Hematology

Shift cell

"Marrow" reticulocyte. It is the direct precursor to the reticulocyte. They are sometimes released in the peripheral blood when the production of RBCs is especially high (for example in the case of anemia when the bone marrow is trying to compensate for some type of blood loss). Looks bluer than a mature RBC on a Wright stained blood smear.

What is the amount of iron in a 60 kg woman with an Hb of 14 g/100 cc?

1 cc = 1mL (100 cc = 1 dL). Blood volume is approx. 65 cc/kg for a woman and 70 cc/kg for a male. (14 g/ 100 cc) x (3.4 mg Fe/g Hb ) x (60 kg x 65 cc/kg) = 1856 mg Fe + 300 mg storage Fe = 2156 mg Fe.

What is a normal reticulocyte count?

1% (0.5-1.5%) which is 50,000-75,000 reticulocytes.

When a patient's reticulocyte index is > 3%, what are the two things that this indicates?

1. Acute blood loss (hemorrhaging) 2. Hemolysis

What are the three main differential diagnoses for pancytopenia with anemia due to hypoproliferation?

1. Aplastic anemia 2. Myelophthisic anemias 3. Myelodysplastic syndromes

What are additional tests that can be done to distinguish iron deficiency anemia from anemia of chronic disease?

1. Direct assessment of Fe2+ stores by bone marrow biopsy is the GOLD standard for diagnosis of iron deficiency. 2. Measurement of soluble serum transferrin receptor (sTfR). sTfR is proportional to number of erythroid precursors. In uncomplicated iron deficiency sTfR should be elevated since EPO is increased. A decreased sTfR is expected in anemias of chronic inflammation. 3. Free erythrocyte protoporphyrin (FEP) should be elevated in iron deficiency, reflecting the rate limiting effect of the lack of iron on heme synthetase. 4. Measurement of serum/urine hepcidin levels (elevated for anemias of chronic disease).

What are the main causes of hypoproliferative anemia (affecting RBCs only)

1. Iron deficiency 2. Thalassemia minor syndromes 3. Anemias of chronic disease 4. Plumbism/sideroblastic anemias 5. Pure RBC aplasia (acute/chronic).

What are the four major mechanisms involved in anemia of chronic disease?

1. Up-regulated macrophage and splenic function -> shortened RB survival; failure of erythropoietic response 2. Faulty iron reutilization leads to decreased erythropoiesis lactoferrin, hepcidin. 3. Blunted erythropoietin response (EPO) 4. Cytokine inhibition of erythropoiesis (interferon gamma, IL-1, TNF-alpha).

Average lifespan of a RBC is _______days.

120 days.

What are normal values for hemoglobin, hematocrit and RBC count?

15 g/dL, 45% and 5 million RBCs/ uL.

What is the ratio of cells to fat in adult bone marrow?

1:1

1 mg Fe is approximately _______ of whole blood.

2 cc.

What is P50 for normal blood at pH 7.4 and 37 degree Celsius?

27 mm Hg.

How much iron is in normal plasma at one time?

3 mg (bound to transferrin)

What is the amount of iron stored in the body for female and for males?

300 mg Fe is in storage for females and 1000 mg Fe in storage for males. Beyond that, humans do not store iron.

What is the normal M:E (Myeloid:Erythroid) ratio?

3:1. If there is a change in this ratio, indicating that erythroid proportion has increased, this is an indication of erythroid hyperplasia (hyperproliferative anemia).

Hereditary hemorrhagic telangiectasia

A dominant hereditary disorder in which abnormal communications between arteries and veins occurs. Vessels don't form properly and they are more friable and prone to bleeding. Caused by mutations in either the ENDOGLIN gene or ALK-1 gene, which lead to clinically identical illness.

What is the rate-limiting step in heme synthesis in the liver?

ALA synthase (first step). Occurs in the mitochondria

Flipase

ATP-dependent Aminophospholipid translocase prevents flip-flop of PE and PS from the inner to the outer leaflet of the bilayer. (Keeps PE and PS in the INNER leaflet). The presence of PS in the outer leaflet can initiate a coagulation cascade. In sickle cell, a small amount of PS can be found in the outer leaflet of RBCs, which may contribute to the clot formation leading to vasoocclusion.

