The method that I have found useful is to incubate normal platelets with serum/plasma from a patient with anti-platelet antibodies (or commercial serum with anti-platelet reactivity). It is as follows: POSITIVE CONTROLS 1. Collect six EDTA tubes from a known normal individual. Try to always use the same individual in order to minimize the variation in the mean channel results of the quality control files. 2. Isolate platelets as described for patients following steps 1 - 4 only. 3. To the platelet pellet add 500 ul of saturating anti-PLA serum. This is the "Positive" control. 4. Following a 30' incubation on ice, wash the sensitized control x 2 with 10 ml of BSA/Tyrode's. 5. Resuspend the final pellet in 1.8 ml of BSA/Tyrode's and 600 mL of 4% formaldehyde and place the tube in the refrigerator for one hour. 6. Following a 60' incubation in the refrigerator wash the positive control x 2 with 10 ml of BSA/Tyrode's. Resuspend the final pellet in 5 ml of BSA/Tyrode's and measure the platelet count. Adjust the volume so that the final platelet count is 1x108/ml. Store in the refrigerator until the time of assay. 7. Assay the new positive control in parallel with the current positive control. If the new positive control does not fall within the previously established ranges of the quality control files, consult the supervisor for direction. Note: If the same individual's platelets with the same PLA1+ serum are used every time the mean channel of fluorescence should fall within the previously established ranges of the quality control files. 8. When a 50% loss of fluorescence intensity of the positive control is noted, make a new positive control. NEGATIVE CONTROLS: 1. Negative controls are obtained by using the platelet preparations from previously tested negative patient specimens. NORMAL CONTROLS: 1. A normal control is obtained by isolating the platelets from a hematologically normal individual. It should not be prepared more than two days prior to the staining procedure. Regards, Bruce H. Davis, M.D. Maine Medical Center Research Institute 81 Research Drive Scarborough, Maine 04074 USA PHONE: 207-885-8113 FAX: 207-885-8110 Email: davisb@mmc.org >>> "Dr. Michael Brown" <mbrown@ypii.com> 05/31/01 04:55PM >>> We are currently working on bringing a platelet-associated Ig assay by flow on line and are looking for good positive control material. It is unlikely that our initial volume for this test will be sufficient to allow use of previous patient positives from day to day. We are aware of a couple of ELISA kits, but they are quite expensive. Does the group have any suggestions for positive controls for this assay? Thank you in advance. Michael Michael Brown, MD Yellowstone Pathology Institute Billings, MT 406-238-6360
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