the adult?
Restricted to the pelvis, sternum, vertebrae and ends of long bones (femora/humeri)
43
How many RBCs do we have? How many are produced per hour?
- Adults have approximately 4 x 10^12 red cells per litre of blood
- Each RBC has a normal lifespan of 120 days
- 1% of the total need to be replaced daily to maintain a steady state…
- This equates to 10^10 red cells produced per hour
44
RBCs found in the blood
Reticulocytes
| RBCs
45
Stages of RBC found in the bone marrow
- CMP
- CFU
- Proerythroblast
- Basophilic erythroblat
- Intermediate erythroblast
- Late erythroblast
46
What controls the number of RBCs and the amount of haemoglobin? Where is it released from?
EPO (erythropoietin)
| Epo is produced by the interstitial cells of the renal cortex, in response to hypoxia.
47
Stages in RBC production that can go wrong and cause anaemia
Hypoxaemia detected in the kidney (using HIF pathway)
EPO synthesis increased
- Insufficient epo production
Increased red cell synthesis in the marrow
- Lack of the components needed for red cell synthesis
- Defective red cells made?
More oxygen carrying capacity in the circulation
- Red cells lost too soon from the circulation
48
What does EPO do to the bone marrow?
The loss of oxygen-carrying capacity will cause an increase in Epo synthesis, and there will be erythroid hyperplasia in the marrow until a steady state is reached again.
49
Why is a reticulocyte count helpful?
An increased reticulocyte count demonstrates that the process of erythropoiesis is still intact, and that the marrow is responding to the loss of red cells.
This is a useful parameter for determining whether anaemia is due to increased consumption or impaired production.
50
Dietary components needed for RBC synthesis
Iron
Folate
B12
51
How is iron absorbed from the gut?
Binds to divalent metal transporter 1 (DMT1).
Allowed out of the cell by ferroportin.
In the digestive tract, it is located on the apical membrane of enterocytes, where it carries out H+ coupled transport of divalent metal cations from the intestinal lumen into the cell.
DMT1 expression is regulated by body iron stores to maintain iron homeostasis.
Ferroportin is a transmembrane protein that transports iron from the inside of a cell to the outside of the cell. After iron is absorbed into the cells of the intestine, ferroportin allows that iron to be transported out of those cells and into the bloodstream.
52
How is ferroportin inhibited?
Ferroportin is inhibited by hepcidin, which binds to ferroportin and internalizes it within the cell. This results in the retention of iron within enterocytes, hepatocytes, and macrophages with a consequent reduction in iron levels within the blood serum. This is especially significant with enterocytes which, when shed at the end of their lifespan, results in significant iron loss. This is part of the mechanism that causes anaemia of chronic disease; hepcidin is released from the liver in response to inflammatory cytokines, namely interleukin-6, which results in an increased hepcidin concentration and a consequent decrease in plasma iron levels.
53
How does iron deficiency occur?
Not ingesting enough/increased requiremetnt
- limited diet in elderly, vegetarians/vegans (haem iron is more readily asorbed than non-haem iron), pregnancy, toddlers
Not absorbing enough
- Coeliac disease (iron absorbed in duodenum), gastrectomy (HCl needed to solublise iron)
Increased iron loss
- GI bleed (gastric or colon cancer), gynae bleeding (menorrhagia)
54
Characteristic changes in the blood in iron deficiency
- Low MCV (microcytosis)
- Low MCH (hypochromia)
- Variation in the size and shape of the red blood cells (anisocytosis and poikilocytosis).
- Some red cells become pencil shaped.
- The reticulocyte count will not be as high as expected.
55
How to investigate iron deficiency
1. Serum iron
decreased in iron deficiency; however is subject to acute variation due to dietary intake, and also falls in infection/inflammation
2. Transferrin
the circulating iron transport protein and has two binding sites for Fe+++ . The level is raised in iron deficiency. The serum transferrin is frequently reduced in patients with inflammatory arthritis and malignancy.
3. Serum ferritin
levels roughly correlate with the amount of tissue storage iron when the serum ferritin is below 2000 mg/l.
Serum ferritin levels are low in iron deficiency but there is much variation: ferritin also acts as an acute phase protein, rising in infection and inflammation.
56
Function of B12
B12 is needed for nucleic acid synthesis, and is also thought to be needed for myelin maintenance.
57
How and where is B12 taken up?
Absorption is dependent on binding to intrinsic factor, which is produced by the
parietal cells of the stomach.
The B12-IF complex absorbed in terminal ileum.
58
How does vitamin B12 deficiency occur?
Not ingesting enough/increased requiremetnt
- very limited diet (elderly), vegetarians/vegans (B12 found exclusively in animal sources), pregnancy
Not absorbing enough
- pernicious anaemia (autoimmune gastritis of lack of IF)
- gastrectomy or atrophic gastritis (lack of IF)
- pancreatic disease, pancreatectomy
- crohn's disease
- ileal resection
- tropical sprue
59
Characteristic changes in the blood in B12 deficiency:
Anaemia with a high MCV (macrocytosis), red cells often oval
The neutrophil count and platelet count may also be reduced
Hypersegmented neutrophils
The reticulocyte count will
not be as high as expected.
