ABSTRACT

Objective:

To estimate the reference intervals (RIs) of complete blood count parameters in the Brazilian adult population.

Methods:

Cross-sectional study, with data from the National Health Survey (Pesquisa Nacional de Saúde – PNS), between 2014–2015. The final sample consisted of 2,803 adults. To establish the RIs, exclusion criteria were applied, outliers were removed and partitions were made by gender, age, and race/skin color. The non-parametric method was adopted. Differences were assessed using the Mann Whitney and Kruskal Wallis tests (p≤0.05).

Results:

There were statistically significant differences for the following hematological parameters based on gender, red blood cells, hemoglobin, hematocrit, MCH, MCHC, eosinophils and absolute monocytes, neutrophils and platelets (p≤0.05). When analyzed by age, the RIs were statistically different in females for hematocrit, MCV, white blood cells and RDW and in males for red blood cells, white blood cells, eosinophils, mean platelet volume, MCV, RDW, and MCH (p≤0.05). For race/color, there were differences in the RIs for parameters of hemoglobin, MCH, MCHC, white blood cells and mean platelet volume, neutrophils and absolute eosinophils (p≤0.05).

Conclusion:

The differences found in the RIs of some in blood count parameters in Brazilian adults reaffirm the importance of having their own laboratory reference standards. The results can support a more accurate interpretation of tests, adequate identification and disease prevention in Brazil.

Keywords:
Health surveys; Reference values; Blood cell count; Leukocytes; Brazil

INTRODUCTION

Blood count reference intervals (RIs) (red and white series) are important information in clinical practice¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... for screening blood donors, assessing overall health, establishing effective diagnosis²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... , and managing and treating diseases¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... –³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... .

Reliable RIs direct the identification of diseases of important magnitude, such as anemia, infections and neoplasms, and contribute to control and prevention⁴4 Walters MC, Abelson HT. Interpretation of the complete blood count. Pediatr Clin North Am 1996; 43(3): 599-622. https://doi.org/10.1016/s0031-3955(05)70424-7
https://doi.org/10.1016/s0031-3955(05)70... . Anemia represents a global health problem; in 2019, it corresponded to 1.8 billion prevalent cases in the world⁵5 Safiri S, Kolahi AA, Noori M, Nejadghaderi SA, Karamzad N, Bragazzi NL, et al. Burden of anemia and its underlying causes in 204 countries and territories, 1990-2019: results from the Global Burden of Disease Study 2019. J Hematol Oncol 2021; 14(1): 185. https://doi.org/10.1186/s13045-021-01202-2
https://doi.org/10.1186/s13045-021-01202... , and in Brazil, according to data from the National Health Survey (Pesquisa Nacional de Saúde – PNS), between 2014 and 2015, there was a prevalence of 9.9% in adults and the aged⁶6 Machado ÍE, Malta DC, Bacal NS, Rosenfeld LGM. Prevalence of anemia in Brazilian adults and elderly. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190008.SUPL.2. https://doi.org/10.1590/1980-549720190008.supl.2
https://doi.org/10.1590/1980-54972019000... . In 2019, cancer was the second cause of death worldwide (10,079,637), and in Brazil it corresponded to 266,034 deaths. Respiratory tract infections represented the fourth cause of death worldwide (2,493,199), being the third cause in Brazil (88,640)⁷7 Institute for Health Metrics and Evaluation. GBD compare [Internet]. 2019 [cited on Apr. 06, 2021]. Available at: https://vizhub.healthdata.org/gbd-compare/
https://vizhub.healthdata.org/gbd-compar... .

However, establishing RIs is a challenge due to the requirement of methodological rigor with the need for a representative sample of the population; care in collection, transport, biochemical and statistical analysis¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... ,⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... . Thus, determining RIs is not a reality in all countries, being restricted to those that conduct population studies⁹9 Sairam S, Domalapalli S, Muthu S, Swaminathan J, Ramesh VA, Sekhar L, et al. Hematological and biochemical parameters in apparently healthy Indian population: defining reference intervals. Indian J Clin Biochem 2014; 29(3): 290-7. https://doi.org/10.1007/s12291-013-0365-5
https://doi.org/10.1007/s12291-013-0365-... .

