Unusual Response to ABO Incompatible Blood Transfusion D. H. BUCHHOLZA N D J. R. BOVE From rhe Deparrmeni of Laboraiory Medicine, Yale Universiry School of Medicine. New Haven. Connecticui
Three units of group A blood were inadvertently administered to a group 0 recipient during surgery without evidence of hemoglobinemia, hemoglobinuria, hypotension, disseminated intravascular coagulation, acute renal tubular necrosis, or other signs and symptoms of transfusion reaction. The recipient had normal concentrations of IgG, IgA, and I g M as well as complement (Q) prior to transfusion and anti-A agglutinins titered to 64 (titer of 128 by the antiglobulin technique). Seventeen hours following the transfusion, 28 per cent of the circulating red blood cells were group A (equivalent to 475 ml of packed cells); they were eliminated by day 5 without evidence of hemoglobinuria, hemoglobinemia or hyperbilirubmemia. Anti-A titers (antiglobulin) had risen from a posttransfusion low of 4 to 4,096 by day 10. After treatment of serum with 2-mercaptoethanol, however, hemolytic activity which was first noted on day 5 was lost and the antiglobulin titer dropped to 24 which suggested that most of the anti-A produced in response to the transfusion was I g M rather than IgC. The anti-A titer had dropped to essentially pretransfusion levels and the majority of anti-A present was IgM by day 91. The recipient suffered no untoward effects from the transfusion and was in good health three months following the transfusion.
following the surgery and the pulse, blood pressure, and urine output remained normal. The patient was noted to be perspiring and felt “anxious” after surgery but did not complain of symptoms usually associated with transfusion reaction. During the 14 hours following the transfusion, his temperature gradually rose from 37 to 38.3 C and then returned to normal the following day. The discovery that group A blood had been given was not made until 17 hours following the transfusion. A plasma specimen obtained at that time was clear and without visible evidence of hemolysis. Red blood cells obtained from the patient reacted weakly with anti-A and anti-A,B typing serum and the direct antiglobulin test was weakly positive with broad spectrum antihuman serum. Serum electrolytes and BUN were normal and urinalysis did not reveal cells, protein, o r hemoglobin. The patient was given three liters of intravenous fluids on each of the three days following the transfusion; no mannitol was administered. He was discharged ten days following surgery with no apparent sequelae resulting from the transfusion.
INADVERTENT TRANSFUSION of group A, B, or A B red blood cells to recipients with the corresponding alloagglutinins usually leads to a serious and occasionally fatal hemolytic transfusion reaction. This report describes the administration of 1,500 ml of group A whole blood to a healthy group 0 male who displayed no untoward effect from the transfusion.
Materials and Methods Anticoagulated (EDTA) and clotted specimens of the patient’s blood were obtained on days I , 2, 3,5,6, 10, and 9 I following the transfusion. Small amounts of pretransfusion EDTA and clotted blood samples were also available for study. All serum was separated from the red blood cells and stored at -20 C until tested on day 10 or 91. Donor red blood cell survival was estimated by quantitative hemagglutinatione using washed red blood cells that had been maintained in EDTA plasma at 4 C. Titration of agglutinating and antiglobulin reactive anti-A and anti-B was performed using pre- and posttransfusion serum. Quadruplicate serial 0.1 ml doubling dilutions of each of the respective serums were made in saline and 0.1 ml of fresh group AB nonsecretor serum was added to each tube as a source of complement. Each serum was titered against a 2 per cent suspension of A, or B cells in saline (60 minutes incubation at
Case Report A 21-year-old law student complaining of progressive back stiffness and right leg pain exacerbated by walking underwent surgery for L-5 spondylolisthesis. During the procedure the patient, who was group 0, inadvertently received three units of group A whole blood. Total blood lost during the procedure was estimated at 2,000 ml. No unusual bleeding was seen during or Received for publication January 12, 1975; accepted March 8, 1975. Transfusion Nov.-Dec. 1975
577
Volume 15 Number6
578
BUCHHOLZ AND BOVE
Transfusion Nov:Dcc. 1975
Survival of Group A Cells 40c
.c
-a 0
,c
30-
V
.-C
-ul z
V
FIG. 1. Posttransfusion survival of transfused group A cells as determined by quantitative hemagglutination.