60
Clinical features of B12 deficiency
Glossitis
Peripheral neuropathy
Dorsal column involvement
61
Investigations for B12 deficiency
• Serum B12
If pernicious anaemia:
• Anti-intrinsic factor antibodies
• Anti-gastric parietal cell antibodies
• Further GI/pancreatic assessment if needed.
62
What's folate needed for?
Folate is important for the donation of single carbon units, i.e. methyl groups, to make amino acids. It is required in the synthesis of pyrimidines and purines of nucleic acid, and therefore deficiency has
some similarities to B12 deficiency
63
What's folate destroyed by?
Heat
64
Who might require extra folate?
Pregnant or breast feeding women
Clear and important relationship between folate deficiency and neural tube defects in pregnancy – supplementation recommended.
65
Where is folate absorbed?
Jejunum (coeliac patients at risk of deficiency)
66
How does folate deficiency arise?
Not ingesting enough/increased requiremetnt
- very limited diet (elderly)
- patients on dialysis
- increased desquamation (exfoliative dermatitis)
- pregnancy
Not absorbing enough
- coeliac disease
Anti-folate medications
- methotrexate
- anti-convulsants
67
Characteristic changes in the blood in folate deficiency:
Anaemia
High MCV
White count and platelet count may be low
68
What can cause a lack of red cell precursors or marrow space?
Loss of early red cell precursors or haemopoietic stem cells (e.g. aplastic anaemia) can limit erythropoiesis
Obliteration of the marrow space (e.g. by metastatic malignancy) can mean erythropoiesis is suppressed.
69
What can nucleated RBCs in the blood indicate?
Marrow stress e.g infiltration
70
Fate of defective red cells?
Early destruction
e.g thalassaemia myelodysplastic syndromes
71
How can kidney disease cause anaemia?
Lack of EPO production
72
Anaemia of chronic disease and iron
* Also known as the anaemia of inflammation
* Complex mechanism, still not fully understood.
* Characterised by mis-handling of iron
* Increased levels of the master iron regulatory protein hepcidin
* Relatively suppressed epo production
* Relatively suppressed response to epo stimulation
* Frequently seen in chronic inflammatory conditions such as rheumatoid arthritis and in some cancers.
73
Clinical features of anaemia of chronic disease
Often a mild anaemia (Hb usually 80g/L or higher)
Typically normal MCV (sometimes microcytic)
Iron studies can be hard to interpret, but typically:
- Low serum iron
- High or normal ferritin
- Low transferrin
74
What does the following indicate?
Iron low
Transferrin high
Ferritin low
Iron deficiency anaemia
75
What does the following indicate?
Iron low
Transferrin low
Ferritin normal/high
Anaemia of chronic disorders
76
Macrocytic megaloblastic anaemias
B12 deficiency
folate deficiency
Nuclear maturation affected
77
Macrocytic, non-megaloblastic anaemias
Liver disease
| Hypothyroidism
78
Definition of haemolysis
The premature destruction of the mature RBC
79
Life span of a RBC
120 days
80
Lab features of haemolysis
• Elevated unconjugated bilirubin
Haem itself is normally degraded to biliverdin, then bilirubin
Bilirubin transported to liver to be conjugated for excretion– but in haemolytic states this system is overloaded
• Elevated lactate dehydrogenase
• Elevated reticulocyte count
suggests a bone marrow response to falling Hb
may also see nucleated red cells in the peripheral blood
• Mildly macrocytic anaemia
Reticulocytes are slightly larger than normal RBCs
81
What's intravascular haemolysis and what are the lab features?
Haemoglobin released directly into the bloodstream binds to Hb-binding protein haptoglobin
This system rapidly saturated -> free haemoglobin found in the blood
```
Hence in intravascular haemolysis:
• Low serum haptoglobin levels
• Haemoglobinaemia
• Haemoglobinuria
• Haemosiderinuria
```
82
What's haemosiderinuria?
Hemosiderinuria (syn. haemosiderinuria), "brown urine", occurs with chronic intravascular hemolysis, in which hemoglobin is released from RBCs into the bloodstream in excess of the binding capacity of haptoglobin. (Haptoglobin binds circulating hemoglobin and reduces renal excretion of hemoglobin, preventing tubular injury.) The excess hemoglobin is filtered by the kidney and reabsorbed in the proximal convoluted tubule, where the iron portion is removed and stored in ferritin or hemosiderin. The tubule cells of the proximal tubule slough off with the hemosiderin and are excreted into the urine, producing a "brownish" color. It is usually seen 3-4 days after the onset of hemolytic conditions.
83
Extravascular haemolysis
Red cell are lysed mainly in the spleen – so falls in haptoglobin are less marked,and free haemoglobin is not found.