How much of heme synthesis occurs in the erythropoietic marrow?

About 85%. The rest occurs in the liver (mostly for heme in cytochrome p450)

Corrected reticulocyte count

Adjusts for the presence of anemia (ability of the patient to compensate if anemia is present). Corrected reticulocytes = (Patient's retic. count or %) x (Patient's Hb or Hct / Normal Hb or Hct). Compare the corrected reticulocyte count to a normal reticulocyte count.

Hb Constant Spring is a _______________ phenotype due to the instability of the alpha CS mRNA. It is a chain termination mutant.

Alpha thalassemia.

Unstable Hemoglobins

As a result of mutational pressure, or after minor oxidant stress, Hb can precipitate forming Heinz bodies and associated Heinz body hemolytic anemia. Ex. Hb Gun Hill. Many of the mutations occur in the heme binding pocket and involve insertion of polar amino acids.

Lepore hemoglobin

Beta thalassemia phenotype.

How can you treat aplastic anemia?

Bone marrow transplantation (Chemo first given to eliminate diseased marrow, stem cells are infused into bone to regrow healthy marrow) and antithymocyte globulin (immunosuppressant that attacks pathologic T-cells so marrow can recover).

What are main categories of hereditary hemolytic anemias?

CYTOSKELETAL DEFECTS (spherocytosis, elliptocytosis -> blood smear); ABNORMAL HB or decreased HB SYNTHESIS (sickle cell, thalassemia -> blood smear, indices); ENZYMATIC DEFECTS (hexose MP shunt, anaerobic glycolysis -> Heinz bodies, basophilic stippling).

What are main underlying causes of megaloblastic anemia?

Cobalamin (B12) deficiency, folic acid deficiency, hereditary or acquired defects in cobalamin or folic acid transport or metabolism, drugs that interfere with DNA synthesis, acute leukemia/myelodysplasia, congenital dyserythropoietic anemia.

What different would you expect to see in a microscopic examination of this patient's spleen compared to a normal spleen? (Hereditary spherocytosis)

Cord congestion -> splenomegaly. Spherocytes have difficulty getting through cords back into sinuses of the spleen, causing congestion. A splenectomy in hereditary spherocytosis will cause the RBCs to last longer because you are removing the primary site of hemolysis. However this does not eliminate the disease.

Why is the range in hemoglobin different in men and women?

Due to testosterone in men. The difference in iron store between men (1000 mg) and women (300 mg) is due to menstruation/pregnancy.

How much iron is required each day to keep Hb in equilibrium at 15 gm/100 cc?

Each day, about 21 mg Fe is lost from RBC apoptosis. This Fe is recycled to be used for new RBCs. Therefore Fe loss and Fe demand for RBC production balance each other out. About 1.2 mg Fe is required to make up for lost Fe from sloughing of cells, bleeding, etc.

T/F In order to calculate reticulocyte index, you only must know the uncorrected reticulocyte count and the patient's Hb level.

False. Divide by 2 if shift cells are present.

Myeloblast

First morphologically recognized cell in the myeloid series

What are some main causes of aplastic anemia?

HEREDITARY (Falconi's anemia), ACQUIRED - Direct toxicity (Radiation, chemotherapy, toxins); indirect toxicity (idiopathic, drugs [ex. Chloramphenicol], infections [TB, HIV, EBV, CMV, hepatitis], pregnancy, paroxysmal nocturnal hemoglobinuria).

HIF-1

Hypoxia inducible factor. HIF 1 alpha mRNA and protein are induced by hypoxia, leading to transcription and activation of EPO. Comprised of two subunits (HIF-1 alpha and HIF 1 beta).

Reticulocyte index

If the presence of shift cells is noted, divide the corrected retic. count by 2. This corrects for the presence of shift cells, which exist for 2 days while normal reticulocytes last for about one day.

Where is iron absorbed?