Furthermore, RIs are influenced by factors such as race, ethnicity, body mass index (BMI)¹⁰10 Omuse G, Maina D, Mwangi J, Wambua C, Radia K, Kanyua A, et al. Complete blood count reference intervals from a healthy adult urban population in Kenya. PLoS One 2018; 13(6): e0198444. https://doi.org/10.1371/journal.pone.0198444
https://doi.org/10.1371/journal.pone.019... , circadian rhythm, diet, pregnancy, menstrual cycle¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833,¹²12 Ferreira CES, Andriolo A. Intervalos de referência no laboratório clínico. J Bras Patol Med Lab 2008; 44(1): 11-6. https://doi.org/10.1590/S1676-24442008000100004
https://doi.org/10.1590/S1676-2444200800... , menopause¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833, physical activity, stress, smoking and use of medications, alcoholic beverages or caffeine¹²12 Ferreira CES, Andriolo A. Intervalos de referência no laboratório clínico. J Bras Patol Med Lab 2008; 44(1): 11-6. https://doi.org/10.1590/S1676-24442008000100004
https://doi.org/10.1590/S1676-2444200800... . Therefore, it is recommended to determine the RIs for the population in which they will be applied¹⁰10 Omuse G, Maina D, Mwangi J, Wambua C, Radia K, Kanyua A, et al. Complete blood count reference intervals from a healthy adult urban population in Kenya. PLoS One 2018; 13(6): e0198444. https://doi.org/10.1371/journal.pone.0198444
https://doi.org/10.1371/journal.pone.019... ,¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... , as they reflect the real health conditions¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833.

Even though the importance of having an RIs belonging to the population is recognized, international RIs are adopted in Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... ,¹²12 Ferreira CES, Andriolo A. Intervalos de referência no laboratório clínico. J Bras Patol Med Lab 2008; 44(1): 11-6. https://doi.org/10.1590/S1676-24442008000100004
https://doi.org/10.1590/S1676-2444200800... . So far, there is only one study, in which reference values for blood counts in Brazilian adults were calculated using PNS data, using the parametric method¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... .

The population has influences on the RIs values, therefore, advancing in analytical calculation methods can minimize this effect²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . The application of a single approach to RIs calculation can lead to inaccuracy, and it is recommended to test other methodologies¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... . Thus, it is important to carry out studies using the same PNS database, in which different methods of RIs determination are adopted.

This study analyzed, for the first time, the blood count RIs of Brazilian adults with laboratory data from the PNS using the non-parametric method, and according to the recommendations of Guideline C28-A3¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... , a reference widely adopted by laboratories¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... . In addition, it advanced by expanding the exclusion criteria, including hemogram parameters according to race/color and in the analyses used for partitioning in relation to the study previously carried out in Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... .

Thus, the present study aimed to estimate the blood count RIs in the Brazilian adult population.

METHODS

Study design

Cross-sectional study, with PNS data, between 2014 and 2015.

Context and data source

The PNS is a household-based survey carried out by the Brazilian Institute of Geography and Statistics (Instituto Brasileiro de Geografia e Estatística – IBGE)¹⁵15 Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde 2013: percepção do estado de saúde, estilos de vida e doenças crônicas: Brasil, Grandes Regiões e Unidades da Federação. Rio de Janeiro: IBGE; 2014.,¹⁶16 Szwarcwald CL, Malta DC, Souza Júnior PRB, Almeida WS, Damacena GN, Pereira CA, et al. Laboratory exams of the National Health Survey: methodology of sampling, data collection and analysis. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190004.SUPL.2. https://doi.org/10.1590/1980-549720190004.supl.2
https://doi.org/10.1590/1980-54972019000... . In the 2013 PNS, 60,202 adults were interviewed. The collection of tests was planned in a subsample of 25% of the census sectors of the research, and carried out between 2014 and 2015, in 8,952 adults¹⁶16 Szwarcwald CL, Malta DC, Souza Júnior PRB, Almeida WS, Damacena GN, Pereira CA, et al. Laboratory exams of the National Health Survey: methodology of sampling, data collection and analysis. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190004.SUPL.2. https://doi.org/10.1590/1980-549720190004.supl.2
https://doi.org/10.1590/1980-54972019000... . Blood counts were collected at any time of the day in tubes filled with ethylenediaminetetraacetic acid (EDTA). The blood samples were sent to the reference laboratories that ensured the quality control of the Ministry of Health. The samples were examined by an automatic cell analyzer¹⁶16 Szwarcwald CL, Malta DC, Souza Júnior PRB, Almeida WS, Damacena GN, Pereira CA, et al. Laboratory exams of the National Health Survey: methodology of sampling, data collection and analysis. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190004.SUPL.2. https://doi.org/10.1590/1980-549720190004.supl.2
https://doi.org/10.1590/1980-54972019000... .

Due to the complex sampling design of the PNS and the unequal selection probabilities, weights were calculated by post-stratification procedures. Sampling weights were adopted in all analyses¹⁶16 Szwarcwald CL, Malta DC, Souza Júnior PRB, Almeida WS, Damacena GN, Pereira CA, et al. Laboratory exams of the National Health Survey: methodology of sampling, data collection and analysis. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190004.SUPL.2. https://doi.org/10.1590/1980-549720190004.supl.2
https://doi.org/10.1590/1980-54972019000... . Further details on the PNS sampling plan, and the procedures for collecting, sending and storing samples are available in other publications¹⁵15 Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde 2013: percepção do estado de saúde, estilos de vida e doenças crônicas: Brasil, Grandes Regiões e Unidades da Federação. Rio de Janeiro: IBGE; 2014.,¹⁶16 Szwarcwald CL, Malta DC, Souza Júnior PRB, Almeida WS, Damacena GN, Pereira CA, et al. Laboratory exams of the National Health Survey: methodology of sampling, data collection and analysis. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190004.SUPL.2. https://doi.org/10.1590/1980-549720190004.supl.2
https://doi.org/10.1590/1980-54972019000... . The database used is available at: https://www.pns.icict.fiocruz.br/.

