DAT Negative AIHA Dr. Sudipta Sekhar Das TRANSMEDCON 2018 DAT Negative AIHA Dr. Sudipta Sekhar Das MD (Transfusion Medicine), SGPGIMS PDCC (Aphaeresis & Component Therapy), SGPGIMS Senior Consultant & Head, Transfusion Medicine Apollo Gleneagles Hospital, Kolkata Professor (Apollo Hospitals, India)

AIHA in General Characterized by increased RBC destruction and / or decreased red cell survival d/t autoantibodies directed against self RBC antigens Incidence 1 in 80,000 to 100,000 given population/year DAT remains the crucial serological assay in AIHA diagnosis A positive DAT almost always is associated with AIHA & forms hallmark of diagnosis However, positive DAT does not indicate overt disease & combination of clinical and laboratory evidences establish the diagnosis

Pathophysiology Trigger 80% extravascular hemolysis; Spherocytes Genetic (HLA-DQ6) Infections Inflam. Disorders Malignancies Drugs 80% extravascular hemolysis; Spherocytes Less frequently intravascular

Autoimmune Hemolytic anemia Diagnosis is made in the reverse way Anemia Hemolytic Immune Auto

Laboratory Investigations An approach to the probable diagnosis Hematology Hb: 5.4 g/dL Hct: 20% Retic: 8% Plt: 167x103/mm3 TLC: 5.5x103/mm3 MCV: 86fL MCH: 30pg MCHC: 34% RDW Blood smear NCNC Spherocytes + Biochemistry S.Bilirubin: 5.2mg/dL LDH: 1625U/L SGOT: 35U/L SGPT: 30U/L Alk. Phosphate: 350U/L

Probable diagnosis A patient with hemolytic anemia (HA) + Clinical features Weakness, lethargy Pallor Tachycardia Splenomegaly Markers of invivo hemolysis Hb: 5.4 g/dL Hct: 20% Retic: 8% S.Bilirubin: 5.2mg/dL LDH: 1625U/L + A patient with hemolytic anemia (HA)

How to diagnose AIHA Whether immune or non-immune etiology ? Probable diagnosis is hemolytic anemia Detailed blood bank work-up can only diagnose immune HA Detection of RBC antibody by DAT is the hallmark of diagnosis Whether immune or non-immune etiology ? Y Y RBC Y Y 2-4 % cases RBC Y Y Antibody Y Y DAT negative by tube technique DAT positive by tube technique – gold standard

Sensitive technique (CAT) is the demand of the day Various methods of DCT Methods of DAT Sensitivity (molecules / red cell) Conventional tube technique 300 – 500 CAT / Microplate / SPRCA 120 – 180 Enzyme linked anti globulin test 80 – 120 Flow cytometry 30 - 40 Gold standard Most sensitive Sensitive technique (CAT) is the demand of the day

Classification of AIHA Warm AIHA (48-70%) Primary (Idiopathic) Secondary (LPD, AI Dis, immune deficiency states) Cold AIHA Cold Agglutinin Syndrome (16-32%) Secondary (Infections, LPD) PCH (32% in children) Secondary (Syphilis, Viral inf.) Mixed AIHA (7-8%) Primary (Idiopathic) Secondary (Lymphoma, SLE) Drug induced AIHA (12-18%) DAT negative AIHA (2-4%)

DAT Negative AIHA Reasons for DAT negativity Incidence: 2 - 4% 0f all AIHA Reasons for DAT negativity IgG mol / RBC necessary for accelerated in-vivo destruction is lower than number necessary to yield a positive DAT by CTT (Gold standard) RBC coated with IgA autoantibodies or monomeric IgM. Low affinity Ab. causing hemolysis easily dissociate from RBC while washing: Strong IgG in vivo appear to have little / no IgG in vitro.

Techniques implemented Cold wash DAT: retain low affinity IgG CAT / SPRCA / Microplate < 200 IgG/RBC Techniques detecting < 100 IgG / RBC Enzyme linked antiglobulin test (ELAT) Complement-fixation antibody consumption test Immunoradiometric assay (IRMA) Mitogen stimulated – DAT MMA Flow cytometry (Most sensitive 35 IgG /RBC)

Flow cytometric analysis of AIHA patients (N=12) Age/ Sex Hb g% Retic % S.Bil mg% LDH IU/L CTT GT Flow cytomtery Poly IgG C3d MFI Result 22/F 6.8 8.5 2.8 2124 3+ 2+ 1+ 4+ 19.7 Pos 35/F 5.1 25 3.8 1817 21.63 27/F 7.5 16 4.9 1618 17.15 30/F 9.9 2.9 2.3 690 Neg 30.5 18/F 3.2 20 1374 25.7 17/F 7.1 3 937 15.02 31/F 6.5 2 2.4 834 12.9 54/F 9.2 2.5 3.3 695 W+ 17.1 15/F 5 4.8 1013 11.9 62/F 8.3 9 4.1 678 8.9 41/F 6.9 6 0.9 680 8.4 24/m 0.7 710 9.3 Hematology 2006

Flow cytometry in DAT negative AIHA Hematology 2006 Neg control MFI 4..8 Pos control MFI 31.8 DAT Neg AIHA MFI 8.6

Unpublished data, Das et al 2018 DAT positive by polyspecific Under peer review True AIHA suspects N = 353 No conclusion No follow up N = 9 DAT positive by CTT N = 326 DAT negative by CTT N = 27 18 DAT positive by polyspecific CAT N = 8 DAT positive by monospecific CAT N = 2 DAT positive by IgG subclass CAT N = 1 Positive cold wash DAT N = 2 DAT positivity confirmed by elution N = 1 DAT negative by available methods but suspected AIHA responded to therapy N = 4 Figure : Simple methodologies to diagnose DAT negative AIHA

Conclusion All conventional DAT negative patients with strong clinical suspicion of AIHA should be further evaluated Alternate sensitive methods which are otherwise less practiced may be implemented We observed that such techniques are less cumbersome and do not lead to financial constraints Blood bank may establish these useful simple techniques and stick to the defined protocols to diagnose DAT negative AIHA.