In the DUODENUM of the small intestine

Where is ferritin found?

In the liver, spleen, skeletal muscle and bone marrow. A small amount is in the blood.

What are the primary causes of cobalamin deficiency?

Inadequate intake of cobalamin (very rare), inadequate absorption (MOST COMMON), oxidation of cobalamin, defective transport or metabolism.

Acanthocytosis

Increase in CHOLESTROL and SPHINGOMYELIN. RBCs appear rounded with thorny projections. Occurs in alcoholic liver disease where RBCs take up excess cholesterol in the membrane from circulating lipoproteins. RBCs become rigid and hemolysis occurs. Autosomal recessive.

What are some main causes of iron deficiency?

Increased blood loss (Uterus, GI tract), increased requirements (Growth, pregnancy), decreased intake, decreased absorption (gastric atrophy, upper small bowel disease).

Gardos effect

Increased intracellular concentration of calcium in SS RBCs causes opening of K+ channels, resulting in potassium efflux. Sickle cell RBCs thus have increased intracellular calcium and low K+ and is dehydrated.

What is anemia of chronic liver disease characterized by?

Increased plasma volume dilutes RBC mass. Altered lipid metabolism leads to mild macrocytosis => formation of RBCs with "spur" morphology and then hemolysis. Hypersplenism can occur from portal hypertension and splenomegaly. Bleeding from varices can also lead to anemia.

In a post-menopausal woman or a young man, why would iron-deficiency anemia occur?

Indicates a GI blood loss of unknown etiology until proven otherwise. You have to determine the cause of the blood loss. There can also be iron malabsorption deficiencies.

Which form of bilirubin is important to look at when determining the cause of anemia?

Indirect bilirubin because it really reflects the catabolism of hemoglobin. Subtract total bilirubin from direct bilirubin (liver function panel).

What are the two major causes of death with aplastic anemia?

Infection (low neutrophils) and hemorrhage (low platelets)

Hematocrit and EPO are _________________ proportional.

Inversely

Where is hemosiderin found?

It is ALWAYS found within cells and is a complex of ferritin. Iron deposits are poorly available. Most commonly found in macrophages. It can be identified using a Prussian blue stain.

Hemoglobins with high O2 affinity

LEFT shifted Hb O2 association curves. These hemoglobins eagerly bind O2 in alveoli (have low P50), but give up O2 only stingily at peripheral tissue. O2 delivery to tissues is impaired. Patients have a benign clinical course and treatment is generally not indicated. Example is Hb RANIER, Hb HIROSHIMA.

Glucose 6 phosphate dehydrogenase deficiency

Leads to hemolytic anemia. G6P used by NADPH to reduce oxidized agents just as oxidized glutathione (GSSG) or hydrogen peroxide. Individuals with the deficiency do not show clinical manifestations of hemolytic anemia until exposure to infection or taking oxidant drugs (like malarial drugs). The disease is very common and the gene for the deficiency is X-linked.

What are some lab values that you expect to see with iron deficiency anemia?

Low hemosiderin (Marrow Fe), increased total (transferrin) iron binding capacity, decreased plasma ferritin, decreased % saturation of TIBC.

If you suspect spherocytosis, what lab value would you look at expecting it to be high?

MCHC (Hemoglobin per RBC volume) should be high with spherocytes because there are more RBCs (and therefore more hemoglobin) per a given volume.

MCV

Mean cell (corpuscular) volume: the average size (volume) of red blood cells measured in femtoliters. MCV = HCT (%) x 10/ RBC (10^12/L).

MCHC

Mean corpuscular hemoglobin concentration. The mean hemoglobin concentration in RBCs expressed in g/dL. MCHC: Hb (g/dL) x 100 / HCT (%)

MCH

Mean corpuscular hemoglobin. The mean HEMOGLOBIN content of red cells measured in pictograms. MCH: Hb (g/dL) x 10 / RBC (10^12/L)

Hill coefficient (N)

Measure of cooperativity. For myoglobin n = 1.0 and for hemoglobin n = 2.8 - 3.8.

