Transfusion and Apheresis Science 50 (2014) 75–80
Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
ABO blood group discrepancies among blood donors in Regional Blood Transfusion Centre GTB Hospital, Delhi, India Tanya Sharma, Neeraj Garg ⇑, Bharat Singh State Blood Transfusion Council, Guru Teg Bahadur Hospital, Delhi 110 095, India
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 2 October 2013 Accepted 1 November 2013
Keywords: ABO discrepancy Subgroups Alloantibodies
a b s t r a c t Background: Regional Blood Transfusion Centre (RBTC), GTB Hospital, Delhi is providing safe and quality blood to one third of Delhi population. A discrepancy exists when reactions in forward grouping do not match with reverse grouping or if the previous and current results do not match. Aim: To analyze ABO blood group discrepancies in an algorithmic manner, and to access the incidence and causes of ABO discrepancies among blood donors. Material and methods: Retrospective data of blood donors with blood group discrepancies was recorded in Regional Blood Transfusion Centre (East) Delhi, during a period of 3 years from January 2010 to May 2013. DiaMed-ID Card Micro Typing System using Gel Cards (Cressier sur Morat, Switzerland) were used for determination of the ABO/Rh blood groups combined with reverse grouping. A detailed serological workup of these cases was studied for recognition and resolution of the blood group discrepancy. Results: Total number of donors during the study period were 104,010 (30,120; 31,117; 32,173 and 10,600 respectively). Blood group discrepancies were found in 51 cases (0.04%). There were 30 (58.8%) cases with low avidity anti-B Antibodies, 10 (19.6%) cases with weaker expression or subgroups of A, 2 (3.9%) cases with weaker expression or subgroups of B, 5(9.8%) cases with unexpected alloantibodies (Anti-N and Anti-M, Anti-Lea) and one(1.9%) case of Bombay blood group. In 3 cases, discrepancy could not be resolved and were referred to reference laboratory for confirmation by molecular analysis. The most frequent cause of discrepancy in forward grouping was found to be weak A or B antigen expressions and in reverse grouping decreased anti-B titers was most common. Conclusion: All discrepancies reported on ABO cell and serum grouping must be investigated further, so that correct blood group is reported, minimizing the chances of transfusion reaction. A note of caution should be mentioned on the blood group card to prevent ABO incompatibility in case of transfusion. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Regional Blood Transfusion Centre (RBTC), GTB Hospital, Delhi is providing safe and quality blood to one third of Delhi population. RBTC collects more than 30,000 Units of blood annually. A discrepancy exists when reactions in forward grouping do not match with reverse grouping or ⇑ Corresponding author. Address: Department of Pathology, UCMS & GTB Hospital, Delhi 110 095, India. Tel.: +91 8802454756. E-mail address: [email protected] (N. Garg). 1473-0502/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.transci.2013.11.002
if the previous and current results do not match. This may be due to intrinsic problems within red cells or serum, test related problems or technical errors [1]. Until resolved, ABO and Rh group of such cases is not interpreted. 2. Aim To analyze ABO blood group discrepancies in an algorithmic manner, and to access the incidence and causes of ABO discrepancies among blood donors.
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T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80
3. Materials and methods Retrospective data of blood donors with blood group discrepancies was recorded in Regional Blood Transfusion in Centre (East) Delhi), during a period of 3 years from January 2010 to May 2013. The numbers of donors in respective years were 30,120; 31,117; 32,173 and 10,600. DiaMed-ID Card Micro Typing System using Gel Cards (Cressier sur Morat, Switzerland) were used for determination of the ABO/Rh blood groups combined with reverse grouping. ID-Centrifuge 24 S and Saxo ID-Reader (II) Diamed were used to centrifuge the gel cards at 910 rpm for 10 min. The latter can automatically read the gel cards for blood group. Whenever a discrepancy was encountered, errors due to technical problems were first ruled and the test was repeated on same sample using washed red cells. A detailed relevant history was taken and where possible new sample was tested. Cases where discrepancy persisted after ruling out errors related to test or technical errors were included in the study. The reports of ABO and Rh grouping of these cases were held. Discrepancies may be interpreted according to whether negative results are obtained when positive results are expected, or positive results are found when tests should have been negative. These may be divided into red cell weak/missing reactivity, extra red cell reactivity, serum weak/missing reactivity, serum extra reactivity or mixedfield red cell reactivity [1]. Workup was done as appropriate for the individual case. We checked donor details including name, age and registration number among all discrepancies to avoid repetition while analyzing data. Weak/missing antigens are suspected when strength of reaction in forward grouping is weak or negative while in reverse grouping A1, B and O cells are not agglutinated