Transfusion Medicine, 1992, 2, 1 15-1 18
The gel test: some problems and solutions M. de Figueiredo, M. Lima, S . Morais, G. Porto and B. JustiCa SeruiceofHaemarology, Hospital GeraI de Santo Antbnio. 4000 PORTO. Portugal Received 23 May 1991: accepted for publication 16 October 1991
The gel centrifugation test (GT) is a method of transfusion serology, based on the fact that, after centrifugation, unagglutinated red blood cells (RBC) pass easily through a gel, while agglutinated RBC do not. The introduction of the GT to our blood bank transfusion routine [strictly following the manufacturer’s instructions (DiaMed I D Micro Typing System)] resulted in problems with the interpretation of the results. These were overcome after the introduction of modifications, which included; (1) the systema-
tic use of 1 % RBC suspensions; (2) the use of 50 p1 of 1 % RBC suspensions and 25 p1 of serum in all tests; (3) the control of all negative indirect antiglobulin tests (IAT) and direct antiglobulin tests (DAT) by the addition of 50 pl of a 1 % IgG coated RBC suspension followed by centrifugation; and (4) the systematic use of saline-suspended RBC for ABO typing in patients with positive DAT.
The gel centrifugation test (GT) is a method of blood group typing and antibody screening which can be applied to most of the tests used in routine blood group serology (Lapierre, 1989; Lapierre et af., 1990). It was demonstrated that the sensitivity of this test is superior to that of the conventional tube tests (CTT), without loosing specificity (Lapierre, 1989; Lapierre et al., 1990; Hitzler, et al. 1989; Schiitt & Arndt-Hanse, 1989). We have tried to introduce the G T in to routine blood group serology of our blood bank strictly following the instructions of the commercially available kit (DiaMed I D MycroTyping System). However, in a preliminary assay we had difficulty in the interpretation of 5% of the tests due to practical and theoretical problems, the resolution of which was the main purpose of the present work. These problems are outlined below. 1 A large variability in red blood cell (RBC) concentrations for the same test, due to the use of fixed volumes of blood and ID-diluents in the preparation of suspensions. This was the result both of different patients’ haematocrits and of the inconsistent use of coagulated or uncoagulated blood samples. 2 The preparation of RBC suspensions in different concentrations and/or the use of different volumes of
RBC suspensions or serum for different tests is a potential cause of clerical error, with the consequent loss of time and material. 3 The use of unwashed RBC suspensions can theoretically originate both false-positive and falsenegative results: false-positive results in RBC phenotyping by unspecific agglutination of RBC; falsenegative results in direct antiglobulin tests (DAT) and indirect antiglobulin tests (IAT), because without washing antihuman globulin can be neutralized by free immunoglobulins present in the serum (Issit, 1985). 4 The use of enzymes in the RBC suspension medium (ID-diluent 1) for ABO typing made it impossible to ABO type patients with autoimmune haemolytic anaemia correctly, because they systematically originated a ‘false’ AB phenotype. We thus established the best conditions for the application of the G T to routine blood group serology in our blood bank by overcoming the above mentioned problems.
SUMMARY.
Key words: antibody screening; blood typing; gel test.
MATERIALS AND METHODS Blood samples from 197 patients for whom blood transfusions were requested were tested. ABO phenotyping (including A and AB subgroups), Rh and Kell phenotyping, DAT, and antibody screening and compatibility tests by IAT were performed for each sample. M and N phenotyping were also determined in
Correspondence: M. de Figueiredo, Service de Hematologia, Hospital Geral de Santo Antbnio, Largo d o Prof. Abel Salazar, I. 4000 PORTO,Portugal.
115
116
M . de Figueiredo et al.
