Specimen Requirements

Specimen source, date of collection, and an order comment stating the reason for request including pertinent patient history is required at order entry.

Specimen Type: Bronchoalveolar lavage (BAL) + 2 cytospin slides (if cytocentrifuge is available)

Container/Tube: Preferred from Referring Sites: Concentrated BAL sample in enhanced RPMI Flow Cytometry Transport Medium

Note: The vials of enhanced RPMI Flow Cytometry Transport media are shipped to referring sites in gray UV-blocking biohazard bags to prolong the media shelf-life. The media is stored in the refrigerator.

Acceptable: Sterile container

Specimen Volume: 5-10 mL requested (minimum volume 2 mL)

Collection Instructions:

1. It is recommended that greater than or equal to 15% lymphocytes are present on BAL differential for flow cytometry testing for Interstitial Lung Disease (ILD) (sarcoidosis or hypersensitivity pneumonitis). The flow cytometry order will be cancelled per laboratory policy if less than 15% lymphocytes are present. Flow Cytometry testing will be performed on BAL specimens with less than 15% lymphocytes if the patient has a history of lymphoma.

2. Mix specimen well before taking an aliquot for flow cytometry testing.

3. Specimens should be prepared for transport as soon as possible after collection, preferably within 1 hour. See directions below.

4. Forward promptly to Flow Cytometry Lab at ambient temperature. Specimens should be received in the Flow Cytometry Lab within 24 hours of collection or as soon as possible thereafter. Note: It is recommended that testing be completed within 48 hours of specimen collection for optimal cellular viability.

5. Add-on flow cytometry testing to specimens that have been refrigerated is acceptable, however alterations in antigen expression may be seen following refrigeration.

Note: Specimens cannot be frozen.

To concentrate or prepare a bronchoalveolar lavage (BAL) specimen for transport in enhanced RPMI Flow Cytometry Transport Media:

1. If a cytocentrifuge is available, prepare 2 cytospin slides of varying thickness from the BAL to accompany the flow cytometry specimen.

a. Handwrite the slide labels. Do not use labels/stickers as patient identifiers on the slides.

b. Do not stain the slides.

2. Obtain a vial of enhanced RPMI Flow Cytometry Transport Medium (stored in a gray UV-blocking bag in the refrigerator).

3. Check that the enhanced RPMI Flow Cytometry Transport Medium is salmon/pink in color and has an acceptable expiration date. Note: Do not use the media if it is yellow in color.

4. Place 5 to 10 mL of BAL in a large test tube suitable for centrifugation.

5. Centrifuge the fluid for 7 minutes at 2,000 RPM to form a cell pellet in the bottom of the tube.

6. Carefully remove the fluid from the centrifuge. Do not disturb the cell pellet in the bottom of the tube.

7. Using a pipette, remove the supernatant from the tube. Leave the bottom 1 mL of fluid (contains the cell pellet) in the tube.

8. Vortex gently to resuspend the cell pellet in the remaining 1 mL of fluid.

9. Add 1 mL of concentrated BAL to the vial of enhanced RPMI Flow Cytometry Transport Medium.

10. Mix by inverting the vial 3 times.

11. Label the vial with a patient label that includes:

a. The patient’s name

b. The patient’s medical record number

c. The flow cytometry order number (if applicable)

d. The date and time of specimen collection

12. Place the sample in the gray UV-blocking biohazard bag for transport.

13. If cytospin slides were prepared from the BAL specimen, send slides in a slide container in the same biohazard bag as the sample.

West Market laboratories please forward slides to Fargo laboratory for pathology interpretation.

Additional information:

For questions about sample requirements or add-on testing, please call the Flow Cytometry Laboratory at (218)786-5741 for more information (hours of operation 0630 to 1500).