A Flow‐Cytometric Equivalent of the Kleihauer Test

Nelson, Margaret; Zarkos, K.; Popp, H; Gibson, John

doi:10.1046/j.1423-0410.1998.7530234.x

Cited by 39 publications

(21 citation statements)

References 10 publications

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“…Monoclonal antibodies to HbF enable red cells containing HbF to be detected by flow cytometry (FC) for FMH estimation (Davis et al , 1998; Nelson et a l., 1998). Because the intensity of fluorescence of each cell is quantified and FC is very sensitive, accurate determination of fetal cells in maternal blood is possible.…”

mentioning

confidence: 99%

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high‐performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Chambers

Davies

Evans

et al. 2012

Transfusion Medicine

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Background and Objective: All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high‐performance liquid chromatography (HPLC). RhD‐negative women who deliver RhD‐positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). Materials and Methods: The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF‐positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti‐D FC was performed to quantitate fetal D‐positive cells in D‐negative women and compared with FMH estimated by AE and HbF FC. Results: AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. Conclusions: To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti‐D FC are discrepant or when there is maternal and fetal RhD compatibility.

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mentioning

confidence: 99%

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high‐performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Chambers

Davies

Evans

et al. 2012

Transfusion Medicine

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“…Similar reported experiences with the KB assay have also brought into question the clinical validity of this manual method (Duckett & Constantine, 1997;Emery et al, 1995). Flow cytometric methods have recently been developed that improve both sensitivity and precision by using monoclonal antibodies to HbF, i antigen, and D antigen (Campbell, Ware, & Mason, 1999;Chen, Davis, Wood, & Worzynski, 2002;Davis et al, 1998;Lloyd-Evans, Kumpel, Bromeloio, Austin, & Taylor, 1996;Nance, Nelson, Arndt, Lam, & Garratty, 1989;Navenot et al, 1998;Nelson, Zarkos, Popp, & Gibson, 1998;Thorpe et al, 1994). These methods reliably detect low levels of fetal RBCs in maternal circulation (ࣘ0.05%) and can more reliably determine the necessity for and dosage of Rh immune globulin in clinical practice.…”

Section: Fetomaternal Hemorrhage and Fetal Rbc Detectionmentioning

confidence: 99%

“…Current Protocols in Cytometry applicability (Chen et al, 2002;Davis et al, 1998;Nelson et al, 1998;Thorpe et al, 1994). Fetal RBCs are uniquely identified on the basis of their high intracellular content of HbF and can be readily distinguished from F cells (adult RBCs containing both HbF and other adult hemoglobins) and other adult RBCs on the basis of quantitative HbF expression ( Fig.…”

Section: Of 17mentioning

confidence: 99%

Enumeration of Fetal Red Blood Cells, Hemoglobin‐Specific RBC Cells, and F Reticulocytes in Human Blood

Davis

2019

CP Cytometry

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Recent advances in analytical cytometry have improved diagnostic tools for the study of erythropoiesis in anemic patients and resolution of differential diagnosis in diseases of the erythron. This article presents three applications of red blood cell (RBC) analysis-quantitation of fetal red cells, F-cell enumeration, and F-reticulocyte analysis-which improve diagnostic precision, sensitivity, and specificity, and provide better laboratory indicators of therapeutic efficacy in a variety of hematologic and obstetric disorders. Such advances also include the measurement and quantitation of RBC hemoglobins and their relative ribonucleic acid levels. These advances not only promise to improve diagnostic accuracy and laboratory precision over techniques such as the traditional manual reticulocyte counting method and the Kleihauer-Betke stain method for evaluating fetomaternal hemorrhage (FMH), but also serve as tools for newer assays of anemia diagnosis and improved clinical outcomes. In addition to the primary methods, supporting techniques for preparing spiked controls, automating data analysis, setting up a fetal hemoglobin acquisition protocol, and assaying reticulocytes using thiazole orange are also presented. C 2019 by John Wiley & Sons, Inc. Keywords: F cells r F reticulocytes r fetal hemoglobin r fetal red cells r fetomaternal hemorrhage r flow cytometry r hemoglobin levels r red blood cells How to cite this article: Davis, B. H. (2019). Enumeration of fetal red blood cells, hemoglobin-specific RBC cells, and F reticulocytes in human blood.

