Aiha - Dato' Dr Chang [compatibility Mode]

Dr K M Chang MRCP(UK) FRCPA(Aust) Dr Wooi Seong Masters Transfusion Med trainee Hospital Ampang




Definition : autoantibodies ◦ Antibodies vs individual’s own red cells – agglutinate, sensitize or lyse rbcs ◦ Shortening of rbc lifespan

Hemolytic anemia

autoimmune

Warm

Cold

idiopathic

secondary

alloimmune

PCH

Drug-induced







Site and severity of rbc destruction depends on structural and functional characteristics of antibody and efficiency of mechanism of destruction Degree of anemia depends on rate and acuteness of destruction and BM capacity to compensate




3 types ◦ Warm ◦ Cold ◦ mixed








Commonest type (70%) Most active in vitro at 370C Polyclonal IgG usually If specificity determined, usually Rh






IgG1 most commonly found Others in combination IgG3 > IgG1> IgG2>IgG4 in terms of destructive effects





Idiopathic Secondary ◦ ◦ ◦ ◦ ◦ ◦

Autoimmune disease Lymphoproliferative disease Infections Ovarian cysts Ca Drugs











Insidious onset Pallor or icterus Splenomegaly moderate Marked ↓ Hb (acute hemolysis) Mild hyperbilirubinemia (unconjugated) ↑ LDH Positive DAT FBP : sperocytosis and polychromasia, NRBCs, occasionally rbc agglutination





30% Exclude secondary causes
AIHA may precede SLE





Peak incidence in infancy+ early childhood and 3rd decade Female preponderance











Idiiopathic AIHA + immune thrombocytopenia Plt and rbc antibodies – do NOT cross react High incidence of underlying illness (SLE in children and lymphoma in adults) Usu resistant to prednisolone as initial therapy






Frequent association with SLE (young women) Autoantibodies - IgG (IgG and C3d) Also in RA, Sjogren’s syndrome, ulcerative colitis, agammaglobulinemia









Commonest association : B-cell CLL, low grade B-cell non-Hodgkin’s lymphoma, Hodgkin’s disease A/B polyclonal , no specificity Due to immune dysregulation AIHA may precede diagnosis of lymphoma




3 patterns : 1. IgG (20 – 60%) 2. IgG + C3 (24 – 63%) 3. C3 (7 – 14%)




IgG reacts by indirect antiglobulin test against all cells tested

* Need to exclude presence of alloantibodies *1/3 patients with WAIHA have cold-reactive autoagglutinins







Confirms the presence of IgG autoantibody on red cells Reacts with all cells (unless it is complement only)









Apparent simple Rh antigens (D, C, E, c, e) Relative specificity It is rarely necessary to ascertain autoantibody specificity in order to select antigen negative blood for transfusion Most important is to exclude alloantibodies





2nd most common (16 – 32% of AIHA) Types ◦ Acute - in lymphoproliferative dz, mycoplasma

pneumoniae ◦ Chronic – in elderly, sometimes lymphoma, CLL, Waldenstrom’s macroglobulinemia




IgM 40C Agglutinins or lysins (different thermal ranges)




Characterized by clumping of red cells in EDTA at room temp

Clinically : acrocyanosis & hemoglobinuria in cold weather







C’ only




Autoagglutinin is IgM – binds to rbcs at low temp of peripheral circulation causes complement to attach to red cells




Cells circulate to warmer areas, IgM disassociates, complement still attached to rbcs




C3b react with CR1 and CR3 receptors of macrophages in RES




Ig M usu titer> 1000 at 4 C




Rarely react with rbcs > 320C (unless albumin is added) Specimen must be collected and maintained at 37 C until serum and cells separated







Alternatively warm EDTA specimen 15 mins at 370C with repeated mixing then separation from cells.




