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Transfusion Bulletin

High Titer Immunizing anti-A1 in an A2 B patient
Resulting in Hemolytic Transfusion Reaction

Author(s): Dr. Rajendra Chaudhary, MD, DNB; Dr. Atul Sonkar, MD

Vol. 12, No. 2 (2004-08 - 2004-08)

Introduction :

The serologic distinction between A 1 and A2 antigens is based on the reactivity of the red cells with human anti-A 1 serum or with lectin prepared from the seeds of dolicus biflorus. For routine transfusion purposes, it is not necessary to distinguish patients or donors as A1 or A2 except when working with an A 2 or A 2 B individual whose serum contains anti-A 1 antibody. Anti-A 1 antibody appears as an atypical cold agglutinin in the sera of A 2 or A2 B individuals who lack the corresponding antigen. The frequency of occurrence varies with the subgroup. In A 2 it is about 2-8%and in A2 B about 22 to 35%. Most examples ofanti-A1 found in A 2 or A2 B subjects agglutinateA 1 cells only up to a temperature of 25° C and are of no clinical significance. However, they can interfere in routine blood grouping and give an incorrect blood typing results for ABO type. Discrepancies of this sort must be resolved when a transfusion is required. Occasionally, when anti-A1 has been active at 37 °C, extensive destruction of A 1 cells in vivo has been recorded1. We report here a case of hemolytic transfusion reaction in A 2 B patient after transfusion of A1 B red cells.

Case report :

A 31 years old male patient with diagnosis of Non-Hodgkin's Lymphoma was admitted in the division of Hematology at this tertiary care teaching hospital. The patient was being treated for NHL at peripheral hospital where he was transfused with 2 units of AB Rh positive blood 2 months before for correction of anemia. The transfusion was uneventful. Subsequently, the patient was transfused with 1 unit of AB positive red cells about 10 days before the patient was admitted to our hospital. There was history of hemolytic transfusion reaction to the last unit transfused and peripheral blood bank could not find cross match compatible blood for this patient.

The patient was then transferred to our tertiary care hospital for further management. At admission, the patient's Hb was 6.8 gm/dl, Platelet count 1,10,000/ul, TLC of 2400/ul. A requisition along with blood sample was sent to the blood center at our hospital for 2 units of red cells for correction of anemia.

Materials, Methods and Results

ABO & Rh grouping on patient sample: The results are as under:

Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-AB Anti-D A cell B cell O cell
4+ 4+ 4+ 4+ 2+ Neg Neg

ABO discrepancy was observed. The forward grouping was AB while reverse grouping was B. In order to resolve this discrepancy following investigations were performed as per our SOP.

Sub grouping with anti-A 1 lectin (Dolicus Biflorus: The results are as under:

Anti-A Anti-B Anit-AB Anti-A1 lectin
4+ 4+ 4+ negative

Thus it was confirmed that patient was A2B group with suspected anti-A1 in the serum causing agglutination of pooled A 1 red cells.

Confirmation of anti-A1 antibody in the serum:
Three "O", three "A1 " and three "A2 " washed red cells were selected for testing the serum of the patient at different temperature. All tests were performed in test tubes following our SOP. The results are as under:

  4°C 24°C 37°C
"A1" red cells 4+ 4+ 3+
"A2red cells neg neg neg
"O" red cells neg neg neg

Thus it was confirmed that patient had anti-A1 in the serum with wide thermal amplitude from 4 ° C to 37 °o C.

Indirect AGT

In order to rule out any alloantibody reacting with pooled A cells, indirect comb’s test was performed on LISS/coombs card (Diamed) with commercial three cell screening panel (M/S Diamed). It was negative.

Further confirmation of anti-A1 antibody:

Conformation of anti-A1 was performed by "saliva inhibition test" using saliva from a known"A" secretor individual. Briefly, one volume of serum of the patient was added to one volume of known "A" secretor saliva and the tube was incubated at room temperature for one hour. Saline was used as negative control. Finally, one volume of 5% suspension of pooled A1 cells was added to the tube and agglutination was recorded. Absence of agglutination in the test tube with saliva and presence of agglutination in the saline control test tube confirmed the presence of anti-A1 antibody in the serum of the patient.

Characterization of anti-A1 antibody :

  1. Titer of antibody: It was 1:128 at room temperature as well as 37° C
  2. Immunoglobulin (Ig) class of antibody:

In order to determine the class of antibody whether it is IgM or IgG or combined, serum of the patient was treated with thiol reagent (DTT, Dithiothreitol), which cleaves disulfide bonds of IgM antibody leaving behind IgG intact. The procedure was as per SOP. The serial two fold titration was performed in parallel on DTT treated (IgG) serum and untreated (IgM) serum using pooled A1 red cells. DTT treated serum was subjected to AHG test using monospecific IgG coomb's sera. Titers were recorded in both and the results are as under:

  1. Titer on DTT treated serum and with Anti human globulin test 1:16
  2. Titer on untreated serum 1:128

Transfusion Management of the Patient :

All A1 B red cell units were found to be incompatible at 37 ° C and room temp as well. Patient's serum sample was cross-matched with 2 units of A2 B red blood cells and both were found to be compatible. 2 compatible A2 B red blood cells were transfused to the patient. The transfusion was uneventful and the patient subsequently underwent chemotherapy for NHL. Further transfusions were only of A2 B group.

