INTRODUCTION

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Repositories of biologic specimens from cohort studies are valuable resources for medical research. Newly developed assays can take advantage of the specimens in the repository to measure markers that might have been available at the time the samples were obtained. Although many long-term cohort studies invest substantial resources in establishing repositories of specimens that are key for intensive laboratory pathogenesis studies, seldom have reports documenting the quality and utility of those repositories been prepared. The preservation of viable peripheral blood mononuclear cells (PBMC) for virologic and immunologic investigations has been undertaken by several multicenter studies of human immunodeficiency virus (HIV) disease, but few data are available on how long materials can be stored, on typical numbers of cells recovered after different intervals, or on the uses for which such samples are appropriate. Every sample frozen in a cohort study is potentially valuable, and care must be taken to determine the best application for these specimens. Therefore, the random testing of such samples simply to validate the quality of materials is avoided. However, if investigators plan to use samples from repositories for important issues such as genetic research or the investigation of other surrogate markers of disease, measures of the quality and validity of the samples are imperative.

In studying the natural history of HIV, properly preserved PBMC specimens have proven to be invaluable to elucidate HIV pathogenesis. The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective study of the natural history of HIV infection in 5,622 homosexual or bisexual men, conducted since 1984 in four centers located in Baltimore, Md.; Chicago, Ill.; Pittsburgh, Pa.; and Los Angeles, Calif. Details about the recruitment and characteristics of the MACS cohort and the study protocol have been reported previously (2, 10). Applications for the study have included culturing of virus, studies of immune function, immunophenotyping by flow cytometry, and assays of immune function. The purpose of this analysis was to evaluate the viability and recovery of stored PBMC specimens from the MACS that were frozen and thawed over a 12-year follow-up period in conjunction with specific scientific research initiatives utilizing these specimens. This information may be helpful in planning future repositories or as a comparison for other studies.

	MATERIALS AND METHODS

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Cryopreservation, shipment, and storage of lymphocytes. Ninety-milliliter blood samples were collected at 6-month intervals in heparinized tubes (Becton Dickinson Inc., Rutherford, N.J.). The method used was essentially that described previously in detail (8). Whole blood was centrifuged for 15 min at 300 × g. Next, the buffy coats for each sample were placed in a 50-ml polypropylene tube, and an equal volume of isotonic saline was added. This was overlaid onto Ficoll-Hypaque density gradients. Mononuclear cell suspensions were isolated by centrifugation at 700 × g for 10 min. The cell pellets were washed twice at room temperature with Hanks buffered salt solution by centrifugation at 300 × g for 10 min. The final pellets were resuspended in 2 ml of RPMI 1640 containing 20% human AB serum or fetal calf serum, and the suspensions were gently mixed and chilled on ice. Cells were counted on a Coulter Counter in the centers in Baltimore and Los Angeles by using 20 µl of cell suspension in 10 ml of isotonic buffered saline plus 3 drops of Zap-oglobin2 lysing solution or on a hemocytometer in the centers in Chicago and Pittsburgh. Cells were diluted to 2 × 10⁷ cells per ml in cold RPMI 1640 plus 20% serum. Then, an equal volume of RPMI 1640 plus 20% dimethyl sulfoxide (DMSO) as a cryoprotectant was added. Next, the cells were quickly aliquoted into freezing vials (1 ml per vial). Usually six vials were saved for each blood specimen, and each vial contained 10⁷ PBMC. The final freezing solution consisted of RPMI 1640 containing 10% serum and 10% DMSO. All blood samples were processed and frozen by sterile techniques, within 24 h (range, 5 to 24 h) of collection, at the center where they were obtained. For the vast majority of samples, freezing occurred in less than 10 h after collection.

