RBC Lecture 3 - Anemias: Hypoproliferative and Hemolytic

Cobalamin

Vitamin B12 is also known as…

• Hydroxy-cobalamin and cyano-cobalamin

• Methyl-cobalamin and 5’-deoxyadenosylcobalamin

Vitamin B12 has several forms. (—2—) are the versions in food and (—2—) are co-enzymes

• Tetrapyrrole

• Cobalt

• 5,6-dimethylbenzimidazole

Vitamin B12’s structure is a (——) ring with (——) in the middle, attached to a ribonucleotide, (——)

• P separation from CBL

• R separation from CBL

• IF-CBL binding (lack of intrinsic factor)

• Malabsorption

• Competition for available B12 (parasites)

Impaired absorption may occur from… (5)

• Vit B12: Neurological symptoms

• Folate def: Cardiovascular disease (from high serum homocysteine)

Characteristic clinical symptoms that help differentiate between vitamin B12 deficiency and folate deficiency

• Low: Hgb, HCT, pancytopenia

• High: MCV, MCH

CBC characteristics of folate deficiency and vitamin B12 deficiency (5)

• Hypersegmented neutrophils

• Oval macrocytes

• Anisocytosis

• Poikilocytosis

• RBC inclusions (HJ bodies)

Characteristics of PBS morphology in folate and vitamin B12 deficiency (5)

Erythroid hyperplasia (ineffective)

Bone marrow examination characteristics of folate and vitamin B12 deficiency

Low (ineffective erythropoiesis)

Absolute reticulocyte count for folate and vitamin B12 deficiency

High

Serum total and indirect bilirubin for folate def and vitamin B12 def

High

Serum LDH for folate and vitamin B12 def

• Folate: N

• B12: Low

Serum vitamin B12 in folate and vitamin B12 def

• Folate: Low

• B12: N/High

Serum folate for folate and vitmain B12 def

• Folate: Low

• B12: N/Low

RBC folate for folate and vitamin B12 def

• Folate: N

• B12: High

Serum MMA for folate and vitamin B12 def

High

Serum/plasma homocysteine in folate and vitamin B12 def

• Folate: Absent

• B12: Present in pernicious anemia

Antibodies to IF and gastric parietal cells in folate and B12 def

• Folate: N

• B12: Markedly elevated in pernicious anemia

Serum gastrin in folate and vitamin B12 def

• Folate: N

• B12: Achlorhydria in pernicious anemia

Gastric analysis in folate and B12 def

• Folate: N

• B12: Low

Holotranscobalamin assay in folate and vitamin B12 def

• Folate: Negative

• B12: maybe Diphyllobothrium latum

Parasite stool analysis results for folate and vitamin B12 def

Pernicious anemia

An autoimmune disease in which an autoantibody is raised IF or gastric parietal cells

• T CD4

• H+/K-ATPase pump

• achlorhydria

In pernicious anemia, a (——)-mediated response destroys parietal cells over time (significantly decreasing IF secretion into the stomach), and also attacks and destroys (——) pumps on parietal cell membranes, leading to (——)

• Schilling test for absence of IF

• Serum gastrin levels (increased in achlorhydria)

• Detection of Abs against IF or parietal cells

Diagnosis of pernicious anemia (3)

H. pylori

Pernicious anemia can also be developed by (——) infection through parietal cell destruction

• 100-150 fl

• >120

In megaloblastic anemia, MCV could be anything between (——), but is mostly (——)

• High

• Normal

While MCH is (——), MCHC is (——) in megaloblastic anemia

• Dacrocytes

• RBC fragments

• Microspherocytes (high RDW)

While oval macrocytes are a very important clue for megaloblastic anemia, RBC morphology with (—3—) can also be observed throughout a PBS with megaloblastic anemia

• HJ bodies

• Basophilic stippling

• Cabot ring (rare)

RBC inclusions that are associated with megaloblastic anemia (3)

• at least five 5-lobed neutrophils per 100 WBCs

• one 6-lobed neutrophil

When is hypersegmentation reported?