56 randomly seleded blood samples. Blood samples from 10 individuals with weak D (Du), 57 individuals with irregular antibodies and 17 individuals with positive DAT were also tested. These individuals were selected from our blood bank records. In addition ABO/Rh/Kell phenotyping of five blood samples from patients with autoimmune haemolytic anaemia (AIHA) by warm antibodies was also performed. Blood samples were collected in tubes containing EDTA or in tubes without anticoagulant. DAT was performed only on EDTA samples. Serum was separated by centrifugation of the samples for 5 min at 1000 g.All tests were simultaneously carried out by the conventional tube test (CTT), according to the internationally standardized technique (Mollison et al., 1987). The G T was performed using the ‘ID-DiaMed Micro Typing System’ kit (DiaMed AG CH-3280 Murten Switzerland) according to the manufacturers’ instructions, with the following modifications. 1 In order to avoid the variability in RBC concentrations, suspensions were prepared, not with fixed volumes of blood and ID-Diluent, but with the amount necessary to obtain the desired concentration, which was determined by visual comparison with previously prepared standard suspensions. In order to define the best concentration of RBC suspensions, all tests were simultaneously performed with three concentrations: 1, 3 and 6%. 2 Constant volumes of 50 pI of RBC suspensions and 25 p1 of serum were used in all tests. 3 RBC were washed whenever discrepancies occurred between direct and reverse group typing in the presence of a positive reaction in the negative control microtube. In all negative IAT or DAT tests, the results were controlled by the addition of 50 pl of a 1% IgG-(anti-D) coated RBC suspension, followed by centrifugation. This allowed us to confirm the presence and the activity of the antihuman globulin serum. 4 ABO typing was also performed using saline suspended RBC in patients with positive DAT.
RESULTS A BOIRhlKell phenotyping
ABO/Rh/Kell phenotyping was performed in 197 patients. No ABO weak variants were included. All the results were identical to those obtained with the CTT when 1 and 3% RBC suspensions were used in the GT, except in one patient with A, CcDe, k phenotype who had positive reactions in all microtubes (including the negative control one) using both 1 and 3% RBC concentrations, which suggests an AB, CcDEe, K
phenotype. RBC washing with saline was performed and the correct phenotyping achieved. Difficulties were found in the interpretation of seven results with the 6% concentration: three D+ patients showed a + + + reaction, instead of the usual + + + + reaction; two D- patients showed a weak positive reaction (+); one group A phenotype patient showed a positive (+) reaction in the microtube with the anti-B serum; one CcDe phenotype patient showed a positive (+)reaction in the microtube with the anti-E serum. A mixed-field reaction was detected in Rh phenotyping of three recently transfused patients both by the G T and the CTT. The presence of the two RBC populations was much more easily identified in the GT than in the CTT, by completely sedimented RBC (negative reaction) and ge1 surface retained RBC (+ + + +), simultaneously in the same microtube. The presence of ‘rouleaux’ formation interfered with RBC phenotyping by the CTT in two patients. RBC phenotyping was easily performed in the GT with no need for RBC washing. Positive reactions were observed in all microtubes (including the negative control one) in ABO/Rh/Kell phenotyping in the five blood samples of patients with AIHA by warm antibodies. The reactions remained positive despite RBC washing. When RBC were suspended in saline, instead of the enzyme suspension medium (ID-Diluent I), the negative control was no longer positive and ABO typing became possible. Rh and Kell phenotyping remained impossible with saline-suspended RBC because only negative or weak positive reactions were observed as the typing procedure requires enhancement by enzyme tests. A 1 and A 2 subgroups
A 1 and A2 subgroups were determined in I 10 samples with A or AB phenotype. Eighty-five blood samples subtyped as A 1 and 25 as A2. on examination by CTT. Difficulties were found in the interpretation of the results using GT, whatever RBC concentration was used. According to the manufacturer’s instructions, with ‘Lectin A l ’ cards, A1 phenotype RBC should to + reaction and A2 phenotype show a RBC should show a negative reaction. A1 phenotype RBC should show a negative reaction and A2 phenotype RBC a + to + + + reaction with ‘lectin H’ cards. In our study, when ‘Lectin Al’ cards were used, both A l and A2 blood samples showed negative reactions; when ‘Lectin H’ cards were used, A l blood samples showed 57 negative reactions and 28 weak positive reactions (+), while A2 blood samples correctly showed a + + + reaction.