Thawing cryopreserved cells and determining viability. Samples for this study were shipped from BRI in dry-nitrogen shippers (see above) and were then stored continuously in liquid nitrogen or at 135°C until immediately prior to their use. The method used was essentially that described previously (8). Cells were removed from storage and rapidly thawed by immersion in a 37°C water bath with gentle agitation, by trained technical staff in two MACS pathogenesis laboratories. Each tube was thawed individually until only a small amount of ice crystals remained, a procedure that took about 40 s. Samples were immediately transferred to round-bottom tissue culture tubes. Then, 10 to 14 ml of RPMI 1640 containing 10% human AB serum or fetal calf serum was added over a period of 4 min with agitation, and the tubes were centrifuged immediately at 100 to 400 × g for 10 min. After the DMSO-containing liquid had been removed by aspiration, the cells were washed once in medium containing at least 10% serum or an alternate protein source. Next, the cells were resuspended in about 1 ml of buffer appropriate to the assay to be done, and counts and viability were determined with a hemocytometer and the trypan blue dye exclusion technique. With this method, dead cells appear blue and are therefore distinguishable from viable cells. Viable cells for this project were defined as PBMC that had survived the freeze-thaw process. They were used (i) to evaluate cell function, (ii) for immunophenotype analysis by flow cytometry, (iii) to isolate HIV for subsequent analysis, or (iv) for immortalization by Epstein-Barr virus transformation.

Statistical analysis. Data reported to the Center for the Analysis and Management of the MACS included (i) the date the sample was originally collected, processed, and frozen (freeze date); (ii) the date the sample was thawed for laboratory testing (thaw date); (iii) the total number of cells recovered regardless of viability; and (iv) the percent cell viability. The number of viable cells recovered was calculated by multiplying items iii and iv. We addressed two main questions: first, whether cell recovery and viability differed over time according to the duration of freezing, and second, whether cell viability differed by center, the original processing or freeze date, the HIV serostatus of the donor, and the CD4⁺-lymphocyte count of the donor.

	RESULTS

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Viability testing. A total of 596 PBMC samples from 147 individuals were tested for cell viability. They had initially been stored at one of the four MACS centers (179 samples in Baltimore, 170 in Chicago, 88 in Pittsburgh, and 159 in Los Angeles). The center where the sample was originally collected and stored may or may not coincide with the city or laboratory where the sample was thawed, evaluated for cell viability, and used for a pathogenesis study. A total of 197 specimens were from HIV-seronegative donors, and 399 specimens were from HIV-seropositive donors.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Percent cell viability over time by center. (per year) for Baltimore is 0.10%, that for Chicago is 0.47%, that for Pittsburgh is 0.73% (after excluding four specimens with <35% viability, originally stored from 1985 to 1986), and that for Los Angeles is 0.09%.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Viable-cell recovery over time by center. (per year) for Baltimore is 5.1 × 105, that for Chicago is 5.4 × 105, that for Pittsburgh is 1.5 × 105, and that for Los Angeles is 2.3 × 105. Reference lines at 107 mark the number of PBMC stored as suggested by the MACS protocol.

Use of cryopreserved PBMC for pathogenesis studies. One example of a typical use of the samples in this repository for pathogenesis studies is a study that included a subset of the 596 samples, a total of 112 samples from all time points between seroconversion and T-cell inflection (12), a span of up to 10 years. In all cases, one vial per person visit yielded sufficient cells to complete flow cytometry, analysis of cytotoxic-T-lymphocyte precursors, proviral HIV load measurements, and HIV sequencing (16). In another study, 37 of the 596 samples were from donors who were severely immunosuppressed (CD4⁺-cell counts, <50 per mm³). Every sample provided enough cells to obtain immunophenotyping results, and culturable virus was recovered from all except one of the samples (6). In another series of studies (1, 14), 118 samples from HIV-seropositive donors were tested for cell surface markers and immune function.

	DISCUSSION

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The data presented here indicate that 90% of the time, the yield of viable cells from cryopreserved MACS specimens was at least 3.5 × 10⁶, with a viability of 82% or higher. By obtaining cell counts and measuring cell viability for each frozen sample as it was thawed for ongoing research, we established the consistency of the cryopreservation method across time and centers for MACS specimens.

