Evaluation of flow cytometric enumeration of foetal erythrocytes in maternal blood

Abstract

In pregnant women subject to abdominal trauma or other foetomaternal haemorrhage, foetal red blood cells containing haemoglobin-F (HbF) can be found in the circulation. Recently, a monoclonal antibody to HbF has become commercially available, enabling application of a flow cytometric immunofluorescence method for accurately determining the concentration of HbF+ red blood cells. We demonstrate that white blood cells are included in the cluster selected as red blood cells and that these white blood cells exhibit a level of autofluorescence that coincides with the fluorescence signal from HbF+ red blood cells. However, these white blood cells can be excluded from the analysis, thus preventing spuriously increased HbF+ red blood cell counts. We present the results of patient samples containing HbF+ red cells as illustrations of the technique and as a potential interference by HbF-containing cells of nonfoetal origin. Using samples spiked with cord blood, the method is exactly linear with a high coefficient of correlation (r=0.997). Furthermore, the assay has excellent precision (CV < 2.4%), a low limit of detection (0.12% HbF+ RBC), is independent of Rhesus D and can be completed within 1.5 h. This method is suitable for accurate determination of foetomaternal haemorrhage.

Improvement of detection specificity of Plasmodium‐infected murine erythrocytes by flow cytometry using autofluorescence and YOYO‐1

“…It is well known that different subpopulations of erythrocytes show characteristic amounts of autofluorescence upon fixation with glutaraldehyde, with reticulocytes being the most autofluorescent erythrocytic population. This fact has been exploited to quantify reticulocytes in blood of patients with different hematological diseases (19)(20)(21)(22)(23)(24).…”

Estimation of “F‐cell” populations

“…The FL1 signal detects the ''bright'' hemoglobin fluorescence within the HbF cells and the ''dimmer'' fluorescence in the F cells. Although we occasionally confront a problem when analyzing FL1 histogram ''M1'' peaks for HbF cells that we and others have resolved (11,12), we always confront problems quantitating the ''M2'' F cell region. The problem is the ''downward slope'' of the negative/autofluorescent adult erythrocytic population and where to set the left side of the ''M2'' region that separates F cells from these adult erythrocytes.…”

“…Some have proposed using a ''99.95% to the left versus 0.05% events to the right'' FL2 autofluorescence channel approach with unstained erythrocytes for setting the oftentimes ''problematic'' left cursor of an F-cell histogram (5). We describe an evaluation of a previously described ''FL1 Â FL2'' approach to gate HbF cells that may also be applicable for optimally gating F cells (11,12).…”

Improving laboratory practice: Looking at fetal hemoglobin histograms

“…Although the published HbF test shows a FL1 ϫ FL2 dot plot approach to demonstrate a gate on autofluorescent leukocytes (2), the FL1 ϫ FL2 approach published by the Dutch actually shows an FL1 ϫ FL2 technique to purposefully set a gating strategy on the HbF cells (7).…”

“…The HbF test kit allows the flow cytometry laboratory to select its own gating strategy of choice to exclude leukocyte contamination from the analysis using the leukocytes' autofluorescence. Instead of using the FSC versus FL2 dot plot approach, we started in 2001 to evaluate an FL1 (fluorescein isothiocyanate channel with the fluorescent-tagged HbF fetal cells) versus FL2 (phycoerythrin [PE] channel that detects the leukocyte autofluorescence signal) dot plot analysis system for difficult cases with a small bleed, which has been subsequently published elsewhere (7). In contrast to the HbF test's usual clearly negative and positive cases (Fig.…”