84
Clinical features of haemolysis
Pallor and jaundice
Splenomegaly
Chronic haemolysis may also give
- pigment gallstones
- leg ulcers
- marrow expansion
85
Causes of haemolysis
Congenital
(defects in the main components of the red cell; membrane, haemoglobin, enzymes)
Immune
(autoimmune, alloimmune)
Non-immune
(mechanic, MAHA, infection)
86
Congenital cause of haemolysis involving the RBC membrane
- Hereditary spherocytosis
Most common haemolytic anaemia due to a membrane defect (1/5000 in Northern Europeans.
75% of cases inherited in autosomal dominant fashion.
- Hereditary elliptocytosis
Usually autosomal dominant condition
Variety of mutations affecting spectrin / protein 4.1
‘Horizontal connections’ affecting lateral stability of RBC membrane
Often mild in heterozygosity
- Hereditary pyropoikilocytosis
Look like they've been burnt
87
What's hereditary spherocytosis? Defects, etc
Molecularly heterogeneous, but vertical connections in the cytoskeleton thought to be affected.
Primary defects may be in ankyrin, band 3, spectrin, or other components of the red cell membrane, though the common feature is a resultant lack of spectrin
Weakened association between the cytoskeleton and the phospholipid bilayer allows microvesiculation of portions of the bilayer, causing spherocytosis
Spherocytes are unable to distort while passing
through the microvasculature / splenic cords
88
How is Hereditary spherocytosis diagnosed?
- FBC
- reticulocyte count
- film
- negative DAT
- high bilirubin
- high LDH
- SDS-PAGE to look for spectrin, ankyrin, band 3
Used to do an osmotic fragility test – now outmoded
(put RBCs i hypotonic solution. Biconcave RBCs can withstand lower tonic solution as spheres have less space to absorb.
89
What's a DAT?
direct antiglobulin test
90
Treatment of hereditary spherocytosis
Folic acid supplementation
| Consider splenectomy
91
Problems with globin that lead to haemolysis
* Haemoglobin variants that produce rigid RBCs in hypoxic conditions (e.g. HbSS)
* Haemoglobin variants that are unstable to oxidative damage (e.g. Hb Koln)
Lysis is of red cell precursors rather than mature erythrocytes occurs when there is an imbalance of globin chain synthesis (the thalassaemias)
92
Cause of sickle cell anaemia
Single amino acid change in beta globin chain results in marked reduction in solubility in the deoxygenated state.
Hb molecules polymerize in the deoxygenated state and produce rigid ‘sickle’cells.
Patients with HbSS have chronic haemolysis with crises induced by hypoxia /infection.
93
Signs, symptoms and bloods of sickle cell anaemia
- Low Hb
- Increased reticulocytes
- High bilirubin and LDH
- NO splenomegaly (infarcts and ‘autosplenectomy’- shriveled up)
- Pigment gallstones
- Leg ulcers
- other features secondary to vascular occlusion
94
What are the most common enzyme deficiencies resulting in haemolysis?
Glucose 6 phosphate dehydrogenase
(pentose phosphate shunt)
Pyruvate kinase
(glycolytic pathway)
95
What's the role of glucose 6 phosphate dehydrogenase?
It restores NADPH which can then reduce glutathione disulfide bonds and restore the reducing power of the cell.
Without G6PD, the cell runs short of reducing agents. Peroxides inc H2O2 accumulate, with consequent membrane damage and haemolysis
96
What are the different classes of G6PD deficiency?
X-linked. Molecularly heterogeneous
Class I variants
<10% normal G6PD activity; usually have chronic haemolysis (rare)
Class II variants
Severe enzyme deficiency but only intermittent haemolysis
Class III variants
Relatively mild enzyme deficiency – intermittent haemolysis
Common African variant included here
Intermittent haemolysis occurs in response to exposure to oxidants. Drugs (e.g. antimalarials and antibiotics) may be responsible.
97
Which word describes an acute haemolytic crisis after ingestion of fava beans
‘Favism’
98
Pathogenesis of G6PD
Loss of reduced glutathione
• Oxidation / denaturation of Hb (Heinz bodies)
• Oxidation of membrane proteins and increased RBC rigidity
• Haemolysis – both intra- and extra-vascular
99
What would you see on a blood film of G6PD deficiency?
Heinz bodies (intracellular hemoglobin precipitates) form from oxidized, denatured hemoglobin which is no longer soluble.
Bite cells. ‘Bites’ are from splenic macrophages removing the part of the red blood cell with a Heinz body.
Arises when bite cells undergo repair of the cell membrane, resulting in a clearing within the red cell where the Heinz body previous was.
Schistocytes
100
Who suffers from G6PD and when are they symptomatic?
400 million sufferers worldwide – predominantly in Southern Europe, West and central Africa, the Middle East, India, Thailand and Southern China.
Mutation affords some protection against malaria.
May be generally asymptomatic, with haemolytic episodes resulting from exposure to oxidants.
101
Diagnosis and management of G6PDH deficiency
Diagnosis is made by measuring enzyme levels, but can be difficult in the acute phase – enzyme levels are higher in reticulocytes.
Can measure ability of RBCs to generate NADPH with an excess of G-6-P as a screening test.
