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

Bruce H. Davis1, Kathleen Thompson Davis1

1 Maine Medical Center Research Institute, Scarborough, Maine
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.17
DOI:  10.1002/0471142956.cy0617s28
Online Posting Date:  May, 2004
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Keywords: fetal red cells; fetomaternal hemorrhage; flow cytometry; fetal hemoglobin; Freticulocytes

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Table of Contents

  • Basic Protocol 1: Quantitation of Fetal Red Cells by Flow Cytometry
  • Basic Protocol 2: F‐Cell Enumeration by Flow Cytometry
  • Support Protocol 1: Preparation of Spiked Control Blood Samples
  • Support Protocol 2: Setting Up a Fetal Hemoglobin Acquisition Protocol
  • Basic Protocol 3: F‐Reticulocyte Analysis
  • Support Protocol 3: Reticulocyte Assay Using Thiazole Orange
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Quantitation of Fetal Red Cells by Flow Cytometry

  • 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 protocol 3)
  • 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 protocol 4)

Basic Protocol 2: F‐Cell Enumeration by Flow Cytometry

  • Peripheral blood from a healthy nonpregnant adult
  • Alsever's solution (see recipe)
  • Umbilical‐cord or newborn blood
  • Anticoagulant (e.g., EDTA)‐containing vacutainer
  • Additional reagents and equipment for collection of EDTA‐anticoagulated blood and determining blood ABO grouping

Support Protocol 1: Preparation of Spiked Control Blood Samples

  • Blood sample, preferably with high leukocyte count (>25 × 109 cells/liter)
  • Fetaltrol Level 3 or adult blood spiked with washed ABO‐compatible cord cells (see protocol 3)

Support Protocol 2: Setting Up a Fetal Hemoglobin Acquisition Protocol

  • Whole‐blood sample, anticoagulated with EDTA or other suitable anticoagulant (test within 6 hr of collection or store ≤72 hr at 4°C)
  • PE‐Cy5 (Tri‐color)–conjugated anti‐HbF monoclonal antibody (Caltag Laboratories)
  • 1% formaldehyde fixative (see recipe)
  • Retic‐Count kit (thiazole orange; BD Biosciences)
  • Sheath fluid: filtered PBS (0.40 µm pore)
  • Retic‐COUNT solution (BD Biosciences) or an alternative thiazol orange solution in filtered PBS
  • Multiparameter flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence
  • Additional reagents and equipment for staining blood samples (see protocol 1, steps to ) and thiozole orange reticulocyte analysis (see protocol 6).
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Literature Cited

