**1. Introduction**

302 Blood Cell – An Overview of Studies in Hematology

1998;185(3) 256-261.

[29] Murray GI, Duncan ME, O'Neil P, McKay JA, Melvin WT, Fothergill JE. Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer. J Pathol

> Over the last decade, there has been a significant increase in the number of clinical trials taking place in sub-Saharan Africa in a concerted effort to identify safe and effective prevention and treatment strategies to combat the heavy burden of infectious diseases in this region [1-3]. This is because numerous viral, parasitic and bacterial diseases are endemic in this region, including: 66% of the global HIV/AIDS infections, 31% of tuberculosis infections, and 86% of malaria cases [3, 4]. Routine capacity for clinical laboratory testing is also increasing in Africa. Clinical trials and clinical care in sub-Saharan Africa require accurate laboratory reference intervals for appropriate assessment of patients/participants, monitoring disease progression, and reporting of possible toxicity and adverse events.

> This is particularly important in phase I and II clinical trials. Phase I trials often enroll a small group of healthy participants in order to determine the metabolic and pharmacologic actions of drugs, side effects associated with increasing doses and early evidence of efficacy. Phase II trials on the other hand are controlled clinical studies conducted to evaluate efficacy of drug/vaccine for a particular indication in a larger group of participants and to further evaluate its safety. Many HIV vaccine trials are slated for Phase I–III trials in Africa. The inception of the US President's Emergency Plan for AIDS Relief in 2004, with a mandate to treat 2 million HIV infections with anti-retroviral therapy by 2008 has accelerated the implementation of lymphocyte immunophenotyping in urban and rural areas in Africa as initiation of therapy is often predicated on absolute CD4 T- lymphocyte counts. Central to any HIV vaccine and/or care and treatment program is the capability to measure absolute CD4 counts. CD4 counts are important in the context of breakthrough infections during HIV vaccine trials and informing treatment. Correct diagnosis in patient management often involves accurate interpretation of results from laboratory testing [5]. Hence it is critical for medical professionals to have access to an accurate management resource such as reference intervals.

© 2012 Zeh et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Zeh et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Historically, clinical studies as well as routine clinical patient management in most African countries have relied on European-generated automated instrument values, US established reference intervals or the U.S. NIH division of AIDS (DAIDS) toxicity grading tables in assessing clinical parameters in study participants. The US-established reference intervals are obtained from the Massachusetts General Hospital reference values and serve as the standard reference interval comparison for most studies [6]. The DAIDS toxicity tables, also derived from a Caucasian population, are used for grading the severity of adult and pediatric adverse events, whether or not they are considered to be related to the study intervention [7]. DAIDS provides guidelines for estimating severity of adverse events using specific reference intervals (Table 1) as criteria for determining what is 'normal' and among abnormal values, how to grade the severity of the abnormality.

Laboratory Reference Intervals in Africa 305

applying reference intervals in diagnostics as well as in recruitment in clinical trials. According to the Clinical Laboratory and Standards Institute (CLSI) guidelines [8], it is recommended that laboratories establish their own reference intervals from the local population or validate the use of those obtained from a different setting. Despite this, clinicians and researchers in Africa have continued to use reference values of European or North American populations. Our group in Kenya has recently published reference intervals based on the CLSI guidelines and are currently assisting regional laboratories to

In this chapter, we give a brief background on the current status of participant recruitment in clinical trials and patient management in Africa. We will also describe how to select a reference population from which to derive the reference sample group. In addition, we describe various studies advocating the establishment of reference intervals performed in different regions of the African continent including our own. These studies show differences in hematological, biochemical and immunologic parameters between various African populations but these differences are statistically insignificant. However, most hematological, biochemical and immunologic parameters considered in the African studies are significantly different when compared to American and European derived values. This chapter will also discuss the proposed partitioning of adolescent males from adults given their increased recruitment into clinical trials. Adult males have significantly higher values for most hematological and biochemical parameters and we provide an explanation why this is so. While pregnant women and infants undergo physiological processes that alter their hematological and biochemical parameters, the partitioning of these cohorts has been slow. We discuss how pregnancy induces these changes and describe the particular parameters affected. We also highlight the dynamic changes in these parameters during infancy and how they differ from western-derived values. In this chapter, we also illustrate the downside of using inappropriate reference intervals in the recruitment of participants in clinical trials and patient management. We show how the use of such values results in exclusion of clinically healthy participants from clinical trials and may lead to inappropriate reporting of adverse events during the course of these studies. This potentially results in escalation of costs in the conduct of clinical trials. In this book chapter, we also propose the development of laboratory-derived African toxicity grades that, in addition to the already developed reference values, would be used for reporting adverse events in clinical trials and

establish their own reference intervals[9].

for determining critical values in routine health care.