Schilling test

a definitive test useful in distinguishing cobalamin deficiency due to malabsorption, dietary deficiency, or absence of IF

Crystalline vitamin B12

The schilling test measures the amount of an oral dose of radioactively labeled (——) that is absorbed in the gut and excreted in the urine

• ⁵⁷Co-labeled B12

• Unlabeled vitamin B12

• Flushing dose

In the schilling test, the patient is given a dose of (——) orally with or followed within 2 hours by an intramuscular injection of (——). The injection is termed the (——); its purpose is to saturate al cobalamin receptors in the tissues. Thus, any of the labeled oral dose absorbed in the gut and passing into the blood will be in excess of available receptor uptake.

• > 7.5%

• PA and malabsorption syndromes

Excess vitamin B12 in the Schilling test is filtered by the kidney and appears in the urine. Urine is collected for 24 hours, and is radioactively determined. If (——) of the standard oral dose is excreted, absorption is said to be normal. If excretion is low, part 2 of the Schilling test is performed to distinguish between (——)

• Intrinsic factor

• >7.5% excretion

• Pernicious anemia

• Malabsorption defect

In part 2 of the Schilling test, the oral dose of labeled B12 is accompanied by a dose of (——). The remaining of the test is the same as in part 1. If part 2 shows (——) excretion, the absorption is considered normal with the lack of absorption in part 1 due to the lack of IF. The diagnosis is (——). If abnormal, the patient likely has (——)

PNH

Conditions that are acquired are typically extrinsic (and inherited defects are typically intrinsic). The exception is (——), which is an acquired, intrinsic condition.

• Macrophage mediated hemolysis (EV)

• Fragmentation hemolysis (IV)

Hemolytic anemias are due to excessive… (2)

• Free hemoglobin

• Methemoglobin

• Prussian blue staining of urine sediment

Assays with positive results for intravascular hemolytic anemia and negative for extravascular hemolytic anemia (3)

• Schistocytes

• Spherocytes

The RBC morphology associated with intravascular hemolytic anemia is (——) while extravascular hemolytic anemia has (——)

• Total bilirubin

• Indirect bilirubin

• LDH (IV > EV)

• Free Hb (IV > EV)

• Urobilinogen (urine)

Values that are elevated in intravascular and extravascular hemolytic anemia (5)

• Haptoglobin (IV < EV)

• Hemopexin (IV < EV)

• Hemoglobin/HCT/RBC count (WB)

Values that are decreased in intravascular and extravascular hemolysis

Direct bilirubin

Value that is within reference interval for intravascular and extravascular hemolysis

Intrinsic

Hereditary spherocytosis is the outcome of an (——) defect: mutations in proteins which maintain vertical attachments

Mostly autosomal dominant

Hereditary spherocytosis inheritance pattern

Ankyrin and spectrin

Hereditary spherocytosis involves mutations in (—2—)

• Surface area

• Deformability

In hereditary spherocytosis, the RBCs lose (—2—) leading to splenic sequestration (→ EV hemolysis or modification to microspherocytes)

• Anemia

• Splenomegaly

• Jaundice

Triad of symptoms associated with hereditary spherocytosis

• Childhood hemolytic anemia

• Family history of similar abnormalities

• Uniform spherocytosis on PBS

Clinical picture highly suggestive of HS (3)

• Family history of HS

• Splenomegaly

• High MCHC

• Reticulocytosis

• Spherocytes on PBS

Clinical picture for definitive diagnosis of hereditary spherocytosis (5)

• Osmotic fragility (OF) test (↑ fragility)

• eosin-5’-maleimide (EMA) binding test (↓ fluorescence)

Tests that help diagnose hereditary spherocytosis (2)

• ↓ Hb

• ↑ MCHC

• ↑ Retic

• ↑ Hyperdense RBCs

CBC results indicative of hereditary spherocytosis (4)

