PERFORMING ELUTION TEST WITH MONOSPECIFIC ANTIHUMAN GLOBULIN UNMASKS MULTIPLE IMMUNOGLOBULIN COATING; IMPLICATIONS ON IMPROVING MULTIPLE IMMUNOGLOBULIN DETECTION SENSITIVITY IN AIHA SCREENING.
(Abstract release date: 05/19/16)
EHA Library. Bezirgiannidou Z. 06/09/16; 133123; E1574
Dr. Zoi Bezirgiannidou
Contributions
Contributions
Abstract
Abstract: E1574
Type: Eposter Presentation
Background
Autoimmune Haemolytic Anemia (AIHA) basic screening includes: (i) Direct Antiglobulin Test (DAT) using monospecific antihuman globulin (MAHG), namely anti-IgG, anti-IgA, anti-IgM, anti-C3c and anti-C3d, to detect the autoantibody immunoglobulin (Ig) class(es) or complement fragments on erythrocyte membrane; (ii) Indirect Antiglobulin Test (IAT) using polyspecific antihuman globulin (PAHG), comprised of anti-IgG and anti-C3d, to identify autoantibody(ies) in serum or red blood cells (RBCs) elution. Current elution techniques are valuable for allowing the identification of antibody specificity and the elimination of false positive DAT cases. The use of only anti-IgG in IAT implies that: (a) Non-IgG classes causing AIHA will be missed in elution test, rendering false negative results, (b) Elution test in AIHA caused by multiple Ig classes would allow non-IgG classes to be missed, generating misleading results.
Aims
To highlight that in DAT (+) RBCs coated with multiple Igs, DAT is not sufficient to unmask all the putative Ig classes and to suggest a method that potently unmasks multiple Ig coating.
Methods
Acid elution (DiaCidel/BIO-RAD) was performed on 59 patients’ DAT (+) RBCs. The eluates were screened by IAT against a commercially available panel of RBCs (ID-DiaPanel) using PAHG Liss/Coombs gel cards (Biorad, Switzerland). An additional method using MAHG gel cards (DC-Screening I/BIO-RAD) in elution IAT was also applied. Seven patients were excluded from the study since their DAT positivity was due to alloantibodies following recent transfusion or non-specific reactions. Of the remaining 52 patients, only 21 (40.38%) fulfilled the diagnostic criteria for autoimmune haemolysis; 17 presented with AIHA and 4 with Evans syndrome. DAT positivity without haemolysis was observed in 31 (59.61%) patients, representing a random finding during pretransfusion testing.
Results
Initial screening of blood samples by DAT identified 49 warm and 2 cold autoantibodies, as well as one mixed type autoantibody. When AIHA was caused by multiple Ig classes, the use of MAHG in elution IAT allowed the identification of Ig classes that were not detected by the standard DAT. In detail, six DAT (+) and IgM (-) cases by DAT were identified as IgM (+) by elution IAT; three IgG (-) DAT (+) cases were found to be IgG (+) by elution IAT; and five DAT (+) IgA (-) cases by DAT were identified as IgA (+) by elution IAT. Overall, in 14 out of 52 cases (26.92%), the DAT missed to identify multiple Igs coating. Even though elution IAT with PAHG would identify the three IgG (-) DAT (+) cases, still 11 cases with multiple Igs (21.15%) would have been missed, a statistically significant difference (p=0.012). It is notable that 11 of the 14 missed cases (78.57%) presented with severe autoimmune haemolysis.
Conclusion
In cases of AIHA caused by multiple Ig classes, we assume that monospecific antiserums in DAT bind mainly with the dominant Ig class due to the high concentration of bound antibodies on the RBC membrane (like the prozone phenomenon). This constraint is bypassed when Igs are discarded from the RBC membrane in elution, since they are available to react with PAHG by IAT. However, the latter applies only for IgGs, since non-IgGs are not detectable by PAHG. In conclusion, we report an efficient method for unmasking non-IgGs participating in multiple Ig coating, i.e., the use of MAHG in IAT elution. This method may have clinical implications, because AIHA is frequently severe when multiple Ig coating occurs.
Session topic: E-poster
Keyword(s): Autoimmune hemolytic anemia (AIHA), Hemolysis, Immunoglobulin, Monoclonal antibody
Type: Eposter Presentation
Background
Autoimmune Haemolytic Anemia (AIHA) basic screening includes: (i) Direct Antiglobulin Test (DAT) using monospecific antihuman globulin (MAHG), namely anti-IgG, anti-IgA, anti-IgM, anti-C3c and anti-C3d, to detect the autoantibody immunoglobulin (Ig) class(es) or complement fragments on erythrocyte membrane; (ii) Indirect Antiglobulin Test (IAT) using polyspecific antihuman globulin (PAHG), comprised of anti-IgG and anti-C3d, to identify autoantibody(ies) in serum or red blood cells (RBCs) elution. Current elution techniques are valuable for allowing the identification of antibody specificity and the elimination of false positive DAT cases. The use of only anti-IgG in IAT implies that: (a) Non-IgG classes causing AIHA will be missed in elution test, rendering false negative results, (b) Elution test in AIHA caused by multiple Ig classes would allow non-IgG classes to be missed, generating misleading results.