Our case is a rare example of anti-A1 antibody in an A2 B patient causing hemolytic transfusion reaction. The anti-A1 antibody, which is generally a cold agglutinin, had thermal amplitude at 37 ° C and therefore it was clinically significant and resulted in transfusion reaction. Anti-A1 antibody is usually of IgM in nature. In our case it was a mixture of IgG + IgM. We believe that IgG or immunizing anti-A1 might have developed because of previous transfusions with A1 B blood. In India, very few blood centers perform A or AB sub grouping.

Room temperature reacting anti-A1 antibodies are generally test tube nuisances, with little or no in vivo significance. However, there are instances when anti-A1 can cause substantial in vivo hemolysis in a group of A 1 patients. The clinical potential for hemolysis appears relative to the thermal amplitude of the antibody's reactivity. Anti-A1 which is active in vitro at about 28-30 ° C, will bring about the destruction of a proportion of A1 cells in vivo when a small dose of cells is injected. Those antibodies, which are only dubiously active at 37 ° C, would almost certainly fail to produce detectable red cell destruction following the transfusion of therapeutic quantities of blood. On the other hand, in several instances in which anti-A1 has been active at 37 ° C, excessive destruction of A1 cells in vivo has been recorded2. Thus determining the thermal amplitude of the antibody is desirable before assuming the anti-A1 is a nuisance cold reacting, clinically insignificant antibody. Testing at 30° C, and 37° C, and noting both agglutination and hemolysis is sufficient in most instances.

Boorman et al1 reported a case in which a patient of subgroup A2 was transfused with at least 7 units of A1 blood within a period of 4 days. Seven days after the last transfusion, the patient became icteric and anemic and was found to have anti-A1 in the serum active at 37 ° C. Several other examples of the development of anti-A 1 active at 37 °C following transfusions have been described. In a A 2 patient transfused with 5 units of A1 blood followed by 1 unit of group "O" blood containing immune anti-A1 , all the A1 red cells were eliminated within 2 days of transfusion and anti-A1 could be demonstrated in the recipients plasma3. In another case4 in which an A2 patient was transfused with 3 units of group A1 red cells, jaundice and anuria developed following transfusion. In another case report 5 , a patient with acute leukemia who typed as A2 B received 10 packs of platelets of group"O". Subsequent transfusion of A1 B blood resulted in a hemolytic transfusion reaction. Anti-A1 was demonstrated in the serum of the patient. This anti-A1 reacted with the transfused A1 B red cells and other A1 cells but not with the patient’s pre- transfusion red cells. The plasma of the transfused platelets had a high titer immune anti-A1. In yet another report6, an irregular anti-A1 reactive at 37°C was reported. Antibody appeared in an A2 B recipient only two days after massive transfusion of A1 red cells. Transfusion was associated with severe hemolysis and renal failure, which was reversed after exchange transfusion.

Considering the lesser survival of A1 red cells transfused to A2B or A2 persons whose sera contain anti-A1, we propose to distinguish A1 and A2 subgroups in individuals with A and AB blood groups prior to blood transfusion, especially in those with previous history of transfusion reactions following iso group blood transfusion.

References :

  1. Boorman KE, Dodd BE, Loutit JF et al. Some results of transfusion of blood to recipients with "cold agglutinin". Br Med J 1946; 751
  2. Mollison PL. Blood transfusions in clinical medicine. 7th ed. Oxford; Blackwell Scientific Publications; 1997: 127-8
  3. Ervin DM & Young LE. Dangerous universal donors. Observations on destruction of recipient's A cells after transfusion of group O blood containing high titer of A antibodies of immune type not easily neutralized by soluble A substance. Blood 1950; 5: 553
  4. Grove-Rasmussesn M, Soutter L, Marceau E. The use of group O donors as universal donors. Communication, AABB 1951, Minneapolis
  5. Zoes C, Dube VE, Miller HJ, Vye MV. Anti-A1 in the plasma of platelet concentrates causing hemolytic reaction. Transfusion 1977; 17: 29-32
  6. Northoff H, Wolpl A, Sugg U, Junginger W, Meinke J, Welter J, Schneider W. An unusual sample of irregular anti-A1, probably causing an early delayed transfusion reaction. Blut 1986; 52: 317-321

*Additional Professor
** Senior Resident
Department of Transfusion Medicine SGPGIMS
Lucknow 226014

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