+
++ +
1 2 3 4 5
6 7 8 9 10 11
12 13 14 15
16 17 18 19
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
46 41 48 49 50
Antibody anti D anti D anti D anti D anti D anti Cw anti E anti D + C anti D C anti D + C anti D E anti E + c anti D + C + E anti D + C + E anti D + C + E anti K anti K anti K anti Jka anti Jka anti Jka anti Jkb anti Fya anti Lea anti Lea anti Lea anti Leb anti Leb anti M anti M anti N anti PI anti PI anti I anti I anti I anti I anti I anti E + I anti E Fya anti E + K anti D + C + E + F y a anti Lea I anti PI+Lua WAA WAA WAA WAA WAA WAA
+ +
+
+
CTT
+ + + + + + + + + + +* + + + + + + + + + + + + + + + + + + + + + + + + + + -t + -t + + + + + + + + +
1 17
Table 1. Continued
Table 1. Comparison of CTT and GT: identification of 57 antibodies Sample
The gel test
Sample
Antibody
CTT
GT
51 52 53 54
WAA WAA CAA CAA UA UA UA
+ + + + + + +
+ + + + + + +
GT
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
55
56 57
CTT =conventional tube test. GT = gel centrifugation test. WAA =warm autoantibody. CAA = cold autoantibody. UA = unidentified antibody. Anti E not found. t Anti E not found. $ Anti K not found.
M /N phen otyping No differences were found between the results obtained from 1 and 3% RBC suspensions. Difficulties were found in the interpretation of the results using 6% RBC suspensions (data not shown). Antibody screening and identification
Antibody screening and identification was performed with IAT in the 197 blood samples. Irregular antibodies were found in two samples. No differences were seen between the results obtained by both methods. When the 57 blood samples of individuals with irregular antibodies were studied, discrepancies were found in four samples (Table 1). Anti-E antibodies were not identified with the CTT in one anti-E+T and in one anti-E + c associations. One anti-Kell antibody was unidentified with the CTT in one anti-E+Kell association. One anti-Le b antibody was identified only with the CTT. Compatibility tests
Four hundred and three compatibility tests with IAT were performed with the 197 blood samples. No differences were observed between the results obtained with the 1% RBC suspension GT and the CTT (five positive tests and 398 negative tests). These five positive results were positive by the GT with all RBC concentrations used, however, with the 3% and the 6% RBC suspensions, respectively, 120 and 163 weak positive (+) reactions still occurred with the GT, in spite of negative reactions with the CTT and with the 1% RBC suspensions GT. Direct antiglobulin test
One hundred and ninety-seven DAT were performed.
118
M . de Figueiredo et al.
Eleven DAT were positive using both methods. Among the remaining 186 blood samples, nine positive DAT were found only with the GT, with all RBC concentrations used. In addition, four and 23 positive results were found with the GT when 3 and 6% RBC suspensions were used, respectively. Blood samples from 17 patients with previously known positive DAT of different specificities ( 5 IgG, 2 C3d, 9 IgG +C3d and 1 IgG + C3d + IgA) were also studied with the GT. Positive results were obtained using polyspecific antiglobulin serum-containing cards, with all RBC concentrations used. DISCUSSION When we tried to introduce the GT in to our routine blood group serology, following the instructions of the commercially available kit, difficulties were found in the interpretation of about 5% of the results. This occurred because either too high or too low RBC concentrations were obtained. High concentrations led to the spread of RBC through the gel originating false-positive results, and low concentrations led to difficulties in the visualization of RBC. Using 50 p1 of RBC suspensions for all tests, the ideal concentration was 1 % because no problems occurred in the interpretation of the results. More concentrated suspensions (3 and 6%) led to false-positive results. False-positive results were, however, easily identified in RBC phenotyping, as discrepancies occurred between red cell and reverse grouping, and a positive reaction appeared in the ‘negative control’ tube. Although the G T can be routinely performed with no need for previous washing of RBC, this is necessary, however, whenever a positive control is observed. The absence of washing steps in DAT and IAT did not lead to false-negative reactions. The addition of IgG-coated RBC to all microtubes with a negative reaction, followed by centrifugation, demonstrated that the antihuman globulin serum was reactive. As ABO/Rh phenotyping is performed using enzyme-treated RBC, DAT positive samples showed positive reactions in all microtubes, including the control. In the fine tested cases, ABO phenotyping was successfully performed when saline-suspended RBC were used. It is possible that using saline-suspended RBC can cause some weak ABO variants to be missed. This has not been confirmed in this study because no weak ABO variants were included. Rh phenotyping could not be determined in DAT-positive patients,
