Red Blood Cell Disorders

Question 1

Which Gene, which disease? –> Mutation results in replacement of glutamic acid by valine in position 6

Answer 1

Hemoglobin S
Beta globin

Question 2

Hemoglobin S is what?

Answer 2

Beta Globin Gene mutation
*point mutation
*Mutation results in replacement of glutamic acid by valine in position 6
*6Glu–>Val

Question 3

What do hemoglobin S and hemoglobin C have in common?

Answer 3

point mutations in the Beta Globin gene
*Hb S–> 6GLU-VAL
*Hb C–> 6GLU-LYS

Question 4

You see Decreased Hgb A (50-60%), and presence of Hb S (35-45%) on HPLC…

Answer 4

Hemoglobin S TRAIT
*or transfused Hb SS

Question 5

What is the Sickledex test

Answer 5

*(solubility test for Hgb S)
Lyses RBCs, then adds reducing agent
*Reduced Hb S is insoluble
Turbidity of reduced Hb S solution observed in positive tests
*Positive in BOTH sickle cell trait and sickle cell disease

Question 6

What’s the most common hemoglobinopathy in the US?

Answer 6

*Hemoglobin S, and particularly Hb S TRAIT
*Most common hemoglobinopathy in United States
*Present in approximately 9% of African Americans
*Present in 25-45% of population in Western Africa
*Hemoglobin S thought to be protective against Plasmodium infection

Question 7

How do Sickle cell TRAIT patients clinically present?

Answer 7

Patients are usually asymptomatic
*Rarely, symptoms can include vascular occlusion (infarcts) in hypoxic or acidotic conditions (e.g., chronic heart failure, plane travel, respiratory infections, anesthesia)
*Rarely, spontaneous sickling can occur in renal papillae, leading to papillary necrosis, hematuria, and impaired ability to concentrate urine
*Association with exercise-related sudden death and increased risk for renal medullary carcinoma

Question 8

How do HPLC findings change when Hb S is co-inherited with another thalassemia?

Answer 8

If co-inherited with alpha-globin gene deletion(s), Hb S < 35%
If co-inherited with β⁺ thalassemia, percentage of Hb S > percentage of Hb A
If co-inherited with β⁰ thalassemia, almost all hemoglobin is Hb S, same phenotype as sickle cell disease (~ Hb SS)

Question 9

What’s the sickle cell test?

Answer 9

*NOT sicledex!!!
Mixes patient blood and sodium metabisulfite
Deoxygenated red blood cells will sickle and be observed under microscope

Question 10

Sickle cell disorder patients are at risk for which bacterial infections?

Answer 10

They have no functional spleen
*Increased susceptibility to Salmonella and pneumococci

Question 11

What is Hemoglobin C disease?

Answer 11

*Mutation in 1 (“trait”) or both (“disease”) copies of β-globin gene results in substitution of glutamic acid → lysine in sixth amino acid
*Hb C is less soluble than Hb A and crystallizes within RBC
**Hb C crystallizes in oxygenated state as opposed to Hb S, which polymerizes in deoxygenated state
*RBC life span is decreased to 30-35 days

Question 12

Peripheral blood smear with Hemoglobin C disease

Answer 12

Peripheral smear with target cells and in Hb CC polyhedral or rod-shaped “cigar-shaped” crystals (Hb C crystals)

Question 13

What is Hb A2?

Answer 13

Hb A₂ (approximately 3%; α₂δ₂) in normal adults
*elevated when there are no or FEW Beta globin subunits
*Beta-Thal
*point mutations that make Beta globin into C or S will not have A2 elevations, they will have their own peaks

Question 14

What is hemoglobin F?

Answer 14

Hb F (approximately 1%; α₂γ₂) in normal individuals
*increased HbF in Hb SS patients is a good prognostic indicator

Question 15

What do you clinically expect in Hemoglobin C disease?

