The effect of serum pretreatment regimens for the detection of HLA class I antibodies in platelet-refractory patients

Biomedical Excellence for Safer Transfusion (BEST) Collaborative

doi:10.1111/trf.15666

The effect of serum pretreatment regimens for the detection of HLA class I antibodies in platelet-refractory patients

Biomedical Excellence for Safer Transfusion (BEST) Collaborative

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8 Scopus citations

Abstract

BACKGROUND: Single antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.

STUDY DESIGN AND METHODS: Five patients' sera were overnight shipped to 12 laboratories in the United States and internationally. Recipients used their lab's SAB procedure to identify HLA Class I antibodies. The resultant mean fluorescence intensity (MFI) data were compared by instrumentation, bead lot, and pretreatment regimens. Laboratory-specific cutoffs for positive antibodies were applied to the results.

RESULTS: Interlaboratory variability for MFI values appears to be associated with different pretreatment regimens. The coefficient of variation (CV) of MFI from samples pretreated with ethylenediaminetetraacetic acid, dithiothreitol, or heat inactivation (EDHI) were similar, ranging from 14% to 56% (mean, 22%). For samples with no pretreatment, the CVs were significantly higher than EDHI-treated samples, ranging from 25% to 74% (mean, 39%; 95% confidence interval, 12.10-21.90; p < 0.0001). An intralaboratory comparison of pretreatment regimens confirmed these findings. Some positive antibody specificities present in EDHI-treated samples were negative in corresponding samples with no pretreatment when laboratory-specific cutoffs for positive antibodies were applied.

CONCLUSION: Our results show that greater interlaboratory precision can be achieved when samples are pretreated with EDHI as opposed to no pretreatment, likely because these pretreatments eliminate interference from inhibitors. Inhibitors may mask antibodies, leading to missed (or uncalled) specificities when no pretreatment is used.

Original language	English (US)
Pages (from-to)	488-497
Number of pages	10
Journal	Transfusion
Volume	60
Issue number	3
DOIs	https://doi.org/10.1111/trf.15666
State	Published - Mar 2020

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Research Support, Non-U.S. Gov't

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@article{1e70378593df46e4ab246ae335f2ed0d,

title = "The effect of serum pretreatment regimens for the detection of HLA class I antibodies in platelet-refractory patients",

abstract = "BACKGROUND: Single antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.STUDY DESIGN AND METHODS: Five patients' sera were overnight shipped to 12 laboratories in the United States and internationally. Recipients used their lab's SAB procedure to identify HLA Class I antibodies. The resultant mean fluorescence intensity (MFI) data were compared by instrumentation, bead lot, and pretreatment regimens. Laboratory-specific cutoffs for positive antibodies were applied to the results.RESULTS: Interlaboratory variability for MFI values appears to be associated with different pretreatment regimens. The coefficient of variation (CV) of MFI from samples pretreated with ethylenediaminetetraacetic acid, dithiothreitol, or heat inactivation (EDHI) were similar, ranging from 14% to 56% (mean, 22%). For samples with no pretreatment, the CVs were significantly higher than EDHI-treated samples, ranging from 25% to 74% (mean, 39%; 95% confidence interval, 12.10-21.90; p < 0.0001). An intralaboratory comparison of pretreatment regimens confirmed these findings. Some positive antibody specificities present in EDHI-treated samples were negative in corresponding samples with no pretreatment when laboratory-specific cutoffs for positive antibodies were applied.CONCLUSION: Our results show that greater interlaboratory precision can be achieved when samples are pretreated with EDHI as opposed to no pretreatment, likely because these pretreatments eliminate interference from inhibitors. Inhibitors may mask antibodies, leading to missed (or uncalled) specificities when no pretreatment is used.",

author = "{Biomedical Excellence for Safer Transfusion (BEST) Collaborative} and Gizem Tumer and Thomas Gniadek and Jennifer Baye and Ryan Pena and Paul Warner and Mark Fung and Lynette Beaudin and Heather Dunckley and Manish Gandhi and Birgit Gathof and Susan Hsu and Ellen Klohe and Nalaja Marcus and Roberta Bamert and Shona Sims and Minoko Takanashi and Silvano Wendel and Cohn, {Claudia S}",

note = "{\textcopyright} 2020 AABB.",

year = "2020",

month = mar,

doi = "10.1111/trf.15666",

language = "English (US)",

volume = "60",

pages = "488--497",

journal = "Transfusion",

issn = "0041-1132",

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TY - JOUR

T1 - The effect of serum pretreatment regimens for the detection of HLA class I antibodies in platelet-refractory patients

