Clinical Laboratory Parameters
Armonia Bunda
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Background: To date, several clinical laboratory parameters associated with Coronavirus disease 2019 (COVID-19) severity have been reported. However, these parameters have not been observed consistently across studies. The aim of this review was to assess clinical laboratory parameters which may serve as markers or predictors of severe or critical COVID-19.
Conclusions: Relative to non-severe COVID-19, severe or critical COVID-19 is characterised by increased markers of innate immune response, decreased markers of adaptive immune response, and increased markers of tissue damage and major organ failure. These markers could be used to recognise severe or critical disease and to monitor clinical course of COVID-19.
Clinical laboratory reference values from North American and European populations are currently used in most Africans countries due to the absence of locally derived reference ranges, despite previous studies reporting significant differences between populations. Our aim was to define reference ranges for both genders in 18 to 24 year-old Mozambicans in preparation for clinical vaccine trials.
A cross-sectional study including 257 volunteers (102 males and 155 females) between 18 and 24 years was performedat a youth clinic in Maputo, Mozambique. All volunteers were clinically healthy and human immunodeficiency virus, Hepatitis B virus and syphilis negative.Median and 95% reference ranges were calculated for immunological, hematological and chemistry parameters. Ranges were compared with those reported based on populations in other African countries and the US. The impact of applying US NIH Division of AIDS (DAIDS) toxicity tables was assessed.
The immunology ranges were comparable to those reported for the US and western Kenya.There were significant gender differences in CD4+ T cell values 713 cells/L in males versus 824 cells/L in females (p
This study is the first to determine normal laboratory parameters in Mozambique. Our results underscore the necessity of establishing region-specific clinical reference ranges for proper patient management and safe conduct of clinical trials.
Copyright: 2014 Tembe 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 was supported by funding from The Regional HIV/AIDS Team for Africa, Embassy of Sweden, Lusaka jointly funded by Sweden and Norway (Sida contribution number 2150012801). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The use of improper clinical reference ranges to assess participant eligibility and safety for clinical trials may lead to unnecessary exclusion of eligible participants, contribute to over-reporting of adverse events (AEs) [10] and increase the number of referrals for clinical investigations. Laboratory abnormalities based on non-indigenous laboratory parameters and medical abnormalities were reported to be the main reasons volunteers were excluded from two Kenyan HIV vaccine clinical trials [7]. Moreover, studies have suggested that use of the US NIH Division of AIDS (DAIDS) toxicity tables may not be appropriate for African populations [10], [11].
Prior to execution of a phase I/II HIV vaccine trial (TaMoVac 01) in Mozambique, we performed a study to define the prevalence and incidence of HIV and other sexually transmitted viruses in Maputo in a population of young adults. This was also an opportunity to establish clinical reference values in 18 to 24 year-old Mozambicans.
This study establishes reference ranges for immunological, hematological and chemistry parameters in healthy young adults in Mozambique. We determined gender differences and compared values established for Mozambican young adults with those previously reported for the same age group in other African countries and with established intervals from the US (Massachusetts General Hospital, MGH-USA) [12]. Additionally, we applied the division of AIDS (DAIDS) toxicity tables for grading of AEs [13] to evaluate their potential implications for vaccine trials.
A total of 257 healthy individuals between 18 and 24 years old were recruited from a cohort of youths participating in a study of the prevalence and incidence of sexually transmitted viruses at the SAAJ clinic, Maputo Central Hospital. Medical staff performed physical examinations and collected clinical histories. Volunteers who were febrile, pregnant, or seropositive for HIV, syphilis or hepatitis B surface antigens were excluded from the study.
The national algorithm for HIV testing was used to diagnose HIV. HIV testing was performed using two immunochromatographic assays, the Determine HIV-1/2 (Inverness Medical, Bedford, United Kingdom) followed by the UniGold HIV-1/2 (Trinity Biotech, Bray, Ireland). Syphilis testing was performed using SD Bioline Syphilis 3.0(Standard Diagnostics, Suwon City, South Korea). Serum samples were tested for Hepatitis B virus (HBV) using the Hepatitis B Surface Antigen (HBsAg) ELISA Kit (Human, Wiesbaden, Germany).