Answer 15

Hb C disease
Mild to moderate hemolytic anemia
Can have splenomegaly, gallstones
Crystals (rhomboid), target cells
HPLC has it’s own peak with reduced Hb A

Question 16

Tricky thing about Hemoglobin C on acid electrophoresis

Answer 16

Appears identical to Hb EE on acid electrophoresis because Hb E migrates in same position

Question 17

Expected blood smear of Hb SC disease

Answer 17

Sickle cells are not* typically identified
Numerous target cells
Some RBCs may contain precipitated Hb, but hexagonal or rod-shaped crystals associated with Hb CC are not seen
“Boat cells” (plump, angulated cells) are present

Question 18

What’s the unique disease interplay between C and S in Hb SC disease?

Answer 18

C makes S worse
Presence of Hb C causes increased potassium and chloride transport out of cell
**Reduced potassium in red blood cells (RBCs) results in cell dehydration and concentration of Hb S that are thought to increase Hb polymerization
SC cells have decreased life span
***Approximately 27 days vs. 90-120 days for normal RBCs

Question 19

Southeast Asians have an increased risk for what Alpha-Thal?

Answer 19

*NON-deletional
*(generally, Alpha-thal is a deletional problem, B-thal is point mutation problem)
Hemoglobin Constant Spring is the most common nondeletional α-thalassemia in Southeast Asia.

Question 20

Hemoglobin Constant Spring

Answer 20

*type of non-deletional alpha thal
Hemoglobin Constant Spring is the most common nondeletional α-thalassemia in Southeast Asia.
*A point mutation in the stop codon of the α2-globin gene (TAA→CAA) leads to the addition of 31 amino acids to the end of the normal α-globin chain, which renders it unstable.

Question 21

What are the lab findings in Hb CS?

Answer 21

Hemoglobin ‘Constant Spring’
*Hb CS can be missed by all laboratory testing methods due to this instability, which can decrease the variant to undetectable levels. The red cell indices in hemoglobin Constant Spring trait resemble deletional α-thalassemia trait. When coinherited with other α-thalassemia mutations, Hb CS can result in hemoglobin H disease or hemoglobin Bart’s.
*non-deletional Alpha-thalassemia

Question 22

What does del/(-) Alpha3.7 mean?

Answer 22

This is the most common deletional α-thalassemia mutation.
*non-deletional is Hb CS
*another common DELETIONAL mutation is -αTHAI mutatio

Question 23

What is meant by del(-) alpha THAI?

Answer 23

-αTHAI mutation is one of the more common deletional α-thalassemias.

Question 24

What is meant by Hemoglobin Koya Dora?

Answer 24

Hemoglobin Koya Dora is a nondeletional α-thalassemia mutation, but it is not the most common in Southeast Asia.

Question 25

What should you think if you see ALL hemoglobin is Hb A2?

Answer 25

**Hemoglobin E/β-thalassemia
*caused by double heterozygosity for β-thalassemia and the structural hemoglobin variant, Hgb E (G→A substitution in codon 26 of the β-globin gene).
β-Thalassemia results in no hemoglobin being produced from one of the β-globins, and the Hgb E mutation results in a structural variant that is also produced in decreased amounts (thalassemic structural variant). This hemoglobinopathy is found predominantly in Southeast Asia (particularly in Thailand, Laos, and Cambodia). The phenotype is highly variable and can present from mild anemia to transfusion-dependent anemia

Question 26

Southeast asian with thalassemia like indices, has 90+% Hb A2

Answer 26

Hemoglobin E/Beta-thal

Question 27

What is hemoglobin E?

Answer 27

Hgb E (G→A substitution in codon 26 of the β-globin gene).
*elutes as A2 on HPLC

Question 28

what other HPLC hemoglobin peaks are elevated in
beta thal?

