Hi Nicholas, I our experience there are on average 5 n-RBC/100WBC in cord blood, many cords are on the lower side, maybe you were testing those low once Maybe the flow method is not sensitive enough to detect small number of n-RBC. Did you have a positive control? We analyzed 117 normal bone marrow products for transplantation and found that the number of n-RBCs increases significantly with age. Med Wieku Rozw 2007 - see paper paper attached So, the sample of marrow from older donor would be good positive control. Maybe there is a transplant center close to your lab In HPC Lab at CHLA we still use morphological method - Wright-Giemsa slides, I never got a good correlation with automated (Sysmex) analyzer method. Maybe I should try the flow method, where did you get reagents for Solution A and B? Monika E. Monika Smogorzewska PhD, CLS Stem Cell Technologist Hematopoietic Progenitor Cell Laboratory Research Immunology/BMT Childrens Hospital Los Angeles phone (323) 361 2458 fax (323) 361 8154 pager (213) 209 1779 CHLA PROPRIETARY/CONFIDENTIAL: INTERNAL USE ONLY ееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееее NOTICE: This e-mail message is for the sole use of the intended recipient(s) and may contain confidential or privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s), please contact the sender by return e-mail and destroy all copies of the original message. ееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееееее -----Original Message----- From: Nick Greco [mailto:ngreco@clevelandcordblood.org] Sent: Thursday, March 27, 2008 3:32 PM To: Cytometry Mailing List Subject: Determination of nRBC in umbilical cord blood Cytometry Discussion group: I am attempting to determine by flow the number of nucleated red blood cells in cord blood by following the procedure of Tsuji T et al., Cytometry 37:291-301, 1999. Briefly, the protocol uses EDTA-treated PB with anti-CD45-FITC and PI (threshold FSC 52, FACSCalibur) with an acid hypotonic lysis followed by a neutral hypertonic alkaline reagent. I am using a BD Canto II with CPD-treated CB and we are following this protocol but have been unable to see any nRBC in cord blood. I appreciate any suggestions. Nicholas J. Greco, Ph.D. Director of Laboratory Operations Cleveland Cord Blood Center Suite 150 25001 Emery Road Cleveland, OH 44128-0804 216-896-0384 (office) 216-896-0320 (fax) 440-488-9205 (cell) This attachment - 'image002.jpg' - 7.66 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/29dfe10a1b68ae08bec2f81898375724402e07ff.jpg This attachment - 'paper 2007 n-RBC in Marrow.pdf' - 3.11 MBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/228548ea9400fa16290cc989586df4d87eda12e0.pdfReceived on Tue Apr 1 10:58:00 2008
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