either with the GT or with the C’IT because with saline-suspended RBC the reactions were all negative or weakly positive as the typing reagents require enzyme-enhanced tests. Some discrepancies were found between the G T and the CTT when DAT was performed. Nine cases of positive DAT with the GT, which occurred even when a 1 % RBC suspension concentration was used, were negative with the CTT. In these cases the intensity of the reaction with the GT was always + to + f , although the screening of antibodies in the eluate with the CTT was negative. Most of these weak-positive reactions occurred with blood samples of patients who belonged to groups in which a positive DAT was expected (three polytransfused patients, one patient who had been transfused with plasma on the previous day, two patients with systemic lupus erythematosus and one patient who was receiving penicilin treatment). DAT became positive also with the CTT a few days later in this patient, who afterwards developed a penicillin-induced haemolytic anaemia. The apparent increase in sensitivity could be explained by decreased elution of antibodies attached to the RBC membrane due to lack of washing and less manipulation of RBC (Petz, 1980). One problem that remains to be solved is a subgroup phenotyping with the GT. A possible explanation for the results we obtained is that lectins used in the cards were adsorbed by the plastic. REFERENCES Hitzler, W., Schomig-Breckner, H. & Mathias, D. (1989)GeI centrifugation test-a new micro method for blood group typing and antibody screening. Arziliche Laboratorium, 35,89-92. Issit. P.D. (1985) Applied Blood Group Serology, 3rd edn, 72115 . Montgomery Scientfic Publications, Miami, FL. Lapierre, Y. (1989) Le gel test: un nouveau procede en Immuno-Himatologie.Spectra Bioiogie, 89(4), 38-42. Lapierre, Y., Rical, D., Adam, J. et al. (1990) The gel test: a new way to detect red cell antigen-antibody reactions. Transfusion, 30, 109- 1 13.
Mollison, P.L., Engelfriet, C.P. & Contreras, M. (1987) Blood Transfiion in Clinical Medicine, 8th edn, Blackwell Scientific Publication, Oxford. Petz, L.D. (1980) Acquired Immune Hemoiytic Anemias, 1st edn, Churchill Livingstone, New York. Schutt, K.H. & Arndt-Hanser, A. (1989) Erfahrungen mit dem ID-Microtyping System zur Blutgruppen und Antikorperbestimmungin der Routine. Arztliche Laboratorium 35,287-292.
The gel test: some problems and solutions M. de Figueiredo, M. Lima, S . Morais, G. Porto and B. JustiCa SeruiceofHaemarology, Hospital GeraI de Santo Antbnio. 4000 PORTO. Portugal Received 23 May 1991: accepted for publication 16 October 1991
The gel centrifugation test (GT) is a method of transfusion serology, based on the fact that, after centrifugation, unagglutinated red blood cells (RBC) pass easily through a gel, while agglutinated RBC do not. The introduction of the GT to our blood bank transfusion routine [strictly following the manufacturer’s instructions (DiaMed I D Micro Typing System)] resulted in problems with the interpretation of the results. These were overcome after the introduction of modifications, which included; (1) the systema-
tic use of 1 % RBC suspensions; (2) the use of 50 p1 of 1 % RBC suspensions and 25 p1 of serum in all tests; (3) the control of all negative indirect antiglobulin tests (IAT) and direct antiglobulin tests (DAT) by the addition of 50 pl of a 1 % IgG coated RBC suspension followed by centrifugation; and (4) the systematic use of saline-suspended RBC for ABO typing in patients with positive DAT.
The gel centrifugation test (GT) is a method of blood group typing and antibody screening which can be applied to most of the tests used in routine blood group serology (Lapierre, 1989; Lapierre et af., 1990). It was demonstrated that the sensitivity of this test is superior to that of the conventional tube tests (CTT), without loosing specificity (Lapierre, 1989; Lapierre et al., 1990; Hitzler, et al. 1989; Schiitt & Arndt-Hanse, 1989). We have tried to introduce the G T in to routine blood group serology of our blood bank strictly following the instructions of the commercially available kit (DiaMed I D MycroTyping System). However, in a preliminary assay we had difficulty in the interpretation of 5% of the tests due to practical and theoretical problems, the resolution of which was the main purpose of the present work. These problems are outlined below. 1 A large variability in red blood cell (RBC) concentrations for the same test, due to the use of fixed volumes of blood and ID-diluents in the preparation of suspensions. This was the result both of different patients’ haematocrits and of the inconsistent use of coagulated or uncoagulated blood samples. 2 The preparation of RBC suspensions in different concentrations and/or the use of different volumes of
RBC suspensions or serum for different tests is a potential cause of clerical error, with the consequent loss of time and material. 3 The use of unwashed RBC suspensions can theoretically originate both false-positive and falsenegative results: false-positive results in RBC phenotyping by unspecific agglutination of RBC; falsenegative results in direct antiglobulin tests (DAT) and indirect antiglobulin tests (IAT), because without washing antihuman globulin can be neutralized by free immunoglobulins present in the serum (Issit, 1985). 4 The use of enzymes in the RBC suspension medium (ID-diluent 1) for ABO typing made it impossible to ABO type patients with autoimmune haemolytic anaemia correctly, because they systematically originated a ‘false’ AB phenotype. We thus established the best conditions for the application of the G T to routine blood group serology in our blood bank by overcoming the above mentioned problems.
