Vox Sanguinis (2015) 108, 287–293 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12227
ORIGINAL PAPER
Detection of granulocyte-reactive antibodies: a comparison of different methods M. W. Heinzl, M. Sch€ onbacher, E.-M. Dauber, S. Panzer, W. R. Mayr & G. F. K€ orm€ oczi Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria
Background and Objectives 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. Materials and Methods 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. Results Granulocyte aggregation test/GIFT detected 77 positive samples, FlowWIFT found 108 granulocyte-reactive samples. Six Samples were only positive in GAT/GIFT, and 37 samples were only positive in Flow-WIFT (j = 0682). Antibody specification with MAIGA and the microbeads assay confirmed granulocyte-reactivity in 83 cases with 70 matching results (j = 0742). However, out of six detected human neutrophil antigen (HNA) reactivities only two specificities matched in both assays.
Received: 13 July 2014, revised 15 October 2014, accepted 3 November 2014, published online 30 December 2014
Conclusion Flow-WIFT may be a valuable addition to GIFT for granulocyte-reactive antibody screening. MAIGA remains the most reliable laboratory method for antibody specification. Key words: flow cytometry, granulocyte immunology, granulocyte-reactive antibodies, human neutrophil antigens, microbeads assay.
Introduction Granulocyte-reactive antibodies can be a cause for different medical conditions such as transfusion-related acute lung injury (TRALI), ineffectiveness of granulocyte transfusions, autoimmune neutropenia (AIN) and neonatal immune neutropenia (NIN) [1–3]. Such antibodies can be directed against human leucocyte antigens (HLA) class I Correspondence: G€unther K€orm€oczi, M.D., Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, W€ahringer G€urtel 18-20, 1090 Vienna, Austria E-mail: [email protected] Marlies Sch€onbacher and Matthias Heinzl contributed equally to this study.
or human neutrophil antigens (HNA). Granulocyte-reactive alloantibodies are usually formed by pregnant women or transfused patients. Despite their low prevalence, HNA antibodies are especially dangerous in regard to TRALI [4]. The technically challenging and time consuming combination of granulocyte aggregation test (GAT) and granulocyte immunofluorescence test (GIFT) using a panel of typed and freshly isolated granulocytes is currently the recommended laboratory procedure for HNA antibody detection. Panel reactivity can indicate antibody specificity, which can be confirmed with monoclonal antibodyspecific immobilization of granulocyte antigens (MAIGA). MAIGA is a glycoprotein-specific assay using monoclonal
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capture antibodies and has special importance in identifying mixtures of both HLA- and HNA-specific antibodies [5–7]. However, it is important to note that negative results in MAIGA do not necessarily indicate false-positive reactions in the screening tests. Negativity in MAIGA may be due to the non-existence of suitable capture antibodies, sterical hindrance and other effects such as the prozone phenomenon. Although GIFT is known to detect all membranebound granulocyte-reactive antibodies, it is important to additionally perform GAT. GAT has the ability to detect aggregating antibodies like anti-HNA-3a, which are especially important as they are responsible for most of the fatal TRALI reactions [4] and cannot be found in MAIGA because there are no functional capture antibodies available. Furthermore, IgM antibodies are not detected by GIFT using standard secondary antibodies (anti-IgG) but can induce aggregation in GAT. Other techniques such as flow cytometric white blood cell immunofluorescence test (Flow-WIFT, also known as Flow-GIFT) for antibody screening and the microbeads assay LabScreen Multi for antibody specification have emerged more recently and are still subject of evaluation [6, 8, 9]. Flow-WIFT has been reported to be an excellent test for granulocyte antibody screening with great sensitivity for all known HNA antibodies and the potential to be automated. Furthermore, 7-aminoactinomycin D (7-AAD) can be added to identify dead cells to evaluate the cell quality after isolation and the cytotoxicity of antibodies. Importantly, Flow-WIFT can also be used to evaluate antibody binding to monocytes and lymphocytes, which allows the differentiation between granulocyte-specific antibodies (anti-HNA-1 and -2) and antibodies reactive with different leucocyte subsets (anti-HNA-3, -4, -5 and HLA-antibodies) and excludes the necessity of separately performing a lymphocyte immunofluorescence test (LIFT) in addition to GIFT [6, 8]. The commercially available microbeads assay LabScreen Multi detects antibodies to HLA class I and II as well as HNA-1 and -2 antibodies. The true sensitivity and specificity of this rather expensive kit regarding HNA antibodies are not yet entirely clear. Although results have been shown to correlate well with other techniques for the screening of granulocyte-reactive alloantibodies, there were discrepancies reported with respect to antibody specificities and correlation was only moderate for autoimmune antibodies. Furthermore, the highly relevant HNA-3-specific antibodies cannot be detected [6, 9]. Like in all recombinant antigen-based tests, there is a risk that highly variant and conformation-sensitive antibodies may be missed in this assay. Furthermore, there is currently no universally accepted cut-off value to define positive
results. The true value of this bead-based technique for screening as well as specification purposes is thus yet to be explored [6, 9]. To gain more information on the properties of these test systems, Flow-WIFT was compared to the combination of GAT and GIFT as a screening test, and the microbeads assay was assessed for the determination of antibody specificities in comparison with MAIGA.
Materials and methods This study was approved by the Ethical Committee of the Medical University of Vienna. The applied cell panel for antibody screening was assembled from donors previously typed for HNA by sequence-specific priming PCR (HNA Ready Gene, inno-train, Kronberg, Germany) and serologic typing and covered HNA-1a, -1b, -1c, -2, -3a, -3b, -4a and -5a as well as the most important HLA phenotypes. Cells were isolated from freshly drawn EDTA whole blood by dextran sedimentation. For GAT and GIFT, granulocytes were separated by density gradient centrifugation using Ficoll PaqueTM Plus (GE Healthcare, Uppsala, Sweden), erythrocytes were depleted by ammonium chloride lysis, with subsequent paraformaldehyde (PFA) fixation of the granulocytes for microscopic GIFT [4]. For Flow-WIFT, density gradient centrifugation and PFA fixation were not necessary. For all screening methods, cells were adjusted to a concentration of 5 9 103 white blood cells/ll.
