Clsi M45

[Alexandrie Gallup]

Clinicallaboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial.

Equal proportions of men and women were invited to participate in a cross sectional study at seven clinical centers (Kigali, Rwanda; Masaka and Entebbe, Uganda; two in Nairobi and one in Kilifi, Kenya; and Lusaka, Zambia). All laboratories used hematology, immunology and biochemistry analyzers validated by an independent clinical laboratory. Clinical and Laboratory Standards Institute guidelines were followed to create study consensus intervals. For comparison, AE grading criteria published by the U.S. National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) and other U.S. reference intervals were used. 2,990 potential volunteers were screened, and 2,105 (1,083 men and 1,022 women) were included in the analysis. While some significant gender and regional differences were observed, creating consensus African study intervals from the complete data was possible for 18 of the 25 analytes. Compared to reference intervals from the U.S., we found lower hematocrit and hemoglobin levels, particularly among women, lower white blood cell and neutrophil counts, and lower amylase. Both genders had elevated eosinophil counts, immunoglobulin G, total and direct bilirubin, lactate dehydrogenase and creatine phosphokinase, the latter being more pronounced among women. When graded against U.S.-derived DAIDS AE grading criteria, we observed 774 (35.3%) volunteers with grade one or higher results; 314 (14.9%) had elevated total bilirubin, and 201 (9.6%) had low neutrophil counts. These otherwise healthy volunteers would be excluded or would require special exemption to participate in many clinical trials.

To accelerate clinical trials in Africa, and to improve their scientific validity, locally appropriate reference ranges should be used. This study provides ranges that will inform inclusion criteria and evaluation of adverse events for studies in these regions of Africa.

Copyright: 2009 Karita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study and report were made possible in part by the generous support of the American people through the United States Agency for International Development (USAID), the Klingenstein Fund, Gates Challenge Fund, Government of Netherlands TMF, Government of Norway, European Union East Africa Fund, Canadian International Development Agency, Development Cooperation Ireland, Department for International Development, Danish Ministry for Foreign Affairs, Swedish Ministry of Foreign Affairs, and the World Bank. The contents are the responsibility of the International AIDS Vaccine Initiative and do not necessarily reflect the views of USAID or the United States Government.

Clinical trials are increasingly being conducted in Africa, especially trials of preventive interventions for HIV, tuberculosis and malaria. Great strides have been made towards improving the research infrastructure worldwide, especially in Africa [1], [2]. However, laboratory reference intervals used for trial screening and evaluating adverse events (AE) have often been derived from predominantly North American and European (largely Caucasian) populations [3]; use of these reference intervals may lead to unnecessary exclusion of potential participants.. Previous studies from Eastern and Southern African populations indicate differences in hematology and immunology values, including lower values for hemoglobin, hematocrit, red blood cell count (RBC), platelets, mean corpuscular volume (MCV) [4], [5], [6], [7], [8], [9] and neutrophils and increased values for monocytes and eosinophils [5], [7], [9], [10], [11]. Lymphocyte and CD4 T cell counts in Africans have also been reported to be lower than intervals measured in Europe and North America [9], [12], [13]. Other studies have noted hematology and CD4 T cell count variations across different regions of Africa [5], [9], [14]. Within the U.S., lower neutrophil and leukocyte counts have been found to be more common among blacks relative to whites [15]. To date, no studies have assessed laboratory reference intervals in a controlled, systematic manner across multiple African sites among asymptomatic adults who would otherwise be eligible as healthy clinical trial volunteers. Locally appropriate reference intervals are essential for planning and executing trials in a safe, efficient and ethical manner.

This paper presents the results from a cross sectional study in seven African research facilities to: 1) establish values for locally relevant serum chemistry and hematology analytes among healthy African adults in anticipation of future clinical trials of HIV prevention technologies and other interventions, 2) compare these findings to established intervals from the U.S., and 3) determine how many individuals would have been reported as having an adverse event (AE) according to the DAIDS AE Grading Table [16].