Hemoglobin E

Most common Hb variant in the world - prevalent among SE Asians. Point mutation in BETA chain. Heterozygous state is asymptomatic - causes microcytosis (small RBCs, low MCV) without anemia. Homozygous state- shows more severe microcytosis and hypochromia but little anemia. Resembles thalassemia minor.

Most intrinsic hemolytic anemias are genetic EXCEPT

PNH (paroxysmal nocturnal hemoglobinuria)

What type of viral infection can cause you to stop making new RBCs?

Parvovirus B19 infection. (Red cell aplasia).

Sickle cell hemoglobin (properties)

Point mutation (Glutamine -> Valine). Sickling upon deoxygenation. Cause painful crisis with vaso-occlusion.

Extravascular Hemolysis

Primary mechanism for RBC destruction in normal conditions and most hemolytic diseases. Phagocytosis & catabolism by MACROPHAGES in spleen, liver and bone marrow - even lung (if RBCs are severely damaged). Pigment changes are ↑ indirect bilirubin, ↑urinary urobilinogen, ↑stool stercobilinogen. Some free hemoglobin can be released into the circulation and fully scavenged by plasma haptoglobin. Therefore, plasma haptoglobin ↓ but not as dramatically as intravascular hemolysis. LDH released into plasma from destroyed RBCs via passive diffusion.

How do sickle cells block a vessel and create a painful crisis with vaso-occlusion?

RBC that has undergone repeated sickling episodes becomes stiff. Non-flexible RBC becomes caught up in the microvasculature. RBC's with HbS behind the non-flexible RBC give up their O2 to the surrounding hypoxic tissue. As more RBCs with HbS give up their O2 a blockade is formed and as more O2 is released and deoxy Hb forms, there is more sickling.

Intravascular Hemolysis

RBCs lysed in the bloodstream release Hb tetramers into the plasma. Hb free in the plasma produces changes dependent upon the amount of free Hb. Decreased haptoglobin - binds αβ dimers & the complex is cleared by the liver. Decreased hemopexin - binds globin free heme & the complex is cleared by the liver. Increased methemalbumin - Albumin binds globin free heme (oxidized to Fe+++). Free Hb in plasma and/or urine - when binding proteins are saturated. Urine hemosiderin - desquamated renal tubular epithelial cells containing the iron from catabolized Hb; the iron is stored as hemosiderin in the renal tubular epithelial cell.

Hemoglobins with low O2 affinity

RIGHT shifted Hb O2 association curves. Hemoglobins are reluctant to pick up O2 from the lung and have a high P50. O2 delivery to tissues is increased and the decreased O2 affinity can result in anemia and/or cyanosis (with slate gray color of skin and mucous membranes. Clinical intervention not necessary but diagnosis important. Example is Hb KANSAS.

What are some treatment options for thalassemia?

Regular blood transfusions that have been filtered to reduce WBC number (problem with iron overload -> iron chelators), splenectomy if necessary (vaccinations against meningococcal and pneumococcal beforehand with antibiotic prophylaxis). Stem cell transplantation is curative but not common due to donor limitations.

How does lead poisoning lead to anemia?

Shortens RBC lifespan by inhibiting several steps in protoporphyrin metabolism. Produces a microcytic hypochromatic anemia resembling thalassemia minor. Basophilic stippling can also be observed.

What is the mutation associated with sickle cell anemia?

Single point mutation in the beta-globin chain of hemoglobin. A Glutamine -> Valine in the 6 amino acid away from the amino-terminal end of the beta chain.

Hereditary elliptocytosis

Spectrin molecule fails to form tetramers, resulting in weakening skeleton. RBC becomes susceptible to hemolysis during circulation. RBCs appear as elliptocytes. Produces mild hemolytic anemia. Autosomal dominant.

What are the major cytoskeletal proteins in RBCs?

Spectrin, ankyrin, actin and protein 4.1. These cytoskeleton membrane proteins interact with RBC integral proteins (Band 3 and glycophorin) and maintain shape and deformability. Changes in the cytoskeleton membrane proteins lead to alteration in RBC morphology and susceptibility to hemolysis.

