Clinical laboratory testing is the most widely used medical decision-making tool [1] and is crucial for disease screening, diagnosis, monitoring disease progression and treatment efficacy. Correct interpretation of laboratory tests requires accurate reference intervals from an appropriate population [2]. Reference intervals are typically established by assaying specimens from a sample group of people who meet carefully defined criteria [3]. The reference interval is usually defined as the values encompassing the central 95% of specimens; equating to 2 standard deviations on either side of the mean [4]. Producing reference intervals for a general population is a major challenge, as it requires selecting the appropriate reference population and recruiting individuals who represent relevant demographic groups that meet the inclusion criteria; collecting, processing and testing specimens; and finally, calculating reference values with possible stratification of the data into subgroups.

The currently used reference values in Botswana vary by individual clinical laboratory. For example, some laboratories use values supplied in a manufacturer's kit insert but those values are derived from populations outside the country and may not accurately represent normal values for the Botswana population. Without Botswana-based reference values, there could be considerable misclassification of normal and abnormal test results. This is especially important when implementing lifesaving anti-retroviral (ARV) drug therapy for HIV infected persons. ARVs can have adverse effects which must be recognized early on using appropriate laboratory reference values. As the number of HIV prevention and treatment clinical trials in Botswana has increased greatly, accurate reference values are needed to correctly screen volunteers for study eligibility and as well as monitoring for possible adverse events. A few studies reporting immuno-hematological reference values for the Botswana population exist [5], [6] but there is little, if any, local reference information available for clinical biochemistry parameters in Batswana adults.

Our manuscript reports several hematologic and chemistry parameters among a group of healthy Batswana adults. We used those results to create reference intervals and compared our reference intervals to those already in use in Botswana and in neighboring countries.

There is paucity of reference interval data in Botswana with only a few studies conducted on hematological/immunohematological ranges and one on lipids [5]–[6], [9] for the biochemical ranges. Reference intervals for laboratory parameters provide important data for assessment of the health status of an individual. For this reason reference intervals are routinely used in clinical trials at enrollment to determine eligibility, establish baseline measures, and to monitor participants' health during the course of the trial. Moreover, analytes including hemoglobin, bilirubin and neutrophils are used as markers for the presence of disease. Without locally derived reference values for African populations, clinicians and researchers often must use reference values obtained from European or North American populations. However, several African studies have highlighted differences between locally derived values and western derived values. Hence, over the last decade, there has been an increase in the number of studies aimed at establishing locally derived reference intervals for more effective patient management and proper conduct of clinical research in these settings. The use of population based ranges for the TDF study (Table 1) to assess laboratory data, would have led to a reduction in out of range clinical trial volunteers for Amylase, BUN, ALT hemoglobin, inorganic phosphate which could have translated to rapid enrollment of participants and a savings on resources utilized for attending to adverse events. Using population based reference ranges to work out toxicity tables for the study would also have resulted in fewer participants classified as grades 1–4 and requiring management and intensified monitoring.

Similar to other African studies, a substantial number of our TDF2 participants would have been excluded from participating in a clinical trial if the instrument/assay kit-derived values currently in use in most hospitals were applied (Table 3). A study in Uganda reported up to 31% exclusion using western-derived intervals for recruitment into a clinical trial while only 17% of these participants would have been excluded if they had used locally established reference intervals [10]. Similarly, a Kenya study reported up to a 40% exclusion rate if western-derived values were applied compared to population based reference intervals for a rural population [11]. These misclassifications have a negative impact by wasting scarce resources and creating time delays in clinical trials and more importantly in creating misdiagnoses. Therefore, the need to have accurate, locally-derived reference intervals is a fundamental requisite for establishing adequate medical care and conducting more efficiently run clinical trials for the benefit of African populations.

Significant gender differences for hemoglobin and hematocrit levels in our study are consistent with previous reports of higher Hb and Hct levels in males than females and can be explained by the likely effect of androgens on erythropoiesis that increases the number of circulating RBC's with a resultant hemodilution [11]–[15]. The lower Hb levels in women are a common phenomenon in several other African studies [10]–[12], [15]–[19] these levels can further be exacerbated by poor nutritional status resulting in iron deficiency, genetic disorders including thalassemia and sickle cell trait or infections with helminthes, malaria or schistosomiasis for which we did not test. Our findings that males have higher levels of CO2, potassium, AST, ALT, creatinine, blood urea nitrogen, bilirubin (total and direct), and amylase, and lower levels of chloride and phosphate (inorganic) is consistent with previous studies [10]–[11], [15]. Even though we observed significant differences in a majority of parameters, these may not be of medical significance and warrants further investigation.

Several limitations could be cited for our study. Though the study population was from the cities of Francistown and Gaborone with representative populations from most of the areas of Botswana, the ranges established cannot be generalized to the whole of Botswana. Information on other factors that could possibly affect analyte levels such as diet, lifestyle were not collected from the participants. Additionally, although alcohol may affect several biochemical parameters including Amylase, this was not investigated in the present study. The inclusion of women on hormonal contraceptives in this study population means that hormonal effects on biochemical and hematological analytes could not be ruled out. While the study used a single platform for biochemistry and hematology, the CLSI guidelines require that individual laboratories perform evaluations with limited sample size to verify the applicability of such references within their setting. Moreover the platforms used in this study are commonly used in the Botswana public health setting.

Even with the above limitations, our results were still comparable with other studies in the region. Our hemoglobin and hematocrit intervals were comparable to those obtained in a recent Botswana study, [6] but there were variations that were likely due to a larger geographic area and a more ethnically diverse population in our sample. In addition, our intervals came from a larger sample size and they agreed overall with the recently published combined Eastern and Southern Africa consensus intervals, [19] except serum amylase. However, our amylase intervals were similar to the intervals in a study of health Ugandan blood donors [10] indicating that these levels are not uncommon in the African setting. Though the gender differences for some parameters in our study are statistically significant, the absolute value of those differences is relatively small. It may be that these differences are not of sufficient clinical significance to warrant the establishment of different standards between men and women. The accumulation of more reference data from other sources will help to clarify that question.

Given that the frequency of clinical trials and persons receiving clinical services is increasing substantially in sub-Saharan Africa, the introduction of geographic and ethnically valid laboratory intervals is needed for establishing accurate toxicity tables for use in patient management as well as in recruiting and monitoring participants in clinical trials in addition to the DAIDS toxicity grading tables. The established intervals are appropriate for use in young adult population in Botswana and need to be validated using a smaller sample size as indicated in the CLSI guidelines [8]. The accuracy of our tables will increase as more data from representative populations becomes available. As this process of revising the tables unfolds, it will be important to verify that the revised intervals do in fact represent healthy norms for Africa. For that reason, it is important that interventional trials should continue to carefully review their data to ensure that persons whose laboratory values fall within the new ranges are indeed at no greater risk as compared to the current tables. Our reference intervals, in addition to the existing intervals could be used for comparison by the Botswana Ministry of health in establishing Botswana population based reference ranges for standardization of laboratory diagnosis.