Determination of blood count reference intervals

In order to reduce the factors that can influence RIs¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833, and aiming to reach a healthy population, exclusion criteria based on the literature¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... ,⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... ,¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... were applied and the criteria adopted in the national study were expanded¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... . The exclusion criteria used, their references and cutoff points¹⁷17 World Health Organization. Obesity: preventing and managing the global epidemic: report of a WHO consultation [Internet]. Geneva: World Health Organization; 2000 [cited on May 27, 2021]. Available at: https://apps.who.int/iris/handle/10665/42330
https://apps.who.int/iris/handle/10665/4... –²¹21 World Health Organization. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. Vitamin and mineral nutrition information system [Internet]. Genebra: World Health Organization; 2011. [cited on May 27, 2021]. Available at: https://apps.who.int/iris/bitstream/handle/10665/85839/WHO_NMH_NHD_MNM_11.1_eng.pdf?sequence=22&isAllowed=y
https://apps.who.int/iris/bitstream/hand... are in Supplementary Material 1.

For outlier exclusion, visual inspection and the Tukey method were used. Outliers were identified in interquartile ranges (IQR), considering the first quartile (Q1) lower and the third quartile (Q3) higher, according to the formula: (Q1-(1.5xIQR); Q3+(1.5xIQR). At levels of <Q1-1.5 IQR and/or >Q3+1.5 IQR, outliers were discarded¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... .

The sample was partitioned according to gender, age and race/skin color, using statistical tests¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833,¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... and considering the biological conditions that influence RIs¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833.

RIs were estimated considering 95% of healthy individuals⁹9 Sairam S, Domalapalli S, Muthu S, Swaminathan J, Ramesh VA, Sekhar L, et al. Hematological and biochemical parameters in apparently healthy Indian population: defining reference intervals. Indian J Clin Biochem 2014; 29(3): 290-7. https://doi.org/10.1007/s12291-013-0365-5
https://doi.org/10.1007/s12291-013-0365-... , linked to the 2.5 and 97.5¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... percentiles. Samples of over 120 individuals were used in partitioning by gender and age¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... .

Participants

Participants were adults aged 18 years old and older. The PNS database, composed of 8,952 individuals, was used. After exclusion procedures and removal of outliers, the final sample consisted of 2,803 participants.

Variables

The variables included were: sociodemographic and hemogram parameters (red and white series). The complete description of the variables can be found in Supplementary Material 2.

Statistical analyses

Medians were calculated for reference limits. The lower limit (LL) was linked to the 2.5^th percentile and the upper limit (UL) to the 97.5^th percentile of the reference population distribution, according to gender, age, and race/color. RIs were estimated using the non-parametric method, which organizes the observations made by size and classifies them considering the lowest r=1 to the highest r=n. LL corresponded to r=0.025 (n+1) and the UL, the ranking position r=0.975 (n+1)¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... .

Data normality was evaluated using the Shapiro Wilk test and differences were assessed using the Mann Withney or Kruskal Wallis tests, with Dun's post-test with Bonferroni correction, with a significance level of 5%.

Analyses were performed using the Data Analysis and Statistical Software (Stata), version 14, and the Software Package for Social Science (SPSS), version 25.0, using the survey module, which considers post-stratification weights.

Ethical aspects

The PNS was approved by the National Research Ethics Committee of the National Health Council (Opinion 328.159). Adult participation was voluntary, and confidentiality of information was guaranteed¹⁵15 Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde 2013: percepção do estado de saúde, estilos de vida e doenças crônicas: Brasil, Grandes Regiões e Unidades da Federação. Rio de Janeiro: IBGE; 2014..

RESULTS

RIs for red blood cells (millions/mm³), hemoglobin (g/dL), and hematocrit (%) were higher in men (4.3–5.7; median 5.1; 13.2–16.7; median 15.0; 40.4–52.4; median 45.8) than in women (4.0–5.2; median 4.1; 12.0–15.1; median 13.2; 36.8–47.7; median 41.0). The LL of the MCH (pg) were higher in men (26.7–32.3; median 29.8) than in women (26.5–32.3; median 29.6). In males (30.3–34.4; median 32.8), the UL of the MCHC (g/dL) were also higher than in females (30.4–34.1; median 32.6) (p ≤0.05) (Table 1).

RIs of eosinophils (mm³) and monocytes (mm³) were higher in men (17.5–676.4; median 167.5; 52.0–782.0; median 393.6), compared to women (11.7–671.6; median 150.2; 39.6–752.4; median 353.6). RIs of platelets (μl) and neutrophils (mm³) were higher in women (145,000–337,000; median 234,000; 887.0–6,429.6; median 3,374.1) than in men (143,000–315,000; median 210,000; 798.0–6,114.3; median 3,143.7) (p≤0.05) (Table 2).

