Chapter 23: Intracorpuscular Defects Intracorpuscular Defects Leading to

Chapter 23: Intracorpuscular Defects Intracorpuscular Defects Leading to

Chapter 23: Intracorpuscular Defects Intracorpuscular Defects Leading to Increased Erythrocyte Destruction 02/21/20 1 Objectives See Objectives, Chapter 23, Textbook, Rodak. 02/21/20

2 Hereditary Defects of RBC Membrane Biochemistry and Structure of Normal RBC Membrane Outer bi-lipid layer-serves as barrier to separate RBC interior from exterior Protein membrane skeleton- responsible for shape, structure , deformability. Contains pumps and channels for ion movement between RBC interior and plasma. 02/21/20

3 02/21/20 4 Hereditary Spherocytosis Hereditary Spherocytosis (HS)- group of inherited disorders characterized by the presence of spherocytes in PB. Mode of Inheritance and Etiology 75% (Autosomal dominant)- most common

inherited anemia (1 in 5000 northern Europeans) 25% (Autosomal recessive), or spontaneous mutation - neither parents have the disease No homozygotes- may be incompatible with life 02/21/20 5 Pathophysiology 1ry defects are deficiency in membrane skeletal proteins than connects the cytoskeleton to the lipid bi-layer (vertical proteins interactions): Spectrin absolute deficiency

(Attachment deficiency) ankyrin protein 4.2 band 3. In HS, the vertical interactions of proteins are deficient resulting in lipid bilayer destabilization, loss of membrane material See Figure 9-9 handout 02/21/20 6 Clinical and Laboratory Findings:

02/21/20 7 Progressive loss of membrane leading to Spherocytes: decreased surface to volume ratio Spherocytes are rigid and not as deformable as normal RBC. They have decreased survival in the spleen w/c sequesters spherocytes (defective cells).

Splenic conditioning- leads to destruction of RBC in HS 02/21/20 8 Splenic Conditioning in HS Low glucose level in spleen; less ATP produced, Sodium leaks into the interior- cation pump cannot maintain their electrolyte balance, cells take up water, swells, and lyse. Abnormal blebbing- responsible for RBC membrane loss in HS

Splenic Macrophages- entraps the abnormal cells contributing to early RBC hemolysis. Splenectomy (removal of spleen) increases RBC life span and decreases hemolysis of RBC 02/21/20 9 Clinical and Lab Findings 3 key clinical manifestations: Anemia Jaundice Splenomegaly

02/21/20 10 Lab Findings Hallmark of HS is spherocytes in PB Reticulocytosis- RPI >3.0 Elevated MCHC (greater than 36.0), not due to hemolysed specimen Negative Direct Antiglobulin test (DAT)- (indicating cells do not have coated antibodies coating the membrane)

02/21/20 11 02/21/20 12 Osmotic Fragility Procedure Specimen and Normal Control: Draw in green top, Li heparinized blood, drawn same day

when test will be done. Give yourself two days for the entire procedure (involves incubation). Reject: (K2 or K3 EDTA)- too much salt; hemolyzed, or too old (> 24 hrs old) Day 1: Perform Osmotic Fragility procedure on patient and control (Room Temp), then Incubate patient and control green tops overnight at 37oC. Using the incubated bloods, repeat the procedure as in above. Measure hemolysis by spectrophotometer. Plot both results (non-incubated, incubated) on same graph.

02/21/20 13 02/21/20 14 Spherocytes may also be seen in autoimmune hemolytic process. Osmotic Fragility test does not distinguish between spherocytes in HS and spherocytes in acquired autoimmune hemolytic anemia

02/21/20 15 02/21/20 16 Hereditary Elliptocytosis- HE HE- Autosomal dominant (except for rare recessive trait) Occurs (0.02%-0.05% in all races)

Pathophysiology- defect in one of the skeletal proteins that act horizontally. Decreased spectrin dimers to form tetramers due to defective chains Defect in band 4.1 that aids in binding Spectrin to Actin 02/21/20 17 Three Classes of HE: Common HE Silent carrier Mild common HE- 30-100% Elliptocytes- No

symptoms Common HE with Hemolysis- Marked Elliptocytosis Homozygous Common HE- severe fragmentation Spherocytic Hereditary Elliptocytosis Combination of mild HE and mild HS- common among northern European Spherocytes and Elliptocytes present 02/21/20 18 Stomatocytic Hereditary Elliptocytosis or Southeast Asian Ovalocytosis (SAO).- common only among Melanesian and Malaysian population

appear as double stomatocytes? -unusually heat resistant even at 51-52oC (normal is 49oC) Resistant to crenation All patients are Heterozygotes (No homozygotes identified) Band 3 tightly binds with ankyrin and unable to transport Na+ 02/21/20 19 02/21/20