If collected and washed properly at 370C, no immunoglobulin
no reactivity













Usually I specificity, if i – infectious mononucleosis, rarely Pr Auto-I sometimes seen in normal individuals but titers < 64 at 4 C *to detect clinically significant alloantibodies, avoid use of albumin, use IgG specific reagent rather than polyspecific AHG If still continues to interfere with A/B detection, do cold autoadsorption studies

WAIHA CAS

Mixed

PCH

%

48% - 70%

16 – 32%

7-8%

Adults:rare Children 32%

DAT

IgG

C3 only

IgG+C3 (71-100%) C3 only (13%)

C3 only

IgG+C3

C3

Ig

IgG (IgA, IgM rare)

IgM

IgG+ IgM

IgG

Eluate

IgG

IgM agglutinatin g a/b at 4C

IgG IAT reactive a/b + IgM agglutinating a/b react at 30-37C

IgG biphasic hemolysin (Donath Landsteiner a/b)

I>i>Pr

Panreactive or I /i

P

Specificity Panreactiv e Rh

AABB

1.







Drug adsorption Postive anti-IgG (extravascular hemolysis) Penicillin Ampicillin Methicillin Carbenicillin Cephalothin Cephaloridine

2. Immune complex Positive anti- C3 (intravascular hemolysis) - Quinidine - Phanacetin - Hydrochlorothiazide - Rifampin - Sulfonamides - Isoniazid - Quinine - Insulin - Tetracycline - Melphalan - Acetaminophen - Hydralazine - Probenecid - Chlorpromazine - Streptomycin - Fluorouracil - Sulindac

3. Autoantibody Positive anti-IgG (extravascular hemolysis)
alpha methyldopa
Mefenamic acid
L-dopa
Procainamide
Ibubrofen
Diclofenac
Alfa interferon








Anemia Signs of increased RBC destruction Pos DAT











Reticulocytosis ↑ LDH ↑ Unconjugated bilirubin ↓ Haptoglobin Hemoglobinuria Spherocytes Hemoglobinemia Hemosiderinuria












Direct Coombs Test Elution Indirect Coombs Test Autoadsorption Alloadsorption Thermal amplitude test Cold agglutinin titer Donath-Landsteiner test Detection of antibodies to drugs by testing drug-treated cells











First described by Coombs in 1945 Positive DAT is the sine qua non for diagnosis of AIHA Performed by incubating patient’s washed rbcs with polyspecific antibody (AHG) that recognizes human IgG and C3 Agglutination results if there are as few as 200 – 500 IgG / cell




To differentiate immune-mediated hemolysis from other causes







1-2% positive DAT without signs or symptoms of hemolysis 5-10% negative DAT with immune mediated hemolysis




Low number of IgG molecules/ rbc – below the level of detection of standard DAT Low affinity antibodies IgM, Ig A ( when using monoclonal AHG)




Polybrene test / flow cytometry



















rbcs incubated with test sera in low ionic medium at RT (1 min). polybrene (quaternary ammonium polymer) is introduced to cause non-specific rbc aggregation tubes are centrifuged, and supernatant decanted polybrene effect on cells is neutralized by adding dilute sodium citrate-dextrose solution hemaglutination is evaluated wash rbcs, carry out antiglobulin test







Stronger agglutination, more likely associated with hemolysis Direct correlation between immune hemolysis and presence and amount of C3 or IgG on rbc membrane







To confirm positive DAT results is due to autoantibody Methods ◦ ◦ ◦ ◦ ◦

Cold acid elution Heat elution Freeze –thaw Digitonin acid Dichloromethane/methylene chloride










Eluate incubated with panel rbcs to check for specificity Pan-reactive antibody or specificity within Rh system No correlation between the specificity and hemolytic potential







Most AIHA – positive antibody screen, and pan-reactive antibody Residual autoantibody in patient’s serum







To determine concomitant presence of alloantibodies (32% of AIHA) Patient - history of pregnancy, transfusion






Bound antibody eluted from autologous rbcs Incubate with patient’s rbc at 37oC Remove residual circulating autoantibody to allow recognition of alloantibody

Autoadsorption










Insufficient rbc (severe anemia) Aliquots of patient’s serum adsorbed with allogeneic rbcs of known phenotypes After incubation, adsorbed serum samples are tested against panel cells Alloantibody can be identified based on the pattern of reactivity and knowledge of antigens to which each particular aliquot was exposed. Can rule out presence of alloantibodies if all adsorbed serum samples fail to react with reagent cells

