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The Air Force Health Study Specimens Repository

This chapter describes the laboratory specimens collected during the course of the Air Force Health Study (AFHS). It enumerates what type of specimens were collected, the size of the samples, when they were obtained from the study subjects, how they are currently stored, and the ease with which they can be accessed for research. This information is used to draw conclusions concerning whether obstacles exist to retaining and maintaining the materials.

Some of the content in this chapter was previously presented in the committee’s interim letter report (IOM, 2005).

SPECIMEN COLLECTION AND SHIPMENT

Specimen collection was performed as part of the multiday physical examination in each of six cycles carried out by the AFHS during the period 1982–2002.1 These activities were conducted at the Kelsey-Seybold Clinic in Houston, Texas, in Cycle 1 and at the Scripps Clinic in La Jolla, California, by Science Applications International Corporation (SAIC) Inc., a contract service provider to the AFHS, in later cycles. The protocols for collection, shipment, assay, and storage of various biological specimens are documented in respective biomedical

1  

Sera were also collected at American Red Cross Centers in conjunction with the 1987 TCDD assay pilot study, and insulin-sensitivity testing of 60 Cycle 5 participants was performed at the General Clinical Research Center at the University of Arkansas for Medical Sciences/Central Arkansas Veterans HealthCare (Patterson et al., 1987; Kern et al., 2004).



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Disposition of the Air Force Health Study 4 The Air Force Health Study Specimens Repository This chapter describes the laboratory specimens collected during the course of the Air Force Health Study (AFHS). It enumerates what type of specimens were collected, the size of the samples, when they were obtained from the study subjects, how they are currently stored, and the ease with which they can be accessed for research. This information is used to draw conclusions concerning whether obstacles exist to retaining and maintaining the materials. Some of the content in this chapter was previously presented in the committee’s interim letter report (IOM, 2005). SPECIMEN COLLECTION AND SHIPMENT Specimen collection was performed as part of the multiday physical examination in each of six cycles carried out by the AFHS during the period 1982–2002.1 These activities were conducted at the Kelsey-Seybold Clinic in Houston, Texas, in Cycle 1 and at the Scripps Clinic in La Jolla, California, by Science Applications International Corporation (SAIC) Inc., a contract service provider to the AFHS, in later cycles. The protocols for collection, shipment, assay, and storage of various biological specimens are documented in respective biomedical 1   Sera were also collected at American Red Cross Centers in conjunction with the 1987 TCDD assay pilot study, and insulin-sensitivity testing of 60 Cycle 5 participants was performed at the General Clinical Research Center at the University of Arkansas for Medical Sciences/Central Arkansas Veterans HealthCare (Patterson et al., 1987; Kern et al., 2004).

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Disposition of the Air Force Health Study TABLE 4-1 Number and Type of Specimens Collected from AFHS Subjects by Year and Cycle Year 1982 1985 1987 1992 1997 2002 Total # of samples Exam Cycle 1 2 3 4 5 6 Blood serum   # of samples 15,883 6,927 4,588 8,932 8,484 5,853 50,667 # of vials 5–10 3 2 4 4 3   Volume (ml) 2 5 10 7 10 10   Whole blood   # of samples   19,510 19,510 # of vials 10   Volume (ml) 1   Urine   # of samples 2,269 2,309 2,294   6,872 # of vials 1 1 1   Volume (ml) 50 50 10   Semen   # of samples 8,845   8,845 # of vials 1–10   Volume (ml) 1   Adipose tissue*   # of samples   313   313 Weight (g) 10   *A sample of approximately 10 g was extracted by liposuction (AFHS, 2000). SOURCE: J. Robinson, Air Force Health Study, personal communication, September 21, 2005. test plans—created by SAIC—for Cycles 2–6.2 A detailed protocol is not available for Cycle 1. Cycle 1 laboratory methods are discussed in the original protocol (AFHS, 1982); they are also briefly described in the Cycle 2 biomedical test plan (SAIC, 1985) where shift in laboratory techniques or methods between Cycles 1 and 2 is discussed. Table 4-1 provides summary counts of the types and quantities of biological specimens collected in each cycle. Sera were stored in all six cycles and aliquot volumes varied by cycle ranging from 2 to 10 ml per aliquot. The number of aliquots per subject also varied ranging from 1 to 10 vials in Cycle 1, 3 vials in Cycles 2 and 6, 2 vials in Cycle 3, and 4 vials in Cycles 4 and 5. In Cycle 6, ten 1-ml aliquots of whole blood were also stored with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. There was no separation of white blood cells. 2   These documents are now in the public domain (https://www.formrouter.net/forms@NAS/Public_Access_Form.html).

