AUTOIMMUNE HAEMATOLOGICAL DISORDERS DIAGNOSTIC INTRICACIES

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1 AUTOIMMUNE HAEMATOLOGICAL DISORDERS DIAGNOSTIC INTRICACIES Dr.Anjali Kelkar CME in Pathology th - 10th July,

2 INTRODUCTION Spectrum of Autoimmune Haematological Disorders: Autoimmune haemolytic anaemia Immune thrombocytopenia Cold agglutinin disease Paroxysmal cold Haemoglobinuria Autoimmune Lymphoproliferative Syndrome Lupus Anticoagulants Drug induced haemolytic anaemia Autoimmune disorders asso with chronic lymphoproliferations 2

3 CASE 1 26 / F Presented with Anaemia of sudden onset with jaundice No significant past history Since last six months suffering from joint pains, low grade fever on and off; taking NSAIDs On examination: Severe pallor, mild jaundice. No organomegaly or lymphadenopathy 3

4 CASE 1: INVESTIGATIONS Hb 7gm/dl TLC 5.3 x10 9 /L PC 88 x10 9 /L Serum Bilirubin 6.2gm% (D 0.9 / ID 5.3) Retic count 12% LDH 1300 IU/L DCT Positive The peripheral smear showed presence of microspherocytes 4

5 CASE 2 32 / M Gradually noticed that he had 'yellow eyes H/o passing dark urine Felt tired, short of breath: climbing stairs No other symptoms; no itching / fever / bleeding / not taking any drugs On examination: Pallor++ Icterus+ Afebrile No lymphadenopathy, organomegaly 5

6 Hb 5.4 gm/dl CASE 2: INVESTIGATIONS WBC 40 x 10 9 /L (23nRBC/100WBCs) PBS: Polychromasia with nucleated red cells & spherocytes Retic count: 9% Serum bilirubin 9.1, SGPT normal Lactate dehydrogenase: 5721 IU/L Direct Coombs test Positive: IgG and C3 on RBCs 6

7 CASE 2: INVESTIGATIONS The serum contained a warm non-specific autoantibody Antinuclear antibodies and rheumatoid factor tests were negative Immunoglobulin levels were normal; No paraprotein bands in his serum. Large amounts of urinary haemosiderin were detected 7

8 APPROACH TO DIAGNOSIS History and clinical presentation, Signs of haemolysis CBC with PBS Reticulocyte count Serum bilirubin Serum LDH Presence of dark coloured urine: haemoglobinuria Rule out about presence of coexisting diseases; Established haemolysis: Anaemia Reticulocytosis Indirect hyperbilirubinaemia Raised LDH Microspherocytes, polychromasia and nucleated RBCs 8

9 AUTOIMMUNE HAEMOLYTIC ANAEMIA Type of antibody and Optimal temperature for haemolysis Warm Cold Rh I / i Antigenic Stimulus causing immune response Autoimmune Alloimmnue Drug induced 9

10 AUTOIMMUNE HAEMOLYTIC ANAEMIA Immune deregulation Regulatory T cells Pro inflammatory Th17 cells Antigen antibody reactions RBCs lysis Availability of complement, Environmental temperature, Function of the RE system 10

11 Chaudhary RK, Das Sudipta Sekhar. Autoimmune hemolytic anemia: From lab to bedside. Asian J Transfus Sci. 2014;8(1):

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13 ANTI HUMAN GLOBULIN REAGENT Polyspecific: IgG + C3 Tube technique: Ig molecules/ cell Column technique high sensitivity IgA rare, fulminant haemolysis IgM difficult to detect; removed in washing Detection of C3 in absence of IgG: Cold / biphasic Flow cytometry, Potentiators, ELISA to increase sensitivity 13

14 DAT & IAT 14

15 CASE 3 M / 67 Presented with low grade fever, loss of weight, malaise since 4 months On examination: Pallor +, Icterus Mild, Spleen 4 cm palpable below the costal margin Axillary and Cervical lymph nodes palpable. 15

16 CASE 3: INVESTIGATIONS Hb 10 gm/dl TLC 20.8 x 10 9 /L PC 110 x 10 9 /L Serum LDH and uric acid raised Indirect hyperbilirubinaemia DCT Positive BM : Low grade B cell Lymphoproliferative disorder 16