20 Hereditary Pyropoikilocytosis-HPP Subtype of HE Rare disorder- extreme poikilocytosis, present in early childhood resembles blood picture of severe burns & homozygous HE Both -spectrin mutation leading to defective spectrin heterodimers and partial deficiency of spectrin Recessive disease Homozygosity for silent carrier Homozygosity for mild HE, or double heterozygosity

for spectrin defect. RBC morph similar to homozygous HE In 1/3 of HPP, on the parents has mild HE; or silent carrier 02/21/20 21 HPP In homozygous HE and HPP, Osmotic fragility and autohemolysis are markedly increased. Thermal instability- RBC hemolyzes at 45-46oC in 10-15 min. (normal 49oC). Will hemolyze at 37oC in 6 hours.

Severe hemolytic anemia w/ facial bone abnormalities, gallbladder abnormalities, and growth retardation. Severe poikilocytosis, budding RBCs, Elliptocytosis, schistocytes. MCV= very low (25-75 fL) Black population mostly. Some patients respond to splenectomy, but hemolysis still occur due to hemolysis at body temp. 02/21/20 22 02/21/20

23 Inherited Disorders of RBC Cation Permeability and Volume Hereditary Stomatocytosis (Hydrocytosis) Acquired Stomatocytosis Stomatocytosis in Rh null Disease

Hereditary Xerocytosis (HX) 02/21/20 24 Hereditary Stomatocytosis 02/21/20 25 Hereditary Stomatocytosis (Hydrocytosis)

Main defect: Failure of the Na and K pumps to eliminate water from the cell. Na+ influx exceeds the loss of K+ Increased intracellular cation (Na+ and K+): water enters the cell; forms a hydrocyte or a stomatocyte Some are caused by deficiency in Stomatin or Band 7.2b 02/21/20 26 Rh Null Disease

Rh Null= RBC lacking in all the Rh antigens; causes moderately severe anemia with stomatocytes and spherocytes. Rhmod- Stomatocytosis occurs in this disease where Rh antigen is suppressed but not absent. 02/21/20 27 Hereditary Xerocytosis (HX) Rare Autosomal dominant- RBCs are dehydrated (elevated MCHC)

These cells lose K+ due to permeability defect. Stomatocytes, target cells, spiculated RBCs. Hemoglobin puddle in discrete areas 2,3 BPG decreased Splenectomy does not help much 02/21/20 28 Acanthocytosis Acanthocytes- Spur cells- RBC w/ few irregular projections that vary in length

Associated with liver disease abetalipoproteinemia, infantile pyknosis, anorexia nervosa, McLeod and In(Lu) blood groups Abetalipoproteinemia- steatorrhea, retinitis pigmentosa resulting in blindness, progressive neurologic disease- death in 20-s-30s. 02/21/20 29 Acanthocytes of Spur cells 02/21/20

30 RBC Enzymopathies- G6PD def., PK def, Methemoglobin Reductase def. G6PD deficiency- most common RBC enzyme deficiency Asymptomatic G-6-PD located on X chromosome- X-linked pattern: Men can be a normal hemizygote or deficient hemizygote: Women can be normal homozygote, (both alleles normal; deficient homozygote; both alleles abnormal; or a heterozygote (one allele normal and one abnormal).

Female heterozygotes have 2 populations of RBCs (normal and abnormal (G-6-PD deficient) Deficiency results from point mutation on the gene resulting in low G-6-PD activity or function 02/21/20 31 02/21/20 32 02/21/20

33 G-6-PD Deficiency Classes Class I- severe- <20% G6-PD activity- chronic non-spherocytic hemolytic anemia Class II- mild; intermittent activity <10% Class III- mild; 10%-60% activity Class IV- no clinical expression- 100% Class V- no clinical expression, also 100% 02/21/20

34 G-6-PD Variants G-6-PD B- Normal variant G-6-PD A+ - variant common among African descent (40% have this variant) w/ additional amino acid G-6-PD A- - 10% of African Amer. Lack this variant G-6-PD Med - most common variant among whites; G-6-PD activity barely detectable (Class II prototype) G-6-PD Canton- Chinese variant G-6-PD Mahidol- Southeast Asians 02/21/20

35 G6PD - Pathophysiology G-6-PD enzyme reduces NADP to NADPH producing sufficient reduced Glutathione (GSSH). Reduced glutathione protects the RBC from oxidative injury Heinz bodies- denatured hemoglobin that precipitates out. Bite cells- RBCs with a piece cut out (where Heinz bodies were located) Abnormal cells become less pliable; gets trapped in the spleen and destroyed- chronic non-spherocytic

hemolytic anemia Normocytic, normochromic anemia 02/21/20 36 Heinz bodies are denatured globin, and represent the end-product of oxidative degradation of hemoglobin. Heinz bodies may be detected post-splenectomy, with oxidative hemolysis (in normals, but particularly in patients with G-6-PD deficiency) and in patients with unstable hemoglobinopathies.