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Disposition of the Air Force Health Study One aliquot of urine was stored from participants during Cycles 1–3 (1982, 1985, and 1987). Urine aliquot volumes were 50 ml in Cycles 1 and 2 and 10 ml in Cycle 3. The Cycle 1 semen specimens were stored in 1-ml vials with 1–10 aliquots per subject. A separate blood draw was performed to obtain samples for a 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) assay carried out by the Centers for Disease Control (CDC). Table 4-2 delineates the number of assays conducted by exam cycle. Multiple samples were drawn for a subset of the population for use in dioxin half-life studies. The methodology and results of these analyses is presented in Chapter 2 of the Cycle 6 examination report (AFHS, 2005). Most subjects also underwent a glucose tolerance test with bloods and urines collected two hours after the first blood draw. Suspicious skin lesions were biopsied and their pathology determined in all cycles. These samples were not stored in the repository. Specimen aliquots were packaged on dry ice and shipped overnight to the AFHS facilities at Brooks City-Base in San Antonio, Texas, where they have been maintained in –70°C freezers (J. Robinson, Air Force Health Study, personal communication, September 21, 2005). No samples were lost during shipment for any of the six cycles. The current AFHS specimen collection includes portions of frozen whole blood collected originally for dioxin analysis, which were returned to AFHS from CDC. With this exception, no other samples have been returned to the AFHS repository subsequent to distribution to third-party investigators. Of the latter, relatively few have accessed AFHS specimens and, of those who have, some arranged for collection of samples specifically for their own purposes. As an example, adipose tissues were collected in 1997 (Cycle 5) and are held at UC TABLE 4-2 Number of Dioxin Assays Performed on AFHS Subjects by Year and Cycle Year[s] Cycle[s] Ranch Hand Comparison Total 1987 only* 3 277 771 1,048 1992 only 4 76 179 255 1997 only 5 18 95 113 2002 only 6 12 94 106 1987 and 1992 3, 4 30 35 65 1987 and 1997 3, 5 146 0 146 1987, 1992, and 1997 3, 4, 5 218 0 218 Total 777 1,174 1,951   *Includes assays for 150 Ranch Hands and 50 comparisons conducted for pilot study (Patterson et al., 1987). SOURCE: AFHS, 2005.