Sample collection took place between August 2009 and September 2012. Most samples (70%) were collected between November 2009 and August 2010, but the inclusion period was extended to recruit additional males to the study. Blood was collected in 4 ml EDTA Vacutainer tubes (Becton-Dickinson, Franklin Lakes, New Jersey, USA)in preparation for lymphocyte and hematological testing. Whole blood was collected in 10 ml serum Vacutainer tubes (Becton-Dickinson, USA)in preparation for testing chemical parameters and HBV status. Samples were collected in the morning between 8.00 AM and 12.00 noon, kept at room temperature and transferred to the laboratory of the National Institute of Heath in Maputo for analysis.
Immunophenotyping was performed using a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, New Jersey, USA). Samples were analyzed within 24 h of specimen collection. In brief, 20 l of CD3FITC/CD8PE/CD45perCP/CD4APC or CD3 FITC/CD16+CD56 PE/CD45PerCP/CD19 APC MultiTest reagents (Becton-Dickinson, USA) was mixed with 50 l of whole blood and incubated in the dark at room temperature for 15 min. Red blood cells were then lysed by adding 450 l of fluorescence-activated cell sorter lysing solution (Becton Dickinson, USA).The tubes were then incubated at room temperature for another 15 min. MultiSET software (Becton-Dickinson, USA) was used to perform the analysis.
A complete blood count and differential was performed using the Sysmex KX-21N Hematology Analyzer (Sysmex Corporation, Kobe, Japan) as recommended by the manufacturer. The samples were analyzed within 6 h of specimen collection. The machine automatically dilutes a whole-blood sample, lyses and counts the cells, and then gives a printout result. Seventeen parameters were analyzed; leukocytes (WBC), erythrocytes (RBC), platelets (PLT), lymphocytes (LYM), neutrophils (NEUT), hemoglobin concentration (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width measured by standard deviation (RDW-SD), red blood cell distribution width measured by coefficient of variation (RDW-CV), platelet distribution width (PDW), mean platelet volume (MPV), platelet larger cell ratio (P-LCR) and the percentages of lymphocytes (LYM), neutrophils (NEUT), and the mixed population of monocytes, basophiles and eosinophils (MXD). The absolute cell counts were expressed as number of cells [106] per liter.
Serum chemistry was performed using a Vitalab Selectra Junior (Vital Scientific, Dieren, Netherlands) per the manufactureŕs instructions. Serum was separated within 4 h of collection and analyzed within 7 h of blood draw. Each sample was analyzed for creatinine, total bilirubin (T-Bil), albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, urea, uric acid, amylase, HDL cholesterol, triglycerides and alkaline phosphatase (ALP).
In addition to commercial controls run daily with each instrument, the laboratory routinely participates (3 times per year) in external quality assurance testing programs distributed by Thistle (Thistle QA, Bryanston, South Africa) for hematology and clinical chemistry, and the International Quality Assurance Program-QASI (Public Health Agency of Canada, Ottawa, Canada) for lymphocyte typing. In the case of internal control failure, testing was suspended.
Data was entered into Microsoft Office Excel 2007 and analyzed using R version 3.0.0 [17]. Prior to calculation of the reference ranges each parameter was check to be normally distributed using Shapiro-Wilks test; and if a deviation from normality was found the best Box-Cox power transformation was performed. Grubbs tests were performed to check for outliers and box plots and qqnorm plots were used to assess outliers visually. In general outliers were removed but their influence was checked by calculating the reference range with the outlier and comparing the reference range without the outlier.
Median and 95% reference ranges (2.5th- 97.5th percentiles) were established for immunology, hematology and chemistry values. The non-parametric Wilcoxon rank-sum test (Mann-Whitney U test) was used to test for differences by gender. For all analysis the significance was set at 0.05.
For clinical laboratory parameters that did not meet the recommended minimum sample size of 120 individuals, which is recommended by the Clinical and Laboratory Standards Institute (CLSI) [18], percentiles were obtained from a boostrap procedure where 1000 resamples were performed per parameter and gender. On each resample, a percentile was calculated and the final percentile was the averages of the resampled percentiles [19], [20]. The p-values were obtained using a 10000 boostrap resamples per parameter to obtain the distribution of the Wilcoxon rank-sum test under the assumption of no difference between genders and then the original was compared to this distribution and area under curve on the right side of the histogram was considered as p-value.
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