The intracellular cystine content of acid-stabilized white blood cells may be used to evaluate the cystine-depleting effect of Cystagon^TM or to diagnose (or rule out) new cystinosis patients. White cells are used because cystine is not elevated in red cells or plasma of cystinosis patients. (It is elevated in urine also, but this is not diagnostic.) The normal value is generally less than 0.2 nmol half cystine/mg white cell protein. The cystinosis value is generally greater than 2 nmol half cystine/mg white cell protein.

Reminder: Blood must be taken 5-6 hours after a dose of Cystagon^TM and processed immediately for the white cell cystine measurements to be meaningful.

WHITE BLOOD CELL (WBC) PREPARATION
FOR THE DETERMINATION OF CYSTINE

First time requests: Show procedure to laboratory personnel. It will take about an hour and a half of their time. For first time preparers, we would like to have a normal control. For all diagnostic samples a normal control is required.

SUPPLIES FOR WBC PREPARATION(provided with kit)

ACD-Dextran solution

0.9% NaCl and 3.6% NaCl

Distilled water

15 ml centrifuge tubes

Wooden applicator

Return mailing label

0.1 ml 12% 5-sulfosalicylic acid (SSA) in Eppendorf tube

Empty Eppendorf tube with label

Request for Analysis Form and Billing Form- Print this and include with sample(s).

ADDITIONAL ITEMS TO BE PROVIDED BY YOUR LABORATORY

Refrigerated centrifuge set at 5° C and capable of spinning at 450 x g

Vortex type mixer

Timer

Dry ice/ethanol bath (styrofoam cup with walnut size piece of dry ice)

Warm water

Transfer pipets

Pipets for dispensing 0.8, 2.4 and 0.3 ml

Dry ice for shipping sample

Styrofoam insulated container, strapping tape

Payment for shipping sample

Request for Analysis Form and Billing Form

Illustrated WBC PREP

We especially need to know the time of the patient's last medication and the time of the blood draw. Optimally the blood is obtained 5-6 hours after the medication is taken.

If there are any questions concerning sample preparation, sample shipment or if any items are missing from the kit, please feel free to call Sara Albanil, supervisor of Cystine Determination Laboratory at (619) 471-0426. Ship no later in the week than Wednesday and if possible phone to let us know courier, airbill number, and when to expect the sample. The sample should be sent to the following address if the mailing label is not used:

Sara Albanil

University of California, San Diego

Cystine Determination Laboratory

CTF - Bldg A Room 202 (Building & room number are essential)

210 Dickinson Street

San Diego, CA 92103

CENTRIFUGE SETTINGS FOR PREPARATION OF WHITE CELL PELLET

PROCEDURE:     Illustrated WBC Prep Manual

1. ACD-Dextran should be at room temperature before using. The 0.9% NaCl, 3.6% NaCl, and distilled water should be placed on ice. Determine the correct setting for your centrifuge from the chart above.

2. Draw 5 mls of heparinized blood (0.1 ml heparin, Na or Li, in syringe or a green top vacutainer.) If blood must be allowed to stand before mixing with ACD-dextran, keep at room temperature. Standing markedly decreases the yield of wbc.

3. As soon as possible transfer blood from syringe to the 15 ml centrifuge tube, add an equal volume of ACD-Dextran, cap and invert to mix. Then remove cap or trapped blood may dribble down later. Set on ice for 30-45 minutes. The RBC's clump together and "settle" much faster than the WBC's.

4. Transfer supernatant to a clean 15 ml centrifuge tube. It is more important to avoid taking red cells than to take all of the supernatant. The final value is based on protein, not volume. Centrifuge for 10 minutes at 5° C at your centrifuge setting.

5. Discard supernatant. The white cell pellet will look red.

6. Add 0.8 ml of 0.9% NaCl and 2.4 ml of distilled water. Start timing and vortex continuously at moderate speed for 90 seconds and then add 0.8 ml of 3.6% NaCl. If there is a small red cell clot, remove it. Spin for 3 minutes at 5° C at your centrifuge setting.

