ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

Revised Version 2.0 10 May 2004

APOPTOSIS- PROPIDIUM IODIDE (PI) METHOD 1. PRINCIPLES AND PURPOSE OF THE TEST 1.1 Peripheral blood mononuclear cells (PBMC) isolated from HIV-1-infected persons undergo spontaneous apoptosis upon in-vitro cultivation. The method using propidium iodide staining (PI) and measurement of DNA content from PBMC isolated from HIV-1-infected persons to determine percentage apoptosis relies on the fact that PI will stoichiometrically stain cellular DNA following methanol fixation and RNase A treatment. Apoptotic cells have more fragmented DNA, which will be lost from the cell following the methanol fixation step. Thus, after staining with PI, these apoptotic cells will have a lower DNA content than non-apoptotic cells and will appear as a peak or smear with less fluorescence. Reduction in the value of percent apoptosis in PBMC obtained from HIV-1-infected persons over a time period after treatment with certain agents can aid to evaluate the effect of therapy and the progression of the disease.

1.2

2.

SPECIMENS 2.1 Patient preparation: 2.1.1 There are no specific instructions for the patient. An HIV seronegative person should be included as well

2.2

Specimen Collection Blood Collection: 2.2.1 Blood collected by venipuncture using heparin as an anticoagulant. Specimen Labeling: 2.3.1 Patient Identification number 2.3.2 Date and time of draw 2.3.3 Protocol Number Transport and storage: 2.4.1 Blood should be stored at room temperature and should be processed within 6 hours. Blood can be processed within 24 hr, however it results in higher baseline values as compared to 6 hr values. Thus, samples should be processed consistently within each protocol either within 6 hrs. or after 24 hrs. of storage.

2.3

2.4

3.

REAGENTS 3.1 Pooled Human AB Serum PHS (Suggested vendors: Gemini Bio-Products or NABI). Specific ACTG protocols will require a specific vendor or lot of serum. Verify with protocol immunologist or check the PIIS for each protocol found on the ACTG website. RPMI 1640 with L-glutamine should be used. If media is not used within 30 days, fresh L-glutamine must be added before use to a final concentration of 2 mM. (Suggested vendor: Biowhittaker)
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3.2

ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

Revised Version 2.0 10 May 2004

3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 4.

L-glutamine 200 mM (Suggested vendor: Biowhittaker or Gibco BRL Life Technologies) Penicillin-Streptomycin 100X stock solution (Suggested vendor: Gibco BRL Life Technologies) Ficoll-Hypaque (Suggested vendor: Amersham Pharmacia Biotec Inc.) or Histopaque-1077 (Suggested vendor: Sigma Chemical Co.) Bleach Methanol, stored at -20oC (Reagent Grade) Heparin vacutainer tubes (Suggested vendor: Becton Dickenson #6480) Dulbeccos Phosphate-Buffered Saline (D-PBS, pH 7.4 without calcium and magnesium) EDTA, Disodium Salt (Sigma #ED2SS) DNase-free RNase A (Sigma #R-4875) Sodium Azide Propidium Iodide (Sigma #P-4170) Igepal CA-630 (Sigma #I 3021)

EQUIPMENT 4.1 50 ml Conical tubes 4.2 Pipettes (serological and transfer) Plastic, sterile, disposable 4.3 12 x 75 mm sterile polypropylene, round-bottom tubes (Falcon#2063) 4.4 Laminar Flow hood (BL-II specifications) 4.5 Centrifuge, refrigerated (4C) 4.6 37oC humidified incubator with 5% CO2 4.7 Flow cytometer with an Argon laser REAGENT PREPARATIONS 5.1 PI Stain:

5.

