ENUMERATION OF LYMPHOCYTES SUBSETS (IMMUNOPHENOTYPING-IPT) (CD3, CD4, CD8, CD19 & CD16/56)

Transcription

1 Hospital Universiti Sains Malaysia for ENUMERATION OF LYMPHOCYTES SUBSETS (IMMUNOPHENOTYPING-IPT) (CD3, CD4, CD8, CD19 & CD16/56) Prepared by: Checked by: Approved by: En. Jamaruddin Mat Asan Date: Dr. Noor Suryani Mohd Ashari Date: PM Dr. Che Maraina Che Hussin Date: Amendment / Review No. Date Page Description Checked by Approved by / 7 Reviewed document / 7 Amendment 1 : Add procedure

2 Page 2 / 7 Enumeration of Lymphocytes Subsets ( Immunophenotyping IPT) (CD3, CD4, CD8, CD19 & CD16/56) CONTENTS: 1. Technique used 2. Principle of the test method 3. Clinical significance 4. Preparation of specimen 5. Equipment requirement 6. Reagent requirement 7. Test procedure 8. Report and interpretation 9. Internal quality control 10. Reference

3 Page 3 / 7 1. Technique used Immunophenotyping using direct immunoflourescence (read with flowcytometer) 2. Principle When whole blood is added to the reagent, the flourochrome labeled antibodies in the reagent bind specifically to leukocyte surface antigens. The stained samples are treated with lysing solution to lyse erythrocytes. During acquisition, the cells travel past the laser beam and scatter the laser light. The stained cells fluoresce. These scatter and fluorescence signals, detected by the instrument, provide information about the cell s size, internal complexity and relative fluorescence intensity 3. Clinical significance 1. Immunodeficiency cases 2. Human Immunodeficiency Virus cases 4. Specimen ml whole blood in EDTA container 2. Paediatric samples minimum 1 ml 5. Equipment and materials requirement 1. Flow cytometer machine 2. Micropipette (20µl, 50µl, 450µl) 3. Disposable 12x75mm polystyrene test tubes 4. Haematology Counter 5. Vortex

4 Page 4 / 7 6. Reagent requirement BD Multitest IMK kit containing - Fluorochrome- conjugated monoclonal antibody - 10X FACS Lysing Solution * Dilute FACS lysing solution 1:10 with distilled water 7. Procedure Staining direct immunoflourescence of whole blood using lyse 1. For each patient sample, label two 12x75mm tubes with number 1 and 2 2. Pipette 20uL of BD Multitest CD3/CD8/CD45/CD4 reagent into the bottom of each tube labeled 1 3. Pipette 20uL of BD Multitest CD3/CD16+CD56/CD45/CD19 reagent into the bottom of each tube labeled 2 4. Pipette 50uL of well-mixed, anticoagulated whole blood into the bottom of each tube 5. Cap the tubes and vortex gently to mix. Incubate for 15 minutes in the dark at room temperature (20 0 C C) 6. Add 450 ul of 1X BD Multitest lysing solution to each tube 7. Cap the tubes and vortex gently to mix. Incubate for 15 minutes in the dark at room temperature (20 0 C C) Samples are now ready to be analyzed on the flow cytometer

5 Page 5 / 7 Amendment 1 Haematology Counter 1. Turn on the printer, CPU and main unit 2. Log on user name [husm], password [husm] and press [OK] 3. Click [F6] and followed by [F9] to register sample 4. Insert sample number (lab number), test required (CBC + DIFF), patient ID, patient name and click [OK] 5. Click [F2], insert sample number, patient ID and click [OK] 6. Mix the sample blood thoroughly by inversion several times 7. Insert the probe to the bottom of the tube and press start button 8. The green LED flashes during sample aspiration, then buzzer beeps 2 times to indicate end of aspiration. Remove the tube 9. Click [F7] to display the result 10. Go to file and select print to print the results 11. Click [F4] and double click the [shutdown]. Click [execute] 12. After the shutdown sequence is completed, turn off the power of main unit and computer

6 Flow cytometer FACSCanto II Page 6 / 7 1. Turn on the main power of the flow cytometer, computer and printer 2. Insert password BDIS 3. Click [BDFACSCanto] on the desktop to open the BD FACSCanto software. 4. Insert password husm. After opening the software, fluidics startup will be automatically processed. (If not, go to cytometer bar and select fluidics startup) 5. Wait for the processing finish, then click [OK] 6. Insert the [PATIENT NAME],[PATIENT ID(lab no)], [CASE NUMBER (test quantity)], [PANEL (choose 4 colour TBNK)] and [LYMPHS (lymphocyte count)]. 7. Click [RUN] from main menu. Click [YES] for the message would you like to save the changes you ve made to the current work list 8. Use the of the test done for the [FILE NAME] box 9. Then click [SAVE] 10. When the message show you may load the tube CD3/CD4/CD45/CD8, insert the 1 st tube at the Sample Injection Tube (SIT), then click [OK] 11. Remove the tube from SIT after the message shows you may now unload the tube 12. Insert the 2 nd tube when the message shows you may load the tube CD3/CD16+56/CD45/CD Remove the tube from SIT after the message shows you may now unload the tube 14. Double click to the bar [STATUS] to see the result on the screen 15. Click [FILE] and [PRINT] to print out the result 16. Click [FILE], then [EXIT] 17. Choose [RUN FLUIDICS SHUTDOWN AND EXIT] for the messages please select how you want to exit 18. Then click [OK] 19. Switch off the computer and main power of flow cytometer

7 Page 7 / 7 8. Internal Quality Control 1. Percentage of CD3 difference must be less then 5% 2. Percentage of T-Sum must be less then 10% 3. Percentage of Lymph sum count (T Cells, B Cells, NK Cells) must be % 9. Reporting and interpretations of result Normal values lymphocyte populations as a function of age These values are derived from studies on the Caucasian population. Therefore they can only be used as a rough guide for the local population Absolute count expressed in 10 3 cells mm -3 Data are expressed as median together with percentile P25 and P75 values. Previous studies have shown that the distributions of many lymphocyte subsets are asymmetric 10. Reference 1. Pamphlet of the BD Multitest IMK kit 2. Flow cytometer manual 3. Hematology counter manual