17 AUTOIMMUNE HAEMOLYTIC ANAEMIA: ASSOCIATED WITH CHRONIC LYMPHOPROLIFERATIONS (CLL/B CELL TYPE) Autoimmune haemolytic anaemia Autoimmune thrombocytopenia Autoimmune neutropenia Pure red cell aplasia Association with Chronic Lymphocytic leukemia and other lymphoproliferations typically in elderly 17

18 CASE 4 M / 67 Presented one winter with malaise, very cold hands and feet Non-smoker, not on any medication O/E: some bruising on the shins. Mildly jaundiced fingers, toes: cold with cyanotic tinge Bilateral axillary and inguinal LN. No organomegaly Hb 10.0 gm/dl PBS: Rouleaux formation, autoagglutination, polychromasia 18

19 Auto-agglutination 19

20 COLD AGGLUTININ DISEASE 20

21 COLD AGGLUTININ DISEASE Median Age: 65 years Acrocyanosis: Due to stasis of the agglutinated RBCs Underlying B cell disorder/mycoplasma/ebv/ideopathic Extravascular haemolysis in the liver, by the Kupffer cells having the receptors for IgM. IgM antibody can readily activate the complement; thus, intravascular haemolysis can occur. IgM Antibody Anti I / i Fixes Complement 4 0 C 21 DAT Positive for C3 IgM elutes in vitro Poly-specific Ig reagent

22 CASE 5 M / 8 Presented with fever and cervical lymphadenopathy Hb 11.2 gm/dl, TLC 7.4 x 10 9 /L, Platelets 130 x 10 9 /L PBS: Many reactive lymphocytes suggestive of virocytes Got better with symptomatic Rx Day 15: Hb 7.8 gm/dl, Pale, Icterus+ ID Hyperbilirubinemia DAT Positive History of exposure to cold and passing dark coloured urine 22

23 PAROXYSMAL COLD HAEMOGLOBINURIA Children: after viral infections / immunisation Acute attack of intravascular haemolysis Donath-Landsteiner antibody: Biphasic antibody, IgG, Anti P specificity Binds complement at 4 0 C Haemolysis at 37 0 C DAT weak positive : Papain sensitivity 23

24 Potentiator Action Remarks 22% Alb Reduces zeta potential High MW protein LISS PEG Polybrene Enzymes (Papain, Trypsin, Ficin, Bromelin) Low ionic environment. zeta potential. antibody uptake. Removes water from the test system. Concentration of antibody Antibody uptake Aggregates normal RBCs Addition of sodium citrate disperses the aggregates Remove sialic acid residues from the red cell membrane surface charge. Split polypeptide chains antigen exposure 24 NaCl+Albumin+Glycine Low ionic strength medium. Aggregation if centrifuged. Incubation at 37deg C. Washing step important to avoid false positivity. A positive polymer; for resolving ABO discripancies due to polyagglutination. Proteolytic substances, hemolytic activity of complement dependent antibodies.

25 CASE 6 F / 72 With osteoarthritis Suffered acute haemolysis after right hip replacement No organomegaly or lymphadenopathy No explanation for the episode: Warm and cold antibody tests were negative She required prophylactic antibiotic for her next surgery when she haemolysed again Prophylactic antibiotic Cephalosporin: suspected as the causative agent, serum reacted with red cells coated with Cephalosporin Advised to avoid this antibiotic in the future 25

26 DRUG INDUCED HAEMOLYSIS Cefotetan, Ceftriaxone, Piperacillin RBCs Surface for loose attachment of drug Structural damage Antigenic stimulus Careful history of drugs Remission with discontinuation of drug 26

27 RBC Lysis Three Mechanisms of Action 27

28 CASE 7 Neonate / Day 2 FTND Presented with pallor and jaundice Respiration, Feeding, Cry normal, No hyper/hypothermia Hb 12 gm/dl TCL /L DC WNL Platelets 170 x 10 9 /L S Bil153.9 mmol/l D13.7 mmol/l ID140.2 mmol/l SGPT30 CRP6 mcg Reticulocyte count 8%, DCT Negative 28

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30 HAEMOLYTIC DISEASE OF NEWBORN Uncompensated haemolysis Antigen from father Commonest: Rh and ABO Anaemia, Hyperbilirubinemia, Erythroblastosis 30

