Red Blood Cell Disorders

Question 1

What is the structure of hemoglobin (Hgb)? What is/are the consequence(s) of compromise in production of/obtaining any of these components?

Answer 1

Hemoglobin = heme + globin
Heme = Iron (Fe) + protoporphyrin 

Result = microcytic anemia

Pathoma, page 41

Question 2

What is the main problem for each of the following causes of microcytic anemia?

1) Iron deficiency
2) Anemia of chronic disease
3) Sideroblastic anemia
4) Thalassemia

Answer 2

1) Iron deficiency means decreased iron levels –> unable to form heme and therefore Hgb molecule.
2) Anemia of chronic disease is a condition in which chronic inflammation leads to iron being “hidden” or sequestered in macrophages that prevents it from being used.
3) Sideroblastic anemia is an anemia as a result of decreased levels of protoporphyrin.
4) Thalassemias involve impaired production of globin.

Pathoma, page 41

Question 3

What are the different forms in which iron can be obtained from diet? How is the iron then absorbed, transported, and stored? Outline the cells, transporters, etc. involved.

Answer 3

Iron can be consumed in either the heme (meat-derived, Fe2+) more-readily absorbed form or non-heme (vegetable-derived, Fe3+ non-oxygen binding) form.

The site of absorption is within the duodenum:

1) Enterocytes lining the villi uptake the iron from the intestinal lumen via heme or non-heme (DMT1) transporters.
2) The enterocytes then transport the iron across the cell membrane and into the blood via ferroportin.
3) Transferrin transports the iron throughout the blood and delivers it to liver and bone marrow macrophages for storage.
4) Stored intracellular iron is bound to ferritin, which prevents it from forming free radicals via Fenton reaction!

Pathoma, page 41

Question 4

The following four lab values help assess iron status. What do each of the following measure?

1) Serum iron
2) Total iron binding capacity (TIBC)
3) % saturation (what is normal?)
4) Serum ferritin

Answer 4

1) Serum iron measures iron in the blood
2) TIBC measures number of tranferrin molecules in the blood, regardless of whether or not they are bound
3) % saturation measures number of transferrin molecules actually bound to iron (normal is 33%).
4) Serum ferritin reflects iron stores in macrophages and in the liver.

Pathoma, page 42

Question 5

In elderly adults, what are the main causes of iron deficiency in Western world vs. developing world?

Answer 5

Western world - colon polyps or carcinoma
Developing world - hookworm, or Ancylostoma duodenale and Necator americanus

Pathoma, page 42

Question 6

How can gastrectomy result in iron deficiency?

Answer 6

Stomach acid aids in maintaining iron in the Fe2+ state, which is more readily absorbed than the Fe3+ state.

Pathoma, page 42

Question 7

What are the four stages of iron deficiency [anemia]?

Answer 7

1. Storage iron is depleted - decreased ferritin, increased TIBC from liver to “find” more iron
2. Serum iron is depleted - decreased % saturation (normally 33%, or one in every 3 transferrin is bound)
3. Normocytic anemia - early stage is normocytic as bone marrow compromises quantity for fewer quality RBCs
4. Microcytic, hypochromic anemia - expansion of central pallor due to less Hgb in each RBC

Pathoma, page 42

Question 8

Some clinical features of iron deficiency, besides anemia, include koilonychia and pica. Lab findings also include increased FEP. What are they?

Answer 8

Koilonychia - spooning of nails

Pica - psychological drive to abnormally chew on things; can be thought a means of trying to attain iron

FEP - free erythrocyte protoporphyrin; decreased iron with normal protoporphyrin levels

Pathoma, page 42

Question 9

Plummer-Vinson Syndrome

Answer 9

Iron deficiency anemia with esophageal web (due to outfolding of mucosa) and atrophic glossitis, so that patients present with anemia, dysphagia (due to web), and beefy-red tongue (due to glossitis).

Pathoma, page 42

Question 10

Pathophysiology of anemia of chronic disease? Labs?