Management focuses on avoiding oxidant stressors where possible.
Folate supplementation
102
Pattern of inheritance of PK deficiency
Classically autosomal recessive inheritance
103
Mechanism of PK deficiency
Not exactly known
Reduced ATP generation will impact upon ATP-dependent processes such as active transport across the cell membrane -> Loss of cellular K+ and water -> Increased RBC rigidity -> Haemolysis
104
Clinical features of PK deficiency
Clinical features are very variable – depending on degree of enzyme activity
Anaemia, jaundice and splenomegaly are common
Pigment gallstones are increased in frequency
105
Management of PK deficiency
Supportive
| Severe cases may require splenectomy
106
Capsulated bacteria
Gosh Some Nasty Killers Have Serious Capsule Protection
```
Group B strep
Strep pneumoniae
Neisseria meningitides
Klebsiella pneumoniae
Haemophilus influenza type B
Salmonella typhi
Cryptococcus neoformans (fungi)
Pseudomonas aeruginosa
```
107
What's WAIHA?
IgG: “warm” AIHA – antibodies react best with red cells at 37°C
IgG coats circulating RBCs; phagocytosis by splenic macrophages.
Often idiopathic; may be associated with other AI disease (e.g. SLE).
May be found with lymphoproliferative diseases
108
What's cold AIHA?
IgM: “cold” AIHA – antibodies react best with red cells at <32°C
IgM coats red cells in the peripheries in cool conditions. Pentameric Ab causes red cell agglutination, and complement activation. Lysis may occur intravascularly.
May be associated with lymphoma or certain infections (mycoplasma).
109
What's seen on a blood film on warm AIHA?
Spherocytes
| Reticulocytes
110
What's the direct antiglobulin test?
- Take blood
- Add anti human globulin (recognises the Fc region of Ig) -> crosslinking
- Positive -> agglutinates
111
How do you diagnose AIHA?
Coombs test
112
Who gets AIHA and how do you treat it?
Idiopathic form commoner in women and in middle age.
Presentation variable, but may be of acute onset with severe anaemia.
Investigation should consider the possibility of an underlying predisposing cause.
Treatment for warm AIHA usually starts with steroids – other forms of immunosuppression may need to be considered.
Blood transfusion is avoided if possible.
Folate supplementation is given.
113
Drugs causing AIHA – various mechanisms
drug acts as a hapten
(e.g. penicillin)
drug permits complement activation on the red cell membrane
(e.g.quinidine)
drug prevents T-suppressor inhibition of autoantibody production (e.g. methyldopa)
Withdrawal of the drug is the first therapeutic step – but antibodies may persist for some time.
114
Allo-immune haemolytic anaemia
Antibodies against from red cells come from another source –
| • e.g. Maternal antibodies to paternal antigens on fetal red cells (RhD haemolytic disease of the newborn)
115
Haemolytic disease of the newborn
RhD+ fetal cells may cross the placenta (after trauma, or naturally in the third trimester) with resulting immunisation of the mother.
IgG antibodies cross the placenta and react with fetal red cells in subsequent affected pregnancies.
Clinical features in affected pregnancies range from mild jaundice in the infant to intra-uterine death from hydrops.
Anti-D prophylaxis is now routinely administered to all RhD negative mothers during pregnancy to limit the chance of primary immunisation.
116
Examples of Non-immune causes of haemolytic anaemia
* Mechanical stress on the red cell
* Microangiopathic Haemolytic Anaemia (MAHA)
* Some infections
* Drugs
* Severe burns
117
Blood film and biochemical test results of mechanical haemolysis
- Schistocytes
- Low serum haptoglobin
- Haemoglobinuria
- Haemosiderinuria
- High LDH
- High bilirubin
118
Which diseases is microangiopathic haemolytic anaemia observed?
```
disseminated malignancy
eclampsia
malignant hypertension
SLE
TTP/HUS (Thrombotic Thrombocytopenic Purpura/ Haemolytic uremic syndrome)
DIC
```
119
Infections that cause haemolytic anaemia
- malaria can result in severe intravascular haemolysis
(‘blackwater fever’)
- clostridium perfringens
120
What's Paroxysmal nocturnal haemoglobinuria?
A rare condition in which a clone of cells arises which lack glycosyl-phoshoinositol, an anchor for several membrane proteins, including some involved in complement regulation.
Red cells in PNH are very sensitive to complement mediated intravascular haemolysis.
121
How many globin chains does a haemoglobin molecule have?
4
122
Where is haem synthesised and what is needed?
* Synthesized partly in a mitochondrion and partly in the cytosol
* Initiation of haem synthesis requires the presence of iron
123
Where are the globin genes located?
The various globin chains are encoded by two clusters of globin genes:
1. The α cluster on chromosome 16. Each chromosome 16 has one ζ gene and two α genes, α2 and α1.
2. The β cluster on chromosome 11. Each chromosome 11 has one ε gene, two γ genes (γG and γA), one δ gene
and one β gene.