20-
U a
$
10-
W c 0) C
? TX
Days Following Transfusion
ambient temperature) or by the high protein/antiglobulin (60 minutes incubation at 37 C) technique. Titers were expressed as the reciprocal of the highest doubling dilution of serum that produced a 2+ reaction (macroscopic clumps with many free cells in the background) with the cell being tested. Appropriate controls showed the absence of alloantibodies other than those of the ABH system. Similar titrations using pretransfusion, day 10, and day 91 samples were performed after serum inactivation with 2-mercaptoethanoP and following absorption with an excess of group B cells. Pre- and posttransfusion sera were also neutralized with equine-porcine group specific A and B substance and the titer of nonneutralized anti-A and B determined. Pretransfusion serum immunoglobulins G, A, M, and complement (C3) were quantitated by radial immunodiffusion. Urine was collected for 72 hours beginning at the time the incompatibility was recognized and was examined for cells, hemoglobin, and protein. Direct and total bilirubin were determined daily following transfusion and the BUN and creatinine were determined on days 1, 10, and 91.
Results Two of the three transfused units of blood were group A, while the third was group A*. The secretor status of the donors was not determined. Figure 1 shows the posttransfusion survival of the infused cells as determined by quantitative hemagglutination. The patient’s blood volume was estimated to be 4,475 ml based on his height and weight. The day following surgery his hematocrit was 38 vol/dl and 28 per cent of the circulating red blood cells were group A, corre-
sponding to a total of approximately 475 ml of group A cells. Although the hematocrit values of the three donor units could not be determined accurately, it was calculated that the total volume of red blood cells infused could have ranged from 495 to 765 ml assuming all donors had hematocrits of 37 or 50 vol/dl, respectively. If the average donor hematocrit were 43 vol/dl, approximately 600 ml of group A cells would have been infused. Although some transfused cells were undoubtedly lost during surgery, it is apparent that the bulk of transfused cells were still circulating the following day. The number of circulating group A cells fell progressively (Table 1) until none was detectable on day 5, coincident with the first appearance of an anti-A hemolysin (Figure 2). Prior to the infusion of the group A cells, antiA and anti-B titers were 128 and 64 respectively and no hemolysins were present. On the day following the transfusion, the anti-A had dropped to a titer of 4 although the anti-B remained at 64. There was no agglutination of A, or B cells by pretransfusion serum that had been neutralized with blood group specific A and B substance, which suggested that all antibody present in a titer greater than 4 was of “naturally occurring” type. Figures 2 and 3 illustrate the change in anti-A and B titers following the transfusion. Anti-A fell to low levels on day I but increased to titers of 2,048 (direct agglutination) and 4,096 (antiglobulin) by day 10, at which time a hemolysin with a titer of 32 was present. By day 9 I the hemolysin had fallen to a titer of 4 and the antiglobulin titer had returned to a level only slightly higher than the pretransfusion titer. Anti-B also rose following transfusion to a titer of 256 on day 10
Volume 15 Number6
579
ABO INCOMPATIBILITY
but then dropped to a titer of 64 (direct agglutination) and 128 (antiglobulin) by day 91. Pretransfusion serum lost all anti-A and anti-B reactivity greater than a titer of 12 (lowest titer tested) following 2-mercaptoethanol treatment, which suggested that most of the blood group specific antibody present was IgM (Table 2). The titer of agglutinating and antiglobulin reactive anti-A had increased greatly by day 10; however, treatment of serum with 2-mercaptoethanol resulted in loss o f hemolytic activity and a marked diminution of antiglobulin reactivity. A similar though less marked pattern was observed in serum obtained three months following the transfusion. This is in contrast to what was seen when five control serums from unselected nonimmunized group 0 persons were tested at the same time. In those serums, titers were higher by the antiglobulin technique than by direct agglutination. When retitered after treatment with 2mercaptoethanol, the antiglobulin titer remained virtually unchanged although, as expected, direct agglutinating reactivity was lost. IgM anti-A appeared to be the primary type of blood group
Table 1. Removal of Group A R e d Blood Cells Following Transfusion
Days Following Transfusion 1 2 3 4 5
Per Cent Group A Hematocrit Cells (Vol/dl) Circulating
Calculated Volume Group A Cells Circulating (ml packed RBC)
475 315
33
28 20 9
32
0
0
38 36
-
130
-
-
specific immunoglobulin produced in response to the immunizing stimulus. When day 9 1 serum was neutralized with blood group specific substance, the “immune” or nonneutralizable anti-A titered to only 5 and no “immune” anti-B could be detected. Although there appeared to be little crossreactive antibody present in pretransfusion and day 10 serum; there was sufficient crossAnti-A Production After Transfusion x Hemolysin 0 Saline Reactive A Antiglobulin Reactive
819240962048-
1024512256-
FIG.2. Anti-A titers before and after transfusion of group A whole blood.