Answer 10

Chronic disease/inflammation results in increased production of acute phase reactants from the liver, including hepcidin.

Hepcidin:

1) sequesters iron in storage sites as ferritin to limit transfer from macrophages phages to erythroid precursors
2) Suppresses EPO production–important to provide exogenous EPO especially for cancers.

Labs:

1) Increased ferritin/ decreased TIBC
2) Decreased serum iron/% saturation since BM cannot used stored iron from macrophages
3) Increased free erythrocyte protoporphyrin due to decreased iron (decreased heme)

Pathoma, page 43

Question 11

Name the key steps, enzymes, and cofactors involved with the production of protoporphyrin.

Answer 11

Occurs within mitochondria of erythroid precursors:

1) Succinyl CoA converted to ALA by ALAS with cofactor Vitamin B6.
2) ALA converted to porphobilinogen by ALAD.
3) Porphobilinogen converted to protoporphyrin.
4) Ferrochelatase binds irons (which accumulates in mitochondria to form ring around nucleus) to protoporphyrin to form heme.

Pathoma, page 43

Question 12

What are some of the common causes of sideroblastic anemia, both congenital and acquired? How would you viusalize the sideroblasts on bone marrow biopsy? What would labs reveal?

Answer 12

Sideroblasts form as a result of accumulating iron to form ringed sideroblasts in the absence of protoporphyrin production.

Congenital defects are usually due to a deficiency in ALAS, the enzyme catalyzing the rate-limiting step.

Acquired causes include:

1) Alcohol - mitochondrial poisoning
2) Lead poisoning - inhibits ALAD and ferrochelatase (denature)
3) Vitamin B6 deficiency - most commonly seen as side effect of isoniazid for TB treatment

Would visualize with Prussian Blue stain, which stains irons, and would reveal rings of iron within mitochondria that is formed around the nucleus.

Labs would reveal:
1) Increased ferritin/decreased TIBC
2) Increased serum iron
3) Increased % saturation (iron-overloaded state)
As iron accumulates in cells, it also produces free radicals that can eventually lead to death of erythroid precursors to result in leakage of iron into blood, leading to increased serum iron. Iron then accumulates and it consumed by bone marrow macrophages to increase ferritin.

Pathoma, page 43

Question 13

What are the 3 normal types of hemoglobin produced in human beings?

Answer 13

HbF - alpha2 and gamma2
HbA - alpha2 and beta2
HbA2 - alpha2 and delta2

Pathoma, page 43

Question 14

What are the consequences of one and two gene deletions in alpha-thalassemias?

Answer 14

Normally, we would have four copies of the alpha genes on chromosome 16 (two alleles).

One deletion is asymptomatic.

Two deletions often present with mild anemia with increased RBC count. Cis deletions are worse than trans because of increased risk of severe thalassemia in offspring. Cis deletions more commonly seen in Asians and trans in Africans.

Pathoma, page 44

Question 15

What happens with three gene deletions in alpha thalassemia? Clinical presentation in utero vs. after birth?

Answer 15

Severe anemia.

In the absence of alpha chains, beta dimers can polymerize to form tetramers (HbH) that can be seen on electrophoresis.

In utero, fetus is still producing HbF, which is composed of alpha and gamma dimers. The alpha chain production is sufficient so no significant clinical consequence.

Pathoma, page 44

Question 16

What happens with four gene deletions in alpha thalassemia?

Answer 16

Gamma chains form tetramers called Bart bodies that damage RBCs, resulting in hydrops fetalis and death in utero.

Pathoma, page 44

Question 17

What is the common cause of beta-thalassemias and potential genotypes?

Answer 17

Beta-thalassemias usually due to gene mutations on chromosome 11 (usually two copies, one on each copy
of chromosome) seen in those of African or Mediterranean descent.

B/B+ - minor
B0/B0 - major

Pathoma, page 44

Question 18

Symptoms and labs seen with beta-thalassemia minor?

Answer 18

Mildest form of disease and usually asymptomatic.