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Disposition of the Air Force Health Study Davis (as indicated in Table 4-1). According to documentation provided to the committee (J. Robinson, Air Force Health Study, personal communication, September 21, 2005), past uses of AFHS specimens that have resulted in removal of samples from the collection include 200 samples of sera from the Cycle 1 collection provided for infectious disease antibody testing at the University of Cincinnati in 1984, 626 samples of sera from Cycle 1 used for dioxin half-life studies by AFHS and CDC in 1987, 4 samples of sera from Cycle 4 from individuals with consistently low immunoglobulin A levels provided to Duke University in 1993, 4 samples of sera from Cycle 4 provided to the University of Virginia in 1993, and 100 samples of sera (cycle not specified; described as “late 1980s”) used by CDC to test for Legionella antibodies. OBSTACLES AND LIMITATIONS TO FURTHER USE OF AFHS SPECIMENS Limitations Inherent to the Nature of Specimens Collected and Their Subsequent Storage and Preservation As indicated in Table 4-1, the type and quantity of specimens collected varied by cycle, with some specimen types being collected only once or for only a subset of study participants. Limitations in specimen volumes and the number of aliquots archived would necessitate the prudent restriction of future uses of AFHS specimen assets with appreciable biomaterial requirements. Although the stored specimens are suitable for many current assays and potentially for new assays as yet unidentified, there are clearly some analytes (such as volatile organic compounds in blood) that require different methods of handling and preservation than those employed in the AFHS in order to ensure analytic viability. There also exist other analyte classes for which the adequacy of the storage conditions over the duration of storage is generally not well established. It is clear that some analytes will not be measurable as special precautions that are sometimes instituted for specific studies were not taken with the specimens. For example, some blood processing protocols require specimen protection from light to prevent degradation of carotenoids. The addition of metaphosphoric acid is a standard practice for serum ascorbic acid measurement (Paoli, 2005). Other analyses require the addition of antioxidants to protect certain chemical species. Ascorbic acid—an antioxidant—is sometimes added to urine specimens as an analyte preservative. Although whole blood has been stored, it was not done in the presence of a dimethyl sulfoxide containing freezing medium. Thus, the white blood cells

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Disposition of the Air Force Health Study (WBC) are not viable. Additionally, without intact cells it is currently not feasible to assay for gene expression in WBCs. However, emerging technologies may make these types of studies possible. Thus, it is not possible for the committee to make an exhaustive prediction of what assays could be run in the future, especially given the rapid development of instrument technologies and assay methods with improved analytical sensitivity and specificity. However, it is reasonable for the committee to conclude that there will be an appreciable demand for these specimens given the accompanying physical exam and laboratory data collected over the 20-year course of the AFHS. The National Health and Nutrition Examination Survey (NHANES)3 repository provides a practical example for determining the utility of AFHS specimens for any specific future study. The committee feels that researchers should bear the burden of demonstrating the suitability of specimens for their particular studies, and these data must be provided before specimens are released. Logistical Limitations Logistical limitations to the future use of AFHS specimens include deficiencies in or absence of documentation regarding the methods of collection and processing of specimens,4 the need to reaccomplish inventory controls through the reorganization of specimens, the improvement of documentation of specimen locations, and, where feasible, the annotation of specimen history. More importantly, preserving the specimens for future uses will require continued support of a specialized facility. The working group responsible for issues regarding specimens during the committee’s May 2005 site visit (Drs. Hankinson and Kalman) interviewed program staff and conducted exercises to evaluate access to and preservation of specimen whole blood, serum, urine, and semen. Their findings are presented in the committee’s interim letter report (IOM, 2005) and recapitulated here. Storage Specimens are stored at −70°C in a total of 21 freezers in a single room in a building that also houses other AFHS functions. This storage temperature was the accepted state of the art in the 1980s.5 A few smaller collections of AFHS 3   Information on NHANES can be found at http://www.cdc.gov/nchs/nhanes.htm. 4   Variability in laboratory procedures notwithstanding, the ability to detect true variation in selected laboratory endpoints is predicated on the elimination or control of variation in specimen collection and processing. 5   The practice in 2005 is to store biologic samples at least to −80°C; liquid nitrogen storage—which maintains samples at still lower temperatures (below −130°C)—is used in some studies (Paoli, 2005).