AU - Biomedical Excellence for Safer Transfusion (BEST) Collaborative

AU - Tumer, Gizem

AU - Gniadek, Thomas

AU - Baye, Jennifer

AU - Pena, Ryan

AU - Warner, Paul

AU - Fung, Mark

AU - Beaudin, Lynette

AU - Dunckley, Heather

AU - Gandhi, Manish

AU - Gathof, Birgit

AU - Hsu, Susan

AU - Klohe, Ellen

AU - Marcus, Nalaja

AU - Bamert, Roberta

AU - Sims, Shona

AU - Takanashi, Minoko

AU - Wendel, Silvano

AU - Cohn, Claudia S

PY - 2020/3

Y1 - 2020/3

N2 - BACKGROUND: Single antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.STUDY DESIGN AND METHODS: Five patients' sera were overnight shipped to 12 laboratories in the United States and internationally. Recipients used their lab's SAB procedure to identify HLA Class I antibodies. The resultant mean fluorescence intensity (MFI) data were compared by instrumentation, bead lot, and pretreatment regimens. Laboratory-specific cutoffs for positive antibodies were applied to the results.RESULTS: Interlaboratory variability for MFI values appears to be associated with different pretreatment regimens. The coefficient of variation (CV) of MFI from samples pretreated with ethylenediaminetetraacetic acid, dithiothreitol, or heat inactivation (EDHI) were similar, ranging from 14% to 56% (mean, 22%). For samples with no pretreatment, the CVs were significantly higher than EDHI-treated samples, ranging from 25% to 74% (mean, 39%; 95% confidence interval, 12.10-21.90; p < 0.0001). An intralaboratory comparison of pretreatment regimens confirmed these findings. Some positive antibody specificities present in EDHI-treated samples were negative in corresponding samples with no pretreatment when laboratory-specific cutoffs for positive antibodies were applied.CONCLUSION: Our results show that greater interlaboratory precision can be achieved when samples are pretreated with EDHI as opposed to no pretreatment, likely because these pretreatments eliminate interference from inhibitors. Inhibitors may mask antibodies, leading to missed (or uncalled) specificities when no pretreatment is used.

AB - BACKGROUND: Single antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.STUDY DESIGN AND METHODS: Five patients' sera were overnight shipped to 12 laboratories in the United States and internationally. Recipients used their lab's SAB procedure to identify HLA Class I antibodies. The resultant mean fluorescence intensity (MFI) data were compared by instrumentation, bead lot, and pretreatment regimens. Laboratory-specific cutoffs for positive antibodies were applied to the results.RESULTS: Interlaboratory variability for MFI values appears to be associated with different pretreatment regimens. The coefficient of variation (CV) of MFI from samples pretreated with ethylenediaminetetraacetic acid, dithiothreitol, or heat inactivation (EDHI) were similar, ranging from 14% to 56% (mean, 22%). For samples with no pretreatment, the CVs were significantly higher than EDHI-treated samples, ranging from 25% to 74% (mean, 39%; 95% confidence interval, 12.10-21.90; p < 0.0001). An intralaboratory comparison of pretreatment regimens confirmed these findings. Some positive antibody specificities present in EDHI-treated samples were negative in corresponding samples with no pretreatment when laboratory-specific cutoffs for positive antibodies were applied.CONCLUSION: Our results show that greater interlaboratory precision can be achieved when samples are pretreated with EDHI as opposed to no pretreatment, likely because these pretreatments eliminate interference from inhibitors. Inhibitors may mask antibodies, leading to missed (or uncalled) specificities when no pretreatment is used.

U2 - 10.1111/trf.15666

DO - 10.1111/trf.15666

M3 - Article

C2 - 31951028

SN - 0041-1132

VL - 60

SP - 488

EP - 497

JO - Transfusion

JF - Transfusion

IS - 3

ER -

The effect of serum pretreatment regimens for the detection of HLA class I antibodies in platelet-refractory patients

Abstract

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