Immunohematological Reference Values for Healthy Adults in Burkina Faso

Abstract

Reference ranges for peripheral blood lymphocyte subsets were generated for 186 healthy adults in Burkina Faso using single-platform flow cytometry. CD4⁺ T-cell counts ranged from 631 to 1,696 cells μl⁻¹; they were lower in males (n = 97) than in females (n = 89), whereas natural killer cell counts were higher.

Immunohematological parameters may be influenced by genetic and environmental factors, differ between sexes (2), and change with age (11). Recent studies reveal important differences in lymphocyte normal ranges between different populations in Asia (3), Europe (5), and Africa (6, 7, 9, 12, 13). We therefore undertook a population-based study in Nouna, Burkina Faso, in order to generate site- and gender-specific reference values for immunohematological parameters in healthy adults.

The study was part of an ongoing longitudinal prevention of mother-to-child transmission trial which is embedded in the National Programme for AIDS Prevention in Burkina Faso. It was approved by the Institutional Ethics Committees of the University of Heidelberg, Germany, and of the Nouna Health Research Centre, Burkina Faso.

Donors were recruited from female clients of the mother and infant clinic at the local hospital and during repeated blood donation campaigns in the community of Nouna town. Donors were classified as “clinically healthy” if they did not show or report signs and symptoms of illness (>5 kg weight loss in the preceding month, fever during the last 2 weeks, asthma, diabetes, and cardiovascular and renal diseases). The presence of antibodies against the human immune deficiency virus (HIV) or syphilis or the presence of the surface antigen of the hepatitis B virus in serum led to subsequent exclusion of the individual from data analysis.

Donors were excluded from analysis irrespective of their health status if they had used drugs or traditional treatments within the preceding month, had undergone any surgical treatment, had received any blood transfusions in the past, or could not document their age or if they reported ear piercing, tattooing, and acupuncture during the last 3 months or unprotected sex with multiple partners. Female donors were excluded if they were pregnant or had delivered within the past 6 months. “Nonpregnant” status was defined as having had a menstrual bleed within the past 30 days.

Venipuncture was performed in the morning between 7 and 11 a.m., and samples were accessed for hematology analysis and single-platform flow cytometry (FCM) staining within 6 h following venipuncture. Samples were prepared at the climatized room temperature (range, 20 to 30°C) according to the instructions of the manufacturers.

Total leukocyte counts and absolute numbers of lymphocytes were determined by using an automated device (Sysmex KX21N; Sysmex Corporation, Kobe, Japan). Flow cytometric analysis was performed on a BD Biosciences three-color instrument (BD FACScan). All reagents, hard- and software, and consumables were generously provided by BD. Instrument setup was accomplished by using analysis of BD CaliBRITE beads and BD FACSComp software (initially in daily intervals, later twice a week, and then weekly). Analyses were interpreted according to the Centers for Disease Control and Prevention guidelines (8). Instrument settings were manually controlled by visual inspection of typical staining patterns of peripheral blood lymphocytes.

Eligible donors were retrospectively selected from 364 individuals originally recruited. Eighty-one study participants were excluded because the hematology analyser indicated sample agglutination during measurement. Of the remaining 283 donors, 232 were seronegative for HIV, hepatitis B virus, and syphilis. FCM (MultiSET and TruCount) was performed on 223 samples, 186 of which were ultimately included in the calculation of reference values for healthy adults (20 samples had to be excluded because of incomplete FCM measurements due to temporary lack of reagents, and 17 samples did not pass the MultiSET internal quality control and visual inspection by the operator did not allow manual analysis).

The donors' ages ranged from 18 to 78 years: 28 donors were younger than 20 years (15%), 99 were between 20 and 29 years (53%), 31 were between 30 and 39 years (17%) and 28 were 40 or more years old (15%). No statistically significant influence of age on the distribution of lymphocyte subpopulations was seen, either in terms of absolute or of relative cell numbers.

Table 1 shows the reference values of the peripheral blood lymphocyte subsets for the total study group, males (n = 89; 48%) and females (n = 97; 52%). Male blood donors had significantly lower CD4⁺ T-cell counts than females (mean difference, −140 cells μl⁻¹; 95% confidence interval, −43 to −238 cells μl⁻¹; P < 0.005), whereas their NK cell counts were significantly higher (mean difference, 163 cells μl⁻¹; 95% confidence interval, 83 to 242 cells μl⁻¹; P < 0.0001). Similar gender-specific differences in lymphocyte subpopulations have been reported from Singapore and Tanzania (2, 3, 13).

TABLE 1.