5.1.1 D-PBS pH 7.4 supplemented with: 5.1.1.1 100 g/ml Propidium Iodide 5.1.1.2 0.1% Igepal CA-630 5.1.1.3 0.1% Sodium Azide 5.2 Rnase A:

5.2.1 D-PBS ph7.4 supplemented with: 5.2.1.1 250ug/mL Dnase-free Rnase A(Sigma) 5.2.1.2 10nM EDTA

5.3

Culture Medium: 5.3.1 RPMI 1640 supplemented with: 5.3.1.1 10% Heat inactivated (30 min. at 56oC) PHS 5.3.1.2 100 Units/mL Penicillin 5.3.1.3 100ug/mL Streptomycin 5.3.1.4 10mM Hepes buffer

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ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

Revised Version 2.0 10 May 2004

5.3.1.5 2mM L-Glutamine 6. INSTRUMENTATION CALIBRATION 6.1 Each laboratory must have a comprehensive quality assurance program that will assure proper instrument operation and calibration.

7.

ASSAY PROCEDURE:

NOTE: SUBSEQUENT PROCEDURES SHOULD BE PREFORMED IN A CLASS 2 BIOSAFETY LAMINAR FLOW HOOD USING STERILE TECHNIQUE AND ADHERING TO CDC/NIH STANDARDS (INCLUDING THE USE OF GLOVES AND LAB COATS). 7.1 PBMC Preparation

Check the consensus protocol for preparation of PBMCs found in the Virology Manuel for HIV Laboratories, Preparation of PHA-stimulated uninfected donor peripheral blood mononuclear cells on the web at: http://www.niaid.nih.gov/daids/vir_manual/. Patient and seronegative control PBMCs should be isolated by this consensus method. 7.2 Apoptosis setup 7.2.1 After ficoll separation and washing, set PBMCs at a concentration of 106 cells/ml in PHS medium. Add one ml to 12 x 75 mm polypropylene round-bottom tubes, for a total of 106 PBMC per tube. (Please Note: Set up duplicates tubes of each patient sample. In addition, set up duplicate tubes of a seronegative donor for each assay run.)

7.2.2

7.2.3 Incubate tubes for 48 hours at 37oC with 5% CO2 in a humidified incubator. 7.3 Fixing and Staining of Cells for the Detection of Apoptotic Cells Note: Cells should be kept cold (4C) during centrifugation and methanol treatment. 7.3.1 7.3.2 Centrifuge tubes for 5 minutes at 400-600 x g at 4oC. Aspirate and discard supernatant and wash cells by adding 1 ml cold PBS to each cell pellet and finger-flicking the tube to resuspend the cells. Centrifuge tubes for 5 minutes at 400-600 x g at 4oC Aspirate supernatant and wash as before with cold PBS. Centrifuge tubes for 5 minutes at 350 x g at 4oC.

7.3.3 . 7.3.4 7.3.5

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ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

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7.3.6

Resuspend cells in 50 l cold PBS and finger flick the tube. Add 450 l of -20oC Methanol. Store tubes at -20oC for a minimum of 1 hour. Cells may be stored at this stage up to a maximum of three weeks. Wash and centrifuge as above two times using cold PBS to remove all traces of methanol. Resuspend cells in 100 l PBS and finger-flick the tube. Add 10 l RNase A solution.

7.3.7

7.3.8

7.3.9

7.3.10 Incubate tubes at 37oC for 30 minutes. 7.3.11 After incubation, add an equal volume (110 l) of PI stain to each tube and incubate the tubes at 4oC for at least 30 minutes. 8. Instrument Setup 8.1 Two companies (Coulter and Becton Dickinson) flow cytometers are presently being used in the ACTG laboratories across the country. The following directions are to be used in combination with the standard practices recommended by the manufactures. Use an argon laser at 15 mW (excitation wavelength 488 nm) and collect red fluorescence from PI above 640 nm. Coulters Flow Cytometer