124
What is HbA? Normal proportion in adults
α2β2
98%
125
What is HbF? Normal proportion in adults
α2γ2
~1%
126
What is HbA2? Normal proportion in adults
α2δ2
<3.5%
127
Types of haemoglobinopathies
Two types:
Synthetic (Quantitative) disorders:
e.g. Thalassaemia
```
Structural Variants (Qualitative) disorders:
e.g. Sickle Cell Anaemia
```
128
α thalassaemia
α-globin chain production decreased.
β globin chains are normal.
129
β thalassaemia
β globin chain production decreased.
α-globin chains are normal.
130
β0 thalassaemia
No β-globin chain is made
131
β+ thalassaemia
decreased β-globin chain is made
132
How many α and β-globin genes do we have?
4 α genes and 2 β genes -> there is wide phenotypic variation of thalassaemias.
133
What causes the pathological features of alpha thalassaemia?
Deposition of excess β chains
134
Normal defect of alpha thalassaemia
Deletion of α-globin genes
135
α+ Thalassaemia
One α-globin gene deleted
136
Homozygous α+ Thalassaemia
One α-globin gene deleted on each chromosome
| only 2 functioning α-globin gene
137
α0 Thalassaemia
Two α-globin genes on the same chromosome are deleted.
| only 2 functioning α-globin gene
138
HbH disease
Two α-globin genes on the same chromosome are deleted and one α-globin gene deleted on the other chromosome.
(only 1 functioning α-globin gene)
139
Hemoglobin Barts
No functioning alpha genes
Hydrops Fetalis. Hemoglobin Barts, abbreviated Hb Barts, is an abnormal type of hemoglobin that consists of four gamma globins. It is moderately insoluble, and therefore accumulates in the red blood cells. It has an extremely high affinity for oxygen, resulting in almost no oxygen delivery to the tissues. As an embryo develops, it begins to produce alpha-globins at weeks 5-6 of development. When both HBA1 and HBA2, the two genes that code for alpha globins, are non-functional, only gamma globins are produced. These gamma globins bind to form hemoglobin Barts. It is produced in the disease alpha-thalassemia and in the most severe of cases, it is the only form of haemoglobin in circulation. In this situation, a fetus will develop hydrops fetalis and normally die before or shortly after birth, unless intrauterine blood transfusion is performed.
140
Prevalence, symptoms and treatment for absence of 1-2 alpha chains
– Common
– Asymptomatic
– Does not require therapy
141
Symptoms for absence of 3 alpha chains
– Microcytic anaemia (Hgb 7-10)
| – Splenomegaly
142
Presentation of 4 alpha chains missing
– Hydrops fetalis (non-viable)
143
Laboratory findings in αα/-α
Hgb (g/dl) 12-14
MCV (fl) 75-85
RDW Normal
144
Laboratory findings in α-/α- or - -/αα
Hgb (g/dl) 11-13
MCV (fl) 70-75
RDW Increased
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Laboratory findings in --/- α
Hgb (g/dl) 7-10
MCV (fl) 50-60
RDW Very raised
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RDW
Red blood cell distribution width
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HbH blood film
Hypochromic target cells
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Technique used to investigate haemoglobinopathies
High Performance Liquid Chromatography
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Phenotypes of Beta thalassaemia
1. Beta – Thalassaemia minor (trait)
2. Beta – Thalassaemia intermedia
3. Beta – Thalassaemia major
(2 and 3 are genetically the same)
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Beta Thalassaemia trait
* No symptoms
| * Mild microcytic anaemia
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Beta Thalassaemia Major
* No beta chain produced (no HbA)
* Severe microcytic anaemia occurs gradually in the first year of life
* Marrow expansion (Frontal bone bossing)
* Iron overload
* Growth failure and death
* Life-long transfusion programme with iron chelation
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Symptoms of beta thalassaemia mamjor
* fatigue, weakness, or shortness of breath.
* a pale appearance or a yellow color to the skin (jaundice)
* irritability.
* deformities of the facial bones.
* slow growth.
* a swollen abdomen.
* dark urine.
