Dr.Rajeshwari Basavanna, Dr.Biju George, Dr.Dolly Daniel Transfusion Medicine and Immunohaematology Department of Haematology CMC Vellore

Dr.Rajeshwari Basavanna, Dr.Biju George, Dr.Dolly Daniel Transfusion Medicine and Immunohaematology Department of Haematology CMC Vellore

Background Clinical Severity of disease Autoimmune Haemolytic anaemia - decreased red cell survival / accelerated destruction, secondary to autoantibodies. Wide spectrum - Mild to fulminant life threatening anaemia. Immunoglobulin class Subclass Titer Complement activation Thermal amplitude and Functional ability of the RE system

Objectives To serologically characterize the type of autoantibodies resulting in AIHA and correlate with in vivo haemolysis To study the correlation between the strength of Direct Antiglobulin Test (DAT) and the severity of in vivo haemolysis.

Study was done over a period of 13 months. (May 2014 June 2015) Inclusion criteria Patients, with positive poly specific DAT and also noted to have haemolysis based on clinical and laboratory criteria Exclusion criteria Patients had haemopoietic stem cell transplant Neonates with HDFN

Classification of severity of haemolysis Laboratory parameters 1) Haemoglobin <9gm/dl 2) Bilirubin >2mg/dl 3) Reticulocyte >2% 4) LDH >500IU/ml Severe haemolysis - if all the above parameters were fulfilled, Moderate haemolysis - 2 / 3 of the above laboratory parameters mentioned above are abnormal 1Das SS, Nityanand S, Chaudhary R. Clinical and serological characterization of autoimmune hemolytic anemia in a tertiary care hospital in North India. Ann Hematol. 2009 Aug;88(8):727 32 2.Wheeler CA, Calhoun L, Blackall DP. Warm reactive autoantibodies: clinical and serologic correlations. Am J Clin Pathol. 2004 Nov;122(5):680 5.

Algorithm to demonstrate the methodology of the study DAT positive patients with clinical and laboratory evidence of haemolysis were enrolled for the study Based on laboratory parameters, haemolysis was categorized into severe or moderate Monospecific DAT done to identify the presence of IgM,IgA,C3d and C3c by CAT If IgG was positive, IgG subtyping was done using anti IgG1 and IgG3 by CAT

STATISTICAL ANALYSIS Median, mean, Standard deviation, and range was calculated for all continuous variables. Pearson and Fisher s exact chi square was used for comparison of categorical data. Mann-Whitney test is used to calculate p value of LDH, bilirubin and reticulocytes. Binary logistic regression analysis was used for comparing the categorical data and to calculate the odds ratio with 95% confidence interval (CI). P value <0.05 was considered significant.

Percentage Percentage Results Age distribution of patients in Primary and Secondary AIHA Gender distribution in Primary and Secondary AIHA 70 68.4 65.9 80 74.4 60 50 40 30 31.6 52.9 47.1 34.1 70 60 50 40 30 41.2 58.8 25.6 20 20 10 10 0 0 <20 years 21-40 years >40years Primary AIHA Secondary AIHA Primary Secondary Male Female

Percentage Patients with Severe and Moderate haemolysis. 60 52(55.3%) 50 42(44.7%) 40 30 20 10 0 Severe Moderate

Type of AIHA Causes of secondary AIHA Out of 94 AIHA patients, Numbers Percentage Primary AIHA- 51 (54.3%) patients Secondary AIHA - 43(45.7%) patients Autoimmune disease 28 65.1 Lymphoproliferative disorder 6 14.0 Infection 2 4.7 Other causes 7 16.3 Total 43 100

Correlation of severity of haemolysis with 1. Type of AIHA 2. Antibody profile identified on monospecific DAT 3. IgG subtypes 4. Strength of DAT

Severity of haemolysis and type AIHA 52 patients with severe haemolysis Primary AIHA - 37(71.2% ) Secondary AIHA -15(28.8% ) The association of primary AIHA with severe haemolysis was statistically significant with p< 0.001 (OR = 4.76, 95% CI 1.97-11.48 )

Distribution of autoantibodies Immunoglobulin type IgG Only Number(%) 27,(28.7%) IgG + C3d 29(30.9%) C3d alone 8.5%(8) IgG with combination 62.8%(59) IgG alone 28.7%(27) IgG + IgM + C3d 15 (16.0%) IgG + IgM + C3c + C3d 8( 8.5%) IgG + IgM 1(1.1%) IgG + IgA + C3d IgG + IgA + IgM+C3d IgG + IgA 2 (2.1%) 2 (2.1%) 1, (1.1%) IgG + IgA + IgM 1(1.1%) C3d only 8 (8.5%)