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Disposition of the Air Force Health Study biospecimens have been dispersed to collaborating investigators and would need to be recombined, destroyed, or be handled by some other custodial arrangement prior to the termination of the AFHS. Specimen Access Specimens have been maintained in lots, reflecting the manner in which they were received; in some cases several vials from multiple individuals were in zipper-sealed plastic bags, and in other cases several vials were held together with rubber bands. Labels on stored samples are not uniform but in general contain the subject’s case number, last and first name, and middle initial. Some aliquots also indicate the subject’s age, the sample type (e.g., SER3), collection time, and date. Multiple steps were required to locate particular specimens. First, the subject’s case number and the first four letters of his last name are keyed into a master (computer-based) database that identifies the box in which the sample is stored and the freezer in which that box is located. However, boxes have been moved over time, and locations stored in this database have not been updated and are thus not reliable. The AFHS investigators recently completed a physical inventory of all boxes in the repository and were able to locate all boxes listed in the database. To locate a particular box, one must refer to the hard copy list generated in the physical inventory to determine the current freezer and shelf location of a particular specimen. For example, the database may indicate that a subject’s serum specimen from Cycle 2 is located in freezer 19, drawer 2, box 4, but the inventory hard copy might indicate that the actual location of the specimen is freezer 14, drawer 2, box 4.6 Despite the intricacy of the inventory records, staff was able to locate specimens requested by the working group with little difficulty. Neither the existing database nor the hard copy reinventory provide data regarding the number of aliquots or volumes stored. It appears that these data were not recorded consistently. For those cycles that had quantities recorded, the current volumes in the record reflect the original quantities received. These have not been updated to indicate removal from the original amounts received. Specimen Integrity and Condition No documentation exists for the thermal history of the specimens. However, the physical condition of the freezers appears to be adequate. All had external labels indicating that they had been inspected at 6-month intervals for at least the past three years. The freezers have been monitored for failure throughout the course of the study—in earlier years by physical inspection and later through 6   The working group was told that drawer and box numbers were maintained in moves.

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Disposition of the Air Force Health Study electronic monitoring and alarm systems. Three people are on call at all times to move samples in case of a freezer failure. Empty freezers maintained at −70°C are available for immediate transfer of samples in case of freezer breakdown. In the earlier years of the study, there was one instance where specimens were compromised by an equipment failure. This occurred in 1986 when 656 urine samples were lost because of a freezer outage. Thus, the biorepository has been well preserved and certainly appears comparable in quality to repositories housed by other large epidemiologic studies (e.g., the Physicians’ Health Study7 and the NYU Women’s Health Study8). Retrieval and inspection of three specimens from randomly selected subjects showed no signs of leakage or major thaws. The appearance of one urine specimen examined by the working group was open to interpretation: the position of the fluid in the vial was consistent with either minor thawing and refreezing, or the initial freezing of the specimen occurring in two phases. Some residuals from serum samples sent to CDC for dioxin analysis were returned to the AFHS repository. These samples are kept separate from the others, and they are marked as returns in an inventory document. With this exception, all samples in the repository appear to have remained frozen since collection. As noted above, specimens have been sent to Duke University, the University of Cincinnati, and the University of Virginia for various analyses over the course of the study. None of these were returned to the inventory. During the Cycle 5 physical examination, 313 adipose tissue samples were collected; they are being held by a collaborating investigator at the University of California-Davis. The committee does not have any information on the condition and storage circumstances of these samples. During the committee’s first meeting, the AFHS investigators mentioned that they planned to conduct a reassay experiment to evaluate the analytical viability of the specimens. The plan called for analyzing specimens from a small sample of subjects over the six study cycles. These samples will be assayed for endpoints for which historical values are available. Results are expected to generally indicate the current condition of preserved specimens, although their interpretation is not straightforward, and the suitability of these samples for specific analytes will need to be determined in some cases. A major reinventory and physical reorganization of the specimens is planned for the final year of the study and was recommended in the Committee’s letter report (IOM, 2005). This effort will reconcile the inventories and create a new 7   The Physicians’ Health Study is a randomized trial. Phase I began in 1982 and Phase II is scheduled for termination in 2007. Over 26,000 blood specimens were collected during Phase I, and more than 11,000 blood specimens have been collected in Phase II (http://phs.bwh.harvard.edu/). 8   The New York University Women’s Health Study is an observational study in which blood samples from more than 17,000 participants have been collected. Specimens are stored in −80°C freezers (http://mcrcr4.med.nyu.edu/womenshealthstudy).