8.2

8.2.1 Create the following histograms with the linear scale set at 1024 (Example on the last page of this protocol): 8.2.1.1 Linear Forward Scatter (X axis) versus Log Side Scatter (Y axis) 8.2.1.2 Linear PI signal (X axis) versus Linear PI peak (Y axis, FL3) 8.2.1.3 Linear PI signal (X axis) versus Count (Y axis) 8.2.2 Set a discriminator on the histogram drawn in Section 8.0112 (PI signal vs. PI peak) low and adjust the voltage so that singlets are at a 45 angle. 8.2.3 Draw a gate on the histogram created according to the instructions in Section 8.0112 around the apoptotic, G0/G1 Phase, S-Phase, and G2+M Phase cells excluding any doublets present. Doublets appear as cells in the lower right-hand area of the dot plot. Avoid gating on these cells. 8.2.4 Anchoring on the above gate, acquire 5,000 10,000 events with the flow rate on low. Adjust G0/G1 peak to channel 200 (2) and the G2+M peak to channel 400 (4). 8.2.5 Draw 3 cursors:

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ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

Revised Version 2.0 10 May 2004

8.2.5.1

Draw a tight cursor (B) around the 200 peak so that the coefficient of variation (CV) is 3. It may be necessary to tighten the cursor to reduce the CV. A second cursor (C) should be placed before the G0/G1 peak to quantitate the percentage of apoptotic cells. This cursor should extend from the lower discriminator up to, but not overlapping, the left cursor of the G0/G1 peak. The third cursor (D) should be placed around the G2+M peak at approximately channel 400.

8.2.5.2

8.2.5.3 8.2.6

Percent of apoptotic cells is reported as the percentage of gated cells under the 2cd or C cursor.

8.3 Becton Dickinson Flow Cytometry 8.3.1 To check linearity, resolution and the doublet discrimination module (DDM) on a BD Flow Cytometer, the company provides a DNA QC Particles Kit (Cat. No. 349523) that will aide in setting up the instrument. This kit is run on a CELLQuest DNA Experiment document. 8.3.2 Using a linear scale, set up the following histograms (Example on the last page of this protocol): 8.3.2.1 8.3.2.2 Dot plot - Forward Scatter (X axis) versus Side Scatter (Y axis). Dot plot - FL2-Width (X axis) versus FL2 Area (Y axis). An FL2Area versus FL2-Height is acceptable, however Becton Dickinson recommends the first dot plot to better determine doublets.

8.3.2.3 Histogram plot FL2-Area (X axis) versus Counts (Y axis). 8.3.3 Set a threshold on the histogram drawn in Section 8.0222 low at approximately 50. This threshold eliminates debris. 8.3.4 Draw a gate (R1), on the histogram drawn in Section 8.0222 around the apoptotic, G0/G1 Phase, S Phase, and G2+M Phase cells excluding any doublets present 8.3.5 Anchoring the R1, acquire 5,000 10,000 events with the flow rate on low. Adjust G0/G1 peak to channel 200 (2) and the G2+M peak channel should be at 400 (4) if the instrument is linear. 8.3.6 Draw 3 cursors: 8.3.6.1 Draw a tight cursor (M1) around the 200 peak so that the coefficient of variation (CV) is 3. It may be necessary to tighten the cursor to reduce the CV. The second cursor (M2) should be placed around the G2+M peak at approximately channel 400.

8.3.6.2

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ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

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8.3.6.3

A third cursor (M3) should be placed before the G0/G1 peak to quantitate the percent of apoptotic cells. This cursor should extend from the lower threshold up to, but not overlapping, the left cursor of the G0/G1 peak.

8.3.7 Percent of apoptotic cells is reported as the percentage of gated cells under the M3 cursor. 9. QUALITY CONTROL 9.1 9.2 9.3 10. A normal HIV-1 seronegative control should be set up with each assay in duplicate. Normal values from healthy controls are usually less than 10% apoptotic cells. Patients should be set up in duplicate and their values may vary from 5% to 60-70% apoptotic cells. Flow cytometric data should be stored as a list mode file for later analysis.

