Spherocytes significance and differential diagnosis

Spherocytosis

1. SPHEROCYTES- SIGNIFICANCE AND DIFFERENTIAL DIAGNOSIS Dr. SADAF KHAN

2. What are spherocytes ? Erythrocytes that have lost their biconcavity. Appear as densely stained spheres smaller in size and lacking central area of pallor. Can be called hyperchromic .

3. MCV- normal or slightly decreased (77-87 f L ) MCHC- increased (more than 35 g/ dL ) RDW > 14

4. HOW ARE THEY FORMED? Depending on the pathology it can be due to : - membrane defect or - immune mediated lysis of membrane

5. RBC MEMBRANE

6. MEMBRANE DEFECT E.g. HS Involves the interplay between an intrinsic erythrocyte membrane protein defect and an intact spleen.

7. ROLE OF SPLEEN An intrinsic red cell defect leads to RBC destruction, but only in the presence of an extrinsic factor—an intact spleen . splenic conditioning Splenic entrapment ( pitting) (culling )

8. IMMUNE MEDIATED LYSIS OF MEMBRANE E.g. immune mediated hemolytic anemias

9. SPHEROCYTES IN PERIPHERAL SMEAR 1.Membrane abnormalities -Hereditary Spherocytosis - Spherocytic E lliptocytosis -Hereditary Pyropoikilocytosis -Rh null phenotype 2.Oxidant damage -G6PD deficiency -Unstable Hb 3.Immune hemolytic anemias 4.Non immune hemolytic anemias 5.Thermal injuries- burns 6.IV water infusion or drowning 7.Toxins - snake bite -clostridium sepsis - Bartonellosis 8.Hyposplenism

10. AGT + - IHA OFT Family study + - HS Spider venom, clostridium sepsis, burns Thalassemia Hb Electrophoresis

11. HEREDITARY SPHEROCYTOSIS Anemia, intermittent jaundice and splenomegaly. History of gall stones. Family history of cholelithiasis in the second or third decade of life.

12. CASE REPORT 0yrs/MALE HEMOGLOBIN – 6.0g/dl MCV – 78.7fl MCHC – 38.4g/dl (high) RDW – 24.1% PBS – RBC – Predominantly spherocytes , NCNC, Occasional fragmented forms (+), Polychromasia (+) WBC, PLT - normal

13. Laboratory features Anemia – mild, Reticulocytosis , Low MCV, Increased mean corpuscular hemoglobin concentration (MCHC), Spherocytes on the peripheral blood smear- UNIFORM IN SIZE AND DENSITY, Hyperbilirubinemia, and An abnormal OF test.

14. It gives an indication of the surface area/volume ratio of the erythrocytes. As this ratio falls cells become more sensitive to osmotic lysis . Red blood cells that are spherocytic , for whatever cause, take up less water in a hypotonic solution before rupturing than do normal blood cells. Does not distinguish between spherocytes due to HS and AIHA Thalassemia – increased surface to volume ratio – decreased osmotic fragility OSMOTIC FRAGILITY TEST

15. Principle of OFT When RBCs are placed in hypotonic solution water is drawn into RBC This result in swelling of cell, which acquire a spherical shape After a critical volume is reached membrane first leaks small molecules like potassium ions Membrane pores increase in diameter, larger molecules like Hb leave the cell The amount of Hb in supernatant is measured colorimetrically and compared with sample of completely lysed cells

16. Most common of the hereditary hemolytic anemias 75%- Autosomal dominant inheritance 25%- Non dominant inheritance

17. Pathogenesis Membrane protein defect First identified- spectrin Most common- combined spectrin and ankyrin > β- spectrin > α- spectrin > Band-3> Protein 4.2 Loss of lipid bilayer resulting in decreased surface area to volume ratio- spherocytes . Deformed RBCs undergo destruction in spleen.

18. The most sensitive test to detect spherocytes is the incubated OF test performed after incubating RBCs 18 to 24 hours under sterile conditions at 37°C.