Negative

In hereditary spherocytosis, a DAT test is…

• ↓ serum haptoglobin

• ↑ serum lactate dehydrogenase

• ↑ serum indirect bilirubin

Test results indicative of hemolysis in hereditary spherocytosis (3)

⍺ and β spectrin genes (horizontal)

95% of hereditary elliptocytosis has a mutation in (——) genes

Hereditary pyropoikilocytosis

Severe form of hereditary elliptocytosis in which RBCs are fragmented on heating due to low stability at 41-45°C

• Extreme poikilocytosis

• MCV low

• Fragmentation

• Microspherocytosis

• Elliptocytosis

Hereditary pyropoikilocytosis characteristics on PBS (5)

• Stomatin

• RhAG

In overhydrated hereditary stomatocytosis, RBC membranes are highly permeable, where a deficiency in (——) proteins or a mutation in (——) protein has been reported

Xerocytosis

Dehydrated hereditary stomatocytosis or hereditary (——) is a defect in membrane cation permeability causing RBCs to become dehydrated

• Serum haptoglobin: decreased

• Urine hemoglobin: (+)

• Urine sediment Prussian Blue stain: (+)

What are the expected results for serum haptoglobin, urine hemoglobin, urine sediment Prussian Blue stain for chronic fragmentation hemolysis

• CD55

• CD59

In paroxysmal nocturnal hemoglobinuria, RBCs lack the markers (—2—), rendering RBCs susceptible to spontaneous lysis by complement

Type I

(PNH mosacisism) Type of RBC with normal level of CD55 and CD59, no lysis or little

Type II

(PNH mosacisism) Type of RBC with partial CD55 and CD59 deficiency, relatively resistant to lysis

Type III

(PNH mosacisism) Type of RBC with complete deficiency of CD55 and CD59, highly sensitive to lysis

Type I and III

(PNH mosacisism) most common RBC types in PNH

Detection of CD55 and CD59 by flow cytometry

Confirmatory test for paroxysmal nocturnal hemoglobinuria

G6PD deficiency

Most common RBC enzyme defect; has a large role in the hexose monophosphate shunt (HMS)

NADPH

G6PD is critical to RBCs as it is the only means to produce (——)

X chromosome

G6PD enzyme is encoded on the (——) chromosome

G6PD deficiency (A- variant)

African males with (——) are protected against Plasmodium falciparum malaria

Class I

(G6PD deficiency) Chronic, hereditary nonspherocytic hemolytic anemia; severity is variable; rare

• G6PD-Serres

• G6PD-Madrid

G6PD variants associated with Class I G6PD deficiency (2)

Class II

(G6PD deficiency) Severe, episodic acute hemolytic anemia assocaited with infections, certain drugs and fava beans; not self limited and may require transfusions during hemolytic episodes

• G6PD-Mediterranean

• G6PD-Chatham

G6PD variants associated with class II G6PD deficiency

Class III

(G6PD deficiency) Episodic, acute hemolytic anemia associated with infections and certain drugs; self-limited

• G6PD-A-

• G6PD-Canto

G6PD variants associated with class III G6PD deficiency (2)

• G6PD-B (wildtype)

• G6PD-A+

G6PD variants associated with class IV G6PD deficiency (2)

• Oxidizing drugs

• Infections

• Fava beans

Triggers of acute hemolytic anemia G6PD deficiency (3)

• Usually normo/normo

• Severe → marked aniso

• Severe → marked poik (sphero and schisto)

• Bite cells (nonspecific)

• Heinz bodies (supravital stain)

• Profound reticulocytosis (up to 30%)

PBS characteristics associated with G6PD deficiency (6)

• Very low serum haptoglobin

• High indirect bili

• High LDH

• Increased plasma Hb (IV hemolysis)

Test results for G6PD deficiency (4)

