Abstract
Introduction: An increasing number of medical centers can collect bone marrow, peripheral blood, stem cells. Pathology laboratories should accommodate this trend, but investment in additional equipment may be impractical. In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34cells With this procedure, an absolute CD34 count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-County fluorospheres. To reduce errors inherent to sample washing/centrifugation,
Aims: To compare CD34+ cell counting results by using with and without the use of a separate hematology analyzer (ie, single-platform versus dual-platform methodologies).
Material and Method: A total of 34 samples of peripheral blood, apheresis packs, and bone marrow aspiration were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Dual-platform CD34+ cell counting incorporated data from navious modal 2 laser and eight colour.
Results: Subtle differences in CD34+ cell counting between 2 systems and 2 methods did not achieve statistical significance.
Conclusion: It is the accurate determination CD34+ cell enumeration, properly validated, can support care for patients undergoing transplants setting. These modifications may improve the inter laboratory reproducibility
Keywords: International Society of Hematotherapy and Graft Engineering (ISHAGE), Absolute CD34+ Cell COUNT.
References
- Fritsch G, Stimpfl M, Kurz M, et al. Characterization of hematopoietic stem cells. Ann N Y Acad Sci. 1995;770:42–52.
- Fritsch G, Stimpfl M, Kurz M, et al. The composition of CD34 subpopulations differs between bone marrow, blood and cord blood. Bone Marrow Transplant. 1996;17(2):169–178.
- Allan DS, Keeney M, Howson-Jan K, et al. Number of viable CD34(+) cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2002;29(12):967–972.
- Chin-Yee I, Anderson L, Keeney M, Sutherland DR. Quality assurance of stem cell enumeration by flow cytometry; Canadian QASE Study Group. Cytometry. 1997;30(6):296–303.
- Fritsch G, Printz D, Stimpfl M, et al. Quantification of CD34+ cells: comparison of methods. Transfusion. 1997;37(8):775–784.
- Gratama JW, Orfao A, Barnett D, et al. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells; European Working Group on Clinical Cell Analysis. Cytometry. 1998;34(3):128–142.
- Ikeda K, Ohto H, Kanno T, et al. Automated programs for collection of mononuclear cells and progenitor cells by two separators for peripheral blood progenitor cell transplantation: comparison by a randomized crossover study. Transfusion. 2007;47(7):1234–1240.
- Ikeda K, Ohto H, Nemoto K, et al. Collection of MNCs and progenitor cells by two separators for PBPC transplantation: a randomized crossover trial. Transfusion. 2003;43(6):814–819.
- Kessinger A, Sharp JG. Mobilization of blood stem cells. Stem Cells. 1998; 16(suppl 1):139–143.
- Kessinger A, Sharp JG. The whys and hows of hematopoietic progenitor and stem cell mobilization. Bone Marrow Transplant. 2003; 31(5):319–329.
- Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines; International Society of Hematotherapy and Graft Engineering. Cytometry. 1998;34(2):61–70.
- Barbosa IL, Sousa ME, Godinho MI, Sousa F, Carvalhais A. Single- versus dual-platform assays for human CD34+ cell enumeration. Cytometry. 1999;38(6): 274–279.
- Gajkowska A, Oldak T, Jastrzewska M, et al. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods. Folia HistochemCytobiol. 2006;44(1):53–60.
- Sutherland DR, Nayyar R, Acton E, Giftakis A, Dean S, Mosiman VL. Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers. Cytotherapy. 2009;11(5): 595–605.
- Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry; International Society Of Hematotherapy and Graft Engineering. J Hematother. 1996;5(3):213–226.
- Robinson G, Morgan L, Evans M, et al. Effect of type of haematology analyser on CD4 count. Lancet. 1992;340(8817):485.
- Mercolino TJ, Connelly MC, Meyer EJ, et al. Immunologic differentiation of absolute lymphocyte count with an integrated flow cytometric system: a new concept for absolute T cell subset determinations. Cytometry. 1995;22(1):48–59.
- Strauss K, Hannet I, Engels S, et al. Performance evaluation of the FACS Count System: a dedicated system for clinical cellular analysis. Cytometry. 1996;26(1):52–59.
- Barnett D, Granger V, Kraan J, et al. Reduction of intra- and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training; DK34 Task Force of the European Working Group of Clinical Cell Analysis (EWGCCA). Br J Haematol. 2000;108(4):784–792.
- Gratama JW, Kraan J, Keeney M, Sutherland DR, Granger V, Barnett D. Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study. Cytotherapy. 2003;5(1):55–65.
- Barnett D, Janossy G, Lubenko A, Matutes E, Newland A, Reilly JT. Guideline for the flow cytometric enumeration of CD34+ haematopoietic stem cells: prepared by the CD34+ haematopoietic stem cell working party; General Haematology Task Force of the British Committee for Standards in Haematology. Clin Lab Haematol. 1999;21(5):301–308.
- Gratama JW, Kraan J, Keeney M, Mandy F, Sutherland DR, Wood BL. Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline. Vol 27 (16). 2nd ed. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 2007.
- Sutherland DR, Keeney M, Gratama JW. Enumeration of CD34+ hematopoietic stem and progenitor cells. Curr Protoc Cytom. 2003;chapter 6:unit 6.4.
Corresponding Author
Dr Sachin Sharma
Assistant Professor MGM Medical College with M.Y. Hospital Indore, MP, India
Email: This email address is being protected from spambots. You need JavaScript enabled to view it.
DOI: https://dx.doi.org/10.18535/jmscr/v7i1.101