What is appropriate treatment for Sickle cell anemia?

Supportive care- reducing fever, correcting acidosis and fluids (all of these tend to slow down the rate of polymerization of HbSS), increase HbF in RBC, reduce adherence of SS RBC to endothelium, genetic therapy.

Common myeloid progenitor

This cell gives rise to granulocyte-monocyte progenitors (GMP) which produce neutrophils, eosinophils, basophils, monocytes and megakaryocyte-erythroid progenitors.

What are the two main differential diagnoses when there is a Reticulocyte index >3?

This occurs when there is either HEMOLYSIS or ACUTE BLOOD LOSS. An elevated index indicates that there is not a problem with RBC production.

What is anemia for chronic renal disease characterized by?

Underproduction of EPO. The anemia is evident when renal function (Creatinine clearance) is ~ 50%. The anemia responds to supplement with recombinant EPO in a dose-dependent fashion.

What is the main cause of morbidity from sickle cell disease?

Vaso-occlusion. SS RBCs adheres through specific ligands (integrins) to receptors (like VCAM-1) expressed on endothelial cells in capillaries, leading to vaso-occlusion. Vaso-occlusion may occur acutely, resulting in painful crises, acute chest syndrome, and stroke or may be chronic, resulting in renal failure, bone necrosis and end stage organ damage.

Methemoglobin

When Fe2+ is oxidized to Fe3+ in hemoglobin. It is no longer capable of reversibly binding O2 and is an ineffective carrier of oxygen. MetHb form continuously exists because of Hb auto-oxidation and Fe3+ must be reduced actively by normal red blood cell metabolism. If MetHb level increases to greater than 1% of total Hb, it is an abnormal state -> can be acquired (drug induced) or hereditary.

Erythropoietin (EPO)

a growth factor produced in the kidney in response to tissue hypoxia. A human recombinant form is in clinical use. It stimulates red cell differentiation from stem cells and proliferation of red cells.

Thrombopoeitin

a growth factor that regulates platelet production by megakaryocytes

M hemoglobin

abnormal globin (4/5 known Hb M involve change of distal or proximal HisàTyr in either a- or b-chains). Results in cyanosis. The blue Fugates of Troublesome Creek, KY.

Hemosiderin

an iron storage complex in cells and consists mainly of denatured ferritin. The iron within deposits of hemosiderin is not very available.

2,3-BPG/ 2,3-DPG

an organophosphate that is created in erythrocytes during glycolysis. Created via the shunt pathway in RBCs.

Haptoglobin

binds free Hb released from RBCs. The haptoglobin-hemoglobin complex will then be removed primarily by the spleen. Used to screen for INTRAVASCULAR hemolytic anemia. In intravascular hemolysis, free hemoglobin will be released into circulation and hence haptoglobin will bind the hemoglobin. This causes a DECREASE in haptoglobin levels. With extravascular hemolysis, splenic monocytes, phagocytose the erythrocytes and hemoglobin is not released into circulation; serum haptoglobin levels are therefore NORMAL.

NADH cytochrome B5 reductase

converts NADH -> NAD+ in RBCs. NADH functions to convert methemoglobin (Fe3+), which cannot bind O2 to hemoglobin (Fe2+), which can. Individuals who are heterozygous for a mutation in NADH cytochrome B5 reductase will develop acute methemoglobinemia in response to oxidant drugs (will be cyanotic).

Congenital erythropoietic porphyria

deficiency of uroporphyrinogen III cosynthetase. There is a build up of uroporphyrinogen I and other prophyrins in RBC, marrow, plasma, urine and feces. RBCs are prematurely destroyed and urine of patients is red due to excretion of large amount of uroporphyrin I. Skin is extremely light sensitive and teeth are fluorescent (due to accumulation of porphyrins) and patients are quite anemic. Treat by avoiding exposure to sunlight and intravenous hematin (oxidized form of heme, Fe 3+) can be administered. Think VAMPIRE.

Promonocyte

first morphologically recognized cell in the monocyte series.