128-
‘F
6432168-
4-
20I1
- i t i i i 4 6 s 6” “7 ”8 Tx
Days Following Transfusion
9
lO“91
580
Transfusion Nov.-Dec. 1975
BUCHHOLZ AND BOVE Anti-B Production After Transfusion
xHemolyein *Saline Reactive A Antiglobulin Reactive
512 2 561 128
&
641
FIG. 3. Anti-B titers before and after transfusion of group A whole blood.
32-
.-c 16-
b-
8-
4-
-I
t
II
I
2
3
4
5
6
7
8
9
lO"91
TX
Days Following Transfusion reactive antibody present to reduce the anti-A titer of day 91 serum by two doubling dilutions following absorption with group B cells (Table 3). In view of the unexpected absence of intravascular hemolysis in response to the infusion, patient cells and saliva were tested to rule out the possibility that the patient's blood group was a weak variant of A rather than 0. Incubation of the patient's red blood cells with a potent immune
anti-A,B serum demonstrated no reactivity by direct agglutination or the antiglobulin technique. Heat eluates of similarly treated patient cells did not react with group A cells, and while the patient secreted H substance in his saliva, no A substance could be found. No anti-H specificity was seen in either pre- or posttransfusion serum. Normal amounts of IgG, IgA, and IgM as well as complement (C3) were present and im-
Table 2. Titers o f Pre- and Posttransfusion Serum Before and After Inactivation with 2-Merca~toethanol Titer"
B Test
A , Test Cells Serum Pre-Tx Day 10 Day 91 Control Group 0
2-Mercaptoethanol Inactivation
Direct Agglutination
No Yes No Yes No Yes No Yes
96 0" * 1,536 0 96 12 24 0
Antiglobulin
48 0" 3,072 24 192 48 192 192
Direct Agglutination
48 0"" 96 0 48 0
48 0
'Serum diluted 1 : 6 initially followed by doubling serial dilutions. " " 1 :12 dilution (minimum dilution tested because of limited quantities of pretransfusion serum)
Cells Antiglobulin
48
0"" 48 12 96 48 192 192
Volume
I5
58 1
ABO INCOMPATIBILITY
Number 6
Table 3. Titers of Pie- and Posttransfusion Serum Before and After Absorption with Group B Cells Titer E Test Cells
A , Test Cells Serum Pre-Tx Day 10 Day 91 Control Group 0
Absorbed with Group B Cells
Direct Agglutination
No Yes No Yes No Yes No Yes
128 64 2.048 1,024 128 32 128 16
munoelectrophoresis of pretransfusion serum revealed a normal pattern. During the three days following the transfusion, no hemoglobin, protein or abnormal numbers of cells were noted in the patient’s urine. Specific inquiry of the nursing staff revealed the excretion of only normal colored urine during the 17 hours following the surgical procedure. During the six days following the transfusion, the patient did not become jaundiced and daily bilirubin determinations were consistently within the normal range. Determination of BUN and creatinine on days I , 10, and 91 revealed no detectable evidence of renal damage as a sequelae of the transfusion.