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Haemoglobin analysis of beta thalassaemia minor (trait)
Genotype β/ β+ or β/ β°
HbA 90-94
HbA2 3.5-8
HbF 1-10
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Haemoglobin analysis of beta thalassaemia intermedia
Genotype β+/β+
HbA 5-60
HbA2 2-8
HbF 20-80
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Haemoglobin analysis of beta thalassaemia major
Genotype β+/β°
HbA 2-10
HbA2 1-6
HbF >85
or β°/β°
HbA 0
HbA2 1-6
HbF >94
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Approach to Beta Thalassaemia Major management
* Screening /counselling
* RBC transfusion therapy + chelation therapy
* Agents to increase haemoglobin F (Hydroxycarbamide)
* Better understanding of Haemoglobin switching***
* Bone marrow transplantation
* Gene therapy
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Agent used to increase haemoglobin F
Hydroxycarbamide
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Genetics of sickle cell disease
• Autosomal recessive genetic disease:
• β-globin gene (chromosome 11q) mutation
GAG->GTG at 6th codon
• Glutamic Acid ->Valine at the 6th amino acid along
the β-globin chain
* Α2β2 = normal haemoglobin
* α2βS1 = heterozygote = Sickle trait
* α2S2 = homozygous recessive = Sickle cell disease
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Pathophysiology of sickle cell
- deoxygenated HbS polymerises and forms sickled RBCs
- occlusion of vessels leading to infarction (neuropathy, osteonecrosis -> hip replacements in teens, acute pain, acute chest syndrome, hyposplenism)
- haemolysis (pulmonary hypertension, leg ulcers, cerebrovascular disease x50 risk)
- inflammation (increased expression of VCAM1 and other adhesion molecules, hypercoaguable)
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Sickle cell anaemia blood film
Sickle cells
Erythroblasts
Howell-Jolly body
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Howell-Jolly bodies vs Heinz bodies
Howell-Jolly bodies are little fragments of the red cell nucleus. You see them most commonly in patients with splenectomies (normally, the spleen just bites them out).
Heinz bodies are seen in G6PD deficiency. They represent denatured globin chains. When there’s not enough G6PD around, the bonds between heme and globin are attacked. Heme is just recycled, but the globin chains become denatured, forming a little ball that sticks to the inside of the red cell membrane. This is the Heinz body. You can’t see it unless you do a special stain.
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How do you screen for sickle cell trait
* RBC lysate with concentrated phosphate buffer and sodium hydrosulfite
* Incubate 10-20 min
HPLC
Confirmatory Tests: DNA mutational analysis
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Approach to management of Sickle Cell Anaemia
* Universal antenatal and newborn screening
* Parental education
* Early referral to specialist services – screening /monitoring
* Prophylactic penicillin and folic acid supplements
* Transfusion therapy (including stroke prevention)
* Hydroxycarbamide (Haemoglobin F inducing agent)
* Allogeneic bone marrow transplantation
* Targeted therapies – Crizanlizumab (monoclonal antibody)
* Gene therapy
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Antenatal diagnosis of Haemoglobinopathies
* Test partners of heterozygous or affected individuals
| * Antenatal diagnosis from DNA obtained by chorionic villus sampling, or by amniocentesis
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Screening in sickle cell to prevent risk of stroke
- Annual
- Transcranial doppler (TCD) ultrasound of middle cerebral artery
- Stroke Risk Increases With Elevated TCD Velocities
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What is donated blood screened for?
* HIV
* HTLV (human T-lymphotropic virus)
* Hepatitis B and C
* Syphilis
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Use of blood components
* Red cells
* Most commonly used blood component (approximately 2 million units per year in UK)
* Used to treat acute bleeding or some types of anaemia
* Fresh Frozen Plasma (FFP)
* Most often used to correct clotting factor deficiencies in bleeding patients, or used prophylactically to prevent bleeding
* Platelets
* Used to treat bleeding in thrombocytopenic patients or to prevent bleeding in those perceived to be at risk of bleeding
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Who won the Nobel prize for
| discovery of blood groups ?
Karl Landsteiner in 1901
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What are the ABO blood group phenotypes and genotypes?
4 phenotypes (6 genotypes):
* O (genotype OO)
* A (genotype AA or AO)
* B (genotype BB or BO)
* AB (genotype AB)
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Blood group O
What are the:
- antigens
- antibodies
- safe blood groups for red cell transfusion
- safe blood groups for plasma tranfusion?
- antigens: none
- antibodies: anti-A, anti-B
- safe blood groups for red cell transfusion: O
- safe blood groups for plasma tranfusion: O, A, B, AB
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Blood group A
What are the:
- antigens
- antibodies
- safe blood groups for red cell transfusion
- safe blood groups for plasma tranfusion?
- antigens: A
- antibodies: anti-B
- safe blood groups for red cell transfusion: A or O
- safe blood groups for plasma tranfusion: A or AB
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Blood group B
What are the:
- antigens
- antibodies
- safe blood groups for red cell transfusion
- safe blood groups for plasma tranfusion?
- antigens: B
- antibodies: anti-A
- safe blood groups for red cell transfusion: O or B
- safe blood groups for plasma tranfusion: AB, B
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Blood group AB
What are the:
- antigens
- antibodies
- safe blood groups for red cell transfusion
- safe blood groups for plasma tranfusion?
- antigens: A, B
- antibodies: none
- safe blood groups for red cell transfusion: AB, B, A, O
- safe blood groups for plasma tranfusion: AB
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What's RhD?
- One of the Rh antigens
| - Other Rh antigens include c, C, e, E
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Consequence f a patient with anti-D antibodies is transfused with RhD
positive blood?
they may develop a haemolytic transfusion reaction
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When can you be RhD negative and form anti-D antibodies?
After exposure to blood during pregnancy
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Why do you not get haemolytic disease of the new born with incompatible ABO blood group but you do with RhD incompatibility?
anti ABO are IgM (normally)
anti-D are IgG
IgG can cross the placenta
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What is haemolytic disease of the newborn?