by anti-A or anti-B. In such set of discrepancy, a repeat test after incubation at 4 °C was done. Forward grouping using monoclonal and polyclonal cards with anti-A, anti-B and anti-AB antisera (Cressier sur Morat, Switzerland) was done. Also, agglutination of test cells was done with anti A1 Lectin (Tulip), and anti H Lectin (Tulip). For confirmation of weak A-Subgroup, adsorption & elution studies were performed. For cold adsorption, washed test cells were incubated with Anti-A Monoclonal and Polyclonal Serum for 60 min at 4 °C, washed several times with cold saline and an eluate was prepared. For heat elution, equal volumes of washed test cells and 6% bovine albumin were incubated at 56 °C for 10 min with periodic agitation. Tubes were centrifuged at 1000g for 3 min and an eluate was prepared. The eluate was tested with group A1, B and O cells in both [1]. A weak subgroup is indicated if there is no reaction with Anti-A1 Lectin and positive reaction with Anti-H Lectin. Further eluate will test positive with the cells carrying same antigen as test cell, confirming the group. Molecular analysis and saliva study for secretor status by Hemagluttination inhibition test can further help to confirm and type the subgroups, but these were not available at our centre. In another set of discrepancy cases where reaction in serum was missing or weak while cell grouping showed
strong agglutination, we suspected weak antibodies. The test was repeated after incubation at 4 °C for 20 min. An autocontrol and group O red cells were also tested as control for reactivity of common cold autoagglutinins. Treatment of A1 and B reagent cells with a proteolytic enzyme such as ficin, papain, or bromelin (follow manufacturer’s insert) was also used in some cases. Following this the positive serum test and negative autocontrol indicates the possibility of low avidity antibodies in serum. In cases with unexpected serum reactions, Direct antiglobulin test, autocontrol and a repeat serum testing after treatment of reagent cells (A1, B, O) with enzyme (papain) were performed. Enzyme technique enhances the reaction with certain antibodies notably in Rh, Kidd and Kell system, whereas antibodies to enzyme sensitive antigens may not be detected, notably in Duffy and MNS system. Antibody screening was then performed with three cell panel in Coombs phase, enzyme phase and RT (DiaMed-ID Microtyping system). Subsequently, antibody identification with eleven cell panel (ID-DiaPanel, DiaMed-ID Microtyping system, Cressier sur Morat, Switzerland) was carried out in positive cases. When red cell group appears AB and serum contains anti-B antibodies, acquired B antigen should be suspected. In cell grouping, reaction with anti-B sera is weak or mixed field and acquired B antigen does not react with monoclonal antibodies/anti-B human serum acidified to pH 6. Also, anti-B in patient’s serum does not react with autologous red cells. Cases where discrepancy could not be resolved by serological workup were referred for molecular analysis by reference laboratory. 4. Observations We found 51 cases (0.04%) of ABO discrepancies out of 104,010 donors. Frequency wise cause of ABO discrepancy found at our centre after excluding technical errors were – weak/low avidity anti B antibodies (58.8%), weak A or subgroup of A antigen (19.6%), alloantibodies (9.8%), not resolved (5.8%), weak B or subgroup of B antigen (3.9%) and Bombay blood group (1.9%) (Table 1). To best of our knowledge, this is the first study proving the data on frequency wise occurrence of ABO discrepancies. Ten cases of weak subgroups of A were observed (including three with blood group A (subgroup/weak a antigen) B). One of these is discussed. Forward grouping showed O blood group, whereas, reverse grouping showed A blood group (Table 2). A repeat test after incubation at 4 °C could not resolve the discrepancy. On reverse grouping with A1 Cell, A2 Cell, B Cell and O cell at RT & 4 °C, reaction with A1 Cell and no reaction with A2 Cell in both phases indicated the presence of Anti-A1 in the donor plasma (Table 3). There was agglutination with AB-antisera in polyclonal card but not in monoclonal card, indicating presence of A or B antigen on red cells (Fig. 1). There was no reaction with Anti A1 (human) and Anti A1 (lectin) and agglutination with Anti-H (lectin) indicating that there was no A1 antigen on the red cells. Following cold adsorption and heat elution studies, strong reaction of eluate
T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80 Table 1 Causes of ABO discrepancies. Cause of ABO discrepancy
PERCENT
Weak/low avidity anti B antibodies Weak A or subgroup of A antigen Weak B or subgroup of B antigen Alloantibodies Bombay blood group (Oh) Not resolved Total
30/51 (58.8%) 10/51 (19.6%) 2/51 (3.9%) 5/51 (9.8%) 1/51 (1.9%) 3/51 (5.8%) 51 (100%)
Table 2 According to forward grouping, no reaction is seen with A and B antisera. In reverse grouping no reaction is seen with A1 cells. Missing reaction in forward grouping may be due to weak antigen. Anti-A RT
Anti-B
Anti-DVI 4+
Ctrl
A1
B 4+
from polyclonal and monoclonal anti-A with A1 cells confirmed the presence of A antigen on donor’s red cells (Fig. 2). Results suggested that the blood group is A positive with weaker expression or subgroups of A, most likely to be Ax or Ael. The donor also had Anti A1 in his plasma.
