Q7. My results for the 2-hydroxyestrone look good, but I get consistently higher variability and CVs for the 16a-hydroxyestrone assay. What could be happening?

A. Higher CVs for the 16a-hydroxyestrone assay are often due to plate washing problems. Please make sure that your wash buffer reservoir is free from other buffers or precipitates from previous buffers. Check that all tubing and washer manifolds are free of salt or protein deposition. The Tween-20 in the wash buffer may interact with certain proteins in some wash buffers to give problems. For best results, wash all components of the plate washer as recommended by the washer’s manufacturer before use in these assays.

Q8. How long is the kit good after I receive it?

A. If all kit components are stored correctly following the instructions in the product insert, The kit should perform accurately for at least two months from the date of shipment. Due to differences in storage temperatures, shipping time and conditions, etc., however, we recommend that the kit be used as soon as possible after receipt. (See the product insert).

Q9. I consistently get values for the metabolites slightly higher (or lower) than that on the positive control label. Is this OK?

A. Every individual operator/laboratory will have an individual bias in the assay results. As long as this variability is consistent and not greater than 15% from that given, and your ratios of 2/16-hydroxyestrone are reproducible, your results are acceptable. A 15% inter-laboratory variability is typical of all diagnostic tests. Differences greater than this may be due to operator or equipment problems.

Q10. What is the protocol for the first morning urine collection? What supplies do we need? What instructions do we give subjects?

A. Urine samples collected at the first void in the morning after sleep contain the greatest concentration of estrogen metabolites. This enables accurate measurement of these, especially in postmenopausal women. We highly recommend that samples be collected from "fasted" women, that is women not having eaten within eight hours before collection. The later measure minimizes the concentration of precipitable solids in the urine. The urine (25-100 ml) may be collected in a standard sterile resealable plastic urine collection container. This "spot" urine sample is collected by the Start-Stop-Collect method. The subject begins urination, stops, and then collects the urine sample into the container. The later is not absolutely necessary, but provides a cleaner sample. The sample may be kept at ambient for up to eight hours, or refrigerated at 4-8 deg. C for 24 hours. The metabolites are stable; the danger is bacterial growth. Upon reaching the laboratory, samples are examined, and those containing large amounts of blood or obviously otherwise contaminated are discarded. Ascorbic acid is added to each urine specimen in the amount of about 1 mg/ml.  This prevents oxidation of estrogen metabolites and lowers urine pH. The samples may then be aliquoted (1-5 ml/tube) and stored frozen. Some samples will contain precipitated matter; it is not necessary to remove this at this point, simply pull aliquot from above precipitate. Precipitate must be removed by centrifugation before assay. We recommend saving at least two aliquots of each sample. Store samples at below –20 deg. F (refrigerator freezer) for up to two weeks; store at below –70 deg C. indefinitely. AVOID repeated freeze-thaw cycles.

Supplies:

1.  90-100 ml sterile urine collection tube, able to be resealed tightly (screw cap) with labels for name, collection time, etc.

2.  Ascorbic acid to add to urine (make 100 mg/ml solution in dH2O and add 1% by volume to each urine). Store ascorbic acid solution at 4 deg C. in amber bottle.

1.  Do not eat anything after 8 p.m. the night before collection, drinking juices and soda is OK.

2.  Label the collection tube provided by doctor with your name, date, time.

3.  Collect the urine specimen at the first void of the morning.

4.  To collect urine, start urinating, and then stop after a few seconds. Then collect your sample into the container, and reseal with the cap immediately. You need collect only an ounce or more.

5.  Store container in a cool place and take the sample to the doctor’s that day.

Q11.  Can I alter the assay, such as shaking the plate?

A.  No. The assay has been developed without shaking the plate.  It is important to run the assay as described in the product insert.