* This occurs if a RhD negative pregnant mother has a RhD positive fetus and develops IgG anti-D antibodies
* If a woman is RhD negative then prophylactic anti-D treatment is administered during pregnancy to prevent her developing anti-D
* This is given routinely at 28 and 34 weeks gestation and again at birth.
* This is very effective and rates of haemolytic disease of the newborn have fallen rapidly over the last few decades.
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What is Group and save?
* All recipient blood tested for:
* ABO group
* RhD status
* Screen for antibodies (Indirect anti-globulin test)
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What is cross-matching?
• Donor blood is tested against recipient’s plasma to ensure it is a match
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If blood is needed in an emergency there might not be time for a full compatibility test. What can you do instead?
1) O RhD negative blood (emergency stock) – available immediately
2) Blood of the same ABO and RhD group as the patient -available in 15-20 minutes
3) Fully cross-matched blood -available in 30-45 minutes
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Risks of blood transfusions
* Risk of serious complications 1 in 21,413
* Risk of death from a transfusion reaction or transfusion transmitted infection is 1 in 322,580
* Risk of alloimmunisation 1/50
• Acute transfusion reactions:
• Life-threatening
- Acute haemolytic transfusion reaction
- Anaphylaxis
- Bacterial contamination
- Transfusion-associated acute lung injury
- Transfusion-associated circulatory overload
• Others
- Non-haemolytic febrile transfusion reaction
- Urticaria
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Symptoms and signs to raise suspicion of acute transfusion reaction
* Fevers, flushing or chills
* Urticaria (spots or patches of raised red, or white, skin)
* Rigors (shaking or exaggerated shivering)
* Tachycardia (fast heart rate)
* Hypotension (low blood pressure)
* Collapse
* Respiratory distress
* Loin pain
* Haemoglobinuria
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What do you do if you suspect an acute haemolytic transfusion reaction?
* Call for urgent help
* ABC management
* Stop transfusion!
* Resuscitate
* Call blood bank
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Bacterial contaminations during blood transfusions
* Rare (1 in 1 million transfusions) but life-threatening
* Patients often feverish and shocked
• Blood bag may be broken or show discoloration/clumps
* Call for urgent help
* ABC management
* Stop transfusion!!!
* Resuscitate
* Broad spectrum intravenous antibiotics
* Return blood to blood bank
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Anaphylaxis during a blood transfusion
• Deficient in IgA prone to it
* Often rapid onset after starting transfusion
* Rash, swelling of face/lips
* Airway compromise or hypotension is severe sign
* Call for urgent help (need an anaesthetist present)
* ABC management
* Stop transfusion
* Resuscitate
* Intravenous chlorphenamine and hydrocortisone
* May require intramuscular adrenaline
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What are febrile non-haemolytic transfusion reactions?
• Relatively common (and not serious)
• Due to cytokines in transfused blood
• Temperature rarely rises more than 2°C above baseline.
Always check for evidence of more serious transfusion
reactions.
• Continue transfusion but monitor carefully
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Frequency of transfusion transmitted infections
* Serious infections from transfusion are rare but important
* For comparison, the chance of being struck by lightning is 1:700,000/yr and the chance of winning the UK national lottery is 1:14 million
* What do you think the risks are of contracting the following infections from a blood transfusion?
* 1. Hepatitis B (1:500,000)
* 2. HIV (1:1 million)
* 3. Hepatitis C (1:30 million)
* 4. Creutzfeldt-Jakob disease (CJD) (???)
* 5. New emerging infections (???)
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Complication for patients on lon-tem transfusion programes
Iron overload
* Each unit of blood contains approx. 250 mg iron
* The body is able to excrete approx. 1.5 mg iron per day
* Iron may be deposited in the liver, around the heart and in endocrine glands (amongst others) leading to organ failure
* Caution with transfusion and use of oral iron chelators can reduce this risk
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What are the alternatives to blood transfusions?
* No transfusion!
* Treat underlying cause of anaemia (e.g. iron supplements)
* Erythropoietin (suitable for a subgroup of patients with renal disease and myelodysplastic syndrome)
* Surgery
* Minimize blood loss with surgical technique
* Red cell salvage (intra-operative or post-operative)
* Tranexamic acid
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What's tranexamic acid?
Tranexamic acid (TXA) is a medication used to treat or prevent excessive blood loss from major trauma, post partum bleeding, surgery, tooth removal, nose bleeds, and heavy menstruation. It is also used for hereditary angioedema. It is taken either by mouth or injection into a vein.
Tranexamic acid is a synthetic analog of the amino acid lysine. It serves as an antifibrinolytic by reversibly binding four to five lysine receptor sites on plasminogen or plasmin. This prevents plasmin (antiplasmin) from binding to and degrading fibrin and preserves the framework of fibrin's matrix structure. Tranexamic acid has roughly eight times the antifibrinolytic activity of an older analogue, ε-aminocaproic acid.