SUMMARY.
Key words: antibody screening; blood typing; gel test.
MATERIALS AND METHODS Blood samples from 197 patients for whom blood transfusions were requested were tested. ABO phenotyping (including A and AB subgroups), Rh and Kell phenotyping, DAT, and antibody screening and compatibility tests by IAT were performed for each sample. M and N phenotyping were also determined in
Correspondence: M. de Figueiredo, Service de Hematologia, Hospital Geral de Santo Antbnio, Largo d o Prof. Abel Salazar, I. 4000 PORTO,Portugal.
115
116
M . de Figueiredo et al.
56 randomly seleded blood samples. Blood samples from 10 individuals with weak D (Du), 57 individuals with irregular antibodies and 17 individuals with positive DAT were also tested. These individuals were selected from our blood bank records. In addition ABO/Rh/Kell phenotyping of five blood samples from patients with autoimmune haemolytic anaemia (AIHA) by warm antibodies was also performed. Blood samples were collected in tubes containing EDTA or in tubes without anticoagulant. DAT was performed only on EDTA samples. Serum was separated by centrifugation of the samples for 5 min at 1000 g.All tests were simultaneously carried out by the conventional tube test (CTT), according to the internationally standardized technique (Mollison et al., 1987). The G T was performed using the ‘ID-DiaMed Micro Typing System’ kit (DiaMed AG CH-3280 Murten Switzerland) according to the manufacturers’ instructions, with the following modifications. 1 In order to avoid the variability in RBC concentrations, suspensions were prepared, not with fixed volumes of blood and ID-Diluent, but with the amount necessary to obtain the desired concentration, which was determined by visual comparison with previously prepared standard suspensions. In order to define the best concentration of RBC suspensions, all tests were simultaneously performed with three concentrations: 1, 3 and 6%. 2 Constant volumes of 50 pI of RBC suspensions and 25 p1 of serum were used in all tests. 3 RBC were washed whenever discrepancies occurred between direct and reverse group typing in the presence of a positive reaction in the negative control microtube. In all negative IAT or DAT tests, the results were controlled by the addition of 50 pl of a 1% IgG-(anti-D) coated RBC suspension, followed by centrifugation. This allowed us to confirm the presence and the activity of the antihuman globulin serum. 4 ABO typing was also performed using saline suspended RBC in patients with positive DAT.