Screening for HLA and HNA antibodies For the comparison of screening methods for granulocyte-reactive antibodies, 333 samples from women with a history of pregnancy (second trimenon at least; 143 samples containing anti-red blood cell (RBC) antibodies) were tested. These samples originated from blood withdrawals for the diagnostic routine pretransfusion and anti-RBC antibody testing at the Medical University of Vienna. Data regarding RBC antibodies as a risk indicator for granulocyte alloimmunization in this collective were presented in a previously published study [10]. Samples were collected in a consecutive manner. Furthermore, historic samples containing anti-RBC antibodies were added to increase chances for the presence of granulocyte-reactive antibodies. No further clinical data were available for these samples. Screening for granulocyte-reactive antibodies was performed with a panel of three cells using the classical methods GAT [11] and microscopic GIFT [12] as well as the recently described Flow-WIFT [6, 8]. Flow-WIFT was performed as follows: 50 ll of leucocyte suspension was © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Comparing methods in granulocyte immunology 289
incubated with 50 ll of serum/plasma for 30 min at 37°C. After two washing steps, membrane bound antibodies were labelled with Alexa Fluor 488 conjugated Goat Anti-Human IgG Fab Fragment (Jackson ImmunoResearch, New Market, UK) for 30 min in the dark at room temperature. Before measurement, cells were fixed with 300 ll CellfixTM (Becton Dickinson, Heidelberg, Germany). Samples were defined positive if the fluorescence level was at least double that of the negative control. Flow cytometry was performed on a FACSCaliburTM (Becton Dickinson) and analysed with CELL QUESTTMPRO Software (Becton Dickinson).
Differentiation and specification of HLA and HNA antibodies Granulocyte-reactive samples were further analysed for antibody specificity in MAIGA [6, 13] and the microbeads assay LabScreen Multi. Monoclonal antibody-specific immobilization of granulocyte antigens was performed using the following monoclonal antibody clones: DJ130c (Acris, Herford, Germany) and 3G8 (Beckman Coulter, Krefeld, Germany) for identification of HNA-1a, -1b, -1c antibodies, MEM-166 (Acris) for the detection of HNA-2 antibodies, Bear-1 and HI111 (Becton Dickinson) for antibodies against HNA-4 and -5. HLA class I antibodies were identified using B1G6 (Beckman Coulter) and W6/32 (Sera Laboratories, West Sussex, UK) [10]. The microbeads assay (LabScreen Multi; One Lambda, Canoga Park, CA, USA) for the identification of HNA-1a, -1b, -1c and HNA-2 antibodies as well as antibodies against HLA class I and class II was performed as follows: Samples were incubated with antigen-coated microbeads in a 96-well plate. A negative control serum (One Lambda) was tested to establish background values. After three washing steps, phycoerythrin-conjugated AntiHuman IgG (One Lambda) was added to the antigen-antibody complex. Fluorescence intensity was measured by a Luminex flow analyzer; normalized background (NBG) ratios were calculated using HLA FUSIONTM Software (One Lambda).
Results Comparison of combined GAT and GIFT with Flow-WIFT Of 333 investigated samples, 77 (231%) were positive in GIFT, 23 of these also caused aggregation in GAT, while none was positive in GAT only [10]. Therefore, statistical data are subsequently shown for GIFT only. Antibody screening with Flow-WIFT showed 108 granulocyte-reactive samples (324%). Positive results of both methods corresponded in 71 cases (213% of all tested samples). All GAT-positive samples were also granulocyte-reactive in Flow-WIFT except one sample, which caused very weak aggregation in GAT and was exclusively reactive on lymphocytes but not on granulocytes in Flow-WIFT. In total, 114 samples were tested positive in any screening method. For the comparison of GIFT with Flow-WIFT, Cohen’s j was calculated at 0682, signifying good concordance between these methods (Table 1).
Confirmation of GIFT and Flow-WIFT results in MAIGA and the microbeads assay Overall, 74 and 79 screening-positive samples were confirmed in MAIGA and the microbeads assay, respectively. Of the 77 granulocyte-reactive samples in GIFT, 66 (857%) could be confirmed in MAIGA and the same number of samples in the microbeads assay, 62 reactivities (805%) were confirmed with both methods. Seven samples were negative in both specification methods. Further analysis of the 108 granulocyte-reactive samples in Flow-WIFT confirmed 69 (639%) reactivities using MAIGA and 76 (704%) reactivities using the microbeads assay, with 67 (620%) corresponding positive results in both methods; 30 reactivities were not confirmed by either. Detailed description of the number of reactive samples in all tests is shown in Tables 2 and 3.
