Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry.
Background: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol.Study Design and Methods: D...
| Publicado en: | Transfusion Vol. 40; no. 11; pp. 1376 - 1377 |
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| Autores principales: | , |
| Formato: | CEU research tables/charts Journal Article |
| Publicado: |
Wiley-Blackwell
Nov2000
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=107005535&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 107005535 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00411132 1YB jtl: Transfusion issn: 00411132 maglogo: Y pubinfo: dt: Nov2000 vid: 40 iid: 11 pid: 480 pub: Wiley-Blackwell place: Malden, Massachusetts artinfo: ui: 107005535 107005535 NLM11099668 2001023386 NLM11099668 107005535 ppf: 1376 ppct: 1 formats: tig: atl: Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry. aug: au: Kumpel BM Kumpel, B M affil: International Blood Group Reference Laboratory, Southmead Road, Bristol, United Kingdom sug: subj: Flow Cytometry Maternal-Fetal Exchange Evaluation Quality Control (Technology) Technology, Medical Education, Continuing (Credit) Funding Source Human ab: Background: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol.Study Design and Methods: D-, D+, and artificial FMH (mixtures of D+ and D- RBCs) were stained either by a direct method (using FITC-conjugated IgG3 D MoAb [BRAD-3]), with or without dual labeling with PE-conjugated anti-GPA, or by indirect methods (using polyclonal anti-D followed by FITC- or biotin-conjugated anti-IgG reagents). Cells were selected for flow cytometric analysis on the basis of either forward or side scatter (log FSC/log SSC) characteristics or of GPA+ labeling or were unselected. The numbers of events labeled with anti-D were determined from histograms. For some samples, 10 replicates of 500,000 events each were analyzed.Results: Background fluorescent events in 10 directly labeled gated D- samples ranged from 0.007 to 0.023 percent, equivalent to 0.15- to 0.51-mL FMH. Both the use of a gate on log FSC/SSC or the selection of GPA+ events only resulted in a reduction in FMH of 0.3 mL or less. The intra-assay variation in FMH, or sampling error, was found to be approximately 10 percent at low artificial FMH (<10 mL) but greater (< or =50% with a CV of 15%) with D- samples. Direct staining was quicker and produced a lower background than indirect staining.Conclusion: The inherent sampling error that is due to the random distribution of rare events throughout the blood sample contributed greatly to the variation in the volume of FMH calculated by flow cytometry. The FMH should not be underestimated. For a routine assay, a simplified protocol and calculation will be sufficiently accurate to determine the dose of prophylactic anti-D that should be given to the patient. pubtype: Academic Journal doctype: CEU research tables/charts Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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