LIMITATION OF THE METHOD 10.1 Instrument linearity, resolution, and doublet discrimination is vital to DNA content analysis. The G0/G1 peak should be set at channel 200 and the G2+M peak should be at channel 400 on a linear scale of 1024. Analysis of PI area rather than height enhances linearity analysis. A threshold or discriminator should be set at 50 PI fluorescence to eliminate the events which will be debris, mitochondria, apoptotic bodies, etc. and not whole intact cells with a somewhat reduced DNA content. Good resolution is determined by less peak spread and smaller CVs. Things that effect resolution is instrument cleanliness, sample preparation and flow rate. If the CVs are high, routine cleaning before running might be advisable. Definitively clean the flow cytometer after running cells stained with PI. Poor sample preparation results in an increase number of doublets, triplets, and quadruplets. To avoid their formation: 1) keep the specimen at 4C during the fixing stage and again after the staining phase and 2) make sure that the methanol is completely removed before staining the cells. If however an excessive number of doublets are formed, running the cells through a 45 m nylon mesh helps break some of them up.

11. METHOD VALIDATION 11.1 Intra-assay Variability: The difference between duplicate samples measured in 100 patients and 25 healthy controls was less than 5% (Data combined from the following investigators= laboratories: Dr. P. Blair, Dr. A. Landay, Dr. M.M. Lederman, Dr. T.W. McCloskey, Dr. M. Nokta, Dr. S. Pahwa.) Intra-subject Variability: The difference in % apoptosis seen among 3 blood draws within a week from 10 patients and 5 healthy controls was less than 10%. (Blood was obtained from patients who are on stable antiretroviral therapy and healthy

11.2

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ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

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controls within a week and assay was performed in batches. Data obtained from above referred laboratories.)

12.

REFERENCES

12.1

12.2

12.3

12.4

12.5

12.6

Features of apoptotic cells measured by flow cytometry. Darzynkiewicz, Z., Bruno, S. Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P, and Traganos. Cytometry (1992) 13:795-808. Fixation of mammalian cells for flow cytometric evaluation of DNA content and nuclear immunofluorescence. Schimenti K.J. and Jacobberger, J.W. Cytometry (1992) 13:48-59. HIV-1 Tat protein and its inhibitor Ro 24-7429 inhibit lymphocyte proliferation and induce apoptosis in peripheral blood mononuclear cells from healthy donors. Patki, A. H., and Lederman, M. M. Cellular Immunology (1996) 169:40-46. Use of a flow cytometric assay to quantitate apoptosis in human lymphocytes. McCloskey, T. W., Oyaizu, N., Coronesi, M., and Pahwa, S. Clin. Immunol. Immunopathol. (1994) 71:14-18. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. J. Immunol. Methods (1991) 139:271-279. CD4+-T-cell counts, spontaneous apoptosis, and Fas expression in peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1infected subjects. Patki, A. H., Georges, D., and Lederman, M. M. Clinical and Diagnostic Laboratory Immunology (1997) 4:736-741.

13.

EFFECTIVE DATE 13.1 13.2 Version 1.0 effective date: June 1996 Version 2.0 effective date: June 2000

14.

AUTHORS 14.1 Version 1.0 Abhay H. Patki Ph.D., Michael M. Lederman M.D., and Alan Landay Ph.D. 14.2 Version 2.0 Laboratory Technologists Subcommittee of the ACTG Site Management Committee.

15.

ACKNOWLEDGMENTS 15.1 We would like to acknowledge following Apoptosis focus group members for sharing data and making helpful suggestions in the protocol. Dr. Patrick Blair, Dr. Rebecca Gelman, Dr. Jon Kagan, Ms. Daniella Livnat, Dr. Thomas McCloskey, Dr. Mostafa Nokta, Dr. Savita Pahwa, and Mr. Stephen Perfetto.

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ACTG Laboratory Technologist Committee ACTG Lab Man Apoptosis - PI Method

Revised Version 2.0 10 May 2004

Procedure: ACTG Lab Man Apoptosis- PI Method

Prepared by: ACTG Laboratory Technologist Committee

Preparation Date: 01 June 2004

Date Implemented into the Laboratory: _________________

Updated on: _________________________________________ _________________________________________ Reviewed by: Date:

_________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ Supersedes Archived Manual: DAIDS Virology Manual for HIV Laboratories, Version January 1997

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