**selection of a reference population** 

**2.1. The use of reference intervals** 

**2. Use of reference intervals, consequence of misclassification and** 

Reference intervals are useful both in the clinical and research environment. Medical laboratory reference intervals are primarily used for clinical purposes. They can be used as an indicator of good health. Alternatively, reference intervals/limits can be used to screen for physiological or pathological conditions hence important in routine health assessment,


Adult and pediatric values for age >57 days, HIV-negative from the DAIDS toxicity tables version 1.0, December 2004; clarification August 2009.

**Table 1.** Examples of DAIDS criteria of estimating severity grading based on laboratory parameters.

Reference values, in general, refer to the value or test result obtained by the observation or measurement of a particular type of quantity on an adequate number of persons (reference sample group) selected to represent the general population. Reference values are usually presented as reference intervals which refer to the interval between, and including two reference limits i.e., from the lower reference limit to the upper reference limit defined by a specific percentage (usually 95%). In certain parameters such as absolute counts of monocytes, eosinophils and basophils, only one reference limit (decision limit), more often the upper reference limit is of biological significance hence the lower reference limit assumes a value of zero.

Reference values go hand in hand with toxicity grading or decision limits, which can be defined as specific levels of the analyte that correspond to mild to life threatening clinical situations. Toxicity grading is particularly useful in the decision-making process of interpreting a measured value and assessing the health status of the subject being tested. For this reason reference values or toxicity grading are routinely used in clinical trials at enrollment to determine eligibility, establish baseline measures, and also during the course of the trial to monitor the participants' health. Moreover, several analytes are used either as markers for the possible presence of a disease or as direct evidence for that disease. Reference values, especially hematological and immunologic indices, are influenced by such factors as genetics, dietary patterns, pregnancy, gender, age, ethnic origin and prior exposure to environmental pathogens. Thus, it is important to consider these factors when applying reference intervals in diagnostics as well as in recruitment in clinical trials. According to the Clinical Laboratory and Standards Institute (CLSI) guidelines [8], it is recommended that laboratories establish their own reference intervals from the local population or validate the use of those obtained from a different setting. Despite this, clinicians and researchers in Africa have continued to use reference values of European or North American populations. Our group in Kenya has recently published reference intervals based on the CLSI guidelines and are currently assisting regional laboratories to establish their own reference intervals[9].

In this chapter, we give a brief background on the current status of participant recruitment in clinical trials and patient management in Africa. We will also describe how to select a reference population from which to derive the reference sample group. In addition, we describe various studies advocating the establishment of reference intervals performed in different regions of the African continent including our own. These studies show differences in hematological, biochemical and immunologic parameters between various African populations but these differences are statistically insignificant. However, most hematological, biochemical and immunologic parameters considered in the African studies are significantly different when compared to American and European derived values. This chapter will also discuss the proposed partitioning of adolescent males from adults given their increased recruitment into clinical trials. Adult males have significantly higher values for most hematological and biochemical parameters and we provide an explanation why this is so. While pregnant women and infants undergo physiological processes that alter their hematological and biochemical parameters, the partitioning of these cohorts has been slow. We discuss how pregnancy induces these changes and describe the particular parameters affected. We also highlight the dynamic changes in these parameters during infancy and how they differ from western-derived values. In this chapter, we also illustrate the downside of using inappropriate reference intervals in the recruitment of participants in clinical trials and patient management. We show how the use of such values results in exclusion of clinically healthy participants from clinical trials and may lead to inappropriate reporting of adverse events during the course of these studies. This potentially results in escalation of costs in the conduct of clinical trials. In this book chapter, we also propose the development of laboratory-derived African toxicity grades that, in addition to the already developed reference values, would be used for reporting adverse events in clinical trials and for determining critical values in routine health care.