Comparison of MAIGA and the microbeads assay Of all the 114 screening-positive samples tested in the specification tests, 70 reactivities were confirmed correspondingly with both methods. Four and nine samples were
Statistical analysis Overall results of combined GAT and GIFT were compared with granulocyte reactivity in Flow-WIFT and the results of HNA and HLA class I antibody detection in MAIGA were compared to those of the microbeads assay using descriptive statistics and calculating Cohen’s j. The latter was characterized according to Landis [14]. Statistical analysis was performed using SPSS 20.0 software (Armonk, NY, USA). © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Table 1 Contingency table of the screening tests: granulocyte immunofluorescence test (GIFT) and flow cytometric white blood cell immunofluorescence test (Flow-WIFT)
GIFT + GIFT Total
Flow-WIFT+
Flow-WIFT -
Total
71 (922%) 37 (145%) 108 (324%)
6 (78%) 219 (855%) 225 (676%)
77 (100%) 256 (100%) 333
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Table 2 Comprehensive table of granulocyte-reactive samples in granulocyte immunofluorescence test (GIFT), flow cytometric white blood cell immunofluorescence test (Flow-WIFT), monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) and the microbeads assay. The number of reactive samples in the respective tests (screening tests in rows, specification tests in columns) is shown. Percentages given refer to the total number of reactive samples in the respective row (shown in the last column) and thus mark the percentage of confirmed specificities by MAIGA or the microbeads assay
GIFT Flow-WIFT GIFT and Flow-WIFT Any screening test
MAIGA (%)
Microbeads assay (%)
MAIGA and microbeads assay (%)
MAIGA or microbeads assay (%)
All
66 69 61 74
66 76 63 79
62 67 59 70
73 78 65 83
77 108 71 114
(857) (639) (859) (649)
(857) (704) (887) (693)
Table 3 Reactivity pattern of positive samples clustered according to screening results. The number of reactive samples for different combinations of test results is shown. Of note, 24 samples reactive in flow cytometric white blood cell immunofluorescence test (Flow-WIFT) did not show reactivity in any of the other tests, whereas only one sample was exclusively reactive in granulocyte immunofluorescence test (GIFT). Interestingly, there was not a single sample positive in GIFT and the microbeads assay without reactivity in Flow-WIFT and monoclonal antibodyspecific immobilization of granulocyte antigens (MAIGA). Neither was there a single sample exclusively reactive in the combination of FlowWIFT and MAIGA. Only a single sample that caused aggregation in granulocyte aggregation test (GAT) and was reactive in GIFT and Flow-WIFT could not be specified by MAIGA. One GAT-positive sample was not granulocyte reactive in Flow-WIFT but showed lymphocyte reactivity
(805) (620) (831) (614)
(948) (722) (915) (728)
Table 4 Contingency table of the specification tests: monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) and the microbeads assay. Only the 114 screening-positive samples were tested in these methods
MAIGA + MAIGA Total
Microbeads assay + (%)
Microbeads assay - (%)
Total (%)
70 (946) 9 (225) 79 (693)
4 (54) 31 (775) 35 (307)
74 (100) 40 (100) 114
Antibody specificities Screening pattern
Specification results
Number of reactive samples (n = 114)
GAT
GIFT
Flow-WIFT
MAIGA
Microbeads assay
20 1 1 0 1 0 0 0 2 2 0 1 39 1 3 6 8 0 5 24
+ + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + +
+ + + + + + + + + + -
+ + + + + + + + + + -
only positive in MAIGA and the microbeads assay, respectively. Cohen’s j for the comparison between MAIGA and the microbeads assay was calculated at 0742 (Table 4).
In MAIGA, 73 HLA class I- and four HNA-reactive antibodies were detected, whereas the microbeads assay identified 73 HLA class I, 47 HLA class II and four HNA reactivities. As shown in Table 5, a total of six samples were identified as HNA-reactive in either MAIGA or the microbeads assay. A perfect match of specification results regarding HLA class I and HNA between MAIGA and the microbeads assay was seen in two cases (Y161 and Y316). Three samples (Y26, Y59 and Y316) showed conclusive results regarding panel reactivity and MAIGA. For Y26, panel reactivity matched perfectly with the result in MAIGA, as this HNA-1a-specific antibody was not reactive with the HNA-1b homozygous cell A. One sample (Y1) was negative in MAIGA but panreactive in the microbeads assay, which means that this serum sample showed strong positive results with all HLA- and HNAcoated microbeads but not with negative control beads. One sample (Y57) was reactive with one of three cells in GAT, GIFT and Flow-WIFT and specified as HNA-2-specific in the microbeads assay, but negative in MAIGA. For HNA-2-specific samples, reactivity would be expected on all three cells due to their HNA-2 expression. For Y57, a possible prozone effect in MAIGA could not be excluded due to lacking sample quantity. For Y161, results of MAIGA and the microbeads assay corresponded © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Comparing methods in granulocyte immunology 291
Table 5 Reaction pattern of samples with human neutrophil antigen (HNA) specificity. The cell panel used for the screening tests originated from three different donors. Cell A covered HNA-1b/b, -2, -3a/b, -4a/a and -5a/a; Cell B covered HNA -1a/b, -2, -3a/b, -4a/a, -5a/a; Cell C covered HNA-1a/c, -2, 3a/a, -4a/a, -5a/a. In total, six samples were identified as HNA reactive. One sample (Y1) was negative in monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) but panreactive in the microbeads assay which means that this serum was positive with all human leucocyte antigens (HLA)- and HNA-coated microbeads but not with negative control beads. HLA class II-specific antibodies in the microbeads assay are not shown in this table Screening pattern GAT
GIFT
Specification results
Sample ID Cell
A
B
C
A
B
C
A
B
C
Microbeads assay
MAIGA
Y1 Y26 Y57 Y59 Y161 Y293 Y316
-
+ + -
+ + -
+ + +
+ + + +
+ + + + +
+ +
+ + + + +
+ + + +
Panreactive – HNA-2 HLA class I HNA-2 + HLA class I HNA-1 + HNA-2 + HLA class I HNA-2 + HLA class I
– HNA-1a – HNA-1a + HLA class I HNA-2 + HLA class I – HNA-2 + HLA class I
Flow-WIFT
well, but this HNA-2-specific antibody was missed in GIFT on one cell and, more importantly, was not detected in Flow-WIFT at all. This may indicate that Flow-WIFT cannot entirely replace GIFT as a screening test. On the other hand, Y293 was exclusively reactive with one cell in Flow-WIFT and confirmed in the microbeads assay. Although not reactive in GIFT, GAT or MAIGA, this corresponding result in a cell-based and a bead-based test seems notable. All four HNA-specific antibodies identified by MAIGA were reactive in GIFT, whereas only three were granulocyte-reactive in Flow-WIFT. Both screening methods detected three of the four HNA-reactive samples specified by the microbeads assay (see Table 5). Regarding HLA class I-specific antibodies, 78 were detected by MAIGA or the microbeads assay, with 68 corresponding samples. Of these 78 overall HLA class I-specific samples, 66 (846%) were positive in GIFT and 73 (936%) were granulocyte-reactive in Flow-WIFT.