Reference values of lymphocyte subpopulations and CD4⁺/CD8⁺ T-cell ratios obtained from healthy adults in Nouna, Burkina Faso^a

Percentile	CD3+ CD4+ T cellsb		CD3+ CD8+ T cells		CD4+/CD8+ T-cell ratio	CD3+ T cells		CD19+ B cells		CD16+ CD56+ NK cellsb
	%	μl−1	%	μl−1		%	μl−1	%	μl−1	%	μl−1
Total group (n = 186)
95th	53	1,718	35	1,167	2.8	80	2,921	22	658	28	1,047
75th	45	1,305	29	814	2.2	75	2,364	16	457	19	485
50th (median)	41	1,082	24	600	1.7	70	1,801	13	336	14	352
25th	37	849	20	496	1.3	66	1,503	11	251	10	247
5th	30	631	16	328	0.9	57	1,069	7	148	7	150
Males (n = 97)
95th	48	1,696	37	1,164	2.5	79	2,963	21	633	30	1,294
75th	43	1,231	29	814	2.0	72	2,146	16	428	21	585
50th (median)	39	979	24	595	1.6	68	1,690	13	351	17	408
25th	35	790	21	498	1.2	63	1,417	10	245	13	287
5th	26	546	17	324	0.9	56	1,036	7	141	8	185
Females (n = 89)
95th	53	1,749	35	1,202	3.1	81	2,921	23	716	21	681
75th	48	1,328	29	818	2.2	76	2,275	17	483	16	437
50th (median)	44	1,169	25	620	1.7	73	1,890	13	329	12	313
25th	40	973	20	496	1.4	69	1,589	11	272	8	212
5th	32	716	15	328	1.1	60	1,176	7	181	6	141

^aRelative (%) and absolute (cells μl⁻¹) counts are given.

^bStatistically significant difference between males and females (Student's t test, P < 0.01).

The absolute CD4⁺ T-cell counts of healthy adults living in Nouna, Burkina Faso, are among the highest ever reported in published normal-range studies from African and Asian populations (Table 2). This observation supports the hypothesis that a specific genetic factor influences peripheral blood CD4⁺ T-cell counts (1, 4). Our results, however, may also be influenced by differences in study design and laboratory performance between the different studies. The higher CD4⁺ T-cell counts may reflect the specific selection process of blood donors in our study, since we tried not to include individuals suffering from infectious or other clinically recognizable diseases. Whether clinically inapparent parasitic infestation influences CD4⁺ T-cell counts in Nouna, Burkina Faso, is not known and remains to be studied.

TABLE 2.

Comparison of reference values of relative and absolute counts of lymphocyte subpopulations for several ethnically distinct populations in Burkina Faso (present study), Singapore (3), and Tanzania (13)^a

Region	Total no. (no. male/female)	CD3+ CD4+ T cells		CD3+ CD8+ T cells		CD4+/CD8+ T-cell ratio	CD3+ T cells		CD19+ B cells		CD16+ CD56+ NK cells
		%	μl−1	%	μl−1		%	μl−1	%	μl−1	%	μl−1
Burkina Faso*	186 (97/89)	41 (30-53)	1,082 (631-1,696)	24 (16-35)	600 (328-1,167)	1.7 (0.9-2.8)	70 (57-80)	1,801 (1,069-2,921)	13 (7-22)	349 (148-658)	14 (7-28)	352 (150-1,047)
Singapore	232 (104/128)	35 (23-48)	814 (401-1,451)	27 (13-41)	616 (243-1,206)	1.3 (0.7-2.8)	68 (49-80)	1,550 (796-2,679)	14 (7-28)	335 (133-714)	16 (6-37)	364 (115-1,009)
Tanzania	214 (107/107)	40 (27-55)	833 (405-1,500)	25 (15-41)	499 (261-1,033)	1.5 (0.6-2.7)	70 (54-83)	1,431 (769-2,798)	13 (6-21)	253 (88-654)	16 (5-35)	346 (118-830)
MultiSET reference range (V.1.1.2)		31-60	410-1,590	13-41	190-1,140		55-84	690-2,540	6-25	90-660	5-27	90-590

^aMedian relative (% of lymphocytes) and absolute (cells μl⁻¹) counts of lymphocyte subpopulations, with lower and upper limits of normal given in parentheses (2.5th and 97.5th percentiles, respectively, or 5th and 95th percentiles if indicated by an asterisk). MultiSET reference ranges were provided by BD Biosciences. All data were generated using the MultiSET flow cytometric methodology in combination with TruCount tubes and a lyse-no-wash procedure.

Reference values of lymphocyte subpopulations generated with a single-platform lyse-no-wash procedure should not be used for the interpretation of individual measurements with dual-platform flow cytometry. As already described by Nicholson et al. (10) and confirmed in our study, CD45-based lymphocyte counting with TruCount tubes yields absolute CD4⁺ T-cell counts that are about 15% higher than those obtained with dual-platform flow cytometry using lymphocyte numbers obtained with the Sysmex hematology analyzer.

Elevated normal ranges of absolute CD4⁺ T-cell counts in healthy adults may have an impact on the immunological staging of HIV disease and AIDS. Samples from several other areas in West Africa should therefore be studied to confirm our data. At present, we recommend that treatment decisions be based on repeated measurements of CD4⁺ T-cell percentages and absolute counts and not on absolute counts alone.