In men, the RIs of red blood cells (millions/mm³) were higher between 18 and 39 years of age (4.4–5.8; median 5.1), compared with the age groups from 40 to 59 years (4.3–5.7; median 5.0) and 60 years old or older (4.2–5.8; median 4.8). RIs of MCV (fL) and MCH (pg) were lower in men between 18 and 39 years old (81.8–100.4; median 89.4; 26.4–32.0; median 29.5) and 40 to 59 years old (82.6–101.2; median 91.4; 26.0–32.2; median 30.0) than those aged 60 years old or older (83.9–102.0; median 92.7; 27.2–38.2; median 30.7) (p≤0.05). The median and UL of RDW (%) were higher in men aged 60 years old and older (12.2–15.5; median 13.5) than in men aged 18 to 39 years (12.4–15.3; median 13.3) and 40 to 59 years old (12.3–15.3; median 13.3) (p≤0.05) (Supplementary Material 3).

In women, there were differences for LL and UL, and the median was lower for hematocrit (%) between 18 and 39 years old (36.9–47.3; median 40.7), when compared to 40 to 59 years old (36.6–48.4; median 41.3) and 60 years old or older (36.5–47.1; median 41.2). MCV's (fL) RIs were lower in women aged 18 to 39 years (81.6–100.9; median 90.5) than those aged 40 to 59 years (82.2–100.7; median 91.2). The LL and median of the RDW (%) were lower in women aged 18 to 39 years (12.1–15.2; median 13.4) than in those aged 40 to 59 years (12.3–15.4; median 13.5) and 60 years old or older (12.3–15.2; median 13.7) (p≤0.05) (Supplementary Material 4).

Men had higher white blood cell counts (mm³) between ages 18 and 39 (2,970–9,990; median 6,000), compared with those aged 60 years old and older (2,600–9,400; median 5,640). The LL and median of the eosinophils (mm³) were higher in men aged 18 to 39 years (26.1–661.2; median 190.1), compared with men aged 40 to 59 years (16.8–679.8; median 141.6). RIs for platelets (μl) were lower in men with increasing age (18–39 years: 144,000–314,000; median 215,000; 40–59 years: 143,000–322,000; median 215,000; 60 years old and older: 138,000–306,000; median 203,000; p≤0.05) (Supplementary Material 5).

In women, there were differences for RIs of white blood cells (mm³); the median and LL were more prominent between 18 and 39 years of age (2,600-10,000; median 6,300) and 40 to 59 years (2,800–9,800; median 5,800) than at 60 years old and older (2,000–9,800; median 5,500) (p≤0.05) (Supplementary Material 6).

Hemoglobin (g/dL) and MCH (pg) RIs were higher in white men (13.3–16.8; median 15.1; 27.0–32.4; median 29.9) than in brown ones (13.1–17.3; median 14.8; 26.5–32.2; median 29.7). MCH medians were lower in black men (29.3 pg) than in white men (29.9 pg). The UL and median of the MCHC (g/dL) were slightly higher in white men (30.4–34.6; median 32.8) than in brown (30.3–34.4; median 32.6) and black men (30.5–34.3; median 32.8) (p≤0.05). RIs of eosinophils (mm³) were lower in white (17.1–648.0; median 151.2) than in black (38.5–678.5; median 230.1) and brown men (17.5 –688.1; median 194.0) (p≤0.05) (Supplementary Material 7).

RIs of hemoglobin (g/dL) and MCH (pg) were higher in white women (12.1–15.1; median 13.4; 27.0–32.3; median 29.8) than in brown (12.0–15.0; median 13.2; 26.4–32.2; median 29.4) and black ones (12.0–14.8; median 13.1; 26.3–31.8; median 28.9). The MCV (fL) median and LL was more prominent in white women (82.7–100.4; median 91.0) than in black women (82.4–100.9; median 89.5). The UL of the MCHC (g/dL) was higher in white (30.4–34.2; median 32.7) than in brown (30.3–34.0; median 32.5) and black women (30.5–33.7; median 32.4). White women (2,900–10,000; median 6,300) had higher median and LL values for white blood cells (mm³) than brown (2,500–9,700; median 5,900) and black women (2,650–10,100; median 5,600). In brown (median 3,129) and black (median 2,866) women, median neutrophil (mm³) were higher than in white women (median 2,597). In brown women (9.3–694.4; median 150.0), the median and UL of eosinophils (mm³) were higher than in white women (14.2–660.0; median 140.6). The median and LI of mean platelet volume (fL) was higher in white women (8.4–12.7; median 10.4) than in brown (8.2–12.3; median 10.2) and black women (8.3–12.9; median 10.2) (p≤0.05) (Supplementary Material 7).