Discussion The infusion of group A, B or AB red blood cells to recipients with the corresponding alloantibodies is usually associated with rapid intravascular hemolysis of a large proportion of the transfused cells. Hemoglobinemia and hemoglobinuria are frequent findings and there may be marked hypotension or a severe bleeding diathesis in addition to other signs and symptoms such as chills, fever, urticaria, back pain, or nausea and vomiting. Acute renal tubular necrosis may also occur, probably from a combination of hemoglobin4 and the presence of red blood cell stroma in the circulation.I0 Prolonged survival of ABO incompatible cells has been described in recipients with hypoglobulinemia5*’ and also following the infusion of a large volume of group AB blood to a group A recipient., In the latter instance, it was suggested that red blood cell
Antiglobulin
64 64 4,096 2,048 256 64 256 64
Direct Agglutination
Antiglobulin
64 0 128 0 64 0 64 0
64 0 128 0 128 0 64 0
antigenic heterogeneity was such that a small proportion of transfused AB cells was not cleared from the circulation in spite of the presence of a “potent anti-B antibody.” Neither mechanism seems likely in the patient described in this report. Normal concentrations of immunoglobulins and complement (C3) were present in pretransfusion serum and anti-A agglutinins were present to a titer of 64. While circulation of a small number of group A, cells might be postulated with the selective destruction of group A, cells or the presence of a subpopulation of A cells which displayed antigenic heterogeneity, the presence of large numbers of circulating A cells 17 hours after infusion make these explanations unlikely. A possible explanation of the observed lack of hemolysis may be related to the immunologic type of blood group specific antibody present prior to the transfusion. Pretransfusion serum displayed no agglutination when tested with A, and B cells following neutralization with group specific AB substance, which suggests the presence of only “naturally occurring” agglutinins. This was further confirmed when pretransfusion serum lost both direct agglutinating and antiglobulin reactivity when treated with 2-mercaptoethanol. Although the antiglobulin anti-A titer had increased to 4,096 by day 10, most of the immunoglobulin produced in response to the transfusion stimulus was IgM as reflected by a marked
582
BUCHHOLZ AND BOVE
decrease in activity following 2-mercaptoethanol treatment. Although the presence of a hemolysin in a titer of 32 might suggest the presence of an IgG antibody, loss of hemolytic reactivity was also noted following 2-mercaptoethanol treatment of day 10 serum; this has been reported to be a characteristic finding for hemolytic IgM but not IgG.g The rapid return of anti-A titers to nearly pretransfusion levels by day 91 was somewhat unexpected, since it was anticipated that IgG anti-A would have been produced and maintained in response to the transfusion stimulus, especially since the recipient was group 0.' Although there was evidence of IgG presence on day 91 (titer of 48), this was less than might have been expected when compared with persons deliberately immunized with purified group A substance3 in whom titers remain elevated for a longer period of time.
2.
3.
4.
5.
6.
7.
8. 9. 10.
Transfusion No".-Dec. 1975
Akeroyd, J . H., and W. A. O'Brien. Survival of group AB red cells in a group A recipient. Vox Sang. 3:330, 1958. Allen, P. 2.. and E. A. Kabat: Persistence of circulating antibodies in human subjects immunized with dextran, levan, and blood group substances. J. Immunol. 80:495, 1958. Baker, S. B. de C., and R. L. F. Dawes: Experimental hemoglobinuric nephrosis. J. Pathol. Bacter. 87:49, 1964. Chaplin, H., Jr.: Studies on the survival of incompatible cells in patients with hypogammaglobulinemia. Blood 14:24, 1959. Gibbs, M. B., and J. H. Akeroyd: Quantitative immunohematologic studies of hemagglutination. 11. Assay of the agglutinogen A. J. Immunol. 82577, 1959. Jandl, J. H., and M. E. Kaplan: The destruction of red cells by antibodies in man. I l l . Quantitative factors influencing the patterns of hemolysis in viva J. Clin. Invest. 39:1145, 1960. Mollison, P. L.: Blood Transfusion In Clinical Medicine, 5th ed. London, Blackwell Scientific Publications, 1972, pp. 695-96. -. op. cit., p. 234. Schmidt, P. J., and P. V. Holland: Pathogenesis of the acute renal failure associated with incompatible transfusion. Lancet ii:l 169, 1967.
References I.
Abelson, N. M., and A. J. Rawson: Studies of blood group antibodies. V. Fractionation of examples of anti-B, anti-A,B, anti-M, anti-P, anti-Jka, anti-Lea, anti-D, anti-CD, anti-K, anti-Fya, anti-S and anti-Good. Transfusion 1:116, 1961.