Increased RBC count with microcytic, hypchromic RBCs and target cells on blood smear.

Electrophoresis reveals slightly decreased HbA with [isolated] increased HbA2 and HbF.

Pathoma, page 44

Question 19

What is a target cell and what are the conditions in which we would see production of target cells? How is this achieved in beta-thalassemia?

Answer 19

Target cells are RBCs in which instead of a biconcave RBC with central pallor, we get accumulation of Hgb in the center as a result of a blebbing of membrane.

This occurs in the setting of either excess membrane or reduced cytoplasm. In the setting of beta-thalassemia, the reduced production of HgB is like taking air out of a basketball–it gives us “space” to bleb the membrane where Hgb can accumulate.

Pathoma, page 44

Question 20

B0/B0 clinical presentation? Why does it take babies a few months to develop after birth? Labs?

Answer 20

B0/B0 is the most severe form of beta-thalassemia. The reason why it takes babies a few months to develop after birth is because of the presence of HbF. HbF is temporarily protective of the fetus in utero.

Unpaired alpha chains can precipitate and damage the RBC membrane to result in ineffective erythropoiesis. RBCs can also be removed by splenic macrophages, resulting in extravascular hemolysis.

Normally, erythropoiesis (and hematopoiesis) occurs in the central axial skeleton. However, in the setting of ineffective erythropoiesis such as that in beta-thalassemia massive erythroid hyperplasia results in extramedullary hematopoiesis. This involves both the skull (resulting in a “crew cut” appearance and “chipmunk facies”) and the liver/spleen (hepatosplenomegaly).

This level of hyperplasia puts those affected at risk for aplastic crisis with parvovirus B19 infection of erythroid precursors.

Smear reveals microcytic, hypochromic RBCs with target cells and NUCLEATED RBCs (due to massive hyperplasia, immature RBCs leak out from bone marrow before nuclei are removed). Electrophoresis reveals HbA2 and HbF with little or no HbA.

Pathoma, page 44

Question 21

How are folate and vitamin B12 involved with synthesis of DNA precursors?

Answer 21

Folate first enters the serum as THF and is quickly methylated to form methyl THF. Methyl group is then removed (allows for participation in synthesis of DNA precursors) via transfer to vitamin B12 (cobalamin). From vitamin B12, it is then transferred to homocysteine to form methionine.

Pathoma, page 45

Question 22

Megaloblastic anemia vs. just macrocytic anemia?

Answer 22

Since Vitamin B12 and folate are involved with synthesis of DNA precursors, deficiencies would affect not only RBCs to result in anemia but other cells as well.

Other causes of macrocytic anemia without megaloblastic change would include alcoholism, liver disease, and drugs (e.g. 5-FU).

Question 23

Common causes of folate deficiency? Clinical and laboratory findings?

Answer 23

Dietary folate is obtained from green vegetables and some fruits and absorbed in jejunum. Deficiency develops within months because body stores are minimal.

Causes include:

1) Poor diet, especially in alcoholics and the elderly.
2) Increased demand, especially during pregnancy, cancer, and hemolytic anemia
3) Folate antagonists like methotrexate, which inhibits dihydrofolate reductase

Clinical/laboratory findings:

1) Macrocytic RBCs and hypersegmented neutrophils (> 5 lobes)
2) Glossitis
3) Increased serum homocysteine
4) NORMAL methylmalonic acid–differentiates from Vitamin B12 deficiency!

Pathoma, page 45

Question 24

How is Vitamin B12 obtained and absorbed? What are some common causes of Vitamin B12? Clinical and laboratory findings of deficiency?

Answer 24

Upon consumption of animal proteins, Vitamin B12 is complexed to the animal proteins. Salivary gland enzymes such as amylase liberate Vitamin B12, which is then bound to R-binder produced also from salivary glands and carried through stomach.

Once in the duodenum, pancreatic proteases cleave Vitamin B12 from R-binder. Vitamin B12 then binds intrinsic factor (produced from parietal cells of stomach body) and absorbed in ILEUM.

