Lessons Learned from Biospecimen Shipping Among the Human Heredity and Health in Africa Biorepositories

Introduction

In June 2010, the U.S. National Institutes of Health (NIH) and Wellcome Trust launched the Human Heredity and Health in Africa (H3Africa) Consortium,^1–3 an initiative to build genomics research capacity on the African landscape to improve public health. One of the Consortium goals was to build biobanking expertise, and establish full-scale biorepository (BR) operations to preserve and ethically redistribute biospecimens for future research. Four institutions, Clinical Laboratory Services (CLS), and National Health Laboratory Services/Stellenbosch University Biorepository (NSB) in South Africa (SA); Institute of Human Virology Nigeria- H3Africa Biorepository (I-HAB) in Nigeria; and the Integrated Biorepository of H3Africa Uganda (IBRH3AU) in Uganda, received funding to plan and pilot test H3Africa BR operations, and assess the effectiveness of trans-African shipment of high-quality biospecimens.

Issues with preanalytical processes account for half to nearly 75% of all laboratory errors, and impact downstream analysis and future research.^4,5 Poor shipping and handling are of great concern in Africa, due to a climate that can potentially jeopardize biospecimen integrity.^6,7 Poor infrastructure and lack of regulations affect courier reliability, access, and shipping costs. Furthermore, some cold chain transport boxes are unreliable. ⁵

Biospecimens from H3Africa genomics projects are collected, processed, and temporarily stored at numerous clinical sites before transport to a central laboratory and ultimately the recipient BR. Therefore, during the BR planning phase, it was critical to establish and share minimum shipping requirements to maintain biospecimen integrity.^6–11 Standard operating procedures (SOPs), guidelines, and shipping forms were developed and distributed to the working group members for review, and to the H3Africa Steering Committee for approval. Subsequently, the four BRs pilot tested the shipping process by shipping biospecimens to each other, as shown in Figure 1. This article describes results that

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FIG. 1.

Biospecimen distribution.

Methods

Results

Discussion

Despite overall success, we incurred various challenges particularly related to ELSI and regulatory matters, while shipping delays presented additional challenges or barriers. For example, the turnaround time for ethical approvals, permits, and MTAs postponed the initial timing of the pilot. NHREC had begun a new process of registering biobanks in Nigeria. Thus, I-HAB had to apply for and be registered as a biorepository in Nigeria before initiating ethical review. The MTA process was the most time-consuming, because ethical boards and legal offices from different countries and institutions had to reach terms of agreement. Consequently, the BRs created an H3Africa MTA template, as a reference for direct adoption or modification. Also, the BRs engaged H3Africa investigators early on using the Site Engagement tool, developed to guide activities between the BRs and assigned investigators. The tool covers activities from site initiation through biospecimen deposition, and includes ethical and legal requirements, document submission, pilot activities, and phase II biospecimen submission. The collective strategy has helped to harmonize H3Africa documents and processes, and to implement pilots between BRs and improve the timeliness at which pilot and submission activities occur.

Although shipments were generally delivered within 5 days without interruption to the cold chain, the frozen shipment from I-HAB to IBRH3AU took 10 days due to the inability of the courier to reach the consignee. As World Courier replenishes dry ice, the environment was maintained throughout the delivery process and was ruled out as a factor that could have contributed to the significant difference in DNA purity from I-HAB to IBRH3AU. One dry ice temperature logger was damaged during delivery. It is imperative that temperature loggers containing dry ice probes be properly handled to prevent destruction and to ensure that detailed temperature monitoring is accessible. Credo Cube Shippers should be considered a reliable alternative choice when refrigerant is unattainable, limited, or comparatively expensive. As a future preventative measure, procedures were amended to require shippers and consignees to track shipments daily, from pick up until delivery. An alternative contact is also required for consignees in case the primary contact is unreachable. In addition, permits must be sent to the courier in the country that is sending the shipment as well as the courier in the country that is receiving the shipment, thus aiding faster customs preclearance. The BR also needs to ensure that all couriers have the required permit documentation to avoid customs delays in future.

IBRH3AU's inability to analyze urine biospecimens because they did not receive the intended dipstick tests before the pilot, exemplifies the common challenge of timely access to laboratory products in many parts of Africa and developing counties. Being aware of this challenge, the BRs must forecast consumables, and establish standing orders, or routine orders that enable procurement well in advance of need. It is also crucial for African BRs and investigators to jointly advocate for improved access to laboratory products; and leverage accounts, access, and costs when negotiating with international manufacturers, distributors, and couriers.