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“…Similar reported experiences with the KB assay have also brought into question the clinical validity of this manual method (Emery et al, ; Ducket and Constantine, ). Flow cytometric methods have recently been developed that improve both sensitivity and precision by using monoclonal antibodies to HbF, i antigen, and D antigen (Nance et al, ; Thorpe et al, ; Lloyd‐Evans et al, ; Davis et al, ; Navenot et al, ; Nelson et al, ; Campbell et al, ; Chen et al, ). These methods reliably detect low levels of fetal RBCs in maternal circulation (≤0.05%) and can more reliably determine the necessity for and dosage of Rh immune globulin in clinical practice.…”

Section: Commentarymentioning

confidence: 99%

“…The i antigen is a neoantigen that can be expressed on adult RBCs during some viral infections and paroxysmal nocturnal hemoglobinuria (PNH), as well as related disorders (Cooling, ; Navenot et al, ). The more recent approach to fetal RBC enumeration detects FMH using flow cytometry and anti‐HbF monoclonal antibodies, which, as sold by Caltag Laboratories, are cleared for use by the U.S. Food and Drug Agency, and have broader clinical applicability (Thorpe et al, ; Davis et al, ; Nelson et al, ; Chen et al, ). Fetal RBCs are uniquely identified on the basis of their high intracellular content of HbF and can be readily distinguished from F cells (adult RBCs containing both HbF and other adult hemoglobins) and other adult RBCs on the basis of quantitative HbF expression (Fig.…”

Section: Commentarymentioning

confidence: 99%

Enumeration of Fetal Red Blood Cells, F Cells, and F Reticulocytes in Human Blood

Davis

2004

CP Cytometry

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Recent advances in analytical cytometry have improved diagnostic tools for the study of erythropoiesis in anemic patients and resolution of the differential diagnosis in diseases of the erythron. This unit presents three applications of red blood cell (RBC) analysisquantitation of fetal red cells (see Basic Protocol 1), F cell enumeration (see Basic Protocol 2), and F reticulocyte analysis (see Basic Protocol 3)-that improve diagnostic precision, sensitivity, and specificity, and provide better laboratory indicators of therapeutic efficacy in a variety of hematologic and obstetric disorders. Such advances also include the measurement and quantitation of RBC hemoglobins and their relative nucleic acid levels. These advances not only promise to improve diagnostic accuracy and laboratory precision over techniques such as the traditional manual reticulocyte counting method and the Kleihauer-Betke stain method for evaluating fetomaternal hemorrhage (FMH), but also serve as tools for newer assays of anemia diagnosis and improved clinical outcomes. In addition to the primary methods, supporting techniques for preparing spiked controls (see Support Protocol 1), setting up a fetal hemoglobin acquisition protocol (see Support Protocol 2), and assaying reticulocytes using thiazole orange (see Support Protocol 3) are also presented. BASIC PROTOCOL 1 QUANTITATION OF FETAL RED CELLS BY FLOW CYTOMETRY Flow cytometric methods are more sensitive and precise than routine visual counting and reliably detect low levels of fetal RBCs in circulating maternal blood. The following protocol for the detection of fetal hemoglobin (HbF)-containing RBCs uses a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC), a method for the intracytoplasmic staining of red blood cells, and WinList or any other equivalent Software (Davis et al., 1998; Chen et al., 2000); however, the method can be easily adapted for use with anti-HbF monoclonal antibody reagents conjugated to other fluorochromes and with other data-analysis software. Materials Whole-blood samples anticoagulated with EDTA or other suitable anticoagulant (test within 4 hr of collection or store ≤72 hr at 4°C) Fetaltrol stabilized three-level controls (Caltag Laboratories) or spiked control blood samples (see Support Protocol 1) PBS/0.1% BSA (see recipe) 0.05% glutaraldehyde (see recipe) 0.1% Triton X-100 (see recipe) Anti-HbF antibody dilution (see recipe) 1% formaldehyde fixative (see recipe) 12 × 75-mm disposable polystyrene tubes (Falcon) with rack DAC II automatic cell washer (Baxter) or equivalent (optional) Multiparameter flow cytometer with 488-nm excitation and filters for detection of green and orange fluorescence WinList Software (Verity Software House; http://www.vsh.com) or equivalent Additional reagents and equipment for counting cells (APPENDIX 3A) and acquisition of data (see Support Protocol 2) Supplement 28

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A Flow‐Cytometric Equivalent of the Kleihauer Test

Abstract: The flow-cytometric method may serve to replace the traditional Kleihauer test since it appears to offer improved accuracy and objectivity.

Cited by 39 publications

References 10 publications

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high‐performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high‐performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Enumeration of Fetal Red Blood Cells, Hemoglobin‐Specific RBC Cells, and F Reticulocytes in Human Blood

Enumeration of Fetal Red Blood Cells, F Cells, and F Reticulocytes in Human Blood

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