D. H. Buchholz, M.D., Instructor, Department of Laboratory Medicine. J. R. Bove, M.D., Professor, Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Conn.
Three units of group A blood were inadvertently administered to a group 0 recipient during surgery without evidence of hemoglobinemia, hemoglobinuria, hypotension, disseminated intravascular coagulation, acute renal tubular necrosis, or other signs and symptoms of transfusion reaction. The recipient had normal concentrations of IgG, IgA, and I g M as well as complement (Q) prior to transfusion and anti-A agglutinins titered to 64 (titer of 128 by the antiglobulin technique). Seventeen hours following the transfusion, 28 per cent of the circulating red blood cells were group A (equivalent to 475 ml of packed cells); they were eliminated by day 5 without evidence of hemoglobinuria, hemoglobinemia or hyperbilirubmemia. Anti-A titers (antiglobulin) had risen from a posttransfusion low of 4 to 4,096 by day 10. After treatment of serum with 2-mercaptoethanol, however, hemolytic activity which was first noted on day 5 was lost and the antiglobulin titer dropped to 24 which suggested that most of the anti-A produced in response to the transfusion was I g M rather than IgC. The anti-A titer had dropped to essentially pretransfusion levels and the majority of anti-A present was IgM by day 91. The recipient suffered no untoward effects from the transfusion and was in good health three months following the transfusion.
following the surgery and the pulse, blood pressure, and urine output remained normal. The patient was noted to be perspiring and felt “anxious” after surgery but did not complain of symptoms usually associated with transfusion reaction. During the 14 hours following the transfusion, his temperature gradually rose from 37 to 38.3 C and then returned to normal the following day. The discovery that group A blood had been given was not made until 17 hours following the transfusion. A plasma specimen obtained at that time was clear and without visible evidence of hemolysis. Red blood cells obtained from the patient reacted weakly with anti-A and anti-A,B typing serum and the direct antiglobulin test was weakly positive with broad spectrum antihuman serum. Serum electrolytes and BUN were normal and urinalysis did not reveal cells, protein, o r hemoglobin. The patient was given three liters of intravenous fluids on each of the three days following the transfusion; no mannitol was administered. He was discharged ten days following surgery with no apparent sequelae resulting from the transfusion.
INADVERTENT TRANSFUSION of group A, B, or A B red blood cells to recipients with the corresponding alloagglutinins usually leads to a serious and occasionally fatal hemolytic transfusion reaction. This report describes the administration of 1,500 ml of group A whole blood to a healthy group 0 male who displayed no untoward effect from the transfusion.
Materials and Methods Anticoagulated (EDTA) and clotted specimens of the patient’s blood were obtained on days I , 2, 3,5,6, 10, and 9 I following the transfusion. Small amounts of pretransfusion EDTA and clotted blood samples were also available for study. All serum was separated from the red blood cells and stored at -20 C until tested on day 10 or 91. Donor red blood cell survival was estimated by quantitative hemagglutinatione using washed red blood cells that had been maintained in EDTA plasma at 4 C. Titration of agglutinating and antiglobulin reactive anti-A and anti-B was performed using pre- and posttransfusion serum. Quadruplicate serial 0.1 ml doubling dilutions of each of the respective serums were made in saline and 0.1 ml of fresh group AB nonsecretor serum was added to each tube as a source of complement. Each serum was titered against a 2 per cent suspension of A, or B cells in saline (60 minutes incubation at
Case Report A 21-year-old law student complaining of progressive back stiffness and right leg pain exacerbated by walking underwent surgery for L-5 spondylolisthesis. During the procedure the patient, who was group 0, inadvertently received three units of group A whole blood. Total blood lost during the procedure was estimated at 2,000 ml. No unusual bleeding was seen during or Received for publication January 12, 1975; accepted March 8, 1975. Transfusion Nov.-Dec. 1975
577
Volume 15 Number6
578
BUCHHOLZ AND BOVE
Transfusion Nov:Dcc. 1975
Survival of Group A Cells 40c
.c
-a 0
,c
30-
V
.-C
-ul z
V
FIG. 1. Posttransfusion survival of transfused group A cells as determined by quantitative hemagglutination.