Fig. 1. In forward grouping, weak agglutination with AB-antisera in polyclonal card and not in monoclonal card indicates presence of weak A or B antigen on red cells.
77
Two cases of blood group B with weaker expression of B or subgroup of B was resolved in similar manner. There were two cases where mixed field reactions. In these cases we reviewed the donor’s transfusion history to determine if he has been transfused with non-group specific RBC components in the past 3 months or received an ABO-mismatched stem cell or bone marrow transplant. No such history was present. We suspected and tested for weaker subgroups. Both turned out to be weaker subgroups of A. There were 30 cases with low avidity anti-B Antibodies (Table 4). This was the commonest cause of discrepancy observed. There were 5 cases with unexpected alloantibodies (Anti-N, Anti-M and Anti-Lea). We are discussing one such case. In this case, forward grouping showed B positive blood group, whereas, reverse grouping showed strong reaction with A cells and weak reaction with B cells (Table 5). After treatment of reagent cells with papain, the unexpected agglutination with B cells was not seen. Direct antiglobulin test and autocontrol were negative, ruling out autoantibodies. Antibody screening was positive in Coomb’s phase and at room temperature. Antibody identification using eleven cell panel indicated presence
Fig. 2. Following cold adsorption and heat elution studies, strong reaction of eluate from polyclonal and monoclonal anti-A with A1 cells confirms the presence of A antigen on donor’s red cells.
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Table 3 After incubation, weak reaction with A1 cells indicates the presence of Anti-A1 in sera of donor. Individuals with weak A or Subgroup of A antigen may have anti A1 antibodies in sera. With washed cells
A
B
At RT for 20 min At 40C for 20 min
DVI
Ctrl
4+ 4+
A1 cells
A2 cells
W+ 2+
B cells
O cells
4+ 4+
Table 4 Strong reaction in forward grouping and missing reaction in reverse grouping indicates problem with reverse grouping. Reaction may be enhanced by incubating at 40C for 20 min. The blood group here is A+ with presence of weak/low avidity anti B antibodies. Results after 20 min incubation
A
At RT At 40C
4+ 4+
B
D
Ctrl
A cells
4+ 4+
B cells
O cells
1+
Table 5 A case with alloantibody (anti-N). Forward grouping shows BG B+, whereas in reverse grouping unexpected reaction is seen with B cells indicating presence of unexpected antibodies in donor’s sera. In addition to treating reagent cells with enzyme, auto control, reaction with O cells, Direct Agglutination test, antibody screening and identification helps detection of type of alloantibody present. Results
A
After treatment with papain
B
DVI
4+
4+
Ctrl
A cells
B cells
4+ 4+
wk
Fig. 3. Results of antibody screening and identification using 11 cell panel showing alloantibody M.
0 cells
T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80
79
Fig. 4. Results of antibody screening and identification using 11 cell panel showing alloantibody Lea.
of alloantibody anti-N. We found 2 cases of anti N, 2 cases of anti-M (Fig. 3) and one case of anti-Lea (see Fig. 4). One case of Bombay blood group was resolved with use of anti-H Lectin. Bombay blood group sera reacts with O reagent cells but the test red cells do not react with anti-H Lectin. There were three cases where discrepancy could not be resolved. In all three cases, there were missing reactions in reverse grouping. Two of these showed blood group O in forward grouping and one showed blood group A in forward grouping. We suspected weak or low avidity anti B and anti A antibodies, respectively. Reactions did not occur even after incubation and enzyme treatment. All these cases were referred to reference laboratory for further testing, but, as the test is highly expensive, none of the discrepancy could be finally resolved. In patients with unknown blood group, group 0 Rh( ) red cells and Group AB plasma can be safely given.