Literature Cited
   Atweh, G., Suton, M., Nassif, I., Boosalis, V., Dover, G.J., Wallenstein, S., Wright, E., McMahon, L., Stamatoyannopoulos, G., and Faller, D.V. 1999. Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease. Blood 93:1790‐1797.
   Bianchi, D. 1999. Fetal cells in the maternal circulation: Feasibility for prenatal diagnosis. Br. J. Haematol. 105:574‐583.
   Bischoff, F.Z., Lewis, D.E., Nguyen, D.D., Murrell, S., Schober, W., Scott, J., Simpson, J.L., and Elias, S. 1998. Prenatal diagnosis with use of fetal cells isolated from maternal blood: Five‐color fluorescent in situ hybridization analysis on flow‐sorted cells for chromosomes X, Y, 13, 18, and 21. Am. J. Obstet. Gynecol. 179:203‐209.
   Bohmer, R., Campbell, T., and Bianchi, D. 2000. Selectively increased growth of fetal hemoglobin‐expressing adult erythroid progenitors after brief treatment of early progenitors with transforming growth factor beta. Blood 95:2967‐2974.
   Breymann, C., Fibach, E., Visca, E., Huettner, C., Huch, A., and Huch, R. 1999. Induction of fetal hemoglobin synthesis with recombinant human erythropoietin in anemic patients with heterozygous beta‐thalassemia during pregnancy. J. Matern. Fetal Med. 8:1‐7.
   Bromilow, I.M. and Duguid, J.K. 1997. Measurement of feto‐maternal haemorrhage: A comparative study of three Kleihauer techniques and two flow cytometry methods. Clin. Lab. Haematol. 19:137‐142.
   Campbell, T.A., Ware, R.E., and Mason, M. 1998. Detection of fetal hemoglobin in erythrocytes by flow cytometry. Ann. N. Y. Acad. Sci. 850:446‐448.
   Campbell, T.A., Ware, R.E., and Mason, M. 1999. Detection of hemoglobin variants in erythrocytes by flow cytometry. Cytometry 35:242‐248.
   Chen, J., Bigelow, N., and Davis, B. 2000. Proposed flow cytometric reference method for the determination of erythroid F‐cell counts. Cytometry 42:239‐246.
   Chen, J.C., Davis, B.H., Wood, B., and Worzynski, M.J. 2002. Multicenter clinical experience with flow cytometric method for fetomaternal hemorrhage detection. Cytometry 50:285‐290.
   Cheung, M., Goldberg, J., and Kan, Y. 1996. Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood. Nat. Genet. 14:264‐268.
   Cooling, L. 1997. Increased expression of i on HEMPAS red cells: A flow cytometric study. Transfusion 37:1102‐1104.
   Craig, J.E., Sampietro, M., Oscier, D.G., Contreras, M., and Thein, S. 1996. Myelodysplastic syndrome with karyotype abnormality is associated with elevated F‐cell production. Br. J. Haematol. 93:601‐605.
   Davis, B.H. 2001a. Diagnostic advances in defining erythropoietic abnormalities and red cell diseases. Semin. Hematol. 38:148‐159.
   Davis, B.H. 2001b. Diagnostic utility of red cell flow cytometric analysis. Clin. Lab. Med. 21:829‐840.
   Davis, B.H. and Bigelow, N.C. 1989. Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index. Arch. Pathol. Lab. Med. 113:684‐689.
   Davis, B.H., Olsen, S., Bigelow, N.C., and Chen, J.C. 1998. Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry. Transfusion 38:749‐756.
   Davis, B.H., et al. 2001. Fetal red cell detection; approved guideline. NCCLS Document H52‐A.
   Duckett, J.R. and Constantine, G. 1997. The Kleihauer technique: An accurate method of quantifying fetomaternal haemorrhage? Br. J. Obstet. Gynaecol. 104:845‐846.
   Emery, C.L., Morway, L.F., Chung‐Park, M., Wyatt‐Ashmead, J., Sawady, J., and Beddow, T.D. 1995. The Kleihauer‐Betke test. Clinical utility, indication, and correlation in patients with placental abruption and cocaine use. Arch. Pathol. Lab. Med. 119:1032‐1037.
   Garner, C., Tatu, T., Reittie, J.E., Littlewood, T., Darley, J., Cervino, S., Farrall, M., Kelly, P., Spector, T.D., and Theun, S.L. 2000. Genetic influences on F cells and other hematologic variables: A twin heritability study. Blood 95:342‐346.
   Giacoia, G.P. 1997. Severe fetomaternal hemorrhage: A review. Obstet. Gynecol. Surv. 52:372‐380.
   Hartwell, E.A. 1998. Use of Rh immune globulin: ASCP practice parameter. Am. J. Clin. Pathol. 110:281‐292.
   Iyamu, W.E., Adunyah, S.E., Fasold, H., Horiuchi, K., Elford, H.L., Asalsura, T., and Turner, E.A. 2000. Enhancement of hemoglobin and F‐cell production by targeting growth inhibition and differentiation of K562 cells with ribonucleotide reductase inhibitors (didox and trimidox) in combination with streptozotocin. Am. J. Hematol. 63:176‐183.
