In response to numerous requests for a FLAER - PNH method... here are our notes. May be I will write it up. --------------------------------------------------------------- We follow the product insert from the Protox Biotech, Victoria, BC, Canada (http://protoxbiotech.com/flaer.htm), with following provisos: FLAER FL1- Protox Biotech Victoria (developmental product)- Refer to preparation suggestions on the package insert. FLAER is received in freeze-dried form. Add 375 mL of PBS to dissolve. Thoroughly mix and spin at 1200 rpm for 5 minutes. Remove the supernatant and store in 30 m L aliquots in the -70° C freezer. The concentration in the frozen aliquots is 10-6 M when cells are added. Can't stress how important it is to limit exposure to light when working with FLAER! We aliquot new lots in a dark/lights-off room. Also, there will be up shift in staining when you open a new lot, so need to compare with standards. We run FLAER by itself. Gate on neutrophils, monos and lymphs using FS/SS. Lymphs staining is quite variable and essentially useless. Monos and neutrophils almost always correlate (in my experience of 30+ positives and 300+ negatives). It is usually possible to differentiate partial expression from absence. FLAER does not work on RBCs. Normal FLAER expression in neutrophils and monocots excludes PNH. I have seen ambiguous CD55 and 59 results with normal FLAER in patients clinically not PNH. We have not correlated this discrepancy with genetics. CD55 and 59 results may sometimes be ambiguous but FLAER is always night or day. Hope this helps. Bakul --------------------------------------------- Bakul I. Dalal MD FRCPC FCAP FASCP Clin Prof, Fac Medicine Hematopathologist, Vancouver General Hospital Suite JPPN1557, 910 West 10th Avenue Vancouver, BC, Canada, V5Z 4E3 604 875 4496(o)/875 4798(f)/877 5949(p)/484 6005(R) bakul.dalal@vch.ca <mailto:bakul.dalal@vch.ca> I don't want to become immortal through my work. I want to become immortal through never dying. Woody AllenReceived on Mon Mar 3 12:38:00 2008
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