Pronormoblast

first morphologically-recognized cell in the red cell series

Heinz bodies

inclusions seen in red blood cells. Denatured globin bound in Band 3 in the RBCs. Heinz body can be found in Thalassemia, unstable Hbs and G6PD deficiency (all hemolytic).

Hepcidin

increased production by the liver in response to inflammation (IL-1 and IL-6 stimulation) infection or malignancy. Causes iron retention by macrophages via DMT1 (reduces DMT1 on luminal surface of duodenal cells). An acute increase in hepcidin also reduces intestinal absorption and decreased iron absorption into the plasma by villous duodenal cells via down-regulation of ferroportin. Induction of hepcidin by SMAD.

Hemoglobin C

inherited as an autosomal recessive trait. Causes a mild hemolytic anemia (benign hemoglobinopathy). Point mutation in the BETA chain (6 Glutamate -> Lysine). HbC is less soluble than HbA and can precipitate in red blood cells to form crystals. RBC with HbC are less deformable and have shorter survival. HbC is associated with resistance to malaria in parts of Africa. Homozygotes may have some abdominal and joint pain, enlarged spleen and mild jaundice.

Transferrin

iron is delivered to erythroid precursor cells in the bone marrow by transferrin, which binds to the transferrin receptor on these cells and is internalized by receptor mediated endocytosis. After delivering iron, transferrin is recycles and re-enters the blood. Transferrin transports Fe in the ferric state (Fe3+) but when iron is released in the erythroid precursor cells, it is in the ferrous state (Fe2+).

Therapy for iron deficiency

liquid iron salt, iron salts (pills), parenteral iron salts, RBC transfusion.

Pyruvate kinase deficiency in RBCs

most common enzyme abnormality in the glycolytic pathway. Most patients exhibit life-long hemolytic anemia. RBCs of these patients typically have 5-25% normal PK activity. Their rate of glycolysis is decreased, resulting in less production of ATP. Consequently, the cell is not able to maintain structural integrity of the membrane, leading to hemolysis.

Characteristics of megaloblastic anemia

pancytopenia with decreased reticulocyte count. Increased MCV, increased RDW. Hyperplastic marrow with INEFFECTIVE hematopoiesis -> HIGH indirect bilirubin, and HIGH LDH (due to increased destruction of cell in marrow in blood).

Acute intermittent porphyria

porphobilinogen deaminase is depressed and there is a compensatory increase in ALA synthase. In the liver there is an increase in ALA and porphobilinogen and large amounts of both of these are found in the urine. They disease is seen clinically as intermittent abdominal pain and neurological disturbances. Enzyme deficiency occurs in all tissues. Approximately 90% are clinically unaffected if they have the heterozygous disease. Treat by intravenous hematin (oxidized form of heme, Fe 3+).

Hepcidin

principle regulator of iron hemostasis. Produced by LIVER cells. Found (along with ferroportin) in hepatocytes, enterocytes and macrophages. Hepcidin inhibits cellular iron export by binding directly to ferroportin and inducing its internalization and degradation. Synthesis INCREASES with iron loading, infections or inflammation (via IL-6) and DECREASES with hypoxia or iron deficiency.

Hereditary spherocytosis

reduced content of SPECTRIN, which affects its interaction with cytoskeletal proteins causing membrane instability and hemolysis. RBCs appear as spherocytes. Produces mild hemolytic anemia. Autosomal dominant.

Bohr effect

refers to a right shift in the hemoglobin binding curve when pH is lowered. This means that the P50 is decreased at a higher concentration of H+ -> Hb demonstrates a lower affinity for O2. Therefore hemoglobin gives up its oxygen with greater ease at the tissues where pH is decreased (H+ concentration elevated) due to presence of CO2.

Lactoferrin

released from the cytoplasmic granules of polymorphonuclear leukocytes, bind iron more avidly than transferrin and preferentially delivers that iron to macrophages.

Pyropoikilocytosis

severe form of hereditary ellipsocytosis.

Ferritin

storage protein for iron

Poikilocytosis

variation in SHAPE of RBCs

Anisocytosis

variation in SIZE of RBCs