This study was approved by the National Ethics Committee of Rwanda, the Uganda Virus Research Institute Science and Ethics Committee, the Uganda National Council for Science and Technology, the Kenya Medical Research Institute Ethics Committee, Kenyatta National Hospital Ethics and Research Committee at the University of Nairobi, the University of Zambia Biomedical Research Ethics Committee and the Emory University School of Public Health Ethics Committee. All EC/IRBs are registered with the U.S. Office of Human Research Protection. Written informed consent was obtained prior to conducting any study procedures, literacy was not a requirement for participation.

All laboratory staff received equipment training and were required to pass an independent quality review before enrolling volunteers. Analyte results were validated throughout the course of the study through a central reference laboratory (Central Laboratory Services [CLS], Johannesburg, South Africa) external quality assurance (EQA) program provided by the South African National Health Laboratory Services [18], [19]. Results were compared across technicians and across laboratories. The study was conducted according to the principles of Good Clinical Laboratory Practices [20].

Data were directly recorded on Case Report Forms which were then faxed to a central server using DataFax software (Clinical DataFax Systems Inc., Hamilton, Canada). Data analyses were conducted using Stata (v9.1 College Park, TX, USA) and SAS (v9.1, Cary, NC, USA) software. The Clinical and Laboratory Standards Institute (CLSI, www.clsi.org, formerly NCCLS) terms and guidelines for defining reference intervals [21] were followed. Briefly, we evaluated intervals by clinical centers and gender. If the overall F-test from an ANOVA on mean values was statistically significant (p

The hematology results are shown in Table 2. Due to significant gender and site variability, the complete data set could not be used to construct study consensus intervals for four of the 12 analytes (hematocrit, RBC, eosinophils and basophils). Women had lower hematocrit (median 39.7% vs. 45.1%, p

There was no significant gender or site variability for complete WBC counts across our study populations. The study consensus interval was lower than the U.S.-based comparison interval with nearly 29% of values OOR. Neutrophil counts also tended to be low (Figure 2), with a similar proportion of OOR values. Eosinophil values tended to be high (Figure 3). The eosinophil counts did not vary significantly by site or gender. Because eosinophil percent were significantly higher among men in Masaka, these values were not included in the study consensus interval. Basophil counts and percent varied by site, and the study consensus intervals do not include values from Lusaka. The study consensus interval for basophil percent also excludes values from Entebbe and women from Kilifi.

The chemistry results are shown in Table 3. Due to significant gender and site variability, we were unable to create study consensus intervals using the complete data set for three of the 12 analytes (direct bilirubin, total immunoglobulin gamma [IgG], and lactate dehydrogenase [LDH]). Our total and direct bilirubin study consensus intervals were considerably wider than the U.S.-based comparison intervals; 31% and 42% of study values were OOR, respectively (Figures 5 and 6). The direct bilirubin interval from women in Kilifi was significantly lower than the other intervals from other sites, so values from women in Kilifi were excluded from the study consensus interval.

Values for IgG, creatinine phosphokinase (CPK) and LDH tended to be higher than their respective U.S.-based comparison intervals. In particular, 83% of IgG values and >99% of LDH values were OOR versus the U.S.-based comparison intervals. The study consensus interval for IgG excluded values from men in Masaka (Figure 7), and the study consensus interval for LDH excluded all Lusaka volunteers and men from KNH (Figure 8).

As the attention of the global health community increasingly turns to the development of preventive and therapeutic interventions for HIV, tuberculosis and malaria, among others, Africa has become an important venue for clinical trials [1], [24]. A thorough understanding of the health status of potential clinical trial volunteers in Africa is essential for planning and executing such trials in a safe, efficient and ethical manner. In this regard, it is essential to use locally appropriate laboratory reference intervals to assess volunteer health, monitor laboratory-based AEs, and assure that healthy individuals who want to volunteer to participate in clinical trials are not unnecessarily prevented from participating if it is safe for them to do so.

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