Distribution of solitary IgG vs IgG in combination with other autoantibodies IgG in combination Solitary IgG Odds ratio 95% CI P value N= 59 N=27 Severe haemolysis 41(89.1%) 5(10.9%) 10.022 Moderate haemolysis 18 (45.0%) 22(55.0%) 1.00 3.276 30.656 <0.001

Percentage Distribution of IgG subtypes in patient s population 60 58.1% 50 40 30 30.2% 20 11.6% 10 0 IgG1 Only IgG1 and / IgG3 Neither IgG1 nor IgG3

Correlation of Ig G subtypes with severity of haemolysis IgG Subtypes Moderate haemolysis Severe haemolysis Total number Odds ratio 95% CI p Value IgG1 19(47.5%) 31(67.4%) 50(58.1%) 3.671 IgG1with IgG3 Absence of IgG1 and IgG3 3(7.5%) 7(15.2%) 10(11.6%) 5.250 18(45.0%) 8(17.4%) 26(30.2%) 1.34-10.08 0.012 1.07-25.70 0.041 Total 40(100%) 46(100%) 86(100%)

IgG Positive 86 Solitarty IgG 27 (31.3%) IgG in combination with other autoantibodies 59 (68.6%) IgG1 alone 9 IgG1 with IgG3 1 Neither IgG1 nor IgG3 17 IgG1 with other auto antibodies 41 IgG1with IgG3 and other auto antibodies 9 Neither IgG1 nor IgG3 with other auto antibodies 9 Distribution of IgG and their subtypes

Impact of IgG subtypes in patient s with solitary IgG Moderate haemolysis Severe haemolysis Total p value IgG1 alone 4 (44.4%) 5(55.6%) 9 IgG1 with IgG3 1(100%) 0 1 Absence of IgG1 and IgG3 17(100%) 0 17 < 0.001 Total 22(81.5%) 5(18.5%) 27

Clinical impact of IgG subtypes in patients with combination of autoantibody. Moderate haemolysis Severe haemolysis Total p value IgG1 15(36.6%) 26(63.4%) 41(100%) IgG1 with IgG3 2(22.2%) 7(77.8%) 9(100%) 0.203 Absence of IgG1 and IgG3 1(11.1%) 8(88.9%) 9(100%)

Impact of complement fixation and IgG subtypes on severity of haemolysis Moderate haemolysis Severe haemolysis Total p value Patients with IgG1 and/or IgG3 with complement fixation 16(33.3%) 32(66.6%) 48(100%) 0.413 Patients with neither IgG1 nor IgG3 with complement fixation 1(12.5%) 7(87.5%) 8(100%)

Impact of complement fixation in patients with IgG1 and/or IgG3 subtype Moderate haemolysis Severe haemolysis Total p value Patients with IgG1 and /or IgG3 without complement fixation 5(50%) 5(50%) 10 IgG1 and /or IgG3 with complement fixation 16(32.6%) 33(67.3%) 49 0.306

Clinical impact of complement fixation in patients with absence of IgG1 nor IgG3 Moderate haemolysis Severe haemolysis Total p value Patients with neither IgG1 nor IgG3 Patients who fixed complement with neither IgG1 nor IgG3 17(100%) 0 17 1(12.5%) 7(87.5%) 8 <0.001

Percentage Strength of Polyspecific DAT with severity of haemolysis 90 80 80.8 70 60 52.4 50 40 30 20 10 0 23.8 23.8 15.4 3.8 4+ 3+ 2+ Strength of polyspecific DAT Severe Moderate

Discussion The factors, which had the greatest impact on severity of haemolysis are Diagnosis of primary AIHA Presence of antibodies with complement fixation Presence of IgG1 and IgG3 subtypes Strength of DAT.

CONCLUSION Recommended algorithm Presence of IgG1 and IgG3 subtypes Polyspecific DAT Strength of DAT Severe haemolysi s complement fixation Diagnosis of primary AIHA Monospecific DAT In presence of IgG, and in the absence of complement fixation IgG subtyping is recommended

Variability is the law of life, and as no two faces are the same, so no two bodies are alike, and no two individuals react alike and behave alike under the abnormal conditions which we know as disease. William Osler

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