• Pyruvate kinase

• phosphoenolpyruvate → pyruvate

(——) is a key enzyme of the glycolytic pathway, where it catalyzes the conversion of (——) forming ATP

Pyruvate kinase deficiency

(——) causes premature destruction of RBCs from the depletion of ATP and therefore lack of membrane integrity

• Anemia

• Jaundice

• Splenomegaly

• Gallstones (chronic hemolysis)

Clinical presentation of pyruvate kinase deficiency (4)

• Anisocytosis

• Poikilocytosis

• Polychromasia

• Burr cells

PBS characteristics of pyruvate kinase deficiency (4)

• PK activity assay

• Genetic testing

Pyruvate kinase deficiency confirmatory tests (2)

Immune hemolytic anemia

Conditions where red cell survival is shortened because of antibody-mediated mechanisms

• Macrophages (EV)

• Complemented mediated hemolysis (IV)

• Combination of extravascular and intravascular

In immune hemolytic anemia, RBCs are removed by… (3)

• IgM

• IgG

In immune hemolytic anemia, (——) mediated hemolysis can result in both extravascular and intravascular hemolysis, while (——) mediated hemolysis is predominantly extravascular (macrophages in the spleen/liver)

DAT positive

Confirmatory test for immune hemolytic anemia

Autoimmune hemolytic anemia

Premature RBC destruction and anemia caused by autoantibodies that bind to the RBC surface with or without complement activation

Warm autoimmune hemolytic anemia

The most common type of AIHA

• Immunoglobulin: IgG

• Optimal temp: 37°C

• DAT: IgG or IgG + C’

• Complement activation: variable

• Hemolysis: EV

• Ab specificity: Panreactive

• Other: Polychromasia and spherocytosis

WAIHA characteristics (immunoglobulin, iptimal temp, DAT, complement activation, hemolysis, Ab specificity, other (2))

Hepatic macrophages (IV can occur if full complement activation)

In CAIHA, hemolysis is predominantly by…

• Immunoglobulin: IgM

• Optimal temp: 4°C

• DAT: Complement

• Complement activation: Yes

• Hemolysis: EV (some IV)

• Ab specificity: I (most) > i > Pr

• Other: RBC agglutination, hemoglobinuria

Cold Agglutinin Disease characteristics (immunoglobulin, iptimal temp, DAT, complement activation, hemolysis, Ab specificity, other (2))

• Immunoglobulin: IgG

• Optimal temp: 4°C

• DAT: Complement

• Complement activation: Yes

• Hemolysis: IV

• Ab specificity: P

• Other: Polychromasia, spherocytes, schistocytes, nRBCs, aniso, poik, hemoglobinuria

Paroxysmal cold hemoglobinuria characteristics (immunoglobulin, iptimal temp, DAT, complement activation, hemolysis, Ab specificity, other (7))

• Cold temp

• Warm temp

(PCH) Auto anti-P binds to the P antigen on RBCs in (——) and partially activates complement BUT fully activates complement at (——), exhibiting intravascular hemolysis

Young children after viral respiratory infection

When is PCH most commonly seen?

• Complement mediated intravascular hemolysis

• Activation of coagulation

Outcomes of acute hemolytic transfusion reactions (2)

• Hemoglobinemia and hemoglobinuria

• Decreased hemoglobin

• Increased serum indirect bili

• Decreased serum haptoglobin

• DAT positive

Test results for acute hemolytic transfusion reactions (Hb (3), bili, haptoglobin, DAT)

• Minutes to hours

• Days to weeks

Acute hemolytic transfusion reactions occur within (——) of the initiation of transfusion while delayed hemolytic transfusion reactions may occur (——) after a transfusion

• inadequate posttransfusion increase in Hb

• Positive DAT - IgG/complement

• Morphologic evidence of hemolysis (polychromasia, spherocytes, etc.)

• Increase in serum unconjugated bilirubin

Signs DHTR has occurred (4)