Cleveland Family Study

4.4 Urine Processing

4.4.1 Procedure

1. Label two containers with the Participant ID number.
   • The 7 digit ID number followed by a 4 denotes urine samples.
   • One container is just urine (no additives) and the other container has 25 mL of 1M acetic acid to preserve the urine (final pH < 3).
   • The containers should also be clearly marked with labels for the plain urine and the urine+acid. This can be done with colored labels if desired, a yellow label for the plain urine and a green label for the urine+acid.
   • To ensure safe handling of both the urine and acid preservative, the containers that will hold the overnight urine collections should be adequately sized (1–2 L) and have a wide mouth to facilitate addition of samples.
   • Containers with covers are advised.
2. Record the urine volume and time of collection each time the participant voids during the overnight urine collection. The volume measurement can be approximate as this measurement does not need to be precise.
3. Divide the urine into two containers (1–2 L) and refrigerate (4°C) promptly (within 15 minutes) after removal from the participant’s room.
   • Urine and acid should be carefully decanted down the side of the containers to avoid splashing.
4. Once the first collection is received, measured and divided, swirl the urine and acid in the second container to mix the contents. Take care to avoid splashing.

   Note: We recommend that this procedure be carried out in a fume hood. While not toxic at the levels used for this study, acetic acid has a strong, unpleasant odor and the addition of acid to the urine may generate a small amount of heat.

5. Once the acid is added and the container is capped, the container can be safely removed for storage in the refrigerator.
6. The addition of subsequent urine samples to this container should also take place in the fume hood. The container can be removed from the refrigerator and the urine added in the hood. The container can then be re-capped and stored in the refrigerator until the next urine addition or processing.

4.4.2 Aliquoting Procedure and Scheme

Aliquot Scheme:

NOTE: DO NOT OVERFILL the urine aliquots. There needs to be adequate space for the urine to expand when frozen without displacing the cryovial cap.

1. Pipette one 3.0 mL sample from Container 1 (plain urine) into the 4.0 mL clear-capped cryovial #36.
2. Place on ice.
3. Pipette two 9.0 mL samples of urine from Container 1 into 10.0 mL yellow-capped tubes #37–38.
4. Place on ice.
5. Pipette two 9.0 mL samples of urine from Container 2 (urine+acid) into 10.0 mL green-capped tubes #39–40.
6. Place on ice.
7. Freeze the samples at -70°C or on dry ice within 10 minutes of aliquot preparation.
8. Discard left-over urine (unless the participant has been selected as a blind duplicate for urine processing).
9. Check-off on Processing Form the number of urine aliquots made (#s).
10. Send the completed Urine Collection/Processing Form to the LCBR.

4.4.2.1 Blind Duplicates

Aliquot Scheme:

Blind duplicates will also be processed for urine. At least 35 participants will be selected for urine blind duplicates. Each time an EDTA tube is drawn for a blood blind duplicate, urine blind duplicates will be processed as well. The urine QC ID numbers are the same as the blood QC ID numbers except that the final digit is a 5. The 2 digit extension denotes the cryovial number: 99930005-01. The QC number is matched to the Participant ID at Case Western Reserve University.

The urine blind duplicates will be aliquoted after the regular urine samples are added to tubes #36–40. They are color-coded in the same way as the regular urine samples.

It is important to complete the Blind Duplicate Shipping Log and include a copy in the shipping box with the frozen samples. Ship frozen urine blind duplicates at least one month after the original participant’s samples are shipped to LCBR. Blind duplicate samples may be shipped individually or several samples may be stored and shipped at the same time.