Aims
To highlight that in DAT (+) RBCs coated with multiple Igs, DAT is not sufficient to unmask all the putative Ig classes and to suggest a method that potently unmasks multiple Ig coating.
Methods
Acid elution (DiaCidel/BIO-RAD) was performed on 59 patients’ DAT (+) RBCs. The eluates were screened by IAT against a commercially available panel of RBCs (ID-DiaPanel) using PAHG Liss/Coombs gel cards (Biorad, Switzerland). An additional method using MAHG gel cards (DC-Screening I/BIO-RAD) in elution IAT was also applied. Seven patients were excluded from the study since their DAT positivity was due to alloantibodies following recent transfusion or non-specific reactions. Of the remaining 52 patients, only 21 (40.38%) fulfilled the diagnostic criteria for autoimmune haemolysis; 17 presented with AIHA and 4 with Evans syndrome. DAT positivity without haemolysis was observed in 31 (59.61%) patients, representing a random finding during pretransfusion testing.
Results
Initial screening of blood samples by DAT identified 49 warm and 2 cold autoantibodies, as well as one mixed type autoantibody. When AIHA was caused by multiple Ig classes, the use of MAHG in elution IAT allowed the identification of Ig classes that were not detected by the standard DAT. In detail, six DAT (+) and IgM (-) cases by DAT were identified as IgM (+) by elution IAT; three IgG (-) DAT (+) cases were found to be IgG (+) by elution IAT; and five DAT (+) IgA (-) cases by DAT were identified as IgA (+) by elution IAT. Overall, in 14 out of 52 cases (26.92%), the DAT missed to identify multiple Igs coating. Even though elution IAT with PAHG would identify the three IgG (-) DAT (+) cases, still 11 cases with multiple Igs (21.15%) would have been missed, a statistically significant difference (p=0.012). It is notable that 11 of the 14 missed cases (78.57%) presented with severe autoimmune haemolysis.
Conclusion
In cases of AIHA caused by multiple Ig classes, we assume that monospecific antiserums in DAT bind mainly with the dominant Ig class due to the high concentration of bound antibodies on the RBC membrane (like the prozone phenomenon). This constraint is bypassed when Igs are discarded from the RBC membrane in elution, since they are available to react with PAHG by IAT. However, the latter applies only for IgGs, since non-IgGs are not detectable by PAHG. In conclusion, we report an efficient method for unmasking non-IgGs participating in multiple Ig coating, i.e., the use of MAHG in IAT elution. This method may have clinical implications, because AIHA is frequently severe when multiple Ig coating occurs.
Session topic: E-poster
Keyword(s): Autoimmune hemolytic anemia (AIHA), Hemolysis, Immunoglobulin, Monoclonal antibody
Abstract: E1574
Type: Eposter Presentation
Background
Autoimmune Haemolytic Anemia (AIHA) basic screening includes: (i) Direct Antiglobulin Test (DAT) using monospecific antihuman globulin (MAHG), namely anti-IgG, anti-IgA, anti-IgM, anti-C3c and anti-C3d, to detect the autoantibody immunoglobulin (Ig) class(es) or complement fragments on erythrocyte membrane; (ii) Indirect Antiglobulin Test (IAT) using polyspecific antihuman globulin (PAHG), comprised of anti-IgG and anti-C3d, to identify autoantibody(ies) in serum or red blood cells (RBCs) elution. Current elution techniques are valuable for allowing the identification of antibody specificity and the elimination of false positive DAT cases. The use of only anti-IgG in IAT implies that: (a) Non-IgG classes causing AIHA will be missed in elution test, rendering false negative results, (b) Elution test in AIHA caused by multiple Ig classes would allow non-IgG classes to be missed, generating misleading results.
Aims
To highlight that in DAT (+) RBCs coated with multiple Igs, DAT is not sufficient to unmask all the putative Ig classes and to suggest a method that potently unmasks multiple Ig coating.
Methods
Acid elution (DiaCidel/BIO-RAD) was performed on 59 patients’ DAT (+) RBCs. The eluates were screened by IAT against a commercially available panel of RBCs (ID-DiaPanel) using PAHG Liss/Coombs gel cards (Biorad, Switzerland). An additional method using MAHG gel cards (DC-Screening I/BIO-RAD) in elution IAT was also applied. Seven patients were excluded from the study since their DAT positivity was due to alloantibodies following recent transfusion or non-specific reactions. Of the remaining 52 patients, only 21 (40.38%) fulfilled the diagnostic criteria for autoimmune haemolysis; 17 presented with AIHA and 4 with Evans syndrome. DAT positivity without haemolysis was observed in 31 (59.61%) patients, representing a random finding during pretransfusion testing.