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Disposition of the Air Force Health Study database that would not only provide the actual current location of all specimens but would also indicate the volumes and types of biological material in storage for each individual. Based on the specimen storage method employed in the NIOSH/FAA Working Women’s Health Study, the planned AFHS reorganization will collect all specimens for an individual in one or more sequential boxes. Currently, AFHS specimens are stored in freezers by cycle and specimen type, not by individual. This effort will greatly decrease the effort associated with the retrieval of specimens. However, if the request is for a single type of specimen (e.g., urine), many more boxes will have to be pulled than if the inventory were organized by biospecimen type. Organization by subject will, however, facilitate the withdrawal of a subject’s entire specimen archive should he not consent to research in the future.9 Combining of the specimens from various cycles will necessitate very careful pulling of requested specimens in the future so that not only the correct specimen type but also the correct collection time period for an individual is selected. It is not clear that each vial has the cycle number or date of collection on the label. Thus, any vials without this information should have it added during this reorganization. Many of the specimens are stored in volumes (e.g., 5-ml to 50-ml vials) much larger than what is required for most assays. It would be desirable to realiquot specimens (e.g., 100-µl to 500-µl aliquots) before sending them out for analysis. This would minimize additional freeze–thaw cycles. While the collaborating laboratory may be able to perform the realiquoting, this is not an ideal solution because the repository would lose control over the specimens and their freeze–thaw history. Realiquoting has cost implications—supervision and technician time and processing and freezer space needs—but is a means to help maintain the specimen integrity for future use. Procedural Limitations: Privacy and Other Ethical, Legal, and Social Issues Individual specimens are currently labeled by individual name among other identifiers. This requires that managers of the specimen repository be bound by privacy and confidentiality agreements and that specimens provided to any future users either be relabeled10 and realiquoted or that privacy requirements be otherwise assured. The operation of a future specimen repository would need to have the capability to satisfy such requirements. Related issues, including informed consent, are discussed in Chapter 6. 9   Specimen control issues are discussed in Chapter 6. 10   The current state-of-the-art is to use bar codes for specimens held in repositories. It is not known if relabeling with bar codes was planned in the AFHS reorganization of specimens.

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Disposition of the Air Force Health Study FINDINGS, CONCLUSIONS, AND RECOMMENDATIONS On the basis of its review of AFHS reports, its site visit of the study facility, the scientific literature, and other information presented in this chapter, the committee has reached the following findings regarding the laboratory specimens and other study data collected during the AFHS. As noted in the interim letter report, the committee finds that: The laboratory specimens collected in the course of the AFHS have been properly maintained. However, they are not currently organized and documented in a manner that allows them to be easily understood, evaluated, managed, or analyzed by persons outside of the AFHS. The committee also finds that: Obstacles, including privacy concerns, exist to retaining and maintaining the laboratory specimens. The letter report noted that there were two primary options for the disposition of the laboratory specimens collected by the AFHS: disposal or maintenance for possible future use. Regardless of what decision is made concerning their disposition, the committee believes that there is value in fully documenting and reorganizing the laboratory specimens prior to the study’s presently scheduled termination date. This would be helpful in auditing their proper disposal if that decision were taken and would be vital to facilitating any possible future use. The committee’s interim letter report offered several recommendations for addressing the obstacles to retaining and maintaining the AFHS laboratory specimens that related to the organization and documentation of these materials. These are listed in the following section. Approaches to addressing the other obstacles identified here are presented in Chapter 6 in the context of the committee’s recommendations for the further study. Recommendations Contained in the Interim Letter Report The interim letter report (IOM, 2005) explicates the committee’s conclusion that the present state of the documentation and organization of AFHS laboratory specimens was an obstacle to retaining and maintaining these materials after the currently scheduled termination date of the study. The committee therefore recommended that: Action should be taken prior to the currently scheduled termination date of the AFHS to reorganize and document the study’s laboratory specimens in a form and format that allows them to be easily understood, evaluated, managed, or analyzed by persons outside of the AFHS.