DISCUSSION

In this study, blood count RIs of Brazilian adults was estimated by PNS tests, using the non-parametric method. Statistically significant differences were observed for some components of the red and white series blood count when analyzed according to gender, age, and race/color. RIs were calculated using a methodology that had not yet been tested, following recommendations in the literature, in order to obtain increasingly accurate and reliable values¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... . Therefore, it differed from the only existing national study in which reference values were calculated for Brazilian adults, by using a non-parametric approach and also by the tests applied for sample stratification, application of the Tukey method to remove outliers and the expansion of the exclusion criteria of the study by Rosenfeld et al.¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... .

The non-parametric methodology adopted here is in line with studies carried out in Ghana³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... , Canada⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... , India⁹9 Sairam S, Domalapalli S, Muthu S, Swaminathan J, Ramesh VA, Sekhar L, et al. Hematological and biochemical parameters in apparently healthy Indian population: defining reference intervals. Indian J Clin Biochem 2014; 29(3): 290-7. https://doi.org/10.1007/s12291-013-0365-5
https://doi.org/10.1007/s12291-013-0365-... , Kenya¹⁰10 Omuse G, Maina D, Mwangi J, Wambua C, Radia K, Kanyua A, et al. Complete blood count reference intervals from a healthy adult urban population in Kenya. PLoS One 2018; 13(6): e0198444. https://doi.org/10.1371/journal.pone.0198444
https://doi.org/10.1371/journal.pone.019... , and Korea²²22 Jeon K, Kim M, Han J, Lee J, Lee JS, Kim HS, et al. Establishment of sex-specific reference intervals for automated haematology analyser-delivered research parameters in healthy Korean adults: a retrospective database review. BMJ Open 2020; 10(10): e036887. https://doi.org/10.1136/bmjopen-2020-036887
https://doi.org/10.1136/bmjopen-2020-036... . This method is recommended due to many analytes not having a normal distribution, and because it is simpler, depending only on the classifications of the reference data arranged in increasing order of size¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... . The literature describes that the results found in parametric and non-parametric methods are usually similar¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... . As can be seen from the median values found in this study according to gender and the mean values identified in the national study for red and white blood cells were 5.0 million/mm³ and 6,142 mm³ in men and 4.5 million/mm³ and 6,426 mm³ in women, respectively¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... .

For selection of healthy individuals, exclusions were defined according to Guideline C28-A3¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... and based on studies¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... ,⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... –¹⁰10 Omuse G, Maina D, Mwangi J, Wambua C, Radia K, Kanyua A, et al. Complete blood count reference intervals from a healthy adult urban population in Kenya. PLoS One 2018; 13(6): e0198444. https://doi.org/10.1371/journal.pone.0198444
https://doi.org/10.1371/journal.pone.019... ,²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... . The procedures adopted here for removing outliers were used in studies in Canada⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... and Oman²⁴24 Al-Mawali A, Pinto AD, Al-Busaidi R, Al-Lawati RH, Morsi M. Comprehensive haematological indices reference intervals for a healthy Omani population: first comprehensive study in Gulf Cooperation Council (GCC) and Middle Eastern countries based on age, gender and ABO blood group comparison. PLoS One 2018; 13(4): e0194497. https://doi.org/10.1371/journal.pone.0194497
https://doi.org/10.1371/journal.pone.019... . Tukey's method was used because it is more useful and indicated in the presence of more than one outlier¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... , which occurred in this study, and visual inspection because it is considered effective¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... .

Considering partitioning as a power tool for diagnosing RIs⁷7 Institute for Health Metrics and Evaluation. GBD compare [Internet]. 2019 [cited on Apr. 06, 2021]. Available at: https://vizhub.healthdata.org/gbd-compare/
https://vizhub.healthdata.org/gbd-compar... , this investigation used statistical tests to verify its need, as well as in other studies²²22 Jeon K, Kim M, Han J, Lee J, Lee JS, Kim HS, et al. Establishment of sex-specific reference intervals for automated haematology analyser-delivered research parameters in healthy Korean adults: a retrospective database review. BMJ Open 2020; 10(10): e036887. https://doi.org/10.1136/bmjopen-2020-036887
https://doi.org/10.1136/bmjopen-2020-036... –²⁵25 Bakrim S, Motiaa Y, Benajiba M, Ouarour A, Masrar A. Establishment of the hematology reference intervals in a healthy population of adults in the Northwest of Morocco (Tangier-Tetouan region). Pan Afr Med J 2018; 29: 169. https://doi.org/10.11604/pamj.2018.29.169.13042
https://doi.org/10.11604/pamj.2018.29.16... . The partitioning adopted is set out in Guideline C28-A3¹⁵15 Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde 2013: percepção do estado de saúde, estilos de vida e doenças crônicas: Brasil, Grandes Regiões e Unidades da Federação. Rio de Janeiro: IBGE; 2014., and the physiological changes of adulthood were also considered¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833. When calculating RIs, the physiological aspects are as important as the statistical ones¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833,¹⁴14 Clinical and Laboratory Standards Institute. EP28-A3C defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition [Internet]. Wayne: CLSI; 2008 [cited on Apr. 06, 2021]. Available at: https://clsi.org/media/1421/ep28a3c_sample.pdf
https://clsi.org/media/1421/ep28a3c_samp... , as RIs differ in children, adults, the aged, men and women; in puberty, pregnancy and menopause, due to organic changes throughout life¹¹11 Sikaris KA. Physiology and its importance for reference intervals. Clin Biochem Rev 2014; 35(1): 3-14. PMID: 24659833.