19. AUTOHEMOLYSIS TEST ACIDIFIED GLYCEROL LYSIS TEST CRYOHEMOLYSIS TEST ANTIHUMAN GLOBULIN TEST – helps to distinguish AIHA and HS – negative in HS

20. IDENTIFICATION OF DEFICIENT / DEFECTIVE MEMBRANE PROTEIN – SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis DENSITOMETRIC QUANTITATION &/or ELISA PCR – molecular diagnosis FLOWCYTOMETRY – recently introduced ; EMA (eosin-5-malemide) fluorescent dye to label RBC Dye binds to band -3 ; decrease in fluorescence - + ve high specificity and high sensitivity for HS – screening NO TEST CAN DIAGNOSE ALL CASE OF HS : COMBINATION NEEDED

21. SPHEROCYTIC ELLIPTOCYTOSIS Dominant inheritance Mild to moderate anemia Mild to moderate hemolysis Splenomegaly INVSETIGATIONS Peripheral smear- ovalocytes (rounded elliptocytes ) and spherocytes Osmotic fragility- increased.

22. HEREDITARY PYROPOIKILOCYTOSIS Severe anemia Severe hemolysis Splenomegaly Low MCV MCHC- Normal P/S- poikilocytosis RBC budding with fragments; elliptocytes ; microspherocyte Increased osmotic fragility Thermal instability of HPP erythrocytes, susceptibility to budding and fragmentation upon heating to 46°C.

23. GLUCOSE 6 PHOSPHATE DEHYDROGENAS DEFICIENCY H/O recent infection, administration of drugs, ingestion of fava beans Unexplained hyperbilirubinemia in neonates Clinical manifestation: Chills, fever, headache, nausea, backpain and abdominal pain, jaundice and dark urine

24. Indicator of hemolysis : ↓serum haptoglobin , ↑serum LDH, ↑ serum indirect bilirubin, hemoglobinemia and hemoglobinuria PBS finding: Polychromasia , RBC morphology varies from normal to marked anisocytosis, poikilocytosis , spherocytosis or schistocytes , bite cells and H einz bodies DAT result: Negative

25. Important intracellular enzyme for protecting hemoglobin & other cellular protein and lipids from oxidative denaturatation Gene located on X chromosome Deficiency is the most common RBC enzyme defect G6PD-deficient RBCs can’t generate sufficient NADPH to reduce glutathione and thus can’t effectively detoxify hydrogen peroxide

26. T ests for G6PD deficiency

27. Q uantitative Spectrophotometric Assays Gold standard for determining G6PD deficiency Based on direct measurement of NADPH generated G-6-P+ NADP G6PD 6-Phosphogluconate + NADPH Rate of NADPH formation ≈ G6PD activity and measured as increase in absorbance at 340nm Activity is measured as a ratio of G6PD activity per gram of hemoglobin(g Hb ) Cut off point for G6PD deficiency set as < 20% of normal activity (< 4.0 IU/g Hb )

28. F luorescent Spot Test Based on the principle that NADPH generated is fluorescent Blood and glucose-6-phosphate/NADP reagent are incubated and spotted on filter paper at timed intervals Normal G6PD activity – moderate to strong fluorescent spots under UV light Decreased or no activity – display weak or no fluorescence

29. D ye Reduction Assay NADPH produced by enzymatic reaction reduces a dye, giving a visually observed colour change In normal G6PD activity, NADPH generated reduces the dye to formazan product, brown black in colour Senstivity of 98% in detecting deficient specimen with G6PD deficiency < 4% IU/g Hb

30. Immune H emolytic Anemia Autoimmune Warm antibody type Cold antibody type a.Idiopathic b.Secondary -Lymphoproliferative disorders - CLL,NHL -Autoimmune Disorders -Viral infections c.Drug Induced Mixed type -Autoimmune type - Hapten a. Cold agglutinin disease -Idiopathic -Secondary Infections -Infectious Mononucleosis -Mycoplasma pneumonia Autoimmune disorders CLPD b . PCH -Syphilis, Mumps, measles Alloimmune - --- - HemolyticTransfusion reaction -Hemolytic disease of newborn

31. COLD AUTOIMMUNE HEMOLYTIC ANEMIA Exacerbation of hemolysis in winters- Acrocyanosis in cooler vessels of the extremities, nose and ears. Livedo reticularis over limbs reversible upon warming of the affected area.