5. Discussion ABO discrepancies can result from errors made not only by the medical staff during phlebotomy but also to by the clerical staff during registration and identification. These findings emphasize the need to standardize data transmission between health care personnel [2]. Mislabeled specimens collected for cross matching procedures are
common, and are responsible for approximately one third of transfusion-related deaths [3,4]. Whenever a discrepancy is encountered, repeat tests are done on same sample using washed red cells before carrying out additional investigations. Quality assurance of reagents, correct technique, careful observation and interpretation of results resolves many problems. Where discrepancy persists, tests are done on new blood specimens obtained from the donor unit or patient to resolve discrepancies due to mislabeled or contaminated specimens. History of medication, disease, transfusion or transplantation should be obtained [1]. We found 51 cases of ABO discrepancies (0.04%) among total donations. In our study we found low avidity antibodies as most common cause of blood group discrepancy (58.8%). In some individuals, Anti-A and anti-B in the sera are too weak to agglutinate red cells without centrifugation or prolonged incubation [1]. This may occur due to weakening of antibodies with to age or due to disease state. At 4 °C, Anti-A and Anti-B are enhanced since they are saline, cold-acting antibodies. Overall, weak subgroups were found in 12 (0.01%) cases in our study, accounting for second most common cause of discrepancies. Thakral et al. from India found overall incidence of weak subgroups as 1:5100 or 0.02% in their donor population [5]. Adsorption elution studies facilitate detection of weak antigens. Subgroups of A were more common than of B group. Three out of ten subgroup A
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cases were blood group AB. From 1% to 8% of A2 and 22% to 35% of A2B individuals produce anti-A1 antibodies in their serum. Similarly Ax individuals almost always have antiA1 antibody in their serum whereas A3, Aend, and Ael may occasionally have this antibody [1]. Weakening or loss of antigens on cell may occur due to old age and diseases like leukemias and other malignancies. A positive reaction may occur with the reagent A1 or B cell due to a room-temperature antibody reacting with an antigen other than A or B on the cells. Cold alloantibodies yield unexpected reactions in ABO/Rh typing and create a nuisance for blood group serologists. Anti M and anti N antibodies are IgM antibodies that are not active at 37 °C and the discrepancy can be resolved by performing the testing at strict warm temperatures [1]. Thus, these are not clinically significant and can be ignored in transfusion practice [6,7]. However, sometimes, anti M or anti N is reactive at 37 °C and at the antihuman globulin phase; in this case, the importance of these antibodies cannot be ignored because of the potential to cause hemolytic transfusion reactions and HDN. Hence, the test results must be interpreted cautiously. Acquired B antigen may result in extra red cell reactivity leading to blood group discrepancy. It is usually found in A1 individuals with in colorectal carcinoma, intestinal obstruction, gram negative septicemia. We did not find any case of discrepancy due to acquired B antigen. The importance of clinical details and associated disease states cannot be underscored and should be gathered in all cases of discrepancies. ABO genotyping is useful for differentiating the ABO discrepancies that are difficult to resolve by serological tests. Duck Cho et al found results similar to our study by doing genotyping. They found that most frequent unusual red cell reactions were weak A and B antigen expressions
and the most frequent unusual serum reactions were caused by decreased anti-B titers [8]. Molecular techniques (PCR), family studies and saliva studies further confirm the blood group. 6. Conclusion All discrepancies reported on ABO cell and serum grouping must be investigated further, so that correct blood group is reported, minimizing the chances of transfusion reaction. A note of caution should be mentioned on the blood group card to prevent ABO incompatibility in case of transfusion. We found causes of discrepancy among largest number of donors ever studied. Still, a further study advised which includes molecular techniques, secretor status and family study. References [1] Brecher ME editor. AABB technical manual. 15 th ed. Bethesda: American Association of Blood Banks; 2005. p. 289–03. [chapter 13] [2] Chiaroni J, Legrand D, Dettori I, Ferrera V. Analysis of ABO discrepancies occurring in 35 French hospitals. Transfusion 2004;44:860–4. [3] Sazama K. Reports of 355 transfusion-associated deaths: 1976 through 1985. Transfusion 1990;30:583–90. [4] Linden JV, Wagner K, Voytovich AE, Sheehan J, et al. Transfusion errors in New York State: an analysis of 10 years experience. Transfusion 2000;40:1207–13. [5] Thakral B, Saluja K, Bajpai M, Sharma RR, Marwaha N. Importance of weak ABO subgroups. Lab Med 2005;36:32–4. [6] Khalid S, Dantes R, Varghese S, Hakawati IA. Naturally occurring anti M complicating ABO grouping. Indian J Pathol Microbiol 2011;54:170–2. [7] Tondon R, Kataria R, Chaudhry R, Anti M. Report of two cases and review of literature. Asian J Transfus Sci 2008;2:81–3. [8] Cho D, Lee JS, Park JY, Jeon MJ, Song JW, Kim SH, et al. Resolution of ABO Discrepancies by ABO Genotyping. Kor J Lab Med 2006;26:107–13.
Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
ABO blood group discrepancies among blood donors in Regional Blood Transfusion Centre GTB Hospital, Delhi, India Tanya Sharma, Neeraj Garg ⇑, Bharat Singh State Blood Transfusion Council, Guru Teg Bahadur Hospital, Delhi 110 095, India
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 2 October 2013 Accepted 1 November 2013
Keywords: ABO discrepancy Subgroups Alloantibodies
a b s t r a c t Background: Regional Blood Transfusion Centre (RBTC), GTB Hospital, Delhi is providing safe and quality blood to one third of Delhi population. A discrepancy exists when reactions in forward grouping do not match with reverse grouping or if the previous and current results do not match. Aim: To analyze ABO blood group discrepancies in an algorithmic manner, and to access the incidence and causes of ABO discrepancies among blood donors. Material and methods: Retrospective data of blood donors with blood group discrepancies was recorded in Regional Blood Transfusion Centre (East) Delhi, during a period of 3 years from January 2010 to May 2013. DiaMed-ID Card Micro Typing System using Gel Cards (Cressier sur Morat, Switzerland) were used for determination of the ABO/Rh blood groups combined with reverse grouping. A detailed serological workup of these cases was studied for recognition and resolution of the blood group discrepancy. Results: Total number of donors during the study period were 104,010 (30,120; 31,117; 32,173 and 10,600 respectively). Blood group discrepancies were found in 51 cases (0.04%). There were 30 (58.8%) cases with low avidity anti-B Antibodies, 10 (19.6%) cases with weaker expression or subgroups of A, 2 (3.9%) cases with weaker expression or subgroups of B, 5(9.8%) cases with unexpected alloantibodies (Anti-N and Anti-M, Anti-Lea) and one(1.9%) case of Bombay blood group. In 3 cases, discrepancy could not be resolved and were referred to reference laboratory for confirmation by molecular analysis. The most frequent cause of discrepancy in forward grouping was found to be weak A or B antigen expressions and in reverse grouping decreased anti-B titers was most common. Conclusion: All discrepancies reported on ABO cell and serum grouping must be investigated further, so that correct blood group is reported, minimizing the chances of transfusion reaction. A note of caution should be mentioned on the blood group card to prevent ABO incompatibility in case of transfusion. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Regional Blood Transfusion Centre (RBTC), GTB Hospital, Delhi is providing safe and quality blood to one third of Delhi population. RBTC collects more than 30,000 Units of blood annually. A discrepancy exists when reactions in forward grouping do not match with reverse grouping or ⇑ Corresponding author. Address: Department of Pathology, UCMS & GTB Hospital, Delhi 110 095, India. Tel.: +91 8802454756. E-mail address: [email protected] (N. Garg). 1473-0502/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.transci.2013.11.002
if the previous and current results do not match. This may be due to intrinsic problems within red cells or serum, test related problems or technical errors [1]. Until resolved, ABO and Rh group of such cases is not interpreted. 2. Aim To analyze ABO blood group discrepancies in an algorithmic manner, and to access the incidence and causes of ABO discrepancies among blood donors.
76
T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80
3. Materials and methods Retrospective data of blood donors with blood group discrepancies was recorded in Regional Blood Transfusion in Centre (East) Delhi), during a period of 3 years from January 2010 to May 2013. The numbers of donors in respective years were 30,120; 31,117; 32,173 and 10,600. DiaMed-ID Card Micro Typing System using Gel Cards (Cressier sur Morat, Switzerland) were used for determination of the ABO/Rh blood groups combined with reverse grouping. ID-Centrifuge 24 S and Saxo ID-Reader (II) Diamed were used to centrifuge the gel cards at 910 rpm for 10 min. The latter can automatically read the gel cards for blood group. Whenever a discrepancy was encountered, errors due to technical problems were first ruled and the test was repeated on same sample using washed red cells. A detailed relevant history was taken and where possible new sample was tested. Cases where discrepancy persisted after ruling out errors related to test or technical errors were included in the study. The reports of ABO and Rh grouping of these cases were held. Discrepancies may be interpreted according to whether negative results are obtained when positive results are expected, or positive results are found when tests should have been negative. These may be divided into red cell weak/missing reactivity, extra red cell reactivity, serum weak/missing reactivity, serum extra reactivity or mixedfield red cell reactivity [1]. Workup was done as appropriate for the individual case. We checked donor details including name, age and registration number among all discrepancies to avoid repetition while analyzing data. Weak/missing antigens are suspected when strength of reaction in forward grouping is weak or negative while in reverse grouping A1, B and O cells are not agglutinated by anti-A or anti-B. In such set of discrepancy, a