The highest RIs for red blood cells, hemoglobin, and hematocrit identified in men, compared to women, and higher RIs for platelets in women than in men, were also found in Ghana³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... , Oman²⁴24 Al-Mawali A, Pinto AD, Al-Busaidi R, Al-Lawati RH, Morsi M. Comprehensive haematological indices reference intervals for a healthy Omani population: first comprehensive study in Gulf Cooperation Council (GCC) and Middle Eastern countries based on age, gender and ABO blood group comparison. PLoS One 2018; 13(4): e0194497. https://doi.org/10.1371/journal.pone.0194497
https://doi.org/10.1371/journal.pone.019... , and Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... . In this study, as well as in an investigation carried out in Morocco²⁵25 Bakrim S, Motiaa Y, Benajiba M, Ouarour A, Masrar A. Establishment of the hematology reference intervals in a healthy population of adults in the Northwest of Morocco (Tangier-Tetouan region). Pan Afr Med J 2018; 29: 169. https://doi.org/10.11604/pamj.2018.29.169.13042
https://doi.org/10.11604/pamj.2018.29.16... , higher RIs of MCH and MCHC were observed in men compared to women. Differences between genders for erythrocytes, hemoglobin, hematocrit, MCH, MCHC, platelets, and platelet volume¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... have been documented, which can be explained by the effect of menstruation and the increased demand for iron³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... ,²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... , hormonal influences of androgen¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... ,³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... ,⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... and the way in which erythropoiesis and megakaryopoiesis are regulated in men and women³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... .

Our findings agree with studies from Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... , Canada⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... , Korea²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... , and Morocco²⁵25 Bakrim S, Motiaa Y, Benajiba M, Ouarour A, Masrar A. Establishment of the hematology reference intervals in a healthy population of adults in the Northwest of Morocco (Tangier-Tetouan region). Pan Afr Med J 2018; 29: 169. https://doi.org/10.11604/pamj.2018.29.169.13042
https://doi.org/10.11604/pamj.2018.29.16... , in which neutrophil levels were lower in men than in women, possibly due to impacts related to sexual development on immunity, in which estrogen stimulates an immune response, and some androgen hormones, such as testosterone, suppress the response to infection, with neutrophil levels being highest in females during puberty and adulthood⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... ,²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... .

Studies carried out in Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... , Canada⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... , and China²²22 Jeon K, Kim M, Han J, Lee J, Lee JS, Kim HS, et al. Establishment of sex-specific reference intervals for automated haematology analyser-delivered research parameters in healthy Korean adults: a retrospective database review. BMJ Open 2020; 10(10): e036887. https://doi.org/10.1136/bmjopen-2020-036887
https://doi.org/10.1136/bmjopen-2020-036... found differences in monocyte RIs according to gender⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... ,²²22 Jeon K, Kim M, Han J, Lee J, Lee JS, Kim HS, et al. Establishment of sex-specific reference intervals for automated haematology analyser-delivered research parameters in healthy Korean adults: a retrospective database review. BMJ Open 2020; 10(10): e036887. https://doi.org/10.1136/bmjopen-2020-036887
https://doi.org/10.1136/bmjopen-2020-036... , being higher in men, in line with our results. It is known that men can have higher monocyte counts than women, however the physiological and clinical aspects still need to be elucidated⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... . Higher eosinophil RIs in men than in women were also found in studies in Korea²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... , Morocco²⁵25 Bakrim S, Motiaa Y, Benajiba M, Ouarour A, Masrar A. Establishment of the hematology reference intervals in a healthy population of adults in the Northwest of Morocco (Tangier-Tetouan region). Pan Afr Med J 2018; 29: 169. https://doi.org/10.11604/pamj.2018.29.169.13042
https://doi.org/10.11604/pamj.2018.29.16... , China²²22 Jeon K, Kim M, Han J, Lee J, Lee JS, Kim HS, et al. Establishment of sex-specific reference intervals for automated haematology analyser-delivered research parameters in healthy Korean adults: a retrospective database review. BMJ Open 2020; 10(10): e036887. https://doi.org/10.1136/bmjopen-2020-036887
https://doi.org/10.1136/bmjopen-2020-036... , Ghana³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... , and Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... . However, research is needed to investigate the reasons for the biological fluctuations of these leukocyte parameters in specific populations from different locations according to gender⁹9 Sairam S, Domalapalli S, Muthu S, Swaminathan J, Ramesh VA, Sekhar L, et al. Hematological and biochemical parameters in apparently healthy Indian population: defining reference intervals. Indian J Clin Biochem 2014; 29(3): 290-7. https://doi.org/10.1007/s12291-013-0365-5
https://doi.org/10.1007/s12291-013-0365-... .