31 Male child / 11 months CASE 8 Healthy with normal growth and milestones Examined by Paediatrician in the well baby clinic; noticed axillary and inguinal lymphadenopathy splenomegaly 5 cm below the costal margin Mild icterus. Haemoglobin and Platelets normal Lymphocytosis. Absolute lymphocyte count 16000/cmm 31

32 CASE 8 CONTINUED There was a mild indirect hyperbilirubinemia with normal liver function tests The lymphocytosis was persistent on subsequent visits On the flowcytometric analysis the lymphocytes were double negative (CD4-, CD8-) The Paediatrician could elicit the history of similar findings in a paternal cousin 4 years ago 32

33 AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME Failure of apoptosis Persistence of lymphoid mass and accumulation of autoreactive cells Chronic non malignant lymphadenopathy Hepatosplenomegaly Recurring multilineage cytopenias AIHA, ITP, Autoimmune neutropenia 33

34 Required Criteria Chronic (> 6 months), nonmalignant, noninfectious lymphadenopathy and/or splenomegaly Elevated CD3+ TCRαβ+ CD4 CD8 DNT cells (> 1.5% of total lymphocytes or > 2.5% of CD3+ lymphocytes) in the setting of normal or elevated lymphocyte counts Additional Criteria Primary Defective lymphocyte apoptosis in two separate assays Pathogenic mutations in FAS, FASLG, CASP10 34

35 Secondary Elevated plasma sfasl levels (> 200 pg/ml), plasma IL-10 levels (> 20 pg/ml), serum or plasma vitamin B12 levels (> 1500 ng/l) or plasma IL-18 levels > 500 pg/ml Typical immunohistologic findings as reviewed by a hematopathologis Autoimmune cytopenias (hemolytic anemia, thrombocytopenia, or neutropenia) with elevated IgG levels (polyclonal hypergammaglobulinemia) Family history of a nonmalignant/noninfectious lymphoproliferation with or without autoimmunity 35

36 ALPS Definitive diagnosis: Both required criteria + one primary criterion Probable diagnosis: Both required criteria + one secondary accessory criterion 36

37 CASE 9 M / 32 Healthy and Normal till three months back when presented with fever, body ache and petechial rash over body On investigations: Hb and TC, DC normal Platelet count 20,000 / cmm No organomegaly Bone marrow studies confirmed peripheral platelet destruction Immune thrombocytopenia He responded to steroids initially, with waxing and waning of the counts subsequently 37

38 Immune thrombocytopenia (ITP) Clinical diagnosis in routine practice Platelet autoantibodies + suppressed platelet production Primary or secondary (secondary to other disorders including SLE) History of the patient Baseline investigations including complete blood counts and Peripheral smear Giant platelets in the peripheral smear 38

39 Immune thrombocytopenia (ITP) 39

40 Immune thrombocytopenia (ITP) Bone marrow to document adequate megakaryocytes is necessary especially when the patient opts for the steroid treatment The bone marrow study may be avoided when malignancy is ruled out clinically and by laboratory investigations; and a trial of IvIgG is given first before sampling the bone marrow 40

41 Immune thrombocytopenia (ITP) The anti platelet antibodies can be documented by the direct and indirect methods Not mandatory for the diagnosis of ITP and are not routinely recommended Many drugs are documented to cause the Drug-. induced thrombocytopenia Commonly enlisted drugs are quinine and sulphonamides Demonstration of the drug dependant antibodies is carried out in speciality laboratories 41

42 CASE 10 F / 29 Presented with fever, sudden onset weakness, dyspnea on exertion and jaundice Episodes of epistaxis since one week No history of drugs / passing of dark coloured urine / any history suggestive of congenital bleeding disorder On examination: Pale, icteric and had tachycardia Hb 4gm/dl Total WBC count 20,000/cmm with neutrophil leucocytosis. Platelet count 42,000/cmm Indirect hyperbilirubinemia, raised LDH 42

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44 CASE 10 Bone marrow: Hypercellular with erythroid hyperplasia and mild increase in megakaryocytes; consistent with peripheral platelets destruction Patient responded to supportive management and steroids 44

45 EVAN S SYNDROME Rare Simultaneous / subsequent - Autoimmune haemolytic anaemia and thrombocytopenia Non cross reacting antibodies Underlying systemic disorder common 45