Discussion In this study, positive results regarding granulocyte-reactive antibodies were obtained in 231% with GIFT and 324% with Flow-WIFT. This rather high prevalence can be explained by the fact that in this collective of women with a history of pregnancy many samples (n = 143) contained anti-RBC antibodies. Granulocyte-reactive antibodies were shown to be associated with anti-RBC alloimmunization [10]. The prevalence of HNA-specific antibodies in this study detected in MAIGA or the microbeads assay was 6 of 333 samples (18%), which is within the wide range of previously reported HNA antibody frequencies (0–3%) [4, 15, 16]. © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Overall, Flow-WIFT proved to be a valuable and feasible technique to screen for granulocyte-reactive antibodies. It requires less experience than standard GIFT, results are more objective than microscopic evaluation, it has the potential to be automated [17] and lymphocyte-reactive antibodies can be detected simultaneously, thus also replacing the LIFT. The comparison between GIFT and Flow-WIFT as screening tests showed good concordance (j = 0682). Flow-WIFT detected more reactive samples than GIFT, but with considerably lower rates of confirmed antibody specificities in MAIGA as well as in the microbeads assay, as shown in Table 2. Only a single sample reactive in GIFT did not show reactivity in any of the other tests, whereas 24 samples were uniquely reactive in Flow-WIFT (see Table 3). Specificity of Flow-WIFT may thus be considerably lower than that of GIFT. One possible reason for this difference between the two screening tests may be the use of PFA as a fixative agent after cell isolation to prevent unspecific membrane fluorescence in GIFT but not in Flow-WIFT [6, 8]. However, samples that are nonreactive in MAIGA are not necessarily false positive in screening. Such samples may still be granulocyte-reactive but cannot be specified. For example, as there is no capture antibody for CTL2 available, HNA-3 antibodies cannot be detected by MAIGA. Furthermore, sterical hindrance may result in improper antibody immobilization [18, 19]. On the other hand, 13 samples confirmed in MAIGA or the microbeads assay were reactive in Flow-WIFT but negative in GIFT, whereas only five confirmed samples were detected by GIFT but not granulocyte-reactive in Flow-WIFT. Regarding HLA class I-reactive antibodies, Flow-WIFT was superior to GIFT in this study. Flow-WIFT
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detected 936% of all HLA class I-reactive antibodies, whereas GIFT only detected 846%. This may indicate higher sensitivity of Flow-WIFT. This and the ability of Flow-WIFT to detect antibodies at higher levels of dilution were already described by Nguyen et al. [8]. Due to the high number of possibly false-positive results of Flow-WIFT in this study, this assay can currently not entirely replace standard GIFT and requires further evaluation. However, it may serve as a valuable addition to the standard techniques to increase sensitivity. Special attention was paid to the screening for antiHNA-3 antibodies, which are considered a high risk for TRALI due to their strong aggregating potential and are most reliably detected by GAT but cannot be identified by MAIGA or the microbeads assay. In this study, all GAT-positive samples were also reactive in GIFT. In Flow-WIFT, one GAT-positive sample was only reactive with lymphocytes but not with granulocytes. For this and all other samples, however, HNA-3 reactivity was excluded based on the reactivity pattern in the different methods with the cell panel. Microbeads assays are common and feasible standard methods for high-throughput HLA antibody screening and identification. They are highly sensitive and hence particularly suitable for transplant diagnostics [20, 21], but cut-off levels have to be adjusted individually for each diagnostic purpose. Other than MAIGA, microbeads assays offer the possibility of rather rapid and all-time available performance without the necessity of cell isolation. According to our data, the microbeads assay used in this study seems to be less reliable than MAIGA for HNAantibody specification due to doubtful specification results (Table 5), which were sometimes not compatible with screening results regarding the reactivity pattern on the panel cells. This may be due to the fact that antigens
on microbeads are recombinant and might thus have an altered conformation and density in comparison with antigens expressed on natural cells [6]. On the other hand, it shall be noted that MAIGA is a very complex test requiring a considerable level of experience. The selection of monoclonal antibodies for this assay needs to be well considered due to possible steric interferences. Also, large quantities of typed granulocytes are necessary for this test, which can limit the number of realizable tests in one session. However, the use of natural cells guarantees correct conformation of the respective antigens, making specification results more plausible [6].
Conclusion Our data suggest that Flow-WIFT offers a valuable addition to GIFT and GAT for granulocyte-reactive antibody screening and may increase sensitivity. MAIGA remains the most reliable laboratory method to confirm antibody specificities.
Acknowledgements Test implementation was supported by J€ urgen Bux and Angelika Reil and her team from the Blood Service West, German Red Cross, Hagen. Parts of this study contributed to the diploma thesis of Matthias Heinzl (Human Medicine, Medical University of Vienna, Austria) as well as the master thesis of Marlies Sch€ onbacher (Master Program for Biomedical Sciences, FH Campus Wien, Vienna, Austria).
Disclosure of conflict of interests The authors declare no conflict of interests.