We had anticipated the recovery of a minimum of 6 × 10⁶ viable cells per vial from the initial 10⁷ cells stored with a viability of at least 85%. Our results indicate that 519 of the 596 specimens met our goal for viability, but only slightly more than half of them met the goal for numbers of viable cells recovered. A reduction of recovery from 10⁷ to 6 × 10⁶ cells might be expected due to cell loss during the freeze-thawing and subsequent washing process. The reason why substantially fewer than the expected 6 × 10⁶ cells per vial were recovered in some cases is not known but most likely relates in part to variations in protocol with regard to the number of cells originally placed in the vials. There was substantial variation in this protocol, as shown by the recovery of >10⁷ cells from some vials and by the increased cell recovery in relation to the time frozen in the center in Chicago. Since the directionality of changes in cell recovery was not consistent across centers, cell loss in the MACS is probably not due to the length of time frozen.

Although additional data are needed for confirmation, the use of controlled-rate freezers and sample processing within 6 h of blood collection could have contributed to the low variability of data observed in the Los Angeles center. Furthermore, after switching to a controlled-rate freezer in the Pittsburgh center, there was a significant improvement in the maintenance of viability over time. However, it is important to note that the sample size was small (45 samples without and 39 with a controlled-rate freezer), and other possible confounding factors were not addressable.

Some variability in the numbers of cells recovered could be due to variations among technicians who performed the cell counts, but the MACS does not include data on this. In addition, differences in the methods and instruments for cell counting at the time of freezing could contribute to variation, but this possibility was also not assessed. The MACS team developed a method manual that was provided to all the technicians who stored cells. However, our data suggest that future studies may need to incorporate methods to reduce the contribution of technical variables to cell recovery by using cross-center training and standardization programs. Upon thawing, if the cells are used for assays of lymphocyte function, it may be important to determine the composition of the recovered cells, including the percentages of lymphocytes and monocytes, which was not done in the present study.

The experience with the 596 specimens reported here indicates that cells stored for up to 12 years can be used for immunophenotype analysis and for assays of specific anti-HIV immunity, as well as for the characterization of viral phenotype. Cryopreserved lymphocytes from the MACS have been used for the assay of specific HIV-directed memory cytotoxic-T-lymphocyte activity (15, 17), the recovery of HIV by cell culture (7), lymphocyte immunophenotyping by flow cytometry (3, 7), human lymphocyte antigen genetic typing (11), and the detection of inherited resistance mutations (5, 9, 18). An additional 1,562 cell samples, not reported on in detail here, were dispensed in 1996 and 1997 for other ongoing pathogenesis projects by 18 investigators throughout the United States and Australia, both internal and external to the MACS, for studies of a variety of facets of HIV pathogenesis. Although detailed data on recovery are not available for these specimens, their utility for scientific initiatives is documented by publications on the findings from these samples (5, 9, 18).

Our experience suggests several steps to follow and quality control measures to undertake in setting up a cell repository. Some of these have been discussed elsewhere (8). It is important to have enough personnel to allow the separation and storage of blood within 6 h after the blood is drawn. Use of a controlled-rate freezer may be desirable. Nitrogen storage tanks should be fitted with alarms which will alert technicians if the level of nitrogen is not acceptable or the temperature is too warm. Keeping vials in the nitrogen vapor phase is recommended for maintaining a high cell viability during long-term storage, and the use of vials internally threaded with a gasket is generally thought to be best for liquid-nitrogen storage. On-site training by a central technical coordinator to standardize methods for cell counting, freezing, and thawing across sites would be expected to reduce variation in cell recovery. An external quality control program would also be valuable in enabling the optimization of viability and cell recoveries across sites. More predictable yields with high viabilities will maximize the value of PBMC collected and stored for research studies.

This approach to the quality assessment of cryopreserved PBMC may be useful for other studies involving stored specimens, particularly in planning for the recovery of sufficient cells to accomplish the desired research. Successful repositories also require consistent attention to detail by skilled technical staff, including attention to the numbers of cells stored. The quality of specimens should be examined early during the course of a research study, so that any problems in sample storage are recognized and corrected before valuable resources are lost.