An increasing number of clinical trials taking place in sub-Saharan Africa are seeking to identify safe and effective prevention and treatment strategies to combat the heavy burden of infectious diseases in this region [1], [2]. Africa is disproportionately affected by numerous viral, parasitic and bacterial diseases, including: 66% of the global HIV/AIDS infections [3], [4], 31% of the tuberculosis infections, and 86% of the malaria cases [5]. Clinical trials in sub-Saharan Africa need accurate clinical laboratory reference ranges for appropriate screening of volunteers in clinical trials, monitoring disease progression, and evaluating possible clinical trial-associated toxicity and adverse events. Traditionally, normal ranges for clinical laboratory values have been obtained mainly from European and North American populations [2]. However, differences are known to occur between normal Africans values with those of North Americans and Europeans [6]. For example, African populations are reported to have lower hemoglobin (HB), red blood cells (RBCs), hematocrit (HCT), mean corpuscular volumes (MCV), platelets and neutrophils, and higher monocyte and eosinophil levels than their Western counterparts [6]–[9]. Moreover, there are variations in indices between different African ethnic groups [9]–[12]. Factors such as genetics, dietary patterns, gender, age, ethnic origin and environmental pathogens are known to influence hematological and immunologic indices [13]–[16]. Thus, the use of normal laboratory values derived from external populations could produce selection bias leading to exclusions of otherwise healthy volunteers in clinical trials, misclassification of adverse events, and a framework for allowing incorrect patient management in routine clinical care.

Besides the relevant utility of laboratory reference values for clinical trials, such values are also important in routine health assessment, particularly for screening of anemia, blood disorders and diseases of the immune system. Of particular importance is the use of these indices as surrogate markers for disease progression and response to anti-retroviral therapy in HIV-infected individuals [17]. Decisions to initiate, monitor, or change antiretroviral therapy (ART) regimens are determined using CD4+ T-lymphocyte cell (CD4) counts, while drug toxicity is monitored using liver function tests (LFT) and renal function tests (RFT), and complete blood counts (CBC) [18], [19]. Due to differences in these parameters between Western and African populations, it is necessary to develop a range of local values for these indices. In addition, differences in hematological and lymphocyte indices between age groups also suggests the need to develop age-specific reference ranges [8], [14], [15], [20]. However, information about reference values based on age groups is limited for the Africa populace [6], [21]–[23].

The aim of our study was to generate normal ranges of laboratory values for blood chemistry, hematologic and immunologic indices of healthy, HIV-negative females and males aged 13 to 34 years. We also determined gender and age variations in these indicators. In addition, we compared our normal range values with those previously reported from the African continent and with North American-derived reference ranges from the Massachusetts General Hospital [24] and the U.S. NIH Division of AIDS (DAIDS) which are currently used to measure the severity of adverse events in most clinical research studies [25].

A total of 2707 participants were enrolled in the two baseline cross-sectional surveys. Of these 2307 were HIV-seronegative. From these participants a total of two hundred and ninety eight specimens from participants who met the eligibility criteria were included in this study. One hundred and fifty four (51.7%) of these were males while 144 (48.3%) were females. Almost half (46%) of the study population were less than 18 years old, representing 77 (50%) of all males and 62 (47%) of all females.

Clinical laboratory values provide important data to help assess the health of an individual. For this reason they are routinely used in clinical trials at enrollment and also during the course of the trial for monitoring the participants' health. Moreover, several analytes are used either as surrogate markers for indicating the possible presence of a disease or as direct evidence for that disease [32]–[34]. In the absence of locally derived reference values for African populations, clinicians and researchers have had to use reference values of European or North American populations. Previous studies have shown that such values vary with age, ethnic origin, socio-demographic characteristics, and environmental context [6], [8], [9], [21], [35]–[37]. The development of region and age-specific reference values is thus essential for efficient patient management and proper conduct of clinical research. This is especially critical for clinical trials being conducted in Africa to reduce the burden of such diseases as malaria, TB, and HIV in this region. Apart from their use in clinical trials, some laboratory markers are HIV disease surrogate markers and are thus important in care and treatment of HIV-infected patients in sub-Saharan Africa that has the greatest burden of the pandemic [4]–[6]. Our study was carried out in Nyanza Province, western Kenya, the region with the highest HIV prevalence in Kenya (15%) [38]. It is also the region in Kenya where many clinical research studies are being carried out [39]–[41]. Most of the values observed in this population differed with standard US-based reference values (MGH/DAIDS).