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cyte antibody screening of blood donors. Vox Sang 2008; 95:313–317 5 Bierling P, Bux J, Curtis B, et al.: Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury. Vox Sang 2009; 96:266–269 6 Fung YL, Minchinton RM, Fraser JF: Neutrophil antibody diagnostics and screening: review of the classical versus the emerging. Vox Sang 2011; 101:282–290
7 Lucas G, Porcelijn L, Fung YL, et al.: External quality assessment of human neutrophil antigen (HNA)-specific antibody detection and HNA genotyping from 2000 to 2012. Vox Sang 2013; 105:259–269 8 Nguyen XD, Flesch B, Sachs UJ, et al.: Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test. Transfusion 2009; 49:2700– 2708 9 Fromont P, Prie N, Simon P, et al.: Granulocyte antibody screening: evaluation of a bead-based assay in com-
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antigen located on Fcgamma receptor IIIb associated with neonatal immune neutropenia. Transfusion 2013; 53:2145–2151 19 Tamm A, Schmidt RE: The binding epitopes of human CD16 (Fc gamma RIII) monoclonal antibodies. Implications for ligand binding. J Immunol 1996; 157:1576–1581 20 Stroncek DF, Fadeyi E, Adams S: Leukocyte antigen and antibody detection assays: tools for assessing and preventing pulmonary transfusion reactions. Transfus Med Rev 2007; 21:273–286 21 Bux J: Antibody-mediated (immune) transfusion-related acute lung injury. Vox Sang 2011; 100:122–128
ORIGINAL PAPER
Detection of granulocyte-reactive antibodies: a comparison of different methods M. W. Heinzl, M. Sch€ onbacher, E.-M. Dauber, S. Panzer, W. R. Mayr & G. F. K€ orm€ oczi Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria
Background and Objectives 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. Materials and Methods 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. Results Granulocyte aggregation test/GIFT detected 77 positive samples, FlowWIFT found 108 granulocyte-reactive samples. Six Samples were only positive in GAT/GIFT, and 37 samples were only positive in Flow-WIFT (j = 0682). Antibody specification with MAIGA and the microbeads assay confirmed granulocyte-reactivity in 83 cases with 70 matching results (j = 0742). However, out of six detected human neutrophil antigen (HNA) reactivities only two specificities matched in both assays.
Received: 13 July 2014, revised 15 October 2014, accepted 3 November 2014, published online 30 December 2014
Conclusion Flow-WIFT may be a valuable addition to GIFT for granulocyte-reactive antibody screening. MAIGA remains the most reliable laboratory method for antibody specification. Key words: flow cytometry, granulocyte immunology, granulocyte-reactive antibodies, human neutrophil antigens, microbeads assay.
Introduction Granulocyte-reactive antibodies can be a cause for different medical conditions such as transfusion-related acute lung injury (TRALI), ineffectiveness of granulocyte transfusions, autoimmune neutropenia (AIN) and neonatal immune neutropenia (NIN) [1–3]. Such antibodies can be directed against human leucocyte antigens (HLA) class I Correspondence: G€unther K€orm€oczi, M.D., Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, W€ahringer G€urtel 18-20, 1090 Vienna, Austria E-mail: [email protected] Marlies Sch€onbacher and Matthias Heinzl contributed equally to this study.
or human neutrophil antigens (HNA). Granulocyte-reactive alloantibodies are usually formed by pregnant women or transfused patients. Despite their low prevalence, HNA antibodies are especially dangerous in regard to TRALI [4]. The technically challenging and time consuming combination of granulocyte aggregation test (GAT) and granulocyte immunofluorescence test (GIFT) using a panel of typed and freshly isolated granulocytes is currently the recommended laboratory procedure for HNA antibody detection. Panel reactivity can indicate antibody specificity, which can be confirmed with monoclonal antibodyspecific immobilization of granulocyte antigens (MAIGA). MAIGA is a glycoprotein-specific assay using monoclonal
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capture antibodies and has special importance in identifying mixtures of both HLA- and HNA-specific antibodies [5–7]. However, it is important to note that negative results in MAIGA do not necessarily indicate false-positive reactions in the screening tests. Negativity in MAIGA may be due to the non-existence of suitable capture antibodies, sterical hindrance and other effects such as the prozone phenomenon. Although GIFT is known to detect all membranebound granulocyte-reactive antibodies, it is important to additionally perform GAT. GAT has the ability to detect aggregating antibodies like anti-HNA-3a, which are especially important as they are responsible for most of the fatal TRALI reactions [4] and cannot be found in MAIGA because there are no functional capture antibodies available. Furthermore, IgM antibodies are not detected by GIFT using standard secondary antibodies (anti-IgG) but can induce aggregation in GAT. Other techniques such as flow cytometric white blood cell immunofluorescence test (Flow-WIFT, also known as Flow-GIFT) for antibody screening and the microbeads assay LabScreen Multi for antibody specification have emerged more recently and are still subject of evaluation [6, 8, 9]. Flow-WIFT has been reported to be an excellent test for granulocyte antibody screening with great sensitivity for all known HNA antibodies and the potential to be automated. Furthermore, 7-aminoactinomycin D (7-AAD) can be added to identify dead cells to evaluate the cell quality after isolation and the cytotoxicity of antibodies. Importantly, Flow-WIFT can also be used to evaluate antibody binding to monocytes and lymphocytes, which allows the differentiation between granulocyte-specific antibodies (anti-HNA-1 and -2) and antibodies reactive with different leucocyte subsets (anti-HNA-3, -4, -5 and HLA-antibodies) and excludes the necessity of separately performing a lymphocyte immunofluorescence test (LIFT) in addition to GIFT [6, 8]. The commercially available microbeads assay LabScreen Multi detects antibodies to HLA class I and II as well as HNA-1 and -2 antibodies. The true sensitivity and specificity of this rather expensive kit regarding HNA antibodies are not yet entirely clear. Although results have been shown to correlate well with other techniques for the screening of granulocyte-reactive alloantibodies, there were discrepancies reported with respect to antibody specificities and correlation was only moderate for autoimmune antibodies. Furthermore, the highly relevant HNA-3-specific antibodies cannot be detected [6, 9]. Like in all recombinant antigen-based tests, there is a risk that highly variant and conformation-sensitive antibodies may be missed in this assay. Furthermore, there is currently no universally accepted cut-off value to define positive
results. The true value of this bead-based technique for screening as well as specification purposes is thus yet to be explored [6, 9]. To gain more information on the properties of these test systems, Flow-WIFT was compared to the combination of GAT and GIFT as a screening test, and the microbeads assay was assessed for the determination of antibody specificities in comparison with MAIGA.