20-
U a
$
10-
W c 0) C
? TX
Days Following Transfusion
ambient temperature) or by the high protein/antiglobulin (60 minutes incubation at 37 C) technique. Titers were expressed as the reciprocal of the highest doubling dilution of serum that produced a 2+ reaction (macroscopic clumps with many free cells in the background) with the cell being tested. Appropriate controls showed the absence of alloantibodies other than those of the ABH system. Similar titrations using pretransfusion, day 10, and day 91 samples were performed after serum inactivation with 2-mercaptoethanoP and following absorption with an excess of group B cells. Pre- and posttransfusion sera were also neutralized with equine-porcine group specific A and B substance and the titer of nonneutralized anti-A and B determined. Pretransfusion serum immunoglobulins G, A, M, and complement (C3) were quantitated by radial immunodiffusion. Urine was collected for 72 hours beginning at the time the incompatibility was recognized and was examined for cells, hemoglobin, and protein. Direct and total bilirubin were determined daily following transfusion and the BUN and creatinine were determined on days 1, 10, and 91.
Results Two of the three transfused units of blood were group A, while the third was group A*. The secretor status of the donors was not determined. Figure 1 shows the posttransfusion survival of the infused cells as determined by quantitative hemagglutination. The patient’s blood volume was estimated to be 4,475 ml based on his height and weight. The day following surgery his hematocrit was 38 vol/dl and 28 per cent of the circulating red blood cells were group A, corre-
sponding to a total of approximately 475 ml of group A cells. Although the hematocrit values of the three donor units could not be determined accurately, it was calculated that the total volume of red blood cells infused could have ranged from 495 to 765 ml assuming all donors had hematocrits of 37 or 50 vol/dl, respectively. If the average donor hematocrit were 43 vol/dl, approximately 600 ml of group A cells would have been infused. Although some transfused cells were undoubtedly lost during surgery, it is apparent that the bulk of transfused cells were still circulating the following day. The number of circulating group A cells fell progressively (Table 1) until none was detectable on day 5, coincident with the first appearance of an anti-A hemolysin (Figure 2). Prior to the infusion of the group A cells, antiA and anti-B titers were 128 and 64 respectively and no hemolysins were present. On the day following the transfusion, the anti-A had dropped to a titer of 4 although the anti-B remained at 64. There was no agglutination of A, or B cells by pretransfusion serum that had been neutralized with blood group specific A and B substance, which suggested that all antibody present in a titer greater than 4 was of “naturally occurring” type. Figures 2 and 3 illustrate the change in anti-A and B titers following the transfusion. Anti-A fell to low levels on day I but increased to titers of 2,048 (direct agglutination) and 4,096 (antiglobulin) by day 10, at which time a hemolysin with a titer of 32 was present. By day 9 I the hemolysin had fallen to a titer of 4 and the antiglobulin titer had returned to a level only slightly higher than the pretransfusion titer. Anti-B also rose following transfusion to a titer of 256 on day 10
Volume 15 Number6
579
ABO INCOMPATIBILITY
but then dropped to a titer of 64 (direct agglutination) and 128 (antiglobulin) by day 91. Pretransfusion serum lost all anti-A and anti-B reactivity greater than a titer of 12 (lowest titer tested) following 2-mercaptoethanol treatment, which suggested that most of the blood group specific antibody present was IgM (Table 2). The titer of agglutinating and antiglobulin reactive anti-A had increased greatly by day 10; however, treatment of serum with 2-mercaptoethanol resulted in loss o f hemolytic activity and a marked diminution of antiglobulin reactivity. A similar though less marked pattern was observed in serum obtained three months following the transfusion. This is in contrast to what was seen when five control serums from unselected nonimmunized group 0 persons were tested at the same time. In those serums, titers were higher by the antiglobulin technique than by direct agglutination. When retitered after treatment with 2mercaptoethanol, the antiglobulin titer remained virtually unchanged although, as expected, direct agglutinating reactivity was lost. IgM anti-A appeared to be the primary type of blood group
Table 1. Removal of Group A R e d Blood Cells Following Transfusion
Days Following Transfusion 1 2 3 4 5
Per Cent Group A Hematocrit Cells (Vol/dl) Circulating
Calculated Volume Group A Cells Circulating (ml packed RBC)
475 315
33
28 20 9
32
0
0
38 36
-
130
-
-
specific immunoglobulin produced in response to the immunizing stimulus. When day 9 1 serum was neutralized with blood group specific substance, the “immune” or nonneutralizable anti-A titered to only 5 and no “immune” anti-B could be detected. Although there appeared to be little crossreactive antibody present in pretransfusion and day 10 serum; there was sufficient crossAnti-A Production After Transfusion x Hemolysin 0 Saline Reactive A Antiglobulin Reactive
819240962048-
1024512256-
FIG.2. Anti-A titers before and after transfusion of group A whole blood.