repeat test after incubation at 4 °C was done. Forward grouping using monoclonal and polyclonal cards with anti-A, anti-B and anti-AB antisera (Cressier sur Morat, Switzerland) was done. Also, agglutination of test cells was done with anti A1 Lectin (Tulip), and anti H Lectin (Tulip). For confirmation of weak A-Subgroup, adsorption & elution studies were performed. For cold adsorption, washed test cells were incubated with Anti-A Monoclonal and Polyclonal Serum for 60 min at 4 °C, washed several times with cold saline and an eluate was prepared. For heat elution, equal volumes of washed test cells and 6% bovine albumin were incubated at 56 °C for 10 min with periodic agitation. Tubes were centrifuged at 1000g for 3 min and an eluate was prepared. The eluate was tested with group A1, B and O cells in both [1]. A weak subgroup is indicated if there is no reaction with Anti-A1 Lectin and positive reaction with Anti-H Lectin. Further eluate will test positive with the cells carrying same antigen as test cell, confirming the group. Molecular analysis and saliva study for secretor status by Hemagluttination inhibition test can further help to confirm and type the subgroups, but these were not available at our centre. In another set of discrepancy cases where reaction in serum was missing or weak while cell grouping showed
strong agglutination, we suspected weak antibodies. The test was repeated after incubation at 4 °C for 20 min. An autocontrol and group O red cells were also tested as control for reactivity of common cold autoagglutinins. Treatment of A1 and B reagent cells with a proteolytic enzyme such as ficin, papain, or bromelin (follow manufacturer’s insert) was also used in some cases. Following this the positive serum test and negative autocontrol indicates the possibility of low avidity antibodies in serum. In cases with unexpected serum reactions, Direct antiglobulin test, autocontrol and a repeat serum testing after treatment of reagent cells (A1, B, O) with enzyme (papain) were performed. Enzyme technique enhances the reaction with certain antibodies notably in Rh, Kidd and Kell system, whereas antibodies to enzyme sensitive antigens may not be detected, notably in Duffy and MNS system. Antibody screening was then performed with three cell panel in Coombs phase, enzyme phase and RT (DiaMed-ID Microtyping system). Subsequently, antibody identification with eleven cell panel (ID-DiaPanel, DiaMed-ID Microtyping system, Cressier sur Morat, Switzerland) was carried out in positive cases. When red cell group appears AB and serum contains anti-B antibodies, acquired B antigen should be suspected. In cell grouping, reaction with anti-B sera is weak or mixed field and acquired B antigen does not react with monoclonal antibodies/anti-B human serum acidified to pH 6. Also, anti-B in patient’s serum does not react with autologous red cells. Cases where discrepancy could not be resolved by serological workup were referred for molecular analysis by reference laboratory. 4. Observations We found 51 cases (0.04%) of ABO discrepancies out of 104,010 donors. Frequency wise cause of ABO discrepancy found at our centre after excluding technical errors were – weak/low avidity anti B antibodies (58.8%), weak A or subgroup of A antigen (19.6%), alloantibodies (9.8%), not resolved (5.8%), weak B or subgroup of B antigen (3.9%) and Bombay blood group (1.9%) (Table 1). To best of our knowledge, this is the first study proving the data on frequency wise occurrence of ABO discrepancies. Ten cases of weak subgroups of A were observed (including three with blood group A (subgroup/weak a antigen) B). One of these is discussed. Forward grouping showed O blood group, whereas, reverse grouping showed A blood group (Table 2). A repeat test after incubation at 4 °C could not resolve the discrepancy. On reverse grouping with A1 Cell, A2 Cell, B Cell and O cell at RT & 4 °C, reaction with A1 Cell and no reaction with A2 Cell in both phases indicated the presence of Anti-A1 in the donor plasma (Table 3). There was agglutination with AB-antisera in polyclonal card but not in monoclonal card, indicating presence of A or B antigen on red cells (Fig. 1). There was no reaction with Anti A1 (human) and Anti A1 (lectin) and agglutination with Anti-H (lectin) indicating that there was no A1 antigen on the red cells. Following cold adsorption and heat elution studies, strong reaction of eluate
T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80 Table 1 Causes of ABO discrepancies. Cause of ABO discrepancy
PERCENT
Weak/low avidity anti B antibodies Weak A or subgroup of A antigen Weak B or subgroup of B antigen Alloantibodies Bombay blood group (Oh) Not resolved Total
30/51 (58.8%) 10/51 (19.6%) 2/51 (3.9%) 5/51 (9.8%) 1/51 (1.9%) 3/51 (5.8%) 51 (100%)
Table 2 According to forward grouping, no reaction is seen with A and B antisera. In reverse grouping no reaction is seen with A1 cells. Missing reaction in forward grouping may be due to weak antigen. Anti-A RT
Anti-B
Anti-DVI 4+
Ctrl
A1
B 4+
from polyclonal and monoclonal anti-A with A1 cells confirmed the presence of A antigen on donor’s red cells (Fig. 2). Results suggested that the blood group is A positive with weaker expression or subgroups of A, most likely to be Ax or Ael. The donor also had Anti A1 in his plasma.