Specimen: EDTA anticoagulated blood. Do test within 24 hours of collection. Enzyme activity diminishes with time. Older RBCs have less enzymatic activity, will hemolyze first. 02/21/20 37 Causes of Hemolysis in G6PD def.

Oxidizing drugs- Naphthalene (Moth) balls Anti-malarial drugs (Primaquine) Fava beans- seeds Infection Hemoglobinuria 2-3 days after drug is used. Heinz bodies positive- Supravital stain Older RBCs low G-6-PD level will hemolyze first Younger RBCs or Retics will have higher enzyme

activity Reticulocytosis may give false normal screening test Favism- G-6-PD Med- severe intravascular hemolysis 02/21/20 38 G-6-PD Screening Test and Quantitative Test Screening test Qualitative- based on color reduction from Blue to Rust in color. Specimen has to be relatively

fresh. Normal activity within two hours change in color. Abnormal activity- longer time to change the color Reported as either normal activity; low activity Caution: High Retics will give false normal activity, especially after a hemolytic episode. Autohemolysis test- blood is incubated for 48 hrs at 37oC. Hemolysis is increased compared to normal control. Addition of GLUCOSE in the tube reduces hemolysis. 02/21/20

39 G-6-PD Tests Methemoglobin Reduction test- G6PD deficient cells will fail to reduce Methemoglobin in the presence of Methylene Blue Ascorbate-Cyanide Test- not a good test because it will not differentiate between G6PD and PK and other unstable hemoglobins. Fluorescent Spot test: G6PD, NADP, saponin, buffer is mixed with patients blood and placed on filter paper, G6PD converts the NADP to NADPH.

G6PD deficient RBCs will fail to convert the NADP to NADPH and will lack fluorescence. Normal cells will fluoresce. Quantitative test- based on rate of reduction of NADP to NADPH measured at 340 nm. 02/21/20 40 02/21/20 41

Pyruvate kinase (PK) deficiency Most common deficiency in the anaerobic pathway (EM pathway)= Homozygotes manifest Heterozygotes = asymptomatic Catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate with conversion of ADP to generation of ATP.(energy) Inability to maintain sufficient ATP= K loss = RBC dehydration (echinocytes) 2,3 BPG is increased by 2X. Rigid cells are removed by macrophages in spleen and liver. Heinz bodies not found in PK def.

Normocytic normochromic anemia 02/21/20 42 Tests for PK Autohemolysis- increased hemolysis after 48 hrs. Addition of GLUCOSE will not reduce hemolysis. Fluorescent Spot test= based on couples enzyme test. PEP, NADH, ADP, Mg+2, LDH are added to patients sample: If blood lacks PK enzyme NADH will not be oxidized and fluorescence persists up to 1 hour.

If PK is present fluorescence will disappear in 15 minutes, because NAD does not fluoresce 02/21/20 43 Extremely high Retic count Quantitative test involving reduction of NADP to NADPH should follow abnormal screening test. 02/21/20 44

Methemoglobin Reductase Deficiency Methemoglobin reductase , also called "diaphorase," and more properly called cytochrome B(5) reductase, is the only enzyme within the erythrocyte that maintains hemoglobin in the reduced (non-methemoglobin) state. Persons who are heterozygous for methemoglobin reductase deficiency have no clinical or laboratory abnormalities, are not cyanotic, and have normal methemoglobin concentrations in their blood.

02/21/20 45 Persons who are homozygous for methemoglobin reductase deficiency have normal arterial oxygen saturation but have varying quantities of methemoglobin in their blood, generally 15% to 20%, and are quite cyanotic. Paradoxically, homozygotes have normal blood counts; the condition does not cause polycythemia. The reason for this apparent paradox seems to be that the presence of methemoglobin shifts the hemoglobin-O(2) dissociation curve to the right, so that although the

transport of oxygen is diminished, the delivery of oxygen to tissues is normal. The condition is quite benign, but may cause concern to parents of affected children, be a cosmetic embarrassment to the children, and alarm the attending physician. The 02/21/20 46 cyanosis may be treated with methylene blue. Paroxysmal Nocturnal Hemoglobinuria PNH