Because of large hepatic stores, deficiency is less common vs. folate EXCEPT in vegans. Most COMMON cause is due to pernicious anemia, where autoimmune destruction of parietal cells leads to decreased production of intrinsic factor. Other causes include pancreatic insufficiency and damage to terminal ileum (e.g. Crohn’s, Diphyllobothrium latum or fish tapeworm).

Clinical/lab findings include:

1) Macrocytic RBCs with hypersegmented neutrophils
2) Glossitis
3) Increased methylmalonic acid –> impairs spinal cord myelinization. Vitamin B12 is cofactor for conversion of methylmalonic acid to succinyl CoA (important for fatty acid metabolism).
4) Poor proprioception and vibratory sensation

Pathoma, page 46

Question 25

What lab value(s) would you look at to differentiate between normocytic anemia due to peripheral destruction vs. underproduction from the bone marrow? How it reticulocyte count correctly calculated for in the setting of anemia?

Answer 25

Reticulocyte count.

Under normal physiological circumstances, recticulocyte count should be 1-2%. With anemia, a properly functioning (not underproducing) bone marrow should be increasing reticulocyte count > 3% to compensate with the decrease in RBC mass.

RC x (Hct/45) = corrected RC

Pathoma, page 46

Question 26

How do reticulocytes differ from mature erythrocytes in terms of composition and how they appear on smear.

Answer 26

Reticulocytes maintain residual RNA to give it a bluish cytoplasm vs. erythrocytes. They are also larger.

Pathoma, page 46

Question 27

Consequences in extravascular vs. intravascular hemolysis of peripheral RBC destruction?

Answer 27

Extravascular hemolysis involves RBC destruction by reticuloendothelial system (macrophages of spleen, liver, lymph nodes):

1) Macrophages consume RBCs and break down hemoglobin into heme and globin. Globin broken down into amino acids and heme into iron and protoporphyrin.
2) Protoporphyrin turned into insoluble unconjugated bilirubin, which is then bound to serum albumin and shuttled to liver for conjugation and excretion into bile.
3) Splenomegaly due to hypertrophy, jaundice due to increased unconjugated bilirubin that increases risk for gallstones, bone marrow hyperplasia with corrected RC > 3%

Intravascular hemolysis involves RBC destruction within vessels:

1) Hgb gets released into the blood (hemoglobinemia) and is picked up by serum haptoglobin (see decreased serum haptoglobin as a result) and taken to spleen for reprocessing.
2) Hemoglobinuria as Hgb leaks into urine.
3) Hemosiderinuria as renal tubular cells pick up Hgb filtered into urine and break it down into iron, which then accumulates as hemosiderin. Tubular cells eventually shed to result in hemosiderinuria.

Pathoma, page 47

Question 28

What is the pathophysiology behind hereditary spherocytosis? Clinical and laboratory findings? Diagnostic test? Treatment and potential consequences of treatment?

Answer 28

HS is the result of inherited defect of RBC cytoskeleton-membrane tethering proteins (ankyrin, spectrin, band 3) that results in membrane blebbing and formation of spherocytes. Consequently, they are less able to maneuver through splenic sinusoids and are consumed by splenic macrophages to result in anemia.

Clinical/lab findings:

1) Spherocytes with loss of central pallor
2) Increased RDW (macrophages are “nibbling” as membranes) and MCHC (shrinking membrane decreases volume, thereby “increasing” concentration of hemoglobin).
3) Splenomegaly due to hypertrophy of macrophages, jaundice with unconjugated bilirubin and increased risk for gallstones
4) Increased risk for aplastic crisis with B19 infection

Diagnose with osmotic fragility test in hypotonic solution.

Treat with splenectomy:

1) Anemia resolves but spherocytosis persists
2) Howell-Jolly bodies on smear that indicate splenic dysfunction–spleen usually removes “abnormal” RBCs (eg nucleated RBCs)

Pathoma, page 47

Question 29

Mutation responsible for sickle cell disease? Treatment? Conditions that precipitate disease?