No reaction with panel cells

Autoantibody + alloantibody (anti-D)

R0R0 cells

rr cells

Anti-D will react with panel cells with D antigen











To determine the titer of antibody at various temperatures Serial dilutions of serum are incubated at 4oC, 22oC, 30oC, 37oC with adult and umbilical cord rbcs. Adult : I antigen (mycoplasmal infections) Fetal cells : i antigens (infectious mononucleosis)




Ability of patient’s antibody to bind at physiologic temperatures (30oC, 37oC ) confirms its clinical significance









Transfusion may lead to autoantibody production May also stimulate alloantibody production May depress compensatory erythropoiesis If hemolysis
transfusion reaction, hypercoagulability and DIC







Usually chronic, stable Hb > 8 rarely require Tx Tx indicated if : ◦ Anemia progresses ◦ Symptomatic







Consideration : circulatory overload, and increased red cell destruction Blood to Tx ◦ ABO and Rh specific ◦ If alloantibodies – rbcs should lack the corresponding antigens







Transfusion if required is a lifesaving measure Should not be withheld solely because of serologic incompatibility Volume transfused should be the smallest amount Careful monitoring

1.

Does the patient have hemolysis? Markers of hemolysis

2. Is the hemolysis immune mediated? Coombs test

3.

Is it warm or cold AIHA? IgG or complement

4. What is the specificity of the antibody? Eluation & Indirect antiglobulin test

5.

Is an alloantibody present? Autoadsorption or alloadsorption

6.

What is the titer of the antibody? Thermal amplitude test













Reardon et al. Laboratory evaluation and transfusion support of patients with autoimmune hemolytic anemia. Am J Clin Pathol 2006; 125(Suppl 1):S71-S77 The role of Coombs test in evaluating hemolysis in adults. CMAJ January 31, 2006 : 174(3) Schick et al. Hemolytic anemia Jan 29 2007 http://www.emedicine.com/med/topic979.htm Technical Manual 15th Ed, AABB

anemia markers of hemolysis DAT

negative *DAT-negative AIHA

flow cytometry

positive IgG or complement eluate & IAT

autoadsorption / alloadsorption

Thermal amplitude test

Polybrene test/

warm or cold AIHA

specificity

alloantibody









Rarest of AIHA Due to IgG Associated with syphillis in the past Assc viral infections in young children Idiopathic chronic disease in elderly Characteristically ◦ Biphasic hemolysin (Donath Landsteiner hemolysins)







IgG complement fixating A/B In cold areas of body – IgG fixes complement, causes C3 to bind irreversibly to rbcs In warm areas, a/b disassociates So rbcs only coated with complement (unless washed in cold saline and tested with cold anti-IgG – can detect IgG)







Autoantibody : biphasic hemolysin (sensitization at low temp, but hemolysis at 370C) Donath Lansteiner test




Non reactive (because only complement coating rbcs)





P specificity Reacts with all cells by the Donath Lansteiner test







Rarely necessary in adults unless severe hemolysis More common in children (A/B high thermal amplitude – so hemolysis more brisk) P neg red cells rare :1 in 200, 000) P neg red cells indicated if no response to random donor blood

1. 2. 3. 4. 5. 6. 7.

Corticosteroids Cytotoxic immunosuppresives Cyclosporin IvIg Splenectomy Monoclonal a/b Blood transfusion







Avoid exposure to cold Heated gloves, socks Folic acid supplements Drugs 1. 2. 3. 4. 5. 6.

Chlorambucil Corticosteroids Splenectomy Bllod Tx Plasma exchange Monoclonal A/B

1. Chlorambucil 10 mg/d for 14 days – 4 weeks 2. Corticosteroids (should avoid unless a/b in low titers and high thermal range) 3. Splenectomy (not useful – removal of C3b coated cells in liver) 4. Bllod Tx- use blood warmer, in warm environment 5. Plasma exchange may lower titer of cold agglutinin temporarily in severe symptoms 6. Monoclonal A/B – –

Rituximab – in idiopathic and associated with Blymphoproliferative dz bcos CD20 present on mature B-cells Campath -1H





Keep patient warm (preferably 37 C) Blood tx as required, through blood warmer