Materials and methods This study was approved by the Ethical Committee of the Medical University of Vienna. The applied cell panel for antibody screening was assembled from donors previously typed for HNA by sequence-specific priming PCR (HNA Ready Gene, inno-train, Kronberg, Germany) and serologic typing and covered HNA-1a, -1b, -1c, -2, -3a, -3b, -4a and -5a as well as the most important HLA phenotypes. Cells were isolated from freshly drawn EDTA whole blood by dextran sedimentation. For GAT and GIFT, granulocytes were separated by density gradient centrifugation using Ficoll PaqueTM Plus (GE Healthcare, Uppsala, Sweden), erythrocytes were depleted by ammonium chloride lysis, with subsequent paraformaldehyde (PFA) fixation of the granulocytes for microscopic GIFT [4]. For Flow-WIFT, density gradient centrifugation and PFA fixation were not necessary. For all screening methods, cells were adjusted to a concentration of 5 9 103 white blood cells/ll.
Screening for HLA and HNA antibodies For the comparison of screening methods for granulocyte-reactive antibodies, 333 samples from women with a history of pregnancy (second trimenon at least; 143 samples containing anti-red blood cell (RBC) antibodies) were tested. These samples originated from blood withdrawals for the diagnostic routine pretransfusion and anti-RBC antibody testing at the Medical University of Vienna. Data regarding RBC antibodies as a risk indicator for granulocyte alloimmunization in this collective were presented in a previously published study [10]. Samples were collected in a consecutive manner. Furthermore, historic samples containing anti-RBC antibodies were added to increase chances for the presence of granulocyte-reactive antibodies. No further clinical data were available for these samples. Screening for granulocyte-reactive antibodies was performed with a panel of three cells using the classical methods GAT [11] and microscopic GIFT [12] as well as the recently described Flow-WIFT [6, 8]. Flow-WIFT was performed as follows: 50 ll of leucocyte suspension was © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Comparing methods in granulocyte immunology 289
incubated with 50 ll of serum/plasma for 30 min at 37°C. After two washing steps, membrane bound antibodies were labelled with Alexa Fluor 488 conjugated Goat Anti-Human IgG Fab Fragment (Jackson ImmunoResearch, New Market, UK) for 30 min in the dark at room temperature. Before measurement, cells were fixed with 300 ll CellfixTM (Becton Dickinson, Heidelberg, Germany). Samples were defined positive if the fluorescence level was at least double that of the negative control. Flow cytometry was performed on a FACSCaliburTM (Becton Dickinson) and analysed with CELL QUESTTMPRO Software (Becton Dickinson).
Differentiation and specification of HLA and HNA antibodies Granulocyte-reactive samples were further analysed for antibody specificity in MAIGA [6, 13] and the microbeads assay LabScreen Multi. Monoclonal antibody-specific immobilization of granulocyte antigens was performed using the following monoclonal antibody clones: DJ130c (Acris, Herford, Germany) and 3G8 (Beckman Coulter, Krefeld, Germany) for identification of HNA-1a, -1b, -1c antibodies, MEM-166 (Acris) for the detection of HNA-2 antibodies, Bear-1 and HI111 (Becton Dickinson) for antibodies against HNA-4 and -5. HLA class I antibodies were identified using B1G6 (Beckman Coulter) and W6/32 (Sera Laboratories, West Sussex, UK) [10]. The microbeads assay (LabScreen Multi; One Lambda, Canoga Park, CA, USA) for the identification of HNA-1a, -1b, -1c and HNA-2 antibodies as well as antibodies against HLA class I and class II was performed as follows: Samples were incubated with antigen-coated microbeads in a 96-well plate. A negative control serum (One Lambda) was tested to establish background values. After three washing steps, phycoerythrin-conjugated AntiHuman IgG (One Lambda) was added to the antigen-antibody complex. Fluorescence intensity was measured by a Luminex flow analyzer; normalized background (NBG) ratios were calculated using HLA FUSIONTM Software (One Lambda).
Results Comparison of combined GAT and GIFT with Flow-WIFT Of 333 investigated samples, 77 (231%) were positive in GIFT, 23 of these also caused aggregation in GAT, while none was positive in GAT only [10]. Therefore, statistical data are subsequently shown for GIFT only. Antibody screening with Flow-WIFT showed 108 granulocyte-reactive samples (324%). Positive results of both methods corresponded in 71 cases (213% of all tested samples). All GAT-positive samples were also granulocyte-reactive in Flow-WIFT except one sample, which caused very weak aggregation in GAT and was exclusively reactive on lymphocytes but not on granulocytes in Flow-WIFT. In total, 114 samples were tested positive in any screening method. For the comparison of GIFT with Flow-WIFT, Cohen’s j was calculated at 0682, signifying good concordance between these methods (Table 1).
Confirmation of GIFT and Flow-WIFT results in MAIGA and the microbeads assay Overall, 74 and 79 screening-positive samples were confirmed in MAIGA and the microbeads assay, respectively. Of the 77 granulocyte-reactive samples in GIFT, 66 (857%) could be confirmed in MAIGA and the same number of samples in the microbeads assay, 62 reactivities (805%) were confirmed with both methods. Seven samples were negative in both specification methods. Further analysis of the 108 granulocyte-reactive samples in Flow-WIFT confirmed 69 (639%) reactivities using MAIGA and 76 (704%) reactivities using the microbeads assay, with 67 (620%) corresponding positive results in both methods; 30 reactivities were not confirmed by either. Detailed description of the number of reactive samples in all tests is shown in Tables 2 and 3.