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Disposition of the Air Force Health Study The following actions were recommended: Reinventory all laboratory specimens held by the AFHS, verifying their location and ascertaining the number and volume of aliquots and type of sample. Carry out a visual inspection of specimen condition while performing this activity, and identify any problematic samples. Update and create a single specimen database that includes case number, exam cycle, specimen type, and freezer location. Compile all information regarding specimen history (receipt, realiquoting, freeze–thaw cycles, dispersal, and the like) into a single reference database. Where data gaps are present, note their existence. Compile all protocols regarding receipt, maintenance, dispersal, and return of specimens for all cycles into a single reference document. Document the status of all laboratory specimens sent to outside investigators. Ensure that extant specimens are either disposed of using current best practices or reintegrated into the inventory, clearly marked with their history. Perform the currently planned reassay to aid in the evaluation of specimen stability and condition. The interim letter report also noted that the committee was in agreement with a planned reinventory and physical reorganization of their repository that would collect all specimens for a study subject in one or sequential boxes.11 They were told of the plan in their May 2005 site visit and understand that the effort was subsequently initiated. The committee recommended that—if the planned reorganization was carried out—the exam cycle or date of collection be added to any vials that were not already marked with such information. As already noted in Chapter 3, the committee believes that it is incumbent on the U.S. Air Force, as the custodian of the AFHS research materials, to ensure their proper documentation and organization for both historic reasons and for possible future use. It therefore recommends that: If available AFHS program funds are not sufficient to accomplish the actions elucidated above, supplemental funding should be provided to carry out such work in a complete and timely manner. REFERENCES AFHS (Air Force Health Study). 1982. An Epidemiologic Investigation of Health Effects in Air Force Personnel Following Exposure to Herbicides: Study Protocol, Initial Report. Brooks AFB, TX: USAF School of Aerospace Medicine. SAM-TR-82-44. 11   Currently, specimens are stored in freezers by cycle and specimen type.

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Disposition of the Air Force Health Study AFHS. 2000. An Epidemiologic Investigation of Health Effects in Air Force Personnel Following Exposure to Herbicides. 1997 Followup Examination Results. Brooks AFB, TX: Epidemiologic Research Division. Armstrong Laboratory. AFRL-HE-BR-TR-2000-02. AFHS. 2005. An Epidemiologic Investigation of Health Effects in Air Force Personnel Following Exposure to Herbicides. 2002 Followup Examination Results. Brooks AFB, TX: Epidemiologic Research Division. Armstrong Laboratory. AFRL-HE-BR-SR-2005-0003. IOM (Institute of Medicine). 2005. Disposition of the Air Force Health Study—Interim Letter Report. Washington, DC: The National Academies Press. [Online]. Available: http://www.nap.edu/catalog/11483.html [accessed December 1, 2005]. Kern P, Said S, Jackson W, Michalek J. 2004. Insulin sensitivity and serum TCDD in Air Force veterans occupationally exposed to herbicides during the Vietnam War. Organohalogen Compounds 66:3166–3173. Paoli PD. 2005. Biobanking in microbiology: from sample collection to epidemiology, diagnosis and research. FEMS Microbiology Reviews 29:897–910. Patterson DG, Hampton L, Lapeza CR, Besler WT, Green V, Alexander LR, Needham LL. 1987. High-resolution gas chromatography/high-resolution mass spectrometric analysis of human serum on a whole-weight and lipid basis for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Analytical Chemistry 59:2000–2005. SAIC (Science Applications International Corporation). 1985. Biomedical Test Plan. Air Force Contract No. F14689-85-D-0010. SAIC Project No. 2-816-04-195.