History and Etiology Pathophysiology Other Clinical Findings Routine Hematologic Findings Special Diagnostic Tests Therapy and Prognosis 02/21/20

47 PNH History and Etiology Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disorder characterized by nocturnal hemoglobinuria, chronic hemolytic anemia, thrombosis, pancytopenia and, in some patients, acute or chronic myeloid malignancies. Arises from mutation of the stem cell that may affect most stem cells lines (affecting leukocytes, platelets, and RBC) In RBC, the membrane defect renders the

membrane sensitive to lytic action of complement causing the characteristic hemolysis. 02/21/20 48 History I nvestigator Year Gull 1866 Contribution

Described nocturnal and paroxysmal nature of intermittent haematinuria in a young tanner. Strubing 1882 Distinguished PNH from paroxysmal cold haemoglobinuria and march haemoglobinuria. Attributed the problem to the red cells. van den Burgh 1911 Red cells lysed in acidified serum. Suggested a role for complement. Enneking 1928 Coined the name paroxysmal nocturnal haemoglobinuria. Marchiafava

1928- Described perpetual hemosiderinemia in absence of and Micheli 1931 hemolysis. Their names became eponymous for PNH in Europe. Ham 1937- I dentified the role of complement in lysis of PNH red 1939 cells. Developed the acidified serum test, also called the Ham test, which is still used to diagnose PNH. Demonstrated that only a portion of PNH red cells are abnormally sensitive to complement. Davitz 1986 Suggests defect in membrane protein anchoring system responsible

02/21/20& Rosse 49 Hall 1996 Flow cytometry for the diagnosis of PNH 02/21/20 50 Pathophysiology Somatic mutation in PIG-A gene (phosphatydylinositol glycan- complementation group A) located on the X-chromosome in a

clone of stem cells GPI and GPI-linked proteins glycosylphosphatidyl-inositol (GPI) linked membrane proteins are low 2 regulatory proteins: CD55- Decay Accelerating Factor (DAF) CD59- Membrane Inhibitor Reactive Lysis (MIRL) 02/21/20 51 CD55 and CD59 clears the membrane of attached complement and C3 convertase complexes.

Deficiency in either or both proteins result in completion of membrane attack unit - resulting in hemolysis of RBC Acquired membrane abnormality that makes the membrane sensitive to lytic action of complement 02/21/20 52 Subtypes Subtypes- based on surface expression of GPIlinked proteins such as CD16, CD48, CD55, CD59

CD55- Decay Accelerating Factor (DAF) CD59- Membrane Inhibitor of Reactive Lysis (MIRL) PNH I- nearly normal, PNH II-moderate PNH III- devoid of GPI-linked surface protein 02/21/20 53 Clinical Lab Findings Cytopenias (varying in severity)- variability causes delay in diagnosis

Severe aplastic anemia picture Tendency for thrombosis (venous thrombosis in 40% of patients = main cause of morbidity Major cause of death Venous thrombosis Complications from progressive pancytopenia 02/21/20 54 Routine Hematologic Findings Pancytopenia- Leukopenia, anemia, low Pltc

Anemia- varies in degree from low 6 gm/dl to normal Reticulocyte count increased No abnormal RBC morphology associated with PNH Bone marrow RBC hyperplasia Hemosiderinuria- always present 02/21/20 55 02/21/20

56 Special Diagnostic Tests Flow cytometry tests for GPI-linked membrane proteins on RBC & Granulocytes: CD16 CD48 CD55 CD59 02/21/20 57

Therapy and Prognosis -mainly supportive, using transfusions, antibiotics, and anticoagulants (thrombotic complications). Iron therapy- for iron loss (intravascular hemolysis) Steroids help with hemolysis Androgenic steroids appear to be effective with prominent hypoplasia Bone Marrow transplantation can be curative Serious complications do occur Some terminate into acute leukemia 02/21/20

58 Sugar Water test Principle of the test- RBCs hemolyze in low ionic strength solution of sugar-water Clinical significance- PNH cells will hemolyze, normal cells will not Procedure 10% sucrose solution + patients whole blood Apply QC 10% sucrose solution + normal control cells Interpret Results- normal control = no hemolysis

Hemolysis = PNH cells; No hemolysis= neg for PNH 02/21/20 59 Acidified Serum test Principle of the Test- PNH cells are sensitive to acidification and lytic action of complement Clinical Significance Procedure QC Interpretation of the test 02/21/20

60 Heinz Bodies Test Principle of the test Clinical Significance QC procedure

Sources of Error Interpretation of Results 02/21/20 61

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