32. Hg- 5-6 g/Dl RBC count – artifactually low MCV - artifactually high, producing a spuriously high MCHC P/S - agglutination and RBC clumping

33. L aboratory findings in immune mediated hemolytic anemia ↓Hemoglobin ↓Serum haptoglobin level ↑ R eticulocyte count ↑Indirect serum bilirubin and LDH MCV may be ↑ Hemoglobinuria (intravascular hemolysis or severe extravascular hemolysis) PBS- Polychromasia , spherocytes , occasionally RBC agglutination, nucleated RBCs, schistocytes and erythrophagocytosis by neutrophils

34. Warm AIHA Cold agglutinin disease Paroxymal cold hemoglobinuria Mixed-Type AIHA Ig Class IgG IgM IgG IgG , IgM Optimum reactivity temp 37⁰C 4⁰C 4⁰C 4-37⁰C Sensitization detected by DAT IgG or IgG +C3 C3 C3 IgG and C3 Complement activation Variable Yes Yes Yes Hemolysis Extravascular primarily Extravascular and intravascular Intravascular Both Auto Ab specificity Pan reactive or Rh complex I(most), i (some) Pr (rare) P Panreactive ; unclear specificity

35. C oombs Test

36. RBC Clumps Warm sample at 37 degrees and rerun MCV gets reduced DCT at 37degrees and 4 degrees with IgG and C3 + - Cryoglobulins C3 IgG CAD PCH AIHA Antibody ID Medical history Autoimmune disease H/ O Transfusion

37. NON IMMUNE HEMOLYTIC ANEMIA

38. Damage to vascular endothelial cells of Glomerular Capillaries Bacterial toxins(in HUS) BM. Collagen is exposed Platelet adhere to collagen RBC passes thro. Loose fibrin network attached to endothelial cells Platelet aggregation in capillary(hyaline thrombi) RBC get fragmented Damaged membrane of fragmented red cells is sealed Schistocytes Hemolytic anemia(MAHA) Disseminated malignancies Cytokines liberated Activation of coagulation pathway Microthrombi of glomerular capillary Impaired kidney function Release of vWF multimer (TTP) Thrombocytopenia Malignant hypertension Platelet aggregates

39. Schistocytes MAHA Reduced platelets + - Enivironmental factors, Mechanical cardiac valves, V alvulitis , Hypertension Inc. D- dimer Dec. fibrinogen ADAMTS 13 DIC TTP DIC, TTP, HELLP,HUS

40. INVESTIGATIONS Sepsis screen Coagulation profile PT / APTT Fibrinogen D - Dimer Fibrin degradation products Cardiac function test Renal function test

41. Peripheral blood film of microspherocytes seen in Clostridium perfringens sepsis regular spherocytes are usually smaller than normocytic red blood cells, microspherocytes are even smaller than that usually seen in critically ill, septic patients with severe C. perfringens infection

42. HYPOSPLENISM Congenital absence of spleen, sickle cell disease, celiac disease, vasculitis, ulcerative colitis, essential thrombocythemia , and after splenectomy . Blood film- acanthocytes , spiculated spherocytes , target cells, spherocytes , stomatocytes , and Howell-Jolly bodies. Platelet anisocytosis and giant platelets can also be noted in the blood film. Ultrasonography- normal-sized spleen, but a radionuclide liver-spleen scan shows no splenic uptake.

43. Rh NULL DISEASE Absent ( Rhnull ) or markedly decreased ( Rhmod ) Rh antigen expression. P/S- Mild to moderate normocytic, normochromic hemolytic anemia with stomatocytes and occasional spherocytes .

44. REFERENCES Wintrobes Clinical haematology 13th edition Henry’s Clinical Diagnosis and Management 22nd edition manual on hematology Dacie & Lewis practical haematology 11 th edition McKenzie / Williams Clinical Laboratory Hematology 3 rd edition Robbins & Cotran Pathologic Basis of disease Blood cells practical guide Barbara J Bain 4th edition

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