Answer 28

Hb F and Hb A2
*these are alpha2-delta2 or alpha2-gamma2
*no beta globin to make A
*watch for combined Hb C or S and beta thal

Question 29

labs in Hemoglobin C/β⁺ thalassemia

Answer 29

chronic hemolytic anemia
*Hematologic findings present in hemoglobin C/β⁺ thalassemia include jaundice, pallor, mild anemia, microcytosis, target cells, reticulocytosis, and elevated bilirubin due to a chronic hemolytic anemia.
Hemoglobin C crystals may be seen.
*Hemoglobin C/β⁺ thalassemia should be distinguished from homozygous hemoglobin C, which is a much milder disease.

Question 30

clinical presentation of B-thal MAJOR

Answer 30

β-Thalassemia major presents with severe anemia; jaundice; dark urine; failure to thrive or slow growth; delayed puberty; extramedullary hematopoiesis with resulting bone abnormalities; and splenomegaly.

Question 31

What is Hemoglobin H disease?

Answer 31

*chronic hemolytic anemia disease because of 3/4 deleted alpha globin genes
*Hb H disease results in deletion of 3 of the 4 α genes (–/-α)
This leads to reduced production of α chains and excess production of β chains because β chains do not have α chains to polymerize and form Hb A
Excess β chain production leads to formation of β-tetramers, which cause membrane damage and hemolysis
*has a RAPID MIGRATION band on electrophoresis (which is the Beta4 tetramer)

Question 32

HPLC finding on Hemoglobin H

Answer 32

peak at the far LEFT (early eluter)
*same(ish) position as BARTS
*you can see BARTS (gamma4) in neonates with Hemoglobin H disease

Question 33

Hemoglobin H is different than Hb E, S, C…how?

Answer 33

Hemoglobin H is not a Beta globin gene mutation
*the other variant hemoglobins are usually beta globin gene mutations
*Hemoglobin H is 3/4 copies of ALPHA globin lost

Question 34

Hemoglobin Lepore

Answer 34

Hemoglobin Lepore is a δ-β fusion gene that is under control of the δ-globin promoter.

Question 35

Hemoglobin Lepore

Answer 35

hemoglobin Lepore is a thalassemic hemoglobin variant.

In the heterozygous form, the imbalance between α and β globins leads to a β-thalassemia minor phenotype, and patients present with a mild hypochromic, microcytic anemia, which can be associated with a mild elevation of hemoglobin F.

In the homozygous form, which is rare, patients present with a phenotype that ranges between β-thalassemia intermedia and β-thalassemia major.

Question 36

Tricky thing about Hemoglobin Lepore on Acid and Alk electrophoresis

Answer 36

runs with S on one, but not on the other
*On hemoglobin electrophoresis, hemoglobin Lepore runs at the S-position on alkaline cellulose acetate and between hemoglobin A and A2 on cation exchange chromatography. Hemoglobin Lepore represents approximately 5% to 15% of the total hemoglobin in the heterozygous state.

Question 37

What is hemogobin E?

Answer 37

*Beta globin point mutation variant
*runs differently on acid vs alk gel
*elutes as A2 on HPLC
Hgb E mutation causes replacement of glutamic acid by lysine at position 26 in β-globin gene
Hgb E is 2nd most common Hgb mutation globally
Very frequent in Southeast Asia (Malaysia: Incidence up to 40%, Thailand: Incidence up to 70%)
Heterozygotes are asymptomatic, and homozygotes are often asymptomatic, except for mild anemia

Question 38

HPLC has elevated Hb F, and decreased Hb A, but NORMAL A2

Answer 38

CANNOT BE Beta-thal trait
DO NOT CLICK BETA -THAL
*but it looks like Beta thal!!!
*delta-beta thal trait is the right answer
*Deletions of the β-globin–like genes can result in a thalassemic syndrome. Deletional β-thalassemias are less common than point mutations. (δβ)0-Thalassemia trait is caused by a deletion of both the δ and β genes, which are adjacent to one another on chromosome 11.

(δβ)0-Thalassemia trait is generally clinically silent but hematologically resembles β-thalassemia. The hemoglobin A2 is normal, but the hemoglobin F percentage is elevated in (δβ)0-thalassemia trait. The hemoglobin F can represent up to 20% of the total hemoglobin.