   Kleihauer, E., Braun, H., and Betke, K. 1957. Demonstration of fetal hemoglobin in erythrocytes of a blood smear. Klin. Wochenschr. 35:637‐638.
   Lee, D., Contreras, M., Robson, S.C., Rodock, C.H., and Whittle, M.J. 1999. Recommendations for the use of anti‐D immunoglobulin for Rh prophylaxis. Transfus. Med. 9:93‐97.
   Liu, T., Seong, P., and Lin, T. 1997. The erythrocyte cell hemoglobin distribution width segregates thalassemia traits from other nonthalassemic conditions with microcytosis. Am. J. Clin. Pathol. 107:601‐607.
   Lloyd‐Evans, P., Kumpel, B.M., Bromeloio, I., Austin, E., and Taylor, E. 1996. Use of a directly conjugated monoclonal anti‐D (BRAD‐3) for quantification of fetomaternal hemorrhage by flow cytometry. Transfusion 36:432‐437.
   Luna‐Fineman, S., Shannon, K.M., Atwater, S.K., Davis, J., Masterson, M., Ortega, J., Sanders, J., Steinherz, P., Weinberg, V., and Lange, B.J. 1999. Myelodysplastic and myeloproliferative disorders of childhood: A study of 167 patients. Blood 93:459‐466.
   Maier‐Redelsperger, M., de Montalembert, M., Flahault, A., Neonato, M.G., Ducrocq, R., Masson, M.P., Girot, R., and Eiron, J. 1998a. Fetal hemoglobin and F‐cell responses to long‐term hydroxyurea treatment in young sickle cell patients. The French study group on Sickle Cell Disease. Blood 91:4472‐4479.
   Maier‐Redelsperger, M., Elion, J., and Girot, R. 1998b. F reticulocytes assay: A method to evaluate fetal hemoglobin production. Hemoglobin 22:419‐425.
   Mundee, Y., Bigelow, N.C., Davis, B.H., and Porter, J.B. 2000. Simplified flow cytometric method for fetal hemoglobin containing red blood cells. Cytometry 42:389‐393.
   Mundee, Y., Bigelow, N.C., Davis, B.H., and Porter, J.B. 2001. Flow cytometric method for simultaneous assay of foetal haemoglobin containing red cells, reticulocytes and foetal haemoglobin containing reticulocytes. Clin. Lab. Haematol. 23:149‐154.
   Nagel, R.L., Vichinsky, E., Shah, M., Johnson, R., Spadacino, E., Fabry, M.E., Mangahas, L., Abel, R., and Stamatoyannopoulos, G. 1993. F reticulocyte response in sickle cell anemia treated with recombinant human erythropoietin: A double‐blind study. Blood 81:9‐14.
   Nance, S.J., Nelson, J.M., Arndt, P.A., Lam, H.C., and Garratty, G. 1989. Quantitation of fetal‐maternal hemorrhage by flow cytometry. A simple and accurate method. Am. J. Clin. Pathol. 91:288‐292.
   Navenot, J.M., Muller, J.Y., and Blanchard, D. 1997. Expression of blood group i antigen and fetal hemoglobin in paroxysmal nocturnal hemoglobinuria. Transfusion 37:291‐297.
   Navenot, J.M., Merghoub, T., Duarocq, R., Muller, J.‐Y., Krishnamoorthy, R., and Blanchard, D. 1998. New method for quantitative determination of fetal hemoglobin‐containing red blood cells by flow cytometry: Application to sickle‐cell disease. Cytometry 32:186‐190.
   Nelson, M., Zarkos, K., Popp, H., and Gibson, J. 1998. A flow‐cytometric equivalent of the Kleihauer test. Vox Sang. 75:234‐241.
   Paterakis, G.S., Thein, S.L., Fibach, E., and Cappellini, M.D. 1998. Cross evaluation of three flow cytometric F cell counting methods performed by different laboratories. Hemoglobin 22:427‐444.
   Polesky, H. and Sebring, E. 1981. Evaluation of methods for detection and quantitation of fetal cells and their effect on RhIgG. Am. J. Clin. Pathol. 76:525‐529.
   Reinhardt, D., Haase, D., Schoch, C., Wollenweber, S., Hinkelmann, E., V Heyden, W., Lentini, G., Worman, B., Schroler, W., and Pelcrun, A. 1998. Hemoglobin F in myelodysplastic syndrome. Ann. Hematol. 76:135‐138.
   Sebring, E. and Polesky, H. 1990. Fetomaternal hemorrhage: Incidence, risk factors, time of occurrence, and clinical effects. Transfusion 30:344‐357.
   Steele, C.D., Wapner, R.J., Smith, J.B., Haynes, M.K., and Jackson, L.G. 1996. Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: A review. Clin. Obstet. Gynecol. 39:801‐813.
   Thein, S.L. and Craig, J.E. 1998. Genetics of Hb F/F cell variance in adults and heterocellular hereditary persistence of fetal hemoglobin. Hemoglobin 22:401‐414.
   Thorpe, S.J., Thein, S.L., Sampietro, M., Craig, J.E., Mahon, B., and Huehns, E.R. 1994. Immunochemical estimation of haemoglobin types in red blood cells by FACS analysis. Br. J. Haematol. 87:125‐132.
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