7. Discard supernatant and repeat step 6.

8. Add 3.0 ml of 0.9% NaCl. Gently resuspend pellet by vortexing. Pellet does not have to be completely resuspended. Spin for 3 minutes at 5° C at your centrifuge setting.

9. Discard supernatant. If many red cells remain, take a kimwipe, roll it around wooden applicator and "wick up" as many red cells as possible without disturbing the white cell pellet.

10. After discarding supernatant, add as accurately as possible, 0.3 ml of the distilled water. Resuspend gently and transfer all of the cell suspension to a clean, labeled, Eppendorf tube. If pellet is gummy and resists suspension, it may help to cut off the end of a clean blue tip for use in transferring. Transfer EVERYTHING to the Eppendorf tube, even if it remains a glob.

11. Lyse the white cells by freezing in a dry ice/ethanol bath for 2 minutes and then in warm water for the minimum time necessary to thaw. Tube may be supported by a wire loop or strip of aluminum foil. Repeat two more times.

12. Wipe off the tube. Immediately transfer all (0.1 ml) of the SSA solution to the Eppendorf containing the lysed cells and vortex. Label with patient name and date using the provided cloth label and a ball point pen.

13. Freeze sample. The sample can remain frozen up to two weeks.

SHIPPING OF SAMPLE:
1. Make sure the sample is labeled with the patient name and date.

2. Wrap the sample to protect it during shipping and place it in a styrofoam container with a minimum of 5 pounds dry ice. If you are planning to do more samples, keep the leftover solutions frozen. We would like to have the empty bottles back with the sample.

3. Add the COMPLETED forms to a plastic bag and place in the styrofoam container. Seal the container with strapping tape. Send by overnight courier to address above. The room number is essential.

TO LABORATORY PERSONNEL RESPONSIBLE FOR WHITE CELL
PREPARATIONS FOR THE DIAGNOSIS OF CYSTINOSIS

Cystinosis is an inherited disease causing progressive kidney failure, retarded growth, and vision problems. When diagnosed early, these effects can be prevented or delayed by proper management and medication. The metabolic defect is the failure of cellular lysosomes to release cystine. As a consequence the free-cystine in the lysosomes accumulates to many times the normal value. The diagnosis of cystinosis is therefore based in part on the measuremnt of free-cystine in the tissues that accumulate this amino acid. This is most easily done in white (not red) blood cells.

The nature of blood cells and of cystine makes shipment of whole blood to us unreliable for diagnosis. Whole blood contains red cells which are rich in glutathione, a compound which will react with cystine. To prevent this the white cells are separated from the red cells. The white cells are kept cold to slow down reactions, then broken open, acidified, and frozen. This prevents the reaction of cystine with -SH compounds such as glutathione and precipitates the cell protein. These steps stabilize the cystine content of the preparation.

The ACD-Dextran procedure first separates most of the white cells from the red cells by density difference. To remove the rest of the red cells, the solution is made hypotonic for 90 sec. This lyses the RBC's but not the WBC's. Finally the pure white cells are frozen and thawed repeatedly to break open not only the cells but also the lysosomes where cystine is stored in cystinotic patients. The lysate is acidified, frozen, and sent to the Cystine Determination Laboratory for assay. We measure the cystine in the liquid phase and the protein in the precipitate.

Factors which make a good preparation and therefore an accurate cystine/protein value:

1. Prompt processing after blood draw. (If processing must be delayed while other samples are drawn, keep the whole blood at room temperature.)
2. Processing without stopping in the middle.
3. Minimum time between water addition to WBC and SSA addition, especially the thawing time. (During thawing the cystine can react with cell contents, cysteine can oxidize and proteins can hydrolyse.)
4. Minimum red cell contamination.

If you have comments or problems, you can include a note with the samples when you ship them or call Sara Albanil at (619) 471-0426.