Monoclonal Antibody-specific Immobilization Of Granulocyte Antigens Research Articles

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

Leukocyte-Reactive Antibodies in Female Blood Donors: The Austrian Experience

Introduction: Antibody-mediated transfusion-related acute lung injury (TRALI) is caused by antibodies against human leukocyte antigens (HLAs) or human neutrophil antigens (HNAs), and is one of the most serious complications associated with transfusion medicine. Prevention strategies like testing allo-exposed female blood donors have not yet been introduced nationwide in Austria. To assess the need and feasibility of routine leukocyte antibody testing, the prevalence of leukocyte-reactive antibodies in an Austrian female donor population was been determined using classical cell-based methods which were compared with a high-throughput bead-based method. Methods: Sera from 1,022 female blood donors were screened using a granulocyte aggregation test (GAT) and a white blood cell immunofluorescence test (WIFT) after retesting and specification of positive samples by granulocyte immunofluorescence test (GIFT) and monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA). Potential HLA reactivities were confirmed using the microbeads assay LabScreen^TM Mixed. The results in 142 donor sera and 38 well-defined reference sera were investigated by the microbeads assay LabScreen^TM Multi and compared with classical cell-based methods. Results: Reactivity with either granulocytes and/or lymphocytes was detected in 79 sera (7.7%), with the majority being HLA-specific. Antibodies against HNA were obtained in 7 samples (0.7%). The aggregating potential of the detected antibodies was observed in 9 cases (0.9%). Most of the leukocyte-reactive antibodies occurred at a donor age of between 35 and 59 years (n = 61). LabScreen Multi showed good agreement (κ = 0.767) for HNA antibody detection by cell-based assays, but double/multiple specificities (100% of 7 anti-HNA-1b sera) as well as false-negative results (40% of 15 HNA-3-specific sera) occurred. Discussion: Leukocyte-reactive antibody screening is advised in Austrian female donors for safe blood transfusion, including single-donor convalescent plasma treatment of COVID-19 that may be implemented soon. For the introduction of LabScreen Multi, the combination with GAT should be considered to ensure correct anti-HNA-3a detection.

Work‐up in the case of granulocyte antibodies

Granulocyte allo‐ and autoantibodies have been implicated in a variety of clinical conditions such as primary and secondary autoimmune neutropenia (AIN), alloimmune neutropenia (NAIN), transfusion‐related acute lung injury (TRALI) as well as febrile and severe pulmonary transfusion reactions. The classic granulocyte agglutination test (GAT) in combination with the granulocyte indirect immunofluorescence test (GIFT) can detect nearly all relevant antibodies. HNA‐1, ‐2, 4, 5 and HLA class I antibodies are clearly detectable in the GIFT while HNA‐3 antibodies strongly agglutinate neutrophils in the GAT. The glycoprotein‐specific and highly sensitive monoclonal antibody‐specific immobilization of granulocyte antigens (MAIGA) test detects all human neutrophil antigen (HNA) antibodies except for HNA‐3 but is time‐consuming and requires highly skilled personnel. For TRALI diagnostics, laboratory testing is completed by the indirect lymphocyte immunofluorescence test (LIFT), and HLA class I and II ELISAs or fluorescent bead‐based assays (Luminex). The latter also enable faster and more automated HNA antibody detection but to date not all specificities, especially HNA‐3, can be reliably detected so that still the classical tests have to complete the methodological spectrum. HNA allelotyping by PCR methods is the first choice for all HNA except for HNA‐2 because the molecular reason for the HNA‐2null phenotype is not completely understood. Establishing a PCR‐SSP reaction for the main CD177*787A>T polymorphism comprises the risk to miss other causes so that HNA‐2 is still typed serologically. Granulocyte serology even today is widely based on a variety of manual methods, requires experienced laboratory staff and profound knowledge of granulocyte immunobiology.

