INTRODUCTION
Flow cytometry has enabled study of lymphocyte populations and their heterogeneity. In lymphocyte phenotyping, two types of classical T-lymphocyte populations (TCD4+ and TCD8+) were initially reported. These were considered as two unique, mutually exclusive T-cell subpopulations, since early cytometry allowed for only two or three simultaneous markings on a single cell.[1] The TCD3+ lymphocytes that do not express markers CD4 and CD8 are known as “double-negative” T cells (DNT cells). They were recently discovered and are characterized by a mature immunophenotype (CD45⁺/CD3⁺/CD4^–/CD8^–/CD56^–) and express clonotypic T-cell receptors (CTR) of type αβ or type γδ. These cells are found in greater proportion in the blood and lymph nodes, although they reportedly account for 3%–7% of total lymphocytes in peripheral blood.[2–5]

The physiological functions of DNT lymphocytes are not yet fully understood. The immunomodulation attributed to them is based on their ability to suppress the functions of simple positive T cells and their cytotoxicity for tumor cells and cells infected by viruses.[2,3] In some immune-system diseases, variations were found in total numbers of DNT lymphocytes or their functions were altered. Although only small quantities of these lymphocytes are found in blood, they play an important role in various diseases’ immunopathogenesis. Their blood levels provide criteria for diagnosing autoimmune lymphoproliferative syndrome and can provide a prognostic biomarker for cancer or effective therapeutic targets for these diseases.[2–7]

In order to make clinical decisions about autoimmune diseases, viral infections, solid tumors and hematopoietic diseases, transplant rejection, alterations of lymphocytic homeostasis and immunodeficiencies,[3–8] absolute and relative levels of lymphocytes are quantified through flow cytometry, so normal DNT-lymphocyte counts must be determined. This subpopulation can be identified without additional reagent costs because the values can be quantified through conventional analysis of T, B and natural killer (NK) cells.

The purpose of this study was to determine the normal range of values of DNT lymphocytes in a population of healthy Cuban adults and the relation of these values with age and sex—such information could inform diagnosis and prognosis of multiple diseases.

METHODS
Design, subjects and sample selection A cross-sectional study was conducted in 2017–2018 at the Hermanos Ameijeiras Clinical-Surgical Teaching Hospital and The National Oncology & Radiobiology Institute, Havana, Cuba. Subjects included 182 adult residents of Havana, aged 18–80 years (median age 40), 93 women and 89 men, all reportedly healthy, who were accompanying patients at hospital immunology services and who agreed to participate via written informed consent. Persons were excluded who smoked, consumed daily >40 g of alcohol or its equivalent weekly; drank more than 4 cups of coffee daily; were pregnant; had recent or recurring infections in the previous 6 months or autoimmune or neoplastic diseases; or had taken antibiotics, immunostimulators, immunosuppressants or anti-inflammatory drugs in the past 6 months.

Flow cytometry Four mL of peripheral blood was extracted by antecubital venipuncture and deposited in cytometry tubes with the anticoagulant ethylenediaminetetraacetic acid (EDTA). Sample preparation adhered to the manufacturer’s specifications for cell-surface immunophenotyping, using a protocol for no-wash red blood cell lysis with a Versalyse buffer (Beckman-Coulter, France).

An eight-color Beckman-Coulter Gallios flow cytometer (Beckman-Coulter, France) was used. The following fluorochrome-conjugated monoclonal antibodies were used: anti-CD45-AA750/CD3-FITC/CD4-PC5.5/CD8-AA700/CD56-PE (all Beckman-Coulter, France). The DNT lymphocyte population was defined by the CD45⁺/CD3⁺/CD4^–/CD8^–/CD56^– immunophenotype. The data obtained were processed with Kaluza Analysis V1.5a software, with a minimum of 50,000 acquisition events. Absolute values were determined through a double platform and application of the following equation:
absolute value (cells/μL) = (lymphocytes/μL of hemogram) x (% DNT) ÷ 100.

Statistical analysis Variables calculated include descriptive statistics, absolute and relative frequencies, mean, median and percentiles. To define value range, the normal distribution of variables was evaluated by the Shapiro-Wilk test. Most variables did not follow a Gaussian distribution. The effects of sex, age and their interaction were evaluated using a linear model. Reference values were expressed through the median and the 5th and 95th percentiles and stratified by sex and age group (18–25, 26–50, and 51–80 years).

Ethical considerations The project was approved by the research ethics committee of the Hermanos Ameijeiras Clinical-Surgical Teaching Hospital. Written informed consent was obtained from study participants after they received an explanation of the study’s possible risks and benefits. The information was protected under principles of confidentiality without revealing participant identity. Diagnostic methods were selected based on the principle of maximum benefit, international and national standards, and accessibility of materials.

RESULTS
Percentages and absolute values of DNT lymphocytes are presented in Table 1. They were significantly lower in the 51–80 age group.

In both percentage and absolute values, the effect of age was highly significant, as evident in the variations observed between the oldest age group (51–80) and the two younger age groups (18–25 and 26–50).

DISCUSSION
At the time of this writing, there were no studies on reference-value ranges for DNT lymphocytes in the Cuban population. Two publications reported that quantification of these cells is needed to diagnose autoimmune lymphoproliferative syndrome.[6,7] Although it is common for each laboratory to define specific ranges, there should be steady progress toward this goal. Given the dearth of data on reference values, and the urgency to collect such data, this study aims to propose ranges that amount to preliminary approximations for application at the national level.

Multiple factors are known to intervene in the variability of physiological ranges for these lymphocytes and for all immune-system cells. The most studied factors are age and sex.[5] In this study, the effects of both age and sex were evaluated, and it was confirmed that the effect of age was significant, especially differences between those 51–80 years compared to those ≤50. The low sex/age interaction confirms that these differences occur in both men and women and may be due to immunosenescence and thymic involution, the consequences of which become more evident near the sixth decade of life. In a study of healthy Cubans, a negative correlation was found between age and global T-cell count.[9]

The ranges obtained were wider and had higher medians than those reported internationally. These differences could be due to effects of lifestyle, diet and environmental conditions of the populations studied,[5,10] or because, unlike ours, the other studies did not exclude individuals with unhealthy habits or those consuming medications that could have affected immunological variables.[9] More local studies are needed to determine reference values of cells that can be used provisionally until studies with broader population representation can be conducted.

Constraints in our study included the fact that the sampling was based entirely on residents of Havana with no one from other provinces; also, additional serological studies were not conducted, which reduced the possibility of diagnosing some chronic and infectious diseases that could have affected DNT lymphocyte values.

CONCLUSIONS
References were estimated for absolute values and proportions of double-negative T lymphocytes in healthy Cuban adults according to sex and age, and the importance of the effect of age was established. Absolute and relative concentrations of DNT lymphocytes were highest in younger age groups.