Original Article
Evaluation of Screened Blood Donations for Human Immunodeficiency Virus Type 1 Infection by Culture and DNA Amplification of Pooled Cells
N Engl J Med 1991; 325:1-5July 4, 1991
- Abstract
- Abstract
Background.
Reports of transmission of the human immunodeficiency virus type 1 (HIV-1) from transfusions of screened blood and reports of silent, antibody-negative HIV-1 infections in persons at high risk continue to foster concern about the safety of the blood supply. Previous estimates of the risk of HIV-1 range from 1 in 38,000 to 1 in 300,000 per unit of blood but are based on either epidemiologic models or the demonstration of sero-conversion in recipients.
Methods.
We isolated peripheral-blood mononuclear cells from blood that was fully screened and found to be seronegative, combined them into pools of cells from 50 donors, and tested them for HIV-1 by viral culture and the polymerase chain reaction, using protocols specifically adapted for this analysis.
Results.
The 1530 pools of mononuclear cells were prepared from 76,500 blood donations made in San Francisco between November 1987 and December 1989. Of these pools, 1436 (representing 71,800 donations) were cultured successfully; 873 (43,650 donations) were evaluated by the polymerase chain reaction. Only one pool was confirmed as HIV-1—ed by both methods. After adjustment for sample-based estimates of the sensitivity of the detection systems using culture and the polymerase chain reaction, the probability that a screened donor will be positive for HIV-1 was estimated as 1 in 61,171 (95 percent upper confidence bound, 1 in 10,695).
Conclusions.
Silent HIV-1 infections are exceedingly rare among screened blood donors, so the current risk of HIV-1 transmission from blood transfusions, even in high-prevalence metropolitan areas, is extremely low. (N Engl J Med 1991; 325:1–5.)
Article Activity
- Article
VOLUNTARY self-exclusion from blood donation by persons at risk for human immunodeficiency virus type 1 (HIV-1) infection and the implementation of routine screening for HIV-1 antibodies have substantially reduced the risk of HIV-1 transmission through nonautologous blood transfusions.1 , 2 Current transfusions are not risk-free, however, as has been documented by reports of the transmission of HIV-1 from fully screened donors who were later found to have been exposed to the virus at the time of donation.3 4 5 6 7 Concern about the safety of the blood supply has been further heightened by reports of prolonged infection without detectable HIV-1 antibodies preceding seroconversion.8 9 10 These so-called silent infections were detected with the techniques of polymerase chain reaction (PCR) gene amplification and viral culture, neither of which is currently practical for routine donor screening.
Models for estimating the current risk of HIV-1 infection from transfusions require estimates of variables describing both the epidemiology of HIV infection in blood donors and the performance of antibody-screening tests.3 , 4 , 10 Because of uncertainty about these variables, published estimates have varied from a high of 1 infection in 38,000 units of blood transfused3 to a low of 1 in 300,000.11 To study the extent to which HIV-1—infected donations may be missed by current screening procedures, we tested peripheral-blood mononuclear cells (PBMCs) from fully screened donors for HIV-1 infection with both highly specific viral culture12 , 13 and highly sensitive PCR proviral amplification.14 , 15 To achieve a statistically precise estimate of the risk after screening, we developed and validated methods of pooled analysis for the detection of HIV-1—infected cells from an infected donor when the cells were combined with an equivalent number of cells from a total of 50 donors. We report here the results achieved when this investigational approach was used with 76,500 blood donations in San Francisco over a two-year period.