Regarding hematological indices, most of our values were lower than those derived from North American population; this finding is consistent with previous studies in other African regions [8], [21], [22]. However, our combined values for HB and MCV for both young adults and adolescents were higher than those reported from another study in Kericho, Kenya [6], but were slightly lower than those derived from Ethiopia, Kampala in Uganda and Mbeya in Tanzania [8], [21], [22]. The findings of significant gender differences in RBC parameters (RBC, HB, HCT, and MCV) are consistent with previously established evidence that men have higher values than females for these parameters. This difference is partly attributed to the influence of the androgen hormone on erythropoiesis [42], [43] and to menstrual blood loss in women [6], [8], [20], [21], [44]. As previously reported in other studies targeting adolescents [15], [20], older males from this population had significant higher values for RBC, HB and HCT than young males. The difference could be attributed to higher levels of androgen hormones among the older as compared to the younger males. This potential explanation is further supported by the absence of age-related hematological difference among females in our study, which is in agreement with findings obtained from previous studies targeting adolescent cohorts [15].

The lower platelet counts from this population as compared to Western values are synonymous with findings from other African studies [7], [8], [11], [22], [45]. The etiology of low platelet counts in African populations is unknown. However dietary, environmental and genetic factors have been proposed [7], [11], [45]. In agreement with other African studies [6], [8], [28], [35], [46] was the high eosinophil and low WBC and neutrophil values compared to those in North America. The eosinophilia may be attributed to increased parasitemia, since our study area is endemic for schistosomiasis, helminthic infections and perennial malaria [27], [28]. The low neutrophil count observed in our population could possibly be attributed to African genetics, environment or diet [36], [46].

The differences in gender and age in both WBC and CD4 cell counts are in agreement with previous reports [8], [14]. Females generally had higher counts, while young adolescents had equally higher values for both WBC and CD4 cell count as compared to adults. Overall our ranges for the lymphocyte subsets were higher and comparable to the USA reference ranges. The mean CD4 cell count for our population was 857.9, which is consistent with mean CD4 cell counts reported in other HIV-negative populations in Africa.

Liver and renal function tests are also important indicators of patient response to ARTs in the management of HIV/AIDS patients. Clinical chemistry laboratory reference values for LFT and renal function tests are limited in Africa, despite the continuous use of ARTs in this region. Most of our clinical chemistry reference ranges were comparable with the US MGH ranges except for T-bil and blood urea nitrogen. As seen from other studies conducted in Kenya [6], Uganda [8] and Tanzania [21] the upper range for T-bil seen in this study was twice as high as that of the US while the lower range for BUN was twice as low. The etiology of high T-bil in this population is thought to arise from a number of factors like RBC hemolysis caused by malaria or sickle cell disease, malnutrition or physical exertion. However the presence of similar trends among other African populations is suggestive of a common environmental or genetic factor [6], [37].

Analysis of the comparison between the values obtained from this population with those from the MGH ranges used in most clinical research studies revealed high variations for most values. If the US MGH derived ranges were used on this population during screening for any clinical research, over 58% of the volunteers would be screened out of the trial despite having laboratory results consistent with the general population. This erroneous screening would have important implications on study cost, work load and time, as more volunteers would be required for the screening process to meet the required target, even though the screening out process would actually have excluded healthy potential volunteers [47]. Moreover, the fact that the investigational product is designed for use in the same population in which the laboratory values differ with the values being used at the screening process might further complicate post-market analysis or adoption of the product for the general population.