Detection of granulocyte‐reactive antibodies: a comparison of different methods

Granulocyte-reactive antibodies can cause autoimmune and neonatal immune neutropenias as well as transfusion-related acute lung injury. The classical antibody-detection methods granulocyte aggregation test (GAT), granulocyte immunofluorescence test (GIFT) and monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are time-consuming and technically challenging. In recent years, flow cytometric white blood cell immunofluorescence test (Flow-WIFT) and the microbeads assay LabScreen® Multi have emerged and are still subject of evaluation. These serological tests were compared on a screening and specification level. For screening, the combination of GAT/GIFT was compared to Flow-WIFT testing 333 samples. Positive samples were further analysed with MAIGA and LabScreen® Multi. Granulocyte aggregation test/GIFT detected 77 positive samples, Flow-WIFT found 108 granulocyte-reactive samples. Six Samples were only positive in GAT/GIFT, and 37 samples were only positive in Flow-WIFT (κ = 0.682). Antibody specification with MAIGA and the microbeads assay confirmed granulocyte-reactivity in 83 cases with 70 matching results (κ = 0.742). However, out of six detected human neutrophil antigen (HNA) reactivities only two specificities matched in both assays. Flow-WIFT may be a valuable addition to GIFT for granulocyte-reactive antibody screening. MAIGA remains the most reliable laboratory method for antibody specification.

Advances in granulocyte test methodologies

AbstractAlloantibodies to human neutrophil antigens (HNA) are involved in clinical conditions such as neonatal alloimmune neutropenia (NAN), autoimmune neutropenia (AIN) and transfusion‐related acute lung injury (TRALI). For the diagnosis of these conditions, the detection of the causative antibody is essential. Presently, the use of a combination of granulocyte agglutination assay (GAT) and granulocyte immunofluorescence test (GIFT) for the screening of granulocyte antibodies, followed by the determination of antibody specificity by the monoclonal antibody‐specific immobilization of granulocyte antigens (MAIGA) is recommended by the ISBT Working Party on Granulocyte Immunobiology. In Japan, the mixed‐passive hemagglutination (MPHA) assay, a method originally developed for the detection of anti‐platelet antibodies, was adapted for the testing of anti‐granulocyte antibodies. Although various technologies are available, presently, no one of the methods alone is able to detect all clinically relevant antibodies, thus their combination is important, and the development of new methodologies is desired. In this review, we describe the presently available classical methods for anti‐granulocyte antibody detection, including GIFT, GAT, MAIGA and MPHA, and also the new technologies, especially focusing on the advantages and problems of the different methods.

Detection of granulocyte antibodies using simultaneous analysis of specific granulocyte antibodies assay (SASGA)

Granulocyte-associated antibodies can cause several clinical granulocytopenic disorders. The monoclonal-antibody-specific immobilization of granulocyte antigens (MAIGA) is currently used as the standard assay to specify these antibodies. Here we describe an assay for specific analysis of granulocyte antibodies (SASGA) which is able to simultaneously detect and specify granulocyte IgG- and IgM-antibodies using flow cytometry. Bead populations with distinct fluorescence intensities were used as solid phase for immobilization of mAb. Typed granulocytes were incubated with human sera and a mix of three distinct mouse monoclonal antibodies against specific granulocyte antigens (for example CD16, CD11a, HLA class I). After cell lysis and incubation of lysate with beads, goat antibodies against human IgG and IgM antibodies were added. Seventy-one frozen sera of donors and patients previously implicated in transfusion reactions and various underlying disorders were analysed for specific granulocyte-binding antibodies using MAIGA and SASGA. The SASGA assay was able to simultaneously detect granulocyte-specific antibodies for different glycoproteins. Overall, the results of MAIGA and SASGA were concordant in 92·9%. 5 sera containing anti-HNA-1b (n=2) and -HLA class I (n=3) were not detected by MAIGA, but were recognized by the SASGA. In serial dilution tests with sera containing anti-HNA-1a, -1b, -2a and HLA class I, the SASGA assay detected the antibodies at higher dilutions than MAIGA. The SASGA assay permits reliable detection of specific granulocyte antibodies. Six distinct antibodies can be simultaneously determined. This method will potentially open the way to investigations on additional specific antibodies as it facilitates laboratory diagnosis.