46 CASE 11 F / 24 Presented during the second trimester of her first pregnancy with pain and swelling of the left leg CBC and PBS normal Routine biochemical tests normal PT: Test 12 S Control 10.8 S, APTT Test 58 S Control 28 S APTT mixing studies: 1/2 Test + 1/2 Control 54 S DRVVTRatio: 2.3 (LA Positive) 46

47 LUPUS ANTICOAGULANTS Antiphospholipid antibodies In vitro: prolongation of the phospholipid based coagulation tests; mainly APTT. In vivo: thrombosis especially when associated with an additional prothrombotic event. Occur predominantly as part of underlying autoimmune disorders like SLE. Arise due to immunological imbalance are also common during pregnancy. 47

48 LUPUS ANTICOAGULANTS 48

49 LUPUS ANTICOAGULANTS Diagnosis: Laboratory based Patients may present with thrombosis or may be diagnosed accidentally when the test of APTT is found during routine or pre-surgical work-up The deranged APTT value does not correct upon mixing with normal plasma (Not much change in APTT value on equal mixing of the normal control plasma) 49

50 LUPUS ANTICOAGULANTS DRVVT (Dilute Russell Viper Venom Time) is the confirmatory test DRVVT test has two reagents: Screen and Confirm Screen: Reduced amount of phospholipids; leading to prolongation of APTT Confirm: Higher amounts of phospholipids, which neutralise the anti phospholipid antibodies Laboratory should establish own normal range 50

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54 HEPARIN INDUCED THROMBOCYTOPENIA Occurs in 0.5 5% of patients receiving heparin Caused by antibodies against heparin/platelet factor 4 (PF4) Clinical diagnosis: patient develops thrombocytopenia after 5 10 days of exposure to heparin Severe thrombocytopenia is rare Clinical scoring systems: to assess the probability 54

55 HEPARIN INDUCED THROMBOCYTOPENIA 4T scoring system: robust negative predictive value (thrombocytopenia, timing, thrombosis, other causes of thrombocytopenia not evident) Immunoassays: detect the antibodies to heparin- PF4 Functional assays: Serotonin Release Assay is the gold standard: very specific but need technical expertise 55

56 4T SCORE 56

57 NEONATAL ALLOIMMUNE THROMBOCYTOPENIA (NAIT) Caused by maternal allo-antibodies directed against foetal platelet antigens Severely low platelet counts common: bleeding complications 1 in 2000 pregnancies Foetal platelet antigens inherited from the father are targeted and the antibodies (IgG) cross the placenta Commonest antibodies are against 57 the HPA-1a antigen

58 NEONATAL ALLOIMMUNE THROMBOCYTOPENIA (NAIT) H/o previously affected pregnancies: important clinical clue Incompatibility between maternal and paternal (as a representation of possible foetal platelet type) platelets can be documented in the laboratory through platelet genotyping, phenotyping or cross matching 58

59 QUIZ Q1 What are the indicators of established haemolysis? A. Indirect hyperbilirubinemia B. Raised Serum LDH C. Reticulocytosis D. Microspherocytes E. All of the above 59

60 QUIZ Q2 IgM antibodies are difficult to detect by IAT because these: A. Are removed in washing B. Are rare C. Do not cause haemolysis D. Do not react at 37 0 C 60

61 QUIZ Q3 Which of the following is not true? The lupus anticoagulants are: A. Antiphospholipid antibodies B. Are detected in the laboratory generally by APTT based assays C. Can present with thrombotic complications D. Never occur transiently 61

62 QUIZ Q4 Which of the following is not true? The Donath-Landsteiner antibody is A. Biphasic in nature B. Can occur in young children after immunisation C. Causes intravascular haemolysis D. Can be detected in the lab by a simple test 62

63 QUIZ Q5 True or False? B cell lymphoproliferative disorders are associated with autoimmune haemolysis True Haemolysis in cold agglutinin disease is complement mediated True 4T test for HIT has strong positive prediction value Neonatal alloimmune thrombocytopenia is always mild False Drug induced haemolysis stops with discontinuation of the drug False True 63

64 TAKE HOME MESSAGE The diagnosis of autoimmune haematological disorders requires a close interaction between the clinical and laboratory staff Patient s history, clinical findings and results of the baseline laboratory investigations need to be viewed in a comprehensive manner to make an early diagnosis The laboratory person: keen eye and necessary skills to identify the subtle changes in the red cells Better patient care, timely management and interventions 64

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66 Thank you! 66