RESULTS A BOIRhlKell phenotyping
ABO/Rh/Kell phenotyping was performed in 197 patients. No ABO weak variants were included. All the results were identical to those obtained with the CTT when 1 and 3% RBC suspensions were used in the GT, except in one patient with A, CcDe, k phenotype who had positive reactions in all microtubes (including the negative control one) using both 1 and 3% RBC concentrations, which suggests an AB, CcDEe, K
phenotype. RBC washing with saline was performed and the correct phenotyping achieved. Difficulties were found in the interpretation of seven results with the 6% concentration: three D+ patients showed a + + + reaction, instead of the usual + + + + reaction; two D- patients showed a weak positive reaction (+); one group A phenotype patient showed a positive (+) reaction in the microtube with the anti-B serum; one CcDe phenotype patient showed a positive (+)reaction in the microtube with the anti-E serum. A mixed-field reaction was detected in Rh phenotyping of three recently transfused patients both by the G T and the CTT. The presence of the two RBC populations was much more easily identified in the GT than in the CTT, by completely sedimented RBC (negative reaction) and ge1 surface retained RBC (+ + + +), simultaneously in the same microtube. The presence of ‘rouleaux’ formation interfered with RBC phenotyping by the CTT in two patients. RBC phenotyping was easily performed in the GT with no need for RBC washing. Positive reactions were observed in all microtubes (including the negative control one) in ABO/Rh/Kell phenotyping in the five blood samples of patients with AIHA by warm antibodies. The reactions remained positive despite RBC washing. When RBC were suspended in saline, instead of the enzyme suspension medium (ID-Diluent I), the negative control was no longer positive and ABO typing became possible. Rh and Kell phenotyping remained impossible with saline-suspended RBC because only negative or weak positive reactions were observed as the typing procedure requires enhancement by enzyme tests. A 1 and A 2 subgroups
A 1 and A2 subgroups were determined in I 10 samples with A or AB phenotype. Eighty-five blood samples subtyped as A 1 and 25 as A2. on examination by CTT. Difficulties were found in the interpretation of the results using GT, whatever RBC concentration was used. According to the manufacturer’s instructions, with ‘Lectin A l ’ cards, A1 phenotype RBC should to + reaction and A2 phenotype show a RBC should show a negative reaction. A1 phenotype RBC should show a negative reaction and A2 phenotype RBC a + to + + + reaction with ‘lectin H’ cards. In our study, when ‘Lectin Al’ cards were used, both A l and A2 blood samples showed negative reactions; when ‘Lectin H’ cards were used, A l blood samples showed 57 negative reactions and 28 weak positive reactions (+), while A2 blood samples correctly showed a + + + reaction.
+
++ +
1 2 3 4 5
6 7 8 9 10 11
12 13 14 15
16 17 18 19
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
46 41 48 49 50
Antibody anti D anti D anti D anti D anti D anti Cw anti E anti D + C anti D C anti D + C anti D E anti E + c anti D + C + E anti D + C + E anti D + C + E anti K anti K anti K anti Jka anti Jka anti Jka anti Jkb anti Fya anti Lea anti Lea anti Lea anti Leb anti Leb anti M anti M anti N anti PI anti PI anti I anti I anti I anti I anti I anti E + I anti E Fya anti E + K anti D + C + E + F y a anti Lea I anti PI+Lua WAA WAA WAA WAA WAA WAA
+ +
+
+
CTT
+ + + + + + + + + + +* + + + + + + + + + + + + + + + + + + + + + + + + + + -t + -t + + + + + + + + +
1 17
Table 1. Continued
Table 1. Comparison of CTT and GT: identification of 57 antibodies Sample
The gel test
Sample
Antibody
CTT
GT
51 52 53 54
WAA WAA CAA CAA UA UA UA
+ + + + + + +
+ + + + + + +
GT
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
55
56 57
CTT =conventional tube test. GT = gel centrifugation test. WAA =warm autoantibody. CAA = cold autoantibody. UA = unidentified antibody. Anti E not found. t Anti E not found. $ Anti K not found.
M /N phen otyping No differences were found between the results obtained from 1 and 3% RBC suspensions. Difficulties were found in the interpretation of the results using 6% RBC suspensions (data not shown). Antibody screening and identification
Antibody screening and identification was performed with IAT in the 197 blood samples. Irregular antibodies were found in two samples. No differences were seen between the results obtained by both methods. When the 57 blood samples of individuals with irregular antibodies were studied, discrepancies were found in four samples (Table 1). Anti-E antibodies were not identified with the CTT in one anti-E+T and in one anti-E + c associations. One anti-Kell antibody was unidentified with the CTT in one anti-E+Kell association. One anti-Le b antibody was identified only with the CTT. Compatibility tests
Four hundred and three compatibility tests with IAT were performed with the 197 blood samples. No differences were observed between the results obtained with the 1% RBC suspension GT and the CTT (five positive tests and 398 negative tests). These five positive results were positive by the GT with all RBC concentrations used, however, with the 3% and the 6% RBC suspensions, respectively, 120 and 163 weak positive (+) reactions still occurred with the GT, in spite of negative reactions with the CTT and with the 1% RBC suspensions GT. Direct antiglobulin test
One hundred and ninety-seven DAT were performed.
118
M . de Figueiredo et al.