The subtle differences found in hematocrit RIs in women, which are higher after 40 years of age, compared to 18 to 39 years of age, are justified by the variability of this parameter, which is similar throughout life⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... . Slight increase in hematocrit with age was found in Chinese adults. This finding is consistent with lower levels before menopause and higher levels after this period²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . Red blood cell values tended to decrease with age in males, as in Chinese adults²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . The decrease may be due to the gradual loss of androgen²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . The lowest RIs from 60 years of age onward in men may be related to nutritional deficiency, occult malignancy, and anemia²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... .

For the RIs of white blood cells, the results found are similar to those of other studies²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... ,²⁴24 Al-Mawali A, Pinto AD, Al-Busaidi R, Al-Lawati RH, Morsi M. Comprehensive haematological indices reference intervals for a healthy Omani population: first comprehensive study in Gulf Cooperation Council (GCC) and Middle Eastern countries based on age, gender and ABO blood group comparison. PLoS One 2018; 13(4): e0194497. https://doi.org/10.1371/journal.pone.0194497
https://doi.org/10.1371/journal.pone.019... , in which a decline was observed with increasing age⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... . The findings reaffirm that two divisions by age could be made for this parameter, as in the Brazilian study¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... , as there were no differences between the two age groups of 18 to 39 and 40 to 59 years. Higher white blood cell counts in young adults of both genders, when compared to lower counts in the aged, reflect the development of the adaptive and acquired immune response as the immune system is more exposed to pathogens and antigens in the environment⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... ,²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... .

In this study, the slight increase in eosinophil UL with age in men may be related to chronic infections in aged people who were not excluded²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . This finding was present in Chinese adults of both genders²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . Differences in eosinophil values can be attributed to allergic and parasitic diseases in apparently healthy adults²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... .

Studies in Canada⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... and Brazil¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... also identified a slight increase in MCV and RDW throughout life, with small changes in early adulthood, remaining constant after this phase⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... . A study in China identified that aged men and women had higher RIs for RDW ²³23 Nah EH, Kim S, Cho S, Cho HI. Complete blood count reference intervals and patterns of changes across pediatric, adult, and geriatric ages in Korea. Ann Lab Med 2018; 38(6): 503-11. https://doi.org/10.3343/alm.2018.38.6.503
https://doi.org/10.3343/alm.2018.38.6.50... . MCV and RDW provide a classification of erythrocytes based on size and distribution⁴4 Walters MC, Abelson HT. Interpretation of the complete blood count. Pediatr Clin North Am 1996; 43(3): 599-622. https://doi.org/10.1016/s0031-3955(05)70424-7
https://doi.org/10.1016/s0031-3955(05)70... . For MCV values, the LL is approximately 70 fL plus age (in years), and the UL can be obtained by adding 0.6 fL per year to 84 fL beyond the first year of life, until approximately 96 fL is reached in adults⁴4 Walters MC, Abelson HT. Interpretation of the complete blood count. Pediatr Clin North Am 1996; 43(3): 599-622. https://doi.org/10.1016/s0031-3955(05)70424-7
https://doi.org/10.1016/s0031-3955(05)70... , consistent with the findings of this study. RDW is useful in identifying iron deficiency, β-thalassemia trait, inflammatory processes, and chronic infection⁴4 Walters MC, Abelson HT. Interpretation of the complete blood count. Pediatr Clin North Am 1996; 43(3): 599-622. https://doi.org/10.1016/s0031-3955(05)70424-7
https://doi.org/10.1016/s0031-3955(05)70... . It is also a predictor of mortality, and its increase with age is related to organic changes in aging and chronic diseases in this phase¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... .

In this study, significant differences in MCH values with age in men, being higher in the aged, was identified in a previous study¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... . It is noteworthy that the RIs for all age groups in Brazilian men were lower than the international hematrimetric classification (27 to 33.7 pg)²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . The literature shows regional variations for HCM; in Chinese, RIs did not differ according to age and gender²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... , while in Moroccan men there were differences²⁵25 Bakrim S, Motiaa Y, Benajiba M, Ouarour A, Masrar A. Establishment of the hematology reference intervals in a healthy population of adults in the Northwest of Morocco (Tangier-Tetouan region). Pan Afr Med J 2018; 29: 169. https://doi.org/10.11604/pamj.2018.29.169.13042
https://doi.org/10.11604/pamj.2018.29.16... . Although our study found differences in men, the international classification covers the same RIs values for MCH according to gender and age for adults²2 Wu X, Zhao M, Pan B, Zhang J, Peng M, Wang L, et al. Complete blood count reference intervals for healthy Han Chinese adults. PLoS One 2015; 10(3): e0119669. https://doi.org/10.1371/journal.pone.0119669
https://doi.org/10.1371/journal.pone.011... . In this sense, further investigations are desirable to clarify the implications of these differences in clinical practice.