Question 39

TRICKY! the test will give you elevated A2, but it’s not Beta Thal!! What else messes with A2?

Answer 39

*ELEVATE: antiretroviral therapy, hyperthyroidism, folate/B12 deficiency
*Causes of decreased hemoglobin A2 include α-thalassemia, iron deficiency, lead poisoning, sideroblastic anemia, hypothyroidism, and δ-chain variants.

Question 40

Hemoglobin N–Baltimore

Answer 40

Hemoglobin N–Baltimore is a fast-migrating hemoglobin. It is a beta chain variant and composes approximately 45% of the total hemoglobin in heterozygous individuals. By itself, it is not associated with microcytosis or anemia.

Question 41

Hemoglobin J

Answer 41

Hemoglobin J is a fast-migrating hemoglobin variant. It represents up to 45% of the total hemoglobin in heterozygous individuals. By itself, it is not associated with microcytosis or anemia.

Question 42

What is a ZPP test looking for?

Answer 42

NORMAlLY, iron is put into protoporphoryn IX, but if there is no Iron, then ZINC is put in there
*Zinc + protoporphoryn IX = ZPP
*In iron deficiency or anemia of chronic disease, where iron is not available to form heme, ZPP levels increase. ZPP levels can also increase in lead poisoning, erythropoietic protoporphyria, and sideroblastic anemia

Question 43

What can FALSELY elevate ZPP?

Answer 43

Fluorescence method
*ZPP is measured using a fluorescent assay. Therefore, hyperbilirubinemia and increased riboflavin can falsely elevate ZPP.

Question 44

Classically, TTP presents with…

Answer 44

Classically, TTP is accompanied by mental status changes, fever, and renal failure, but these three findings may not always be present
*hemolytic anemia labs

Question 45

What does FLOW show in PNH?

Answer 45

Flow cytometric determination of decreased levels of CD55 and/or CD59 allows a definitive diagnosis of PNH.
*need a clone!

Question 46

What does a deficiency in G6PD cause problems?

Answer 46

*oxidative damage, oxidant hemolysis
*G6PD deficiency is caused by mutations that lead to decreased G6PD activity. G6PD is required to maintain normal NADPH levels. In turn, NADPH is required to regenerate reduced glutathione. In the absence of adequate levels of NADPH, red cell oxidative damage cannot be reversed, leading to red cell damage and increased red cell clearance.

Question 47

False negative qualitative G6PD results

Answer 47

heterozygous woman
*tons of reticulocytes can mask deficiency too, since they overexpress it, and in a hemolytic crisis there are lots of retics

Question 48

hepcidin in Anemia of chronic disease

Answer 48

High hepcidin, so that ferroprotin is destroyed and it’s trapped in macrophages
*prussian blue has lots of color in a spleen with anemia of chronic disease

Question 49

what is TTP?

Answer 49

TTP is a microangiopathic hemolytic anemia that can result from a congenital or acquired deficiency of the enzyme ADAMTS13.
*The normal function of ADAMTS13 is to cleave large von Willebrand factor (vWf) multimers into smaller multimers. A deficiency in ADAMTS13 leads to the presence of large vWf multimers that trap platelets in the microvasculature.
*Red cells become sheared in the microvasculature, which results in a hemolytic anemia.

Question 50

pentose phosphate pathway

Answer 50

G6PD is an enzyme in the pentose phosphate pathway, which is the major pathway to regenerate NADPH in red cells. Because NADPH is required to regenerate reduced glutathione, low NADPH levels can lead to decreased levels of reduced glutathione. Without this mechanism of reversing oxidative damage on red cells, hemolysis can occur under oxidative stress.

Question 51

what can cause megaloblastic anemia

Answer 51

megaloblastic anemias, caused by defects in DNA synthesis that cause asynchronous maturation of the nucleus and cytoplasm in the erythroblast.
**characterized by hypersegmented neutrophils
**deficiencies of vitamin B12, folate, toxins, chemotherapy, and myelodysplasia.