Methods
Specimen Collection
Beginning in November 1987, an extra 10-ml tube of anticoagulated blood was collected from up to 250 nonautologous donations of whole blood and blood for apheresis per day at the collection facilities of the Irwin Memorial Blood Centers in the San Francisco Bay area. The human research committee of the University of California determined that the specific consent of donors for HIV-1 culture and DNA analyses was not required if testing was carried out in a nonlinked fashion that precluded the identification and recall of specific donors. The study tubes were initially labeled with the donors' blood-unit identification numbers and stored at room temperature. Required serologic testing (for syphilis, hepatitis B surface antigen, hepatitis B core antibody, elevated alanine aminotransferase levels, HIV-1 antibody, and beginning in October 1988, antibody to human T-cell lymphotropic virus Type I) was completed, and tubes with positive results were excluded, as were those from patients who had been excluded as blood donors on the basis of their history. Other reasons for the exclusion of tubes from the study are shown in Table 1Table 1
Collection of Tubes of Blood for Study and Preparation of PBMC Pools, November 1987 to December 1989.. Tubes were then handled without any identifiable link to the donors.Separation and Pooling of Cells
Seven milliliters of blood from each eligible study tube was mixed with 7 ml of a PBMC-separation medium (Sepratech, Oklahoma City) and centrifuged at 1500×g for 20 minutes. The PBMC layer, containing a mean of 12×106 PBMCs from each of 10 donor tubes, was transferred to a 50-ml tube containing Hanks' balanced salt solution, and the pooled PBMCs were washed by repeated gentle inversion. After centrifugation, cell pellets from five 10-donor pools were combined into a 10-ml pool containing cells from 50 donors. Duplicate aliquots of 3×106 cells were resuspended in 300 μl of PCR solution A (100 mM potassium chloride, 10 mM TRIS–hydrochloric acid [pH 8.3], and 2.5 mM magnesium chloride)16 and stored at — 70°C. The remaining PBMCs were resuspended in a cryoprotectant medium (RPMI 1640 medium, 10 percent dimethyl sulfoxide, and 20 percent fetal-calf serum) at 1×107 cells per milliliter, and two aliquots of 100×106 PBMCs were frozen in liquid nitrogen for subsequent culture.
HIV-1 Detection by Culture
The pooled cells were brought to 37°C by the addition of prewarmed wash medium (RPMI 1640, 10 percent fetal-calf serum, and 0.1 percent gentamicin). Approximately 75×106 viable PBMCs, or an average of 1.5×106 cells per donor, were recovered. These were cultured with 20×106 PBMCs ("feeder cells") obtained from regular thrombocytapheresis donors negative for HIV-1 antibodies and stimulated for three days after collection with phytohemagglutinin (PHA, 0.5 μg per milliliter; Difco, Detroit). The cells were initially incubated for two hours at 37°C at a high cell density (5×106 cells per milliliter of medium) in the presence of hexadimethrine bromide (Polybrene), followed by the addition of 20 ml of complete medium containing RPMI 1640, 10 percent fetal-calf serum, 0.1 percent gentamicin, interleukin-2 (Cellular Products, Buffalo, N.Y.), and anti—interferon alfa (ICN Immunobiologics, Lisle, Ill.). The cultures were maintained for 28 days, with the weekly addition of 2×106 stimulated feeder cells. The cultured cells were monitored by immunocytochemical techniques for HIV-1 pl8 and p24 antigens, as described elsewhere.17 The culture supernatants were tested for p24 antigen by enzyme immunoassay. At the termination of the analysis, the cultured cells were pelleted and frozen at —70°C in PCR solution A for possible PCR analysis. For a culture to be considered infected, both the immunocytochemical and the supernatant assays for antigen had to be positive on two consecutive days of monitoring. Study pools for which the initial results of culture were positive were cultured again with the duplicate aliquot of PBMCs, and positive results of both cultures were required for the infection to be considered confirmed. The sensitivity and specificity of the pooled-culture system were monitored weekly by the introduction of 1×106 PBMCs from asymptomatic, HIV-1-sero-positive donors into replicate pools and by the routine culture of stimulated feeder cells alone. In addition, a quality-control panel of cells from 10 asymptomatic infected donors was added to the pooled cells at a dilution of 1 to 50 and analyzed in parallel by culture and PCR.