Generally, DNA of good quality was extracted and tested for purity and concentration using NanoDrop. There were nine shipments containing DNA, which were assessed before and after shipment. Overall, 77% of the DNA in this pilot study had acceptable purity before shipment. The two significantly different pairs of results reported above (one for concentration and one for purity) reflected outliers such as negative purity ratios. The negative readings demonstrate that the blank had an OD reading greater than the DNA sample. Possible sources for this error can include using a different solution as a blank than the eluent, using a contaminated blank, unclean sample port, and pipetting error. Other potential contributors include pipetting error, DNA degradation, biospecimen handling, and wavelength accuracy. The ambient and frozen shipments were shipped by different couriers and arrived on different days; thus, they may not have been subjected to the same practices and procedures. The remaining DNA concentration and purity results were consistent among the sites.

There was only one DNA biospecimen extracted from whole blood after shipment that had purity outside the acceptable range. This suggests that EDTA shipped at controlled ambient temperature for a mean of 4 days was sufficient for extracting DNA of acceptable integrity.

To improve on outcomes in DNA comparative analysis, BRs need to carefully examine their preanalytical processes, from biospecimen collection through DNA extraction, to identify problems and preventative procedures. The BRs are designing DNA QC algorithms that incorporate electrophoresis for DNA integrity and Qubit for improved sensitivity and specificity. Our results also highlight the need to conduct pilot, validation, proficiency, and competency exercises to determine weaknesses and devise strategies for improvement.

Visual grading for turbidity and hemolysis was performed for serum and plasma biospecimens. Considering that the protocols are identical, the results for plasma and serum were combined for discussion purposes. Concordance was defined as being equal/identical. As described above, the visual grading is 0–5. For each instance of discordance, the difference was between 0 and 1. The slight differences emphasize the subjectivity of the method and the vulnerability to varying interpretations according to operator. Despite these small variations, visual grading remains an affordable simple method for clinical/collection sites to communicate minimal information about the biospecimen that could be useful to determine appropriateness of downstream analysis. The BRs will investigate appropriateness, benefits, and costs of incorporating more informative QC measures.

Visual grading for turbidity and pH determination were reported for urine. The high concordance between sites for turbidity suggests it is a cost-effective method for sites to provide basic information reflecting biospecimen quality. For pH, the two sites that used similar dipstick methods reported identical results; however, where one site used a dipstick method and the other used an electrode, there was significant differences. It is important to note that pH results varied on an average of 0.59 (range difference 0.2–1) and that the dipstick method consistently provided slightly higher pH than the electrode. This suggests that different testing platforms may vary in reporting, and highlights the need to pilot and/or compare differing testing methods prior to implementation to identify and account for differences. The average pH was 6.22 preshipment and 5.65 post-shipment. This is consistent with the findings of Rist et al. that freezing urine immediately on dry ice causes pH variation, as a result of differences in CO₂ concentration. Rist et al. recommend freezing urine at −20°C and transferring samples to lower storage temperatures within 1 week. ¹² The H3Africa biorepositories are updating relevant SOPs accordingly and exploring other methods for urine: (1) QC such as microalbumin and/or other analytes, (2) processing such as centrifugation and additives, ¹³ and (3) storage ¹³ to ensure high-quality biospecimen for future studies.

Conclusion

Multisite research consortia face numerous challenges for maintaining biospecimen integrity when multiple stages of the preanalytical process occur outside the laboratory. These factors are compounded in Africa where there is poor infrastructure and limited courier access and reliability. ⁵ Thus, it is even more critical to develop and harmonize guidelines, SOPs, and other documents and train staff accordingly to establish minimum requirements. Pilot exercises provide a method to evaluate biobanking processes in the African environment to identify gaps before implementation.

There is a lack of investigations reporting on the validation of standardized processes for international biospecimen exchange among African countries and regions. We have established that African BRs can collect, process, store, and ship biospecimens of good quality within the continent. We have also shown that it is possible to ship biospecimens between different regions of Africa in a reasonable time frame, without compromise to the cold chain and biospecimen integrity. Furthermore, despite country and institution policy differences, we have demonstrated that it is possible to harmonize ethical documents, guidelines, and processes among African BRs and their home institutions to facilitate collaboration.