Eleven DAT were positive using both methods. Among the remaining 186 blood samples, nine positive DAT were found only with the GT, with all RBC concentrations used. In addition, four and 23 positive results were found with the GT when 3 and 6% RBC suspensions were used, respectively. Blood samples from 17 patients with previously known positive DAT of different specificities ( 5 IgG, 2 C3d, 9 IgG +C3d and 1 IgG + C3d + IgA) were also studied with the GT. Positive results were obtained using polyspecific antiglobulin serum-containing cards, with all RBC concentrations used. DISCUSSION When we tried to introduce the GT in to our routine blood group serology, following the instructions of the commercially available kit, difficulties were found in the interpretation of about 5% of the results. This occurred because either too high or too low RBC concentrations were obtained. High concentrations led to the spread of RBC through the gel originating false-positive results, and low concentrations led to difficulties in the visualization of RBC. Using 50 p1 of RBC suspensions for all tests, the ideal concentration was 1 % because no problems occurred in the interpretation of the results. More concentrated suspensions (3 and 6%) led to false-positive results. False-positive results were, however, easily identified in RBC phenotyping, as discrepancies occurred between red cell and reverse grouping, and a positive reaction appeared in the ‘negative control’ tube. Although the G T can be routinely performed with no need for previous washing of RBC, this is necessary, however, whenever a positive control is observed. The absence of washing steps in DAT and IAT did not lead to false-negative reactions. The addition of IgG-coated RBC to all microtubes with a negative reaction, followed by centrifugation, demonstrated that the antihuman globulin serum was reactive. As ABO/Rh phenotyping is performed using enzyme-treated RBC, DAT positive samples showed positive reactions in all microtubes, including the control. In the fine tested cases, ABO phenotyping was successfully performed when saline-suspended RBC were used. It is possible that using saline-suspended RBC can cause some weak ABO variants to be missed. This has not been confirmed in this study because no weak ABO variants were included. Rh phenotyping could not be determined in DAT-positive patients,
either with the GT or with the C’IT because with saline-suspended RBC the reactions were all negative or weakly positive as the typing reagents require enzyme-enhanced tests. Some discrepancies were found between the G T and the CTT when DAT was performed. Nine cases of positive DAT with the GT, which occurred even when a 1 % RBC suspension concentration was used, were negative with the CTT. In these cases the intensity of the reaction with the GT was always + to + f , although the screening of antibodies in the eluate with the CTT was negative. Most of these weak-positive reactions occurred with blood samples of patients who belonged to groups in which a positive DAT was expected (three polytransfused patients, one patient who had been transfused with plasma on the previous day, two patients with systemic lupus erythematosus and one patient who was receiving penicilin treatment). DAT became positive also with the CTT a few days later in this patient, who afterwards developed a penicillin-induced haemolytic anaemia. The apparent increase in sensitivity could be explained by decreased elution of antibodies attached to the RBC membrane due to lack of washing and less manipulation of RBC (Petz, 1980). One problem that remains to be solved is a subgroup phenotyping with the GT. A possible explanation for the results we obtained is that lectins used in the cards were adsorbed by the plastic. REFERENCES Hitzler, W., Schomig-Breckner, H. & Mathias, D. (1989)GeI centrifugation test-a new micro method for blood group typing and antibody screening. Arziliche Laboratorium, 35,89-92. Issit. P.D. (1985) Applied Blood Group Serology, 3rd edn, 72115 . Montgomery Scientfic Publications, Miami, FL. Lapierre, Y. (1989) Le gel test: un nouveau procede en Immuno-Himatologie.Spectra Bioiogie, 89(4), 38-42. Lapierre, Y., Rical, D., Adam, J. et al. (1990) The gel test: a new way to detect red cell antigen-antibody reactions. Transfusion, 30, 109- 1 13.
Mollison, P.L., Engelfriet, C.P. & Contreras, M. (1987) Blood Transfiion in Clinical Medicine, 8th edn, Blackwell Scientific Publication, Oxford. Petz, L.D. (1980) Acquired Immune Hemoiytic Anemias, 1st edn, Churchill Livingstone, New York. Schutt, K.H. & Arndt-Hanser, A. (1989) Erfahrungen mit dem ID-Microtyping System zur Blutgruppen und Antikorperbestimmungin der Routine. Arztliche Laboratorium 35,287-292.