Primary autoimmune neutropenia in children: a study of neutrophil antibodies and clinical course

Primary autoimmune neutropenia (AIN) in children is characterized by severe neutropenia, but mild bacterial infections and a spontaneous resolution. Neutrophil autoantibodies are involved in the disease. The precise relationship between the specificity and level of reactivity of the antibodies with the absolute neutrophil count and frequency of infections is not known. To obtain a better insight into this relationship, we performed a follow-up study in 15 patients with primary AIN. In addition, we performed two different neutrophil antibody tests to evaluate their sensitivity and specificity. Blood samples from 15 children were tested for neutrophil antibodies, at different time-points during the disease, by using the indirect granulocyte immunofluorescence test (GIFT) and the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay. Clinical data related to the occurrence of bacterial infections and treatment, and neutrophil counts were collected. Early in the disease, antibodies with pan-FcRIIIb specificity were detected, and HNA-1a or HNA-1b specificity of the antibodies developed over time. The sensitivity and specificity of neutrophil antibody detection tests were higher in the GIFT than in the MAIGA assay. Variables predicting time of recovery from neutropenia were not found. Prophylactic antibiotics led to the almost complete disappearance of infections. In patients with primary neutropenia, neutrophil antibody specificity changes over time. Prophylactic antibiotics do benefit the patients.

Report on the second international granulocyte serology workshop.

The methods of granulocyte antibody and antigen detection have improved considerably since the First International Granulocyte Serology Workshop. Thirteen laboratories participated in the Second International Granulocyte Serology Workshop. The study was designed to meet five goals: 1) establishment of antigen-typed granulocyte panels, 2) determination of the proficiency of granulocyte antibody detection by laboratory and by technique, 3) identification of granulocyte antibodies present in uncharacterized sera, 4) genotyping of NA antigens by DNA-based techniques, and 5) collection and exchange of information to standardize granulocyte antibody screening. NA1-, NA2- and NB1-specific antibodies were detected by more than two-thirds of the participants with the granulocyte immunofluorescence test (GIFT) but by fewer than two-thirds with the granulocyte agglutination test (GAT). The determination of the NB2-, 5b-, and Fc gamma RIIIb-specific antibodies was the most problematic. Ninety percent of the participants were not able to identify NB2 antibodies in sera with suspected NB2 specificity. The 5b antibodies were only detected by laboratories that performed the GAT. Isoantibodies to Fc gamma RIIIb were identified when the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay or an Fc gamma RIIIb-deficient panel cell was available. In 4 of 11 uncharacterized sera, the presence of NA1, NA2, and Fc gamma RIIIb antibodies could be confirmed by the MAIGA assay. In polymerase chain reaction with sequence-specific primers (PCR-SSP), the NA genotypes of all DNA samples were correctly determined. A combination of the GIFT and GAT is still the best means of antibody detection. The MAIGA assay allows identification of isoantibodies to Fc gamma RIIIb or alloantibodies to NA antigens. The NA genotype can be reliably determined by PCR-SSP. Cell panels should cover NA1, NA2, NB1, 5b, and, if possible, SH.

Characterization of a New Alloantigen (SH) on the Human Neutrophil Fcγreceptor IIIb

AbstractPolymorphic structures of the neutrophil Fcγreceptor IIIb (FcγRIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on FcγRIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody–specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the FcγRIIIb. Nucleotide sequence analysis of the FcγRIIIb coding region from a SH(+) individual showed a single-base C→A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN I showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the FcγRIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the FcγRIIIb. Our findings show the existence of an additional polymorphism of the FcγRIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.