CONSULT WITH ASSAY LAB

You should ALWAYS consult with the Assay Lab prior to starting a series of experiments. There are several reasons for doing so:

1. To ensure that you have the latest version of these instructions (revisions may be in progress - but not yet posted - which could affect your experiments).

2. To ensure that the assay lab has a sufficient stock of critical reagents, such as antibody, to be able to process ALL of your samples from that set of experiments. This is necessary to allow your samples to be compared to each other without the intrusion of errors due to different cross-reactivities, etc.

3. To ensure that other critical reagents and supplies are on hand so that the Assay Lab can supply you with any special buffers or additives you might need when you need them.

4. To ensure that the assay lab has all supplies on hand necessary to process your samples when you submit them.

5. To ensure that the Assay Lab schedules its workload in order to free enough time to be able to process your time-critical samples.

6. To ensure that the Lab understands what you are trying to do, so that they may offer advice that may help you accomplish your goal.

Examples of such advice might be:

a) the organization of samples within and between assays to minimize intrinsic error,

b) the sample volume recommended to maximize sensitivity to the analyte concentrations you expect to find,

c) the use of other assays necessary to correct the requested assay for cross-reactivity,

d) the use of other assays to complement the findings from the assay you requested.

7. Other reasons for consultation are listed below.

EDTA Anticoagulants

There are 3 EDTAs normally employed as anticoagulants: Na2 EDTA, K2 EDTA, and K3 EDTA. Na2 and K2 EDTA are the anticoagulants of choice for samples requiring EDTA as the anticoagulant. (Any EDTA can adversely affect some assays, however. The choice of which, if any, anticoagulant (EDTA, heparin, etc.) always depends upon the analyte, the antibody, and the assay system. If EDTA is an acceptable anticoagulant, however, Na2 and K2 EDTA are almost certain to be acceptable EDTAs. K3 EDTA can adversely affect some antibodies or assays that Na2 and K2 EDTA would not affect. K3 EDTA must be tested with each such antibody and assay system to determine if it is an acceptable anticoagulant. Furthermore, since K3 EDTA is a liquid, it could have a dilutional effect on small sample volumes. (E.g., a 0.5 mL blood sample having a hematocrit of 40 is collected in a 5 mL Vacutainer containing the usual 20 mLs of K3 EDTA. The plasma volume = (0.5 mL * ((100-40)/100)) = 0.3 mLs. The dilution = 0.3 mLs/(0.3 mLs + 0.02 mLs K3 EDTA) = 0.3/0.32 = 0.9375. I.e., use of K3 EDTA would cause the sample's analyte concentration to be diluted by » 6%.)

Sample Type

Samples are routinely performed on plasma, plasma extracts, serum, and cell culture media as indicated herein. Should you need assays on other biological fluids, such as urine samples, not specified herein, or on extracts, please consult the Lab prior to sample collection.

Sample Storage

Generally, you may store, for subsequent assay, plasma, serum, urine, or culture medium samples in plastic or siliconized (silanized) glass tubes. Unsiliconized glass tubes should be avoided since they may adsorb some analytes and lower their concentrations in the sample. Do NOT store blood samples without first removing the RBCs etc. If platelets bind or metabolize the analyte(s) of interest, centrifugation at ³ 2500g for 30 minutes is necessary to remove platelets prior to storage.

Sample Containers

Submit samples in 12 X 75mm tubes if possible. If not possible, or if another tube size is more convenient, consult the Assay Lab before sample collection to be sure that the Lab's automated equipment can accommodate that tube size for that assay.

Arranging Your Samples for Assay

1. Determine which samples should be assayed together in the same assay in order to minimize the effects of random error on your results. (Between-assay inaccuracies and CVs are ALWAYS as large or, usually, larger than within-assay inaccuracies and CVs. Therefore, samples that you wish to compare closely with each other should be assayed in the SAME assay. Example: control, experimental, and recovery samples from the SAME animal should be assayed in the SAME assay. An alternate, but INcorrect, method would be to assay all controls from all animals in one assay, and all experimental samples from all animals in another assay. Sounds good, but this latter method would allow a between-assay error to sum with control vs. experimental differences and perhaps make a non-significant difference seem significant, or vice-versa!)

2. Fill out your Sample Worksheet

3. Arrange your samples in a sample rack in the same order in which they are listed on the Sample Worksheet.

4. Double check that the sample ID on the tube matches that on the Sample Worksheet.

New Assays

If you require an assay of some analyte not listed above, please consult the Lab.

Materials and Methods

Please consult the Assay Lab for technical details and published references when writing "Material and Methods" sections. Since these assays are frequently modified, you should seek the methods used to obtain the results for each paper.