Answer 29

Autosomal recessive mutation in beta globin gene (hydrophilic glutamic acid –> hydrophobic valine). that results in > 90% production of HbS. Need TWO abnormal beta globin genes for abnormal beta dimers in HbS.

HbS polymerizes to cause needle-like structures that results in sickling of RBCs. Increased risk of sickling under conditions of hypoxemia, dehydration, and acidosis.

HbF protective against sickling (reason for being asymptomatic during first few months of life). Treatment with hydroxyurea, which increases HbF.

Pathoma, page 47

Question 30

Consequence of sickle cells passing through microcirculation?

Answer 30

Cells continue to sickle and de-sickle and they pass through microcirculation, resulting in membrane damage.

Damaged membranes results in removal by reticuloendothelial system, leading to anemia via extravascular hemolysis –> jaundice with increased risk for gallstones.

Also get some intravascular hemolysis as RBCs with damaged membranes dehydrate, leading to decreased serum haptoglobin and target cells (loss of cytoplasm with dehydration).

Get massive erythroid hyperplasia: “crew cut” appearance of skull with “chipmunk facies”, extramedullary hematopoiesis, risk of aplastic crisis with B19 infection.

Pathoma, page 48

Question 31

Consequence(s) of irreversible sickling/extensive sickling? Most common cause of death in children and in adults with sickle cell?

Answer 31

Vaso-occlusion.

1) Dactylitis with vaso-occlusion in bones–COMMON presenting sign in infants.
2) Autosplenectomy, resulting in increased risk for infection with encapsulated organisms like Streptococcus pneumoniae and Haemophilus influenzae–the most common cause of death in children (affected children NEED to be vaccinated < 5 years of age)–as well as Salmonella paratyphi. This results in Howell-Jolly bodies on smear.
3) Acute chest syndrome with vaso-occlusion in pulmonary microcirculation, the most common cause of death in adults, with presentations of CP, SOB, lung infiltrates.
4) Pain crisis
5) Renal papillary necrosis –> gross hematuria and proteinuria

Pathoma, page 48

Question 32

Sickle cell trait clinical and lab findings (vs. that of sickle cell disease)? How to screen for trait?

Answer 32

Sickle cell trait is the presence of ONLY ONE mutated beta chain, resulting in production of both HbS ( <50%, so not enough to sickle) and normal HbA.

Generally asymptomatic with no anemia. Sickling does not occur EXCEPT in renal medulla, so under conditions of hypoxia/hypertonicity in medulla sickling occurs to result in microinfarctions. Would lead to microscopic hematuria and evnetually decreased ability to concentrate urine.

On smear, would NOT see sickle and target cells like those in sickle cell disease.

Screen with metabisulfite, which causes cells with ANY AMOUNT of HbS to sickle. Hb electrophoresis can confirm HbS. Would have HbA in trait (55%) vs. none in disease and less HbS vs. disease.

Pathoma, page 48

Question 33

Mutation responsible for Hemoglobin C? Clinical presentation and lab finding?

Answer 33

Autosomal recessive mutation with beta globin chain, where normal glutamic acid is replaced by lysine.

Presentation with mild anemia due to extravascular hemolysis.

CHARACTERISTIC HbC CRYSTALS in RBCs on smear.

Pathoma, page 49

Question 34

Pathophysiology for paroxysmal nocturnal hemoglobinuria (PNH)? Screening and diagnostic tests? Major cause of death and other clinical findings?

Answer 34

Normally, myeloid cells are protected against complement-mediated destruction by the molecule DAF (decay accelerating factor, or CD55), which inhibits C3 convertase. DAF is normally secured to cell membrane by GPI.

In PNH, there is an ACQUIRED defect in GPI, resulting cells of myeloid lineage (RBCs, platelets, WBCs/granulocytes) to become susceptible to complement destruction –> intravascular hemolysis –> hemoglobinemia and hemoglobinuria, with hemosiderinuria developing days after hemolysis.