Results
Initial screening of blood samples by DAT identified 49 warm and 2 cold autoantibodies, as well as one mixed type autoantibody. When AIHA was caused by multiple Ig classes, the use of MAHG in elution IAT allowed the identification of Ig classes that were not detected by the standard DAT. In detail, six DAT (+) and IgM (-) cases by DAT were identified as IgM (+) by elution IAT; three IgG (-) DAT (+) cases were found to be IgG (+) by elution IAT; and five DAT (+) IgA (-) cases by DAT were identified as IgA (+) by elution IAT. Overall, in 14 out of 52 cases (26.92%), the DAT missed to identify multiple Igs coating. Even though elution IAT with PAHG would identify the three IgG (-) DAT (+) cases, still 11 cases with multiple Igs (21.15%) would have been missed, a statistically significant difference (p=0.012). It is notable that 11 of the 14 missed cases (78.57%) presented with severe autoimmune haemolysis.
Conclusion
In cases of AIHA caused by multiple Ig classes, we assume that monospecific antiserums in DAT bind mainly with the dominant Ig class due to the high concentration of bound antibodies on the RBC membrane (like the prozone phenomenon). This constraint is bypassed when Igs are discarded from the RBC membrane in elution, since they are available to react with PAHG by IAT. However, the latter applies only for IgGs, since non-IgGs are not detectable by PAHG. In conclusion, we report an efficient method for unmasking non-IgGs participating in multiple Ig coating, i.e., the use of MAHG in IAT elution. This method may have clinical implications, because AIHA is frequently severe when multiple Ig coating occurs.
Session topic: E-poster
Keyword(s): Autoimmune hemolytic anemia (AIHA), Hemolysis, Immunoglobulin, Monoclonal antibody
Type: Eposter Presentation
Background
Autoimmune Haemolytic Anemia (AIHA) basic screening includes: (i) Direct Antiglobulin Test (DAT) using monospecific antihuman globulin (MAHG), namely anti-IgG, anti-IgA, anti-IgM, anti-C3c and anti-C3d, to detect the autoantibody immunoglobulin (Ig) class(es) or complement fragments on erythrocyte membrane; (ii) Indirect Antiglobulin Test (IAT) using polyspecific antihuman globulin (PAHG), comprised of anti-IgG and anti-C3d, to identify autoantibody(ies) in serum or red blood cells (RBCs) elution. Current elution techniques are valuable for allowing the identification of antibody specificity and the elimination of false positive DAT cases. The use of only anti-IgG in IAT implies that: (a) Non-IgG classes causing AIHA will be missed in elution test, rendering false negative results, (b) Elution test in AIHA caused by multiple Ig classes would allow non-IgG classes to be missed, generating misleading results.
Aims
To highlight that in DAT (+) RBCs coated with multiple Igs, DAT is not sufficient to unmask all the putative Ig classes and to suggest a method that potently unmasks multiple Ig coating.
Methods
Acid elution (DiaCidel/BIO-RAD) was performed on 59 patients’ DAT (+) RBCs. The eluates were screened by IAT against a commercially available panel of RBCs (ID-DiaPanel) using PAHG Liss/Coombs gel cards (Biorad, Switzerland). An additional method using MAHG gel cards (DC-Screening I/BIO-RAD) in elution IAT was also applied. Seven patients were excluded from the study since their DAT positivity was due to alloantibodies following recent transfusion or non-specific reactions. Of the remaining 52 patients, only 21 (40.38%) fulfilled the diagnostic criteria for autoimmune haemolysis; 17 presented with AIHA and 4 with Evans syndrome. DAT positivity without haemolysis was observed in 31 (59.61%) patients, representing a random finding during pretransfusion testing.
Results
Initial screening of blood samples by DAT identified 49 warm and 2 cold autoantibodies, as well as one mixed type autoantibody. When AIHA was caused by multiple Ig classes, the use of MAHG in elution IAT allowed the identification of Ig classes that were not detected by the standard DAT. In detail, six DAT (+) and IgM (-) cases by DAT were identified as IgM (+) by elution IAT; three IgG (-) DAT (+) cases were found to be IgG (+) by elution IAT; and five DAT (+) IgA (-) cases by DAT were identified as IgA (+) by elution IAT. Overall, in 14 out of 52 cases (26.92%), the DAT missed to identify multiple Igs coating. Even though elution IAT with PAHG would identify the three IgG (-) DAT (+) cases, still 11 cases with multiple Igs (21.15%) would have been missed, a statistically significant difference (p=0.012). It is notable that 11 of the 14 missed cases (78.57%) presented with severe autoimmune haemolysis.
Conclusion
In cases of AIHA caused by multiple Ig classes, we assume that monospecific antiserums in DAT bind mainly with the dominant Ig class due to the high concentration of bound antibodies on the RBC membrane (like the prozone phenomenon). This constraint is bypassed when Igs are discarded from the RBC membrane in elution, since they are available to react with PAHG by IAT. However, the latter applies only for IgGs, since non-IgGs are not detectable by PAHG. In conclusion, we report an efficient method for unmasking non-IgGs participating in multiple Ig coating, i.e., the use of MAHG in IAT elution. This method may have clinical implications, because AIHA is frequently severe when multiple Ig coating occurs.
Session topic: E-poster
Keyword(s): Autoimmune hemolytic anemia (AIHA), Hemolysis, Immunoglobulin, Monoclonal antibody
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