Six responses. In summary, two said no loss, four said some loss. It seems that I shall not be using flow cytometry as surrogate for bone marrow differential counts. Many thanks for your input. Bak ------------------------------------------------------------------- We use Whole Blood Lysis technique for our flow all the time, and we do not experience any loss of erythroid precursors. Good luck. Irina Irina Grigorieva, PhD Director, Flow Cytometry Laboratory Northside Hospital, Atlanta, GA (404) 851 6541 e mail: irina.grigorieva@northside.com ---------------------------------------------- Hi Bak that's a question I have been wrestling with for years, and there is no simple answer. It is all gradual, and not black and white. Precursors span a wide range of maturity (and lysability) levels, and it also depends of course on which lysis method/mechanism and protocol details you use. Mature erythrocytes lyse faster than nucleated erythroid cells, so if you lyse "just enough" to blow up the erythrocytes, most precursors will still be there. Their stainability with hemoglobin antibodies goes down before they vanish, but you will probably use surface markers. I would be interested in the responses you get. Could you copy them to me, please ? Greetings Ralph M. Bohmer, Melbourne --------------------------------------------------------------- Yes, erythroid precursors are lost during any lytic procedure. The % loss depends upon many factors but primarily on the number of total nucleated cells present and the quality of the aspirate [age post collection]. The best method is to collect the nucleated cells on a Histopaque 1.119 density interface. Then stain with antibodies while using a cell permeant, vital dye for gating. We use a 4 color, 3 tube protocol with 45/71/glycophorin A [erythroid precursors], 45/38/56 [plasma cells, neuroendocrine CA], and 45/34/HLA DR [myeloblasts, lymphoblasts] in the last. Lymphs, monos, and grans may be calculated from any one tube. We usually see up to 50% RBC contamination. The vital dye is syto13 from Molecular Probes. Good luck. Mark L. Shenkin, Ph.D. Director, Flow Cytometry Ameripath, Inc. mshenkin@att.net ------------------------------------------ From: Brian Newsom <BrianN@OpexaPharma.com> To: "Bakul Dalal [Dr.] [VH]" <bdalal@vanhosp.bc.ca> Date: 2004 December 03 1:13:19 PM Subject: RE: Loss of erythroid precursors from bone marrow during Whole Blood Lysis technic Depends on the type of lysis and what you consider a RBC precursor, however, the earlier the precursor the more protected they are from lysis is they way I have seen it. Brian --------------------------------------- With Tris ammonium chloride treatment for RBC lysis in bone marrow, erythrocytes are lysed, but erythroid precursors are not. Sandra H. Burnett, M.S., Ph.D. Assistant Professor Dept. of Microbiology & Molecular Biology 791 WIDB Brigham Young University Provo, UT 84602 Phone: (801) 422 1310 Fax: (801) 422 0519 sandrab@byu.edu -------------------------------------------- In our hands, the majority of polychromatic and orthochromic erythroid precursors are indeed lost from bone marrow that has undergone lysing using commercially prepared lysing buffers. For enumeration, proerythroblasts and basophillic erythroblasts make it through the procedure relatively unscathed. Renold Capocasale Research Scientist Experimental Pathology, Toxicology and Investigational Pharmacology Centocor, Inc a Division of Johnson & Johnson Inc Ph # 610 651 6421 Fax 610 651 7363 Bakul I. Dalal MD FRCPC FCAP FASCP Director, Flow Cytometry Laboratory Assoc Director, Hematology Laboratory Vancouver General Hospital Clinical Professor, Faculty of Medicine University of British Columbia bdalal@vanhosp.bc.ca 604 875 4496 / 4798(f) Life is not measured by the number of breaths we take, but by the moments that take our breath away.Received on Tue Dec 7 13:58:00 2004
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