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A simplified flow cytometric method for detection of inherited platelet disorders—A comparison to the gold standard light transmission aggregometry

Navred, Kristoffer ; Martin, Myriam LU ; Ekdahl, Lina ; Zetterberg, Eva LU ; Andersson, Nadine Gretenkort LU ; Strandberg, Karin LU and Norstrom, Eva LU (2019) In PLoS ONE 14(1).
Abstract

Background Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score). Methods The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists.... (More)

Background Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score). Methods The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists. Antibodies against GPIIb-IIIa activation epitope (PAC1), P-selectin (CD62P) and lysosome-associated membrane glycoprotein 3 (LAMP3; CD63) were used as platelet activation markers. Sixty-six patients referred to the coagulation unit for bleeding symptoms were included in this single-center observational study. Platelet activation was determined by PACT and LTA. The results of both methods were correlated to BAT score. Results A two-by-two analysis using Cohen’s kappa analysis gave moderate agreement between LTA and PACT (82%, kappa = 0.57), when PACT analysis with ADP and CRP-XL was compared to LTA. Using LTA as reference method, positive predictive value was 70% and negative predictive value was 87%. A substantial number of patients had high BAT score and normal LTA and PACT results. Patients with abnormal LTA or PACT results had higher BAT score than patients with normal results, but the difference was not significant. Conclusions The performance in microtiter plates simplified the PACT method and enabled analysis of more patients at the same time. Our results indicate that with modification of the current PACT assay, a higher negative predictive value can be obtained. Furthermore, with comparable result to LTA the PACT could be used as a screening assay for inherited platelet disorders.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
PLoS ONE
volume
14
issue
1
article number
e0211130
publisher
Public Library of Science (PLoS)
external identifiers
  • pmid:30673773
  • scopus:85060371417
ISSN
1932-6203
DOI
10.1371/journal.pone.0211130
language
English
LU publication?
yes
id
a9032ecb-ed21-4fe9-b1f8-5b8d9b0e1206
date added to LUP
2019-02-01 08:51:16
date last changed
2024-10-01 15:46:18
@article{a9032ecb-ed21-4fe9-b1f8-5b8d9b0e1206,
  abstract     = {{<p>Background Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score). Methods The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists. Antibodies against GPIIb-IIIa activation epitope (PAC1), P-selectin (CD62P) and lysosome-associated membrane glycoprotein 3 (LAMP3; CD63) were used as platelet activation markers. Sixty-six patients referred to the coagulation unit for bleeding symptoms were included in this single-center observational study. Platelet activation was determined by PACT and LTA. The results of both methods were correlated to BAT score. Results A two-by-two analysis using Cohen’s kappa analysis gave moderate agreement between LTA and PACT (82%, kappa = 0.57), when PACT analysis with ADP and CRP-XL was compared to LTA. Using LTA as reference method, positive predictive value was 70% and negative predictive value was 87%. A substantial number of patients had high BAT score and normal LTA and PACT results. Patients with abnormal LTA or PACT results had higher BAT score than patients with normal results, but the difference was not significant. Conclusions The performance in microtiter plates simplified the PACT method and enabled analysis of more patients at the same time. Our results indicate that with modification of the current PACT assay, a higher negative predictive value can be obtained. Furthermore, with comparable result to LTA the PACT could be used as a screening assay for inherited platelet disorders.</p>}},
  author       = {{Navred, Kristoffer and Martin, Myriam and Ekdahl, Lina and Zetterberg, Eva and Andersson, Nadine Gretenkort and Strandberg, Karin and Norstrom, Eva}},
  issn         = {{1932-6203}},
  language     = {{eng}},
  month        = {{01}},
  number       = {{1}},
  publisher    = {{Public Library of Science (PLoS)}},
  series       = {{PLoS ONE}},
  title        = {{A simplified flow cytometric method for detection of inherited platelet disorders—A comparison to the gold standard light transmission aggregometry}},
  url          = {{http://dx.doi.org/10.1371/journal.pone.0211130}},
  doi          = {{10.1371/journal.pone.0211130}},
  volume       = {{14}},
  year         = {{2019}},
}