Fig. 1. In forward grouping, weak agglutination with AB-antisera in polyclonal card and not in monoclonal card indicates presence of weak A or B antigen on red cells.
77
Two cases of blood group B with weaker expression of B or subgroup of B was resolved in similar manner. There were two cases where mixed field reactions. In these cases we reviewed the donor’s transfusion history to determine if he has been transfused with non-group specific RBC components in the past 3 months or received an ABO-mismatched stem cell or bone marrow transplant. No such history was present. We suspected and tested for weaker subgroups. Both turned out to be weaker subgroups of A. There were 30 cases with low avidity anti-B Antibodies (Table 4). This was the commonest cause of discrepancy observed. There were 5 cases with unexpected alloantibodies (Anti-N, Anti-M and Anti-Lea). We are discussing one such case. In this case, forward grouping showed B positive blood group, whereas, reverse grouping showed strong reaction with A cells and weak reaction with B cells (Table 5). After treatment of reagent cells with papain, the unexpected agglutination with B cells was not seen. Direct antiglobulin test and autocontrol were negative, ruling out autoantibodies. Antibody screening was positive in Coomb’s phase and at room temperature. Antibody identification using eleven cell panel indicated presence
Fig. 2. Following cold adsorption and heat elution studies, strong reaction of eluate from polyclonal and monoclonal anti-A with A1 cells confirms the presence of A antigen on donor’s red cells.
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T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80
Table 3 After incubation, weak reaction with A1 cells indicates the presence of Anti-A1 in sera of donor. Individuals with weak A or Subgroup of A antigen may have anti A1 antibodies in sera. With washed cells
A
B
At RT for 20 min At 40C for 20 min
DVI
Ctrl
4+ 4+
A1 cells
A2 cells
W+ 2+
B cells
O cells
4+ 4+
Table 4 Strong reaction in forward grouping and missing reaction in reverse grouping indicates problem with reverse grouping. Reaction may be enhanced by incubating at 40C for 20 min. The blood group here is A+ with presence of weak/low avidity anti B antibodies. Results after 20 min incubation
A
At RT At 40C
4+ 4+
B
D
Ctrl
A cells
4+ 4+
B cells
O cells
1+
Table 5 A case with alloantibody (anti-N). Forward grouping shows BG B+, whereas in reverse grouping unexpected reaction is seen with B cells indicating presence of unexpected antibodies in donor’s sera. In addition to treating reagent cells with enzyme, auto control, reaction with O cells, Direct Agglutination test, antibody screening and identification helps detection of type of alloantibody present. Results
A
After treatment with papain
B
DVI
4+
4+
Ctrl
A cells
B cells
4+ 4+
wk
Fig. 3. Results of antibody screening and identification using 11 cell panel showing alloantibody M.
0 cells
T. Sharma et al. / Transfusion and Apheresis Science 50 (2014) 75–80
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Fig. 4. Results of antibody screening and identification using 11 cell panel showing alloantibody Lea.
of alloantibody anti-N. We found 2 cases of anti N, 2 cases of anti-M (Fig. 3) and one case of anti-Lea (see Fig. 4). One case of Bombay blood group was resolved with use of anti-H Lectin. Bombay blood group sera reacts with O reagent cells but the test red cells do not react with anti-H Lectin. There were three cases where discrepancy could not be resolved. In all three cases, there were missing reactions in reverse grouping. Two of these showed blood group O in forward grouping and one showed blood group A in forward grouping. We suspected weak or low avidity anti B and anti A antibodies, respectively. Reactions did not occur even after incubation and enzyme treatment. All these cases were referred to reference laboratory for further testing, but, as the test is highly expensive, none of the discrepancy could be finally resolved. In patients with unknown blood group, group 0 Rh( ) red cells and Group AB plasma can be safely given.