Equally important is the comparison to the DAIDS toxicity tables; some of the ranges obtained extend between the normal and grade 1–2 toxicity grading. The lower range for HB (8.0 in females), neutrophil counts (0.9), as well as the upper range for eosinophil counts (1.68) and T-bil (40.7) would have been considered as grade 2 adverse events. In addition the observed lower ranges in males for HB (10.8) and the combined values for ALT (61.3) and AST (50.4) would have been classified as grade 1 adverse event. The use of the DAIDS toxicity grading for such populations may lead to inappropriate reporting of adverse events during clinical trials.

In agreement with other published data, we observed age-related variation between the adolescent males as compared to the adults for HB, HCT and RBC levels [8], [15], [20]. The fact that adolescents had lower hematological values is an issue that should be noted whenever clinical trials target this population. However, the observed differences may not be of medical significance, and thus there is need for further research as participation of adolescents in clinical trials increases. We observed no significant age differences in other parameters measured among males or in any parameters measured in females except for creatinine and BUN. This implies that adult values can be used in clinical trials involving adolescents for such parameters for which no differences were reported.

Several limitations could be cited in our study. Clinical Laboratory and Standards Institute (CLSI) guidelines for laboratory indicators recommend the consideration of genetic, environmental and social habits (such as smoking, dietary components, exercise and lifestyle) for which data were not obtained during enrollment of the participants in this study [11], [35], [37], [48]. The sample size was also small in the two age sets and did not meet the recommended CLSI sample size of 120 per every partitioned group [48]. However, a robust bootstrap analysis was used so as to eliminate bias as recommended by the Canadian laboratory initiative on paediatric reference intervals (CALIPER) [49]. In addition, although a thorough medical examination was done, not all sub-clinical conditions such as parasitic infections, hepatitis B infection or nutrition factors known to interfere with the obtained parameters were assessed. However, in the context of resource-limited settings, we assert that our study methods were sufficient to determine reference ranges for use in this population.

The findings from our study confirmed previously published data documenting differences in clinical laboratory reference ranges between African and Western populations. We also assert that there exist age variations in hematological values among males that reinforce the need to establish age-specific reference ranges for use in clinical trials involving such a population. This study presents the first description of biochemistry and hematological reference ranges in western Kenya, and is also the first study in the country that compares two distinct age sets between adolescents and adults. The development of these reference ranges may provide guidelines to be used by local health practioners in patient management within this region and for the design, conduct and evaluation of clinical trials for biomedical interventions. Whilst our study limitations may have influenced the different study parameters, our findings were comparable to those of other studies within Africa [6], [22], [23], [37], [47] and can be used as reference ranges for adolescents and young adults within Kenya.

As clinical trials and anti-retroviral treatment increase in Africa, locally derived clinical laboratory reference ranges are essential to ensure appropriate treatment monitoring, general health assessment and efficient execution of clinical trials. Similarly important is the need for development of toxicity grades for use among African populace in clinical care based on the differences observed between laboratory values from African population and the West. So far clinical studies as well as routine clinical patient management in most African countries are using either the European generated machine values, or the NIH division of AIDS toxicity grading in assessing critical values. The development of the various laboratory-derived African toxicity grades, in addition to the already developed reference values would thus be ideal for use in reporting adverse events in clinical trials as well as in routine health care for determining critical values. Using Western-derived reference ranges, a majority of our study participants would have been misclassified due to the high eosinophil counts and T-Bil as a consequence of regular exposure to endemic pathogens and inherent genetic factors leading to unnecessary treatment. Thus, physicians need to take into consideration the population limitations while attending to patients within this region. This study provides the first age-specific, locally derived clinical laboratory reference ranges in western Kenya for use in health care and clinical research.