The lowest platelet count identified in aged Brazilian men is in line with a study in Canada⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... and was found in men over 40 years of age in Iran⁹9 Sairam S, Domalapalli S, Muthu S, Swaminathan J, Ramesh VA, Sekhar L, et al. Hematological and biochemical parameters in apparently healthy Indian population: defining reference intervals. Indian J Clin Biochem 2014; 29(3): 290-7. https://doi.org/10.1007/s12291-013-0365-5
https://doi.org/10.1007/s12291-013-0365-... . A possible justification is the gradual decline of thrombopoietin, a hormone that regulates the production of platelets in adults. The occurrence of thrombocytopenia is more common in men than in women, and more frequent in the aged⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... .

In this study, RIs was established for 16 hemogram parameters according to race/color. In a previous research, RIs of five parameters were determined, and the values found were close to those described¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... . Although discreet, the differences found according to race/color for RIs of hemoglobin, MCH, MCHC, white blood cells and mean platelet volume, neutrophils and eosinophils, support the need to establish RIs for Brazilians. Differences in the blood count RIs were found in populations from other countries¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... –³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... ,⁸8 Adeli K, Raizman JE, Chen Y, Higgins V, Nieuwesteeg M, Abdelhaleem M, et al. Complex biological profile of hematologic markers across pediatric, adult, and geriatric ages: establishment of robust pediatric and adult reference intervals on the basis of the Canadian Health Measures Survey. Clin Chem 2015; 61(8): 1075-86. https://doi.org/10.1373/clinchem.2015.240531
https://doi.org/10.1373/clinchem.2015.24... ,¹⁰10 Omuse G, Maina D, Mwangi J, Wambua C, Radia K, Kanyua A, et al. Complete blood count reference intervals from a healthy adult urban population in Kenya. PLoS One 2018; 13(6): e0198444. https://doi.org/10.1371/journal.pone.0198444
https://doi.org/10.1371/journal.pone.019... ,²⁴24 Al-Mawali A, Pinto AD, Al-Busaidi R, Al-Lawati RH, Morsi M. Comprehensive haematological indices reference intervals for a healthy Omani population: first comprehensive study in Gulf Cooperation Council (GCC) and Middle Eastern countries based on age, gender and ABO blood group comparison. PLoS One 2018; 13(4): e0194497. https://doi.org/10.1371/journal.pone.0194497
https://doi.org/10.1371/journal.pone.019... , reinforcing the importance of considering geographic and ethnic-racial influences, as they may be related to factors such as genetics, nutrition, socioeconomic, cultural, and lifestyle factors, exposure to allergens, infections and parasitic loads in different locations³3 Abbam G, Tandoh S, Tetteh M, Afrifah DA, Annani-Akollor ME, Owiredu EW, et al. Reference intervals for selected haematological and biochemical parameters among apparently healthy adults in different eco-geographical zones in Ghana. PLoS One 2021; 16(1): e0245585. https://doi.org/10.1371/journal.pone.0245585
https://doi.org/10.1371/journal.pone.024... .

Limitations, such as the possibility of including patients without a previous diagnosis and the failure to obtain samples with 120 individuals for some strata of race/color, must be considered. However, due to the representativeness of the sample, it is emphasized that this study is close to the reality of the health conditions of the Brazilian population. Furthermore, the literature documents that it is possible to establish a RIs of 95% using up to 39 samples¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... . As for the losses attributed to the procedures of exclusion and removal of outliers, this is a conservative bias in clinical practice, as it was possible to estimate the RIs of blood counts for Brazilian adults and predict that the values found were close to the previous national study¹1 Rosenfeld LG, Malta DC, Szwarcwald CL, Bacal NS, Cuder MAM, Pereira CA, et al. Reference values for blood count laboratory tests in the Brazilian adult population, National Health Survey. Rev Bras Epidemiol 2019; 22Suppl 02(Suppl 02): E190003.SUPL.2. https://doi.org/10.1590/1980-549720190003.supl.2
https://doi.org/10.1590/1980-54972019000... , taking into account the recommendations to test other methodological approaches¹³13 Ozarda Y. Reference intervals: current status, recent developments and future considerations. Biochem Med (Zagreb) 2016; 26(1): 5-16. https://doi.org/10.11613/BM.2016.001
https://doi.org/10.11613/BM.2016.001... . The RIs found here confer reliability and allow the generalization of the findings in a relatively safe way. Differences found in some hemogram parameters in Brazilian adults according to gender, age, and race/color show that there is a need to establish RIs that are adequate for the population. The results show the ethnic-racial influences on the RIs and can support the identification and prevention of diseases, as well as future research to validate the RIs of the Brazilian population, contributing to better interpretation, diagnostic accuracy, and quality of care and treatment offered.

ACKNOWLEGMENTS:

To the National Council for Scientific and Technological Development (CNPq) for granting Junior Postdoctoral scholarships received by Sá ACMGN and productivity received by Malta DC. To the Secretariat of Health Surveillance, for the support in TED 142/2018.

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