Intravascular hemolysis occurs often at night, during which mild respiratory acidosis (which activates complement) develops with shallow breathing during sleep.

Screen with sucrose test and confirm with acidified serum test or flow to detect lack of DAF/CD55.

Main cause of death is thrombosis of hepatic , portal, or cerebral veins as destroyed platelets release cytoplasmic contents into circulation to induce thrombosis.

Complications include iron deficiency anemia (chronic loss of hemoglobin in urine) and AML (due to destruction of myeloid cells).

Pathoma, page 49

Question 35

Mechanism and precipitating causes of G6PD deficiency? What are the different variants of the condition? What would you visualize on blood smear? How would you screen and test for the condition?

Answer 35

G6PD deficiency is an X-linked recessive condition that results in increased RBC susceptibility to oxidative stress due to decreased half-life of G6PD (normally lasts throughout whole 120 day life span of RBC). G6PD is necessary for the regeneration of NADPH, which is involved with regeneration of the molecule glutathione that neutralizes H2O2. There are two variants: the more mild African variant and severe Mediterranean variant.

Common precipitating (oxidative) stressors include drugs (sulfa, primaquine, and dapsone), as well as fava beans.

Normal blood smear would reveal bite cells. Under oxidative stress, Hgb precipitate as Heinz bodies (visualized and screened for by Heinz preparation), which are then removed from RBCs by splenic macrophages to form bite cells. Enzymatic studies to confirm deficiency should be done ONLY after resolution of hemolytic episodes, or else deficient cells would all be dead/lysed.

Pathoma, page 50

Question 36

IgG immune hemolytic anemia mechanism? What are potential mechanisms if drug is offending agent? Blood smear? Conditions associated with IgG IHA? Treatment?

Answer 36

IgG IHA involves IgG binding RBCs in relatively warm areas of the body (so IgG is also called “warm agglutinin”) to result in EXTRAVASCULAR hemolysis via splenic macrophages as well as the formation of spherocytes with slow loss of membrane.

One way drugs (e.g. penicillin, cephalosporins) can cause destruction is by attaching to RBC membrane (such as that with penicillin) with subsequent binding of antibody to drug-membrane complex.

Another way if through induced production of autoantibodies (e.g. alpha-methyldopa) that bind self-antigens on RBCs.

Associated with SLE (SLE can involve production of autoantibodies against different cell types, including that of RBCs!) and CLL.

Treatment includes cessation of offending drug, steroids to suppress immune system, IVIG (which would become target of splenic macrophage consumption instead of RBCs), and if necessary splenectomy.

Pathoma, page 50

Question 37

IgM-mediated IHA mechanism, blood smear, and associated conditions?

Answer 37

IgM is a cold agglutinin that fixes complement in the COLD temperature of extremities, like the fingers and toes. In IgM IHA, RBCs can inactivate complement but residual C3b serves as opsonin for splenic macrophages to result in spherocytes, and extreme complement activation can result in INTRAVASCULAR hemolysis.

Associated with Mycoplasma pneumoniae and infectious mononucleosis.

Pathoma, page 50

Question 38

Direct vs. Indirect Coombs test

Answer 38

1) Direct Coombs test involves incubation of PATIENT antibody or complement-bound RBCs with TEST anti-IgG/complement. If RBC is coated with either antibody or complement, agglutination will occur.
2) Indirect Coombs test involves incubation of PATIENT SERUM antibodies with test anti-Ig and test RBCs. Agglutination occurs if serum antibodies present.

Pathoma, page 50

Question 39

Mechanism for microangiopathic anemia? Different etiologies? Blood smear?

Answer 39

Intravascular hemolysis that results from vascular pathology, and RBCs are sheared/destroyed as they pass through circulation to form SCHISTOCYTES.

Different causes include microthrombi (TTP, HUS, DIC, HELLP), prosthetic heart valves, and aortic stenosis.

Pathoma, page 51

Question 40

What test(s) would you run to differentiate HS and IHA?

Answer 40

Osmotic fragility test and/or Coomb’s.