Preparation of Specimen for Testing

The goal of sample preparation is to process a specimen from a patient with suspected leukemia or lymphoma into a representative sample suitable for introduction into the flow cytometer for analysis (5,7).

1. Always prepare slides with your sample for Wright stain preferably.

2. Prepare Wright stain touch imprints on tissue specimens.

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3.1.1. Process Tissue Specimens

The goal of tissue preparation is to maximize the cellular yield while maintaining cell viability; therefore, rapid and gentle processing is imperative.

1. Place specimen between two pieces of 53-pMmesh in a Petri dish.

3. Gently tease cells from tissue using the top of a 12 X 75-glass tube or the bottom of a syringe.

4. Filter cell suspension into a conical centrifuge tube.

5. Wash once with flow PBS.

6. Centrifuge at 804.2g for 5 min.

7. Aspirate supernatant carefully.

8. Resuspend in flow PBS, adjust to a 1+ cell suspension (turbidity standard). Proceed to Subheading 3.1.2., step 9.

3.1.2. Process Fluid Specimens

1. Aliquot the fluids into 50-mL conical screw-cap centrifuge tubes.

2. Centrifuge at 800g for 10 min to obtain the cell pellet. (If the cell count is very low, process as much fluid as necessary to obtain enough cells to work with.)

3. Aspirate the supernatant and vortex the cell pellet.

4. Wash one time each with PBS as follows.

5. Centrifuge at 804.2g for 5 min.

6. Aspirate supernatant carefully.

7. Vortex the cell pellet vigorously.

8. Resuspend in flow PBS and adjust to a 1+ cell suspension.

9. Pipet 100 pL of cell suspension into all 9 (12 X 75 mm) test tubes prepared for each patient Subheading 3.1.5.

10. Mix tubes 1-10 thoroughly, and incubate at room temperature in the dark for 20-30 min.

11. Wash stained cells twice with PBS using the cell washer. Suspend cells in 500 pL PBS and analyze on Coulter XL. If excess red blood cell contamination is present lyse the cells before analysis (proceed to Subheading 3.1.3., step 3).

3.1.3. Process Peripheral Blood

1. Count cells and if >10,000 cell/pL dilute with PBS and make sure that the sample contains no more than 10,000/pL.

2. Use SPA for preparing and staining as instructed.

3. Under reagent rack ID, change to "BD Basic Panel." This will display which antibody goes into which tube.

3.1.4. Processing Bone Marrow

This is the procedure for estimating total white blood count (WBC) for bone marrows. Obtain white cell count slide estimate as follows:

1. Scan the Wright-stained slide with low power first (either xl0 or x20). Look for an area most representative of the total cellularity of the slide.

2. Switch to x40 (high/dry) and count the number of WBCs per field.

3. Examine four to five fields and determine the average number of WBCs per field. Do not truncate. Include smudge cells and degenerated cells in your count. (If there is a significant number of nucleated red blood cells (NRBCs), these can also be noted separately.)

4. Determine WBC by multiplying the average number of WBC per field by 2000 (3000 if using x50 magnification). Refer to sample volume and dilution guide to determine amount of sample to be used.

Follow the whole blood stain then the lyse procedure to stain the cells.

3.1.5. Mononuclear Cells Isolation From Peripheral Blood/Bone Marrow by Ficoll-Hypaque

Follow procedure as recommended by the manufacturer.

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