Comparison of MAIGA and the microbeads assay Of all the 114 screening-positive samples tested in the specification tests, 70 reactivities were confirmed correspondingly with both methods. Four and nine samples were
Statistical analysis Overall results of combined GAT and GIFT were compared with granulocyte reactivity in Flow-WIFT and the results of HNA and HLA class I antibody detection in MAIGA were compared to those of the microbeads assay using descriptive statistics and calculating Cohen’s j. The latter was characterized according to Landis [14]. Statistical analysis was performed using SPSS 20.0 software (Armonk, NY, USA). © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Table 1 Contingency table of the screening tests: granulocyte immunofluorescence test (GIFT) and flow cytometric white blood cell immunofluorescence test (Flow-WIFT)
GIFT + GIFT Total
Flow-WIFT+
Flow-WIFT -
Total
71 (922%) 37 (145%) 108 (324%)
6 (78%) 219 (855%) 225 (676%)
77 (100%) 256 (100%) 333
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Table 2 Comprehensive table of granulocyte-reactive samples in granulocyte immunofluorescence test (GIFT), flow cytometric white blood cell immunofluorescence test (Flow-WIFT), monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) and the microbeads assay. The number of reactive samples in the respective tests (screening tests in rows, specification tests in columns) is shown. Percentages given refer to the total number of reactive samples in the respective row (shown in the last column) and thus mark the percentage of confirmed specificities by MAIGA or the microbeads assay
GIFT Flow-WIFT GIFT and Flow-WIFT Any screening test
MAIGA (%)
Microbeads assay (%)
MAIGA and microbeads assay (%)
MAIGA or microbeads assay (%)
All
66 69 61 74
66 76 63 79
62 67 59 70
73 78 65 83
77 108 71 114
(857) (639) (859) (649)
(857) (704) (887) (693)
Table 3 Reactivity pattern of positive samples clustered according to screening results. The number of reactive samples for different combinations of test results is shown. Of note, 24 samples reactive in flow cytometric white blood cell immunofluorescence test (Flow-WIFT) did not show reactivity in any of the other tests, whereas only one sample was exclusively reactive in granulocyte immunofluorescence test (GIFT). Interestingly, there was not a single sample positive in GIFT and the microbeads assay without reactivity in Flow-WIFT and monoclonal antibodyspecific immobilization of granulocyte antigens (MAIGA). Neither was there a single sample exclusively reactive in the combination of FlowWIFT and MAIGA. Only a single sample that caused aggregation in granulocyte aggregation test (GAT) and was reactive in GIFT and Flow-WIFT could not be specified by MAIGA. One GAT-positive sample was not granulocyte reactive in Flow-WIFT but showed lymphocyte reactivity
(805) (620) (831) (614)
(948) (722) (915) (728)
Table 4 Contingency table of the specification tests: monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) and the microbeads assay. Only the 114 screening-positive samples were tested in these methods
MAIGA + MAIGA Total
Microbeads assay + (%)
Microbeads assay - (%)
Total (%)
70 (946) 9 (225) 79 (693)
4 (54) 31 (775) 35 (307)
74 (100) 40 (100) 114
Antibody specificities Screening pattern
Specification results
Number of reactive samples (n = 114)
GAT
GIFT
Flow-WIFT
MAIGA
Microbeads assay
20 1 1 0 1 0 0 0 2 2 0 1 39 1 3 6 8 0 5 24
+ + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + +
+ + + + + + + + + + -
+ + + + + + + + + + -
only positive in MAIGA and the microbeads assay, respectively. Cohen’s j for the comparison between MAIGA and the microbeads assay was calculated at 0742 (Table 4).
In MAIGA, 73 HLA class I- and four HNA-reactive antibodies were detected, whereas the microbeads assay identified 73 HLA class I, 47 HLA class II and four HNA reactivities. As shown in Table 5, a total of six samples were identified as HNA-reactive in either MAIGA or the microbeads assay. A perfect match of specification results regarding HLA class I and HNA between MAIGA and the microbeads assay was seen in two cases (Y161 and Y316). Three samples (Y26, Y59 and Y316) showed conclusive results regarding panel reactivity and MAIGA. For Y26, panel reactivity matched perfectly with the result in MAIGA, as this HNA-1a-specific antibody was not reactive with the HNA-1b homozygous cell A. One sample (Y1) was negative in MAIGA but panreactive in the microbeads assay, which means that this serum sample showed strong positive results with all HLA- and HNAcoated microbeads but not with negative control beads. One sample (Y57) was reactive with one of three cells in GAT, GIFT and Flow-WIFT and specified as HNA-2-specific in the microbeads assay, but negative in MAIGA. For HNA-2-specific samples, reactivity would be expected on all three cells due to their HNA-2 expression. For Y57, a possible prozone effect in MAIGA could not be excluded due to lacking sample quantity. For Y161, results of MAIGA and the microbeads assay corresponded © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Comparing methods in granulocyte immunology 291
Table 5 Reaction pattern of samples with human neutrophil antigen (HNA) specificity. The cell panel used for the screening tests originated from three different donors. Cell A covered HNA-1b/b, -2, -3a/b, -4a/a and -5a/a; Cell B covered HNA -1a/b, -2, -3a/b, -4a/a, -5a/a; Cell C covered HNA-1a/c, -2, 3a/a, -4a/a, -5a/a. In total, six samples were identified as HNA reactive. One sample (Y1) was negative in monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) but panreactive in the microbeads assay which means that this serum was positive with all human leucocyte antigens (HLA)- and HNA-coated microbeads but not with negative control beads. HLA class II-specific antibodies in the microbeads assay are not shown in this table Screening pattern GAT
GIFT
Specification results
Sample ID Cell
A
B
C
A
B
C
A
B
C
Microbeads assay
MAIGA
Y1 Y26 Y57 Y59 Y161 Y293 Y316
-
+ + -
+ + -
+ + +
+ + + +
+ + + + +
+ +
+ + + + +
+ + + +
Panreactive – HNA-2 HLA class I HNA-2 + HLA class I HNA-1 + HNA-2 + HLA class I HNA-2 + HLA class I
– HNA-1a – HNA-1a + HLA class I HNA-2 + HLA class I – HNA-2 + HLA class I
Flow-WIFT
well, but this HNA-2-specific antibody was missed in GIFT on one cell and, more importantly, was not detected in Flow-WIFT at all. This may indicate that Flow-WIFT cannot entirely replace GIFT as a screening test. On the other hand, Y293 was exclusively reactive with one cell in Flow-WIFT and confirmed in the microbeads assay. Although not reactive in GIFT, GAT or MAIGA, this corresponding result in a cell-based and a bead-based test seems notable. All four HNA-specific antibodies identified by MAIGA were reactive in GIFT, whereas only three were granulocyte-reactive in Flow-WIFT. Both screening methods detected three of the four HNA-reactive samples specified by the microbeads assay (see Table 5). Regarding HLA class I-specific antibodies, 78 were detected by MAIGA or the microbeads assay, with 68 corresponding samples. Of these 78 overall HLA class I-specific samples, 66 (846%) were positive in GIFT and 73 (936%) were granulocyte-reactive in Flow-WIFT.