HIV-1 Detection by PCR
The steps of preparation, amplification, and hybridization and detection of DNA were separated physically, and reagents were prepared and used in a manner designed to minimize exogenous contamination and carry-over of DNA.18 Procedures for lysate preparation, the creation of HIV-specific oligonucleotide primer-probe systems, PCR amplification, and the hybridization and detection of oligomers were modifications of those described by Kellogg and Kwok.16 Aliquots of PBMCs in PCR solution A were thawed weekly and mixed with an equal volume of PCR solution B (10 mM TRIS–hydrochloric acid [pH 8.3], 2.5 mM magnesium chloride, 1 percent Tween-20, and 1 percent NP-40 containing 120 μg of freshly added proteinase K per milliliter), lysed at 60°C for 1 hour, the reaction quenched at 95°C for 10 minutes, and then stored at —20°C. For each sample, a volume of lysate corresponding to 2.5×Xl05 PBMCs was amplified in a 100-μl reaction containing 2 units of Amplitaq DNA polymerase (Perkin—Elmer, Norwalk, Conn.), 50 mM potassium chloride, 10 mM TRIS–hydrochloric acid (pH 8.3), 0.005 percent gelatin, 2.2 mM magnesium chloride, 0.25 percent Tween-20, 0.25 percent NP-40, and 0.2 mM of each deoxynucleotide triphosphate. HIV-1 DNA amplification was accomplished with 50 pmol of each HIV-1 gag primer (SK38 and SK39)16 and 30 cycles of denaturation (95°C, 30 seconds), annealing (55°C, 30 seconds), and primer extension (72°C, 60 seconds), followed by 10 minutes at 72°C in an automated thermal cycling heat block (Perkin—Elmer). Amplified sequences were detected by solution hybridization with a high-specific-activity (3 to 6 Ci per micromole) 32P-labeled SK19 probe, followed by electrophoresis on 10 percent polyacrylamide gels and autoradiography.16 At the same time as the assessment for HIV-1 proviral sequences, the lysates were internally tested for PCR competency by attenuated coamplification19 , 20 of the single copy HLA-DQ-alpha gene with GH26 and GH27 primers and a GH64 probe.21 The initial PCR testing was done in a single aliquot. If the result was positive, a second set of reactions was run in triplicate. If two or more of these four reactions were positive, the initial sample aliquot was considered repeatedly reactive. For all repeatedly reactive samples, the back-up PCR aliquot was tested with a similar algorithm and a second HIV-1 gag primer pair (SK145 and SK101), in concert with probe SK102.22 In every PCR, positive and negative control preparations were included at each step. The rates of false negative and false positive results with the replicate assays of PCR-competent control lysates were determined to be 0 (n = 55) and 4 percent (n = 175), respectively.21 We assessed the sensitivity of the PCR assay weekly in parallel with each routine sample digest, by testing aliquots of a 1:50 dilution of PBMCs from a seropositive donor in feeder cells. A quality-control panel containing fresh PBMCs derived from 10 seropositive persons was also assessed at a dilution of 1:50 in the pooled cells from donors.
Statistical Analysis
The estimates and confidence bounds for probabilities were based on binomial-distribution models for data involving counts. A group-testing estimate was used to estimate the probability that a person would be identified as positive for HIV-1 on assay, given the number of pools tested and the number found positive (see Sobel and Elashoff23 for a general formulation). Exact upper and lower confidence bounds for probabilities were computed24 and combined, with the resulting confidence levels given by the Bonferroni inequality method.25 The upper confidence bound is based on an untestable assumption that the probability of a positive result is constant for all infected donors, and therefore may be lower than estimated. A formulation of the estimate and upper confidence bound for the probability of HIV-1 positivity, with adjustment for estimated sensitivity and specificity as well as a sensitivity analysis, is available from the authors or the National Auxiliary Publications Service.* Because the adjustments for estimated specificity were numerically trivial, they are not referred to further here.
Results
The tubes of blood collected for this study corresponded to 101,314 nonautologous donations made between November 1, 1987, and December 31, 1989. These represented 59.1 percent of the approximately 171,500 donations during this 26-month period. The PBMCs from 76,500 of these samples (75.5 percent) were separated and processed into 1530 pools of donated PBMCs (Table 1). The proportions of study tubes excluded because of a reactive screening test for infectious disease (3.1 percent) and historical or confidential information (1.1 percent) were nearly identical to the corresponding rates of exclusion (3.16 and 1.08 percent, respectively) for all donations during the collection period.