5. Discussion ABO discrepancies can result from errors made not only by the medical staff during phlebotomy but also to by the clerical staff during registration and identification. These findings emphasize the need to standardize data transmission between health care personnel [2]. Mislabeled specimens collected for cross matching procedures are
common, and are responsible for approximately one third of transfusion-related deaths [3,4]. Whenever a discrepancy is encountered, repeat tests are done on same sample using washed red cells before carrying out additional investigations. Quality assurance of reagents, correct technique, careful observation and interpretation of results resolves many problems. Where discrepancy persists, tests are done on new blood specimens obtained from the donor unit or patient to resolve discrepancies due to mislabeled or contaminated specimens. History of medication, disease, transfusion or transplantation should be obtained [1]. We found 51 cases of ABO discrepancies (0.04%) among total donations. In our study we found low avidity antibodies as most common cause of blood group discrepancy (58.8%). In some individuals, Anti-A and anti-B in the sera are too weak to agglutinate red cells without centrifugation or prolonged incubation [1]. This may occur due to weakening of antibodies with to age or due to disease state. At 4 °C, Anti-A and Anti-B are enhanced since they are saline, cold-acting antibodies. Overall, weak subgroups were found in 12 (0.01%) cases in our study, accounting for second most common cause of discrepancies. Thakral et al. from India found overall incidence of weak subgroups as 1:5100 or 0.02% in their donor population [5]. Adsorption elution studies facilitate detection of weak antigens. Subgroups of A were more common than of B group. Three out of ten subgroup A
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cases were blood group AB. From 1% to 8% of A2 and 22% to 35% of A2B individuals produce anti-A1 antibodies in their serum. Similarly Ax individuals almost always have antiA1 antibody in their serum whereas A3, Aend, and Ael may occasionally have this antibody [1]. Weakening or loss of antigens on cell may occur due to old age and diseases like leukemias and other malignancies. A positive reaction may occur with the reagent A1 or B cell due to a room-temperature antibody reacting with an antigen other than A or B on the cells. Cold alloantibodies yield unexpected reactions in ABO/Rh typing and create a nuisance for blood group serologists. Anti M and anti N antibodies are IgM antibodies that are not active at 37 °C and the discrepancy can be resolved by performing the testing at strict warm temperatures [1]. Thus, these are not clinically significant and can be ignored in transfusion practice [6,7]. However, sometimes, anti M or anti N is reactive at 37 °C and at the antihuman globulin phase; in this case, the importance of these antibodies cannot be ignored because of the potential to cause hemolytic transfusion reactions and HDN. Hence, the test results must be interpreted cautiously. Acquired B antigen may result in extra red cell reactivity leading to blood group discrepancy. It is usually found in A1 individuals with in colorectal carcinoma, intestinal obstruction, gram negative septicemia. We did not find any case of discrepancy due to acquired B antigen. The importance of clinical details and associated disease states cannot be underscored and should be gathered in all cases of discrepancies. ABO genotyping is useful for differentiating the ABO discrepancies that are difficult to resolve by serological tests. Duck Cho et al found results similar to our study by doing genotyping. They found that most frequent unusual red cell reactions were weak A and B antigen expressions
and the most frequent unusual serum reactions were caused by decreased anti-B titers [8]. Molecular techniques (PCR), family studies and saliva studies further confirm the blood group. 6. Conclusion All discrepancies reported on ABO cell and serum grouping must be investigated further, so that correct blood group is reported, minimizing the chances of transfusion reaction. A note of caution should be mentioned on the blood group card to prevent ABO incompatibility in case of transfusion. We found causes of discrepancy among largest number of donors ever studied. Still, a further study advised which includes molecular techniques, secretor status and family study. References [1] Brecher ME editor. AABB technical manual. 15 th ed. Bethesda: American Association of Blood Banks; 2005. p. 289–03. [chapter 13] [2] Chiaroni J, Legrand D, Dettori I, Ferrera V. Analysis of ABO discrepancies occurring in 35 French hospitals. Transfusion 2004;44:860–4. [3] Sazama K. Reports of 355 transfusion-associated deaths: 1976 through 1985. Transfusion 1990;30:583–90. [4] Linden JV, Wagner K, Voytovich AE, Sheehan J, et al. Transfusion errors in New York State: an analysis of 10 years experience. Transfusion 2000;40:1207–13. [5] Thakral B, Saluja K, Bajpai M, Sharma RR, Marwaha N. Importance of weak ABO subgroups. Lab Med 2005;36:32–4. [6] Khalid S, Dantes R, Varghese S, Hakawati IA. Naturally occurring anti M complicating ABO grouping. Indian J Pathol Microbiol 2011;54:170–2. [7] Tondon R, Kataria R, Chaudhry R, Anti M. Report of two cases and review of literature. Asian J Transfus Sci 2008;2:81–3. [8] Cho D, Lee JS, Park JY, Jeon MJ, Song JW, Kim SH, et al. Resolution of ABO Discrepancies by ABO Genotyping. Kor J Lab Med 2006;26:107–13.