128-
‘F
6432168-
4-
20I1
- i t i i i 4 6 s 6” “7 ”8 Tx
Days Following Transfusion
9
lO“91
580
Transfusion Nov.-Dec. 1975
BUCHHOLZ AND BOVE Anti-B Production After Transfusion
xHemolyein *Saline Reactive A Antiglobulin Reactive
512 2 561 128
&
641
FIG. 3. Anti-B titers before and after transfusion of group A whole blood.
32-
.-c 16-
b-
8-
4-
-I
t
II
I
2
3
4
5
6
7
8
9
lO"91
TX
Days Following Transfusion reactive antibody present to reduce the anti-A titer of day 91 serum by two doubling dilutions following absorption with group B cells (Table 3). In view of the unexpected absence of intravascular hemolysis in response to the infusion, patient cells and saliva were tested to rule out the possibility that the patient's blood group was a weak variant of A rather than 0. Incubation of the patient's red blood cells with a potent immune
anti-A,B serum demonstrated no reactivity by direct agglutination or the antiglobulin technique. Heat eluates of similarly treated patient cells did not react with group A cells, and while the patient secreted H substance in his saliva, no A substance could be found. No anti-H specificity was seen in either pre- or posttransfusion serum. Normal amounts of IgG, IgA, and IgM as well as complement (C3) were present and im-
Table 2. Titers o f Pre- and Posttransfusion Serum Before and After Inactivation with 2-Merca~toethanol Titer"
B Test
A , Test Cells Serum Pre-Tx Day 10 Day 91 Control Group 0
2-Mercaptoethanol Inactivation
Direct Agglutination
No Yes No Yes No Yes No Yes
96 0" * 1,536 0 96 12 24 0
Antiglobulin
48 0" 3,072 24 192 48 192 192
Direct Agglutination
48 0"" 96 0 48 0
48 0
'Serum diluted 1 : 6 initially followed by doubling serial dilutions. " " 1 :12 dilution (minimum dilution tested because of limited quantities of pretransfusion serum)
Cells Antiglobulin
48
0"" 48 12 96 48 192 192
Volume
I5
58 1
ABO INCOMPATIBILITY
Number 6
Table 3. Titers of Pie- and Posttransfusion Serum Before and After Absorption with Group B Cells Titer E Test Cells
A , Test Cells Serum Pre-Tx Day 10 Day 91 Control Group 0
Absorbed with Group B Cells
Direct Agglutination
No Yes No Yes No Yes No Yes
128 64 2.048 1,024 128 32 128 16
munoelectrophoresis of pretransfusion serum revealed a normal pattern. During the three days following the transfusion, no hemoglobin, protein or abnormal numbers of cells were noted in the patient’s urine. Specific inquiry of the nursing staff revealed the excretion of only normal colored urine during the 17 hours following the surgical procedure. During the six days following the transfusion, the patient did not become jaundiced and daily bilirubin determinations were consistently within the normal range. Determination of BUN and creatinine on days I , 10, and 91 revealed no detectable evidence of renal damage as a sequelae of the transfusion.