The results of the analysis of PBMC pools by HIV1 culture are summarized in Table 2Table 2
Results of Culture in Pools of PBMCs from 76,500 San Francisco Blood Donations.. For 94 pools, the cultures of both aliquots designated for analysis by culture were discarded because of contamination by bacteria or fungus. For 1436 pools, containing PBMCs from 71,800 donations, 28 days of culture were completed with continuous monitoring. Of the 11 pooled cultures that were initially positive, 10 in a single batch were identified as infected with HIV-1 because of contamination of feeder cells in the laboratory. When the second aliquots of these 10 pools of PBMCs were cultured with fresh feeder cells, all were negative for HIV-1. PCR was negative before and after culture in the aliquots of cells from these 10 replicate pools.The results of PCR were complete for 873 pools containing PBMCs from 43,650 donations, including the 94 pools for which no culture results were obtained (Table 3Table 3
Results of PCR in Pools of PBMCs from 43,650 Blood Donations.). No results were available for the 657 pools generated during the first nine months of the study collection, because these cells were either not prepared for PCR (111 pools) or were frozen under conditions later determined not to be optimal for DNA recovery and PCR analysis (546 pools). For 21 of the 873 pools analyzed (2.4 percent), the primary aliquot was repeatedly positive for HIV-1 gag sequences, as compared with 4.0 percent among 74 parallel digests from negative controls. On confirmatory analysis of the second PCR aliquot and testing with the second primer pair, 20 of these pools were negative for HIV-1 by PCR. These 20 pools were also negative by culture analysis.A single pool was confirmed as positive by both culture and PCR analysis. Repeated culture analysis of the replicate aliquot of this pool was also positive, and PCR analysis of the cell pellet after culture confirmed the presence of HIV-1 DNA. Figure 1Figure 1
Detection of HIV Proviral Sequences by PCR Gene Amplification in One Pooled-Donor Preparation of PBMCs. shows the positive PCR result obtained with the two different primer pairs.The sensitivity of the culture and PCR methods using pooled PBMCs was evaluated by routinely simulating the pooling protocol with samples from sero-positive donors. Of 95 cultures routinely "spiked" in this fashion, 76 (80 percent) were positive for HIV-1. The sensitivity of PCR was evaluated as 98.6 percent on the basis of the testing of 73 replicate control digests, largely derived from one seropositive donor. In addition, a coded quality-control panel of PBMCs from 10 asymptomatic seropositive persons at dilutions of 1:50 was assessed in parallel by both culture and PCR. Six of these preparations were positive according to both culture and PCR, one was positive according to culture only, and one was positive according to PCR only, for an overall detection rate of 80 percent.
When we considered the pools for which the culture analysis was complete, we detected a single positive pool among 1436 fully tested pools containing cells from 71,800 discrete donations. Using this figure, we estimated that the probability that more than one seropositive donor was represented in the single positive pool was 0.03 percent. To estimate the sensitivity of the assay, we used the fact that 83 of 105 seropositive control pools (79 percent) were detected as positive by culture. After adjustment for sensitivity, the estimated probability that a single donor would be positive for HIV-1 on culture was 1 in 56,727.* When we extended these results to include detection by either culture or PCR, we detected a single positive pool among 1530 pools of donor cells evaluated by culture, PCR, or both, and 84 of 105 positive control pools (80 percent) were detected as positive by culture, PCR, or both. The estimated probability that a single donor will be positive for HIV-1 by either assay is thus 1 in 61,171, with a 95 percent upper confidence bound of 1 in 10,695.