Discussion In this study, positive results regarding granulocyte-reactive antibodies were obtained in 231% with GIFT and 324% with Flow-WIFT. This rather high prevalence can be explained by the fact that in this collective of women with a history of pregnancy many samples (n = 143) contained anti-RBC antibodies. Granulocyte-reactive antibodies were shown to be associated with anti-RBC alloimmunization [10]. The prevalence of HNA-specific antibodies in this study detected in MAIGA or the microbeads assay was 6 of 333 samples (18%), which is within the wide range of previously reported HNA antibody frequencies (0–3%) [4, 15, 16]. © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 287–293
Overall, Flow-WIFT proved to be a valuable and feasible technique to screen for granulocyte-reactive antibodies. It requires less experience than standard GIFT, results are more objective than microscopic evaluation, it has the potential to be automated [17] and lymphocyte-reactive antibodies can be detected simultaneously, thus also replacing the LIFT. The comparison between GIFT and Flow-WIFT as screening tests showed good concordance (j = 0682). Flow-WIFT detected more reactive samples than GIFT, but with considerably lower rates of confirmed antibody specificities in MAIGA as well as in the microbeads assay, as shown in Table 2. Only a single sample reactive in GIFT did not show reactivity in any of the other tests, whereas 24 samples were uniquely reactive in Flow-WIFT (see Table 3). Specificity of Flow-WIFT may thus be considerably lower than that of GIFT. One possible reason for this difference between the two screening tests may be the use of PFA as a fixative agent after cell isolation to prevent unspecific membrane fluorescence in GIFT but not in Flow-WIFT [6, 8]. However, samples that are nonreactive in MAIGA are not necessarily false positive in screening. Such samples may still be granulocyte-reactive but cannot be specified. For example, as there is no capture antibody for CTL2 available, HNA-3 antibodies cannot be detected by MAIGA. Furthermore, sterical hindrance may result in improper antibody immobilization [18, 19]. On the other hand, 13 samples confirmed in MAIGA or the microbeads assay were reactive in Flow-WIFT but negative in GIFT, whereas only five confirmed samples were detected by GIFT but not granulocyte-reactive in Flow-WIFT. Regarding HLA class I-reactive antibodies, Flow-WIFT was superior to GIFT in this study. Flow-WIFT
292 M. W. Heinzl et al.
detected 936% of all HLA class I-reactive antibodies, whereas GIFT only detected 846%. This may indicate higher sensitivity of Flow-WIFT. This and the ability of Flow-WIFT to detect antibodies at higher levels of dilution were already described by Nguyen et al. [8]. Due to the high number of possibly false-positive results of Flow-WIFT in this study, this assay can currently not entirely replace standard GIFT and requires further evaluation. However, it may serve as a valuable addition to the standard techniques to increase sensitivity. Special attention was paid to the screening for antiHNA-3 antibodies, which are considered a high risk for TRALI due to their strong aggregating potential and are most reliably detected by GAT but cannot be identified by MAIGA or the microbeads assay. In this study, all GAT-positive samples were also reactive in GIFT. In Flow-WIFT, one GAT-positive sample was only reactive with lymphocytes but not with granulocytes. For this and all other samples, however, HNA-3 reactivity was excluded based on the reactivity pattern in the different methods with the cell panel. Microbeads assays are common and feasible standard methods for high-throughput HLA antibody screening and identification. They are highly sensitive and hence particularly suitable for transplant diagnostics [20, 21], but cut-off levels have to be adjusted individually for each diagnostic purpose. Other than MAIGA, microbeads assays offer the possibility of rather rapid and all-time available performance without the necessity of cell isolation. According to our data, the microbeads assay used in this study seems to be less reliable than MAIGA for HNAantibody specification due to doubtful specification results (Table 5), which were sometimes not compatible with screening results regarding the reactivity pattern on the panel cells. This may be due to the fact that antigens
on microbeads are recombinant and might thus have an altered conformation and density in comparison with antigens expressed on natural cells [6]. On the other hand, it shall be noted that MAIGA is a very complex test requiring a considerable level of experience. The selection of monoclonal antibodies for this assay needs to be well considered due to possible steric interferences. Also, large quantities of typed granulocytes are necessary for this test, which can limit the number of realizable tests in one session. However, the use of natural cells guarantees correct conformation of the respective antigens, making specification results more plausible [6].
Conclusion Our data suggest that Flow-WIFT offers a valuable addition to GIFT and GAT for granulocyte-reactive antibody screening and may increase sensitivity. MAIGA remains the most reliable laboratory method to confirm antibody specificities.
Acknowledgements Test implementation was supported by J€ urgen Bux and Angelika Reil and her team from the Blood Service West, German Red Cross, Hagen. Parts of this study contributed to the diploma thesis of Matthias Heinzl (Human Medicine, Medical University of Vienna, Austria) as well as the master thesis of Marlies Sch€ onbacher (Master Program for Biomedical Sciences, FH Campus Wien, Vienna, Austria).
Disclosure of conflict of interests The authors declare no conflict of interests.
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