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Lab features of autoimmune haemolytic anaemia
- Direct Coombs’ test: Usually positive
- LDH: Increased
- Bilirubin: Increased (unconjugated)
- Haptoglobin: Normal/ slight decrease
- Urinary haemosiderin: -
Usually negative
- Blood film findings: Spherocytes, polychromasia
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Lab features of valve-associated haemolysis
- Direct Coombs’ test: Negative
- LDH: Increased
- Bilirubin: Increased (unconjugated)
- Haptoglobin: Decreased/undetectable
- Urinary haemosiderin: Positive
- Blood film findings: Red cell fragmentat
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What's a Direct Coomb’s test and when is it used?
An antibody against human IgG (anti-human globulin, AHG) is added to a sample of the patient’s red
cells; because the anti-human globulin is bivalent, red cells that are coated with IgG will agglutinate in
its presence. This aggregation constitutes a positive test result. Note that the patient’s red cells would
have to be washed first to remove any IgG which might be free in the patient’s serum rather than bound
onto the surface of the red cells: without this step, the free IgG could neutralise the AHG, and give a
false negative result.
The Coombs’ test may be made specific for different IgG subclasses, or may also detect cells coated
with the C3 component of complement. The direct Coombs’ test is used in the investigation of
haemolytic anaemia, although it should be noted that a positive test does not always mean there is
active haemolysis.
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What's a Indirect Coomb’s test and when is it used?
This uses the patient’s serum added to aliquots of test red cells. If there is an antibody present in the
patient’s serum against antigens on the test red cells, these will combine; subsequent addition of antihuman
globulin will result in agglutination and a positive test result, as in the direct Coombs’ test.
This test is used in transfusion medicine, to look for antibodies in the patient’s serum against red cell
antigens in donor blood.
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Why can alpha thalassaemia trait not be detected by high performance liquid chromatography (HPLC)?
Alpha thalassaemia trait cannot be detected by HPLC because there is no new / alternative haemoglobin formed in the context of a minor lack of alpha globin chains (contrast this with the situation of beta thalassaemia, described below – there is no alpha globin equivalent of the delta chain).
Note that in more severe alpha thalassaemia (e.g. the inheritance of only one functioning alpha globin gene, -α/--) it is possible to detect tetramers of beta globin chains (β4). This unstable haemoglobin is
called HbH, and these alpha thalassaemia syndromes are therefore termed HbH disease.
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Why does sickle cell anaemia not present in infancy?
The underlying genetic defect in sickle cell disease is a point mutation in the beta globin gene. In infancy, the predominant haemoglobin is HbF (α2γ2) which does not include the beta globin chain. Once the predominant haemoglobin switches to HbA (α2β2), typically late in the first year of life, sickle cell anaemia becomes clinically apparent.
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Why does beta thalassaemia trait results in an elevated HbA2 level?
Beta thalassaemia trait may be diagnosed by an elevation of HbA2 (α2δ2). The delta chain is encoded alongside beta globin at the beta globin locus on chromosome 11, and when there is a defect in beta globin transcription there is a compensatory increase in delta chain production (though the precise molecular mechanism behind this is unclear).
199
What are the complications of sickle cell anaemia in the central nervous system?
- stroke
(including in children, where transcranial doppler measurements of middle cerebral artery blood flow can be used to assess the potential risk of stroke)
- neuropsychological abnormalities
200
What are the complications of sickle cell anaemia in the hepatobiliary system?
- pigment gallstones
| - hepatic sequestration crises
201
What are the complications of sickle cell anaemia in the cardiovascular system?
- pulmonary hypertension (pathogenesis related to reduced nitric oxide and increased thrombogenicity)
- microvascular occlusion
- transfusion associated cardiac iron overload
202
What are the complications of sickle cell anaemia in the musculoskeletal system?
- painful crises
- dactylitis
- aseptic avascular osteonecrosis (usually of the femoral head)
- salmonella osteomyelitis
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What's dactylitis?
Dactylitis or sausage digit is inflammation of an entire digit (a finger or toe), and can be painful. The word dactyl comes from the Greek word "daktylos" meaning "finger". In its medical term, it refers to both the fingers and the toes.
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Ferritin, serum iron and transferrin in iron deficiency anaemia
Ferritin: low
Serum iron: low
Transferrin: high
205
Ferritin, serum iron and transferrin in anaemia of chronic disease
Ferritin: normal/high
Serum iron: low
Transferrin: low
206
Ferritin, serum iron and transferrin in transfusion-dependent thalassaemia
Ferritin: high
Serum iron: high
Transferrin: normal/low
207
Explain why haemolytic disease of the newborn due to ABO incompatibility between mother and fetus is less common than that due to Rh incompatibility.
There is ABO incompatibility between mother and fetus in a sizeable minority of pregnancies. However, since most anti-A and anti-B antibodies are of the IgM class they are not able to cross the placenta to induce haemolysis in the fetus.
Some cases of ABO-mediated HDN caused by IgG antibodies do occur. Even in this situation, because cells other than erythrocytes also express the A and B antigens, the offending antibody may be sequestered away from red cells, thus limiting haemolysis.