Discussion The infusion of group A, B or AB red blood cells to recipients with the corresponding alloantibodies is usually associated with rapid intravascular hemolysis of a large proportion of the transfused cells. Hemoglobinemia and hemoglobinuria are frequent findings and there may be marked hypotension or a severe bleeding diathesis in addition to other signs and symptoms such as chills, fever, urticaria, back pain, or nausea and vomiting. Acute renal tubular necrosis may also occur, probably from a combination of hemoglobin4 and the presence of red blood cell stroma in the circulation.I0 Prolonged survival of ABO incompatible cells has been described in recipients with hypoglobulinemia5*’ and also following the infusion of a large volume of group AB blood to a group A recipient., In the latter instance, it was suggested that red blood cell
Antiglobulin
64 64 4,096 2,048 256 64 256 64
Direct Agglutination
Antiglobulin
64 0 128 0 64 0 64 0
64 0 128 0 128 0 64 0
antigenic heterogeneity was such that a small proportion of transfused AB cells was not cleared from the circulation in spite of the presence of a “potent anti-B antibody.” Neither mechanism seems likely in the patient described in this report. Normal concentrations of immunoglobulins and complement (C3) were present in pretransfusion serum and anti-A agglutinins were present to a titer of 64. While circulation of a small number of group A, cells might be postulated with the selective destruction of group A, cells or the presence of a subpopulation of A cells which displayed antigenic heterogeneity, the presence of large numbers of circulating A cells 17 hours after infusion make these explanations unlikely. A possible explanation of the observed lack of hemolysis may be related to the immunologic type of blood group specific antibody present prior to the transfusion. Pretransfusion serum displayed no agglutination when tested with A, and B cells following neutralization with group specific AB substance, which suggests the presence of only “naturally occurring” agglutinins. This was further confirmed when pretransfusion serum lost both direct agglutinating and antiglobulin reactivity when treated with 2-mercaptoethanol. Although the antiglobulin anti-A titer had increased to 4,096 by day 10, most of the immunoglobulin produced in response to the transfusion stimulus was IgM as reflected by a marked
582
BUCHHOLZ AND BOVE
decrease in activity following 2-mercaptoethanol treatment. Although the presence of a hemolysin in a titer of 32 might suggest the presence of an IgG antibody, loss of hemolytic reactivity was also noted following 2-mercaptoethanol treatment of day 10 serum; this has been reported to be a characteristic finding for hemolytic IgM but not IgG.g The rapid return of anti-A titers to nearly pretransfusion levels by day 91 was somewhat unexpected, since it was anticipated that IgG anti-A would have been produced and maintained in response to the transfusion stimulus, especially since the recipient was group 0.' Although there was evidence of IgG presence on day 91 (titer of 48), this was less than might have been expected when compared with persons deliberately immunized with purified group A substance3 in whom titers remain elevated for a longer period of time.
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Akeroyd, J . H., and W. A. O'Brien. Survival of group AB red cells in a group A recipient. Vox Sang. 3:330, 1958. Allen, P. 2.. and E. A. Kabat: Persistence of circulating antibodies in human subjects immunized with dextran, levan, and blood group substances. J. Immunol. 80:495, 1958. Baker, S. B. de C., and R. L. F. Dawes: Experimental hemoglobinuric nephrosis. J. Pathol. Bacter. 87:49, 1964. Chaplin, H., Jr.: Studies on the survival of incompatible cells in patients with hypogammaglobulinemia. Blood 14:24, 1959. Gibbs, M. B., and J. H. Akeroyd: Quantitative immunohematologic studies of hemagglutination. 11. Assay of the agglutinogen A. J. Immunol. 82577, 1959. Jandl, J. H., and M. E. Kaplan: The destruction of red cells by antibodies in man. I l l . Quantitative factors influencing the patterns of hemolysis in viva J. Clin. Invest. 39:1145, 1960. Mollison, P. L.: Blood Transfusion In Clinical Medicine, 5th ed. London, Blackwell Scientific Publications, 1972, pp. 695-96. -. op. cit., p. 234. Schmidt, P. J., and P. V. Holland: Pathogenesis of the acute renal failure associated with incompatible transfusion. Lancet ii:l 169, 1967.
References I.
Abelson, N. M., and A. J. Rawson: Studies of blood group antibodies. V. Fractionation of examples of anti-B, anti-A,B, anti-M, anti-P, anti-Jka, anti-Lea, anti-D, anti-CD, anti-K, anti-Fya, anti-S and anti-Good. Transfusion 1:116, 1961.
D. H. Buchholz, M.D., Instructor, Department of Laboratory Medicine. J. R. Bove, M.D., Professor, Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Conn.