Discussion
The only other large-scale prospective study designed to determine the risk of transfusion-transmitted HIV-1 infection monitored patients undergoing cardiac surgery to detect HIV-1—antibody seroconversion.5 , 6 In that study, two cases of probable HIV-1 transmission were identified after the transfusion of 80,630 blood components, whereas two recipients were seropositive before transfusion, confirming a low background rate of infection. In evaluating the feasibility of a similar study in San Francisco, we tested 1058 specimens collected from patients who received transfusions and found 140 (13.2 percent) to be HIV1—positive before transfusion.26 This observation highlights the difficulty of evaluating rates of HIV-1 sero-conversion due to transfusion in cities with high background rates for the prevalence and incidence of infection. Unfortunately, these are also the cities where concern about the persistent risk of transfusion-associated infection is greatest.
In this report, we describe an alternative method of determining how frequently HIV-1-infected donors are missed by routine screening. Mononuclear cells from fully screened donors were isolated, pooled, and probed for occult viral infection with a combination of sensitive techniques of viral culture and DNA amplification. This combination of biologic and physical assays was used to maximize the detection of either slow-growing or variant-sequence viral strains.27 , 28 The two assays also served to confirm each other by identifying potentially false positive results obtained with either. The high specificity of both PCR and culture assays in this study reflects the critical emphasis placed on study design and assay protocols incorporating extensive confirmatory algorithms,29 , 30 as well as the small number of positive samples routinely handled during the pooling process, minimizing the likelihood of carry-over contamination.
It is important to recognize two limitations of our study design. First, all sensitivity studies using culture and PCR were performed with cells from asymptomatic seropositive persons rather than seronegative silent carriers. Although recent studies have demonstrated a high proportion of infected cells immediately before symptomatic HIV-1 seroconversion,31 , 32 it is possible that long-term silent carriers harbor lower levels of HIV-1 infection than those documented during and after seroconversion.9 , 31 32 33 34 If this is the case, our observed sensitivity rate would be higher, and our estimate of risk lower, than the actual rates. Second, because the testing of pools was carried out in an unlinked fashion, we were not able to recall the donors who contributed to the positive pool. Consequently, we cannot assess whether the failure to identify the infected donor was due to a donation given during the antibody-negative phase of infection or to an error in laboratory testing. Although this constitutes a limitation of the study, we thought that the unlinked approach was important to avoid the bias that would result from the refusal of prospective donors to participate.35
After adjusting for the sensitivity of our detection systems and using group-testing estimates, we calculated a point estimate of 1 in 61,171 for the frequency with which an HIV-1—infected donor is missed by current screening measures (upper 95 percent confidence bound, 1 in 10,695). Given the similarity between the rate of HIV-1—seropositive donations in San Francisco today (0.01 percent) and the rates in most other cities around the country,36 the risk we observed is probably comparable to that in other major metropolitan areas. In regions where the prevalence and incidence of HIV-1 among donors are substantially lower, the risk is likely to be proportionately smaller. It is noteworthy that our estimate lies within the confidence intervals of recent projections based on epidemiologic models,3 , 10 as well as within the confidence intervals of a prospective follow-up study of recipients of screened blood.5 , 6 Collectively, these results put into perspective the low risk of HIV-1 transmission by transfusion of fully screened blood.
Supported under a contract (NO1-HB-86–7024) from the National Heart, Lung and Blood Institute.
We are indebted to the nursing staff of the Irwin Memorial Blood Centers for blood-sample collections; to Dr. Melanie Adams for the preparation of the quality-control panel; and to the following people for technical assistance: John Heitman, Jean Shiota, Lillian Lee, Rosalind Endow, Simon Ng, and Angela Evans, all at the Irwin Memorial Blood Centers; and Mark Hurt and Lynn Bobey, at San Francisco General Hospital and the University of California, San Francisco.
Source Information
From the Departments of Laboratory Medicine (M.P.B., B.E.E., H.K.-B., E.L.M., G.N.V.), Epidemiology and Biostatistics (D.H.), and Medicine (H.A.P.), University of California, San Francisco; the Irwin Memorial Blood Centers, San Francisco (M.P.B., H.A.P.); and Cetus Corporation, Emeryville, Calif. (S.K., J.S.). Address reprint requests to Dr. Vyas at the Transfusion Research Program, University of California, San Francisco, CA 94143–0134.
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*
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