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

Abstract

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

Automated Oxygen Gradient Ektacytometry: A Novel Biomarker in Sickle Cell Anemia

“…HbF levels were quantified using capillary zone electrophoresis (Sebia Capillarys 2 Flex Piercing System ® , Lisses, France). F-cell analysis was performed using an adaptation of a previously described method using multiparametric flow cytometry (Davis and Davis, 2004). Whole blood samples were fixed, permeabilized, and labeled with antibodies to HbF (Invitrogen, Waltham, MA, United States) and CD235a (Glycophorin A, BD Biosciences, San Jose, CA, United States).…”

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells

“…Pools of HUDEP-2 cells electroporated with these pairs of deletion-forming Cas9 RNPs were differentiated into erythrocytes to assess HbF expression by intracellular flow cytometry with an HbF-specific antibody. The edited cell pools displayed an increased proportion of cells expressing HbF (Fig 2A, and Figure b in S1 File) [31]. 17.2% of cells expressed HbF when the PRR deletion RNPs were delivered, and 23% of cells expressed HbF when the Corfu deletion RNPs were delivered, compared to 1.9% of cells for untreated cells.…”

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells

“…HUDEP-2 cells that had been electroporated with RNP pairs were differentiated into erythrocytes to assess HbF expression by flow cytometry. The edited cell pools displayed an increased proportion of cells expressing HbF (Figure 2A, and Figure S3) (27). 17.2% of cells expressed HbF when the PRR deletion RNPs were delivered, and 23% of cells when the Corfu deletion RNPs were delivered.…”