Question 52

lab findings B12 deficiency

Answer 52

Vitamin B12 deficiency remains prevalent and difficult to assess clinically (e.g., only 65% of B12-deficient patients show macrocytosis). Measurement of plasma vitamin B12 levels is important in patients with unexplained macrocytosis or those at risk for vitamin B12 deficiency, for example, in autoimmune gastritis.
*Measurement of plasma methylmalonic acid levels is recommended to diagnose vitamin B12 deficiency in patients with borderline vitamin B12 levels.
*mma is elevated
*Increased plasma homocysteine

Question 53

Increased γ-glutamyl transpeptidase

Answer 53

Chronic alcoholism, when it causes macrocytic anemia

Question 54

red cell histogram in β-Thalassemia trait

Answer 54

β-Thalassemia trait is microcytic with a uniform distribution of red cells.

Question 55

Dimorphic red cell histograms

Answer 55

Dimorphic red cell histograms can be caused by treated iron deficiency anemia (microcytic population) accompanied by a second population of normal to macrocytic red cells, which can represent transfused red cells or reticulocytes after iron replacement.
*Early iron deficiency can lead to an emerging population of microcytic cells among normal sized cells.

Other causes of a dimorphic red cell histogram include treated megaloblastic anemia (B12 or folate deficiency), sideroblastic anemia, and hemolytic anemias (warm autoimmune, delayed hemolytic transfusion reactions, hereditary pyropoikilocytosis, and others).

Question 56

hemochromatosis and iron deficiency have what in common?

Answer 56

*low hepcidin
*fundamental problem in hemachromatosis is no production of hepcidin
*ferrorportin isn’t destroyed and macrophages accumulate iron and the body keeps absorbing it
Although hemochromatosis is a disease of iron overload, hepcidin levels are low as if the patient were iron deficient.

Question 57

C282Y mutation

Answer 57

The C282Y mutation in the HFE gene is the most common mutation causing hemochromatosis.

About 1 in 200 northern Europeans carries the C282Y homozygous mutation. However, the disease has variable penetrance, so most cases are asymptomatic.

Question 58

Increased osmotic fragility

Answer 58

Increased osmotic fragility is a useful test for the diagnosis of hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (a subtype of HE).

Question 59

Hereditary pyropoikilocytosis

Answer 59

hereditary pyropoikilocytosis (a subtype of HE).
*increased osmotic fragility

Question 60

sucrose lysis test

Answer 60

*screening test for PNH
*Sucrose provides a medium of low ionic strength that promotes the binding of complement to red cells.

The acidified serum test used to be the gold standard for the diagnosis of PNH; it has been replaced by the flow cytometric measurement of CD55 and CD59 levels on red blood cells.

Question 61

Acute intermittent porphyria and photosensitivity

Answer 61

*this is the one that is NOT photosensitive
*abdominal pain mostly
*Acute intermittent porphyria is associated with neuropsychiatric symptoms. It is caused by a deficiency in the uroporphyrinogen I synthetase enzyme, which catalyzes the conversion of porphobilinogen to pre-uroporphyrinogen. This leads to the accumulation of ALA and porphobilinogen (one ring) so that it is not associated with photosensitivity.

Question 62

What’s the deal with prophyrias?

Answer 62

Porphyrias are inherited or acquired disorders of heme synthesis. Almost all inherited porphyrias are autosomal dominant.

Patients with enzyme defects that lead to the accumulation of tetrapyrrole rings have photosensitivity. The following enzyme deficiencies lead to the accumulation of tetrapyrrole rings: coproporphyrinogen oxidase, ferrochelatase, uroporphyrinogen III cosynthetase, and uroporphyrinogen decarboxylase. They are all associated with photosensitivity.

Question 63

In the acidified serum test…

Answer 63

*old DX of PNH
*In the acidified serum test, the patient’s red cells and serum are mixed with control serum and red cells. Complement is inactivated by heat or activated by acidification, and it is shown that the patient’s hemolysis is caused by an increased susceptibility of his red cells to complement.