Reticulocyte Count and its Immature Fractions as an Early Indicator of Bone Marrow Alteration in Gas Station Workers

Contagem de Reticulócitos e suas Frações Imaturas como Indicador Precoce de Alteração Medular em Trabalhadores de Postos de Combustíveis

Recuento de Reticulocitos y sus Fracciones Inmaduras como Indicador Temprano de Alteración Medular en Trabajadores de Gasolineras




Alain Ferreira Vilhena1; Jamille Douahy Rebelo2; Rubens Corrêa de Souza Neto3; Ana Maria Almeida Souza4; Lacy Cardoso de Brito Junior5


1Laboratório Amaral Costa Medicina Diagnóstica. Belém (PA), Brazil. E-mail: Orcid iD:

2,3Faculdade Integrada Brasil Amazônia (FIBRA). Belém (PA), Brazil. E-mails:; Orcid iD:; Orcid iD:

4Universidade Federal do Pará (UFPA), Instituto de Ciências da Saúde, Faculdade de Medicina. Belém (PA), Brazil. E-mail: Orcid iD:

5UFPA, Instituto de Ciências Biológicas, Laboratório de Patologia Geral – Imunopatologia e Citologia. Belém (PA), Brazil. E-mail: Orcid iD:


Corresponding author: Lacy Cardoso de Brito Júnior. UFPA, Instituto de Ciências Biológicas, Laboratório de Patologia Geral – Imunopatologia e Citologia. Av. Augusto Corrêa, 1 – Guamá. Belém (PA), Brazil. CEP 66075-900. E-mails:;




Introduction: Prolonged occupational exposure to toxic components of gasoline such as benzene for a long time is recognized as an important factor in hematological changes. Objective: Evaluate the count of reticulocytes and their immature fractions (IRF) in samples of gas station workers tested at a private laboratory in Belém, Pará, from January to December 2021. Method: Forty-seven peripheral blood samples were analyzed from male gas station workers, regardless of the time of work, who submitted to periodic tests at a private laboratory in Belém (study group), and 47 samples from male customers who underwent routine blood counts at the same laboratory without previous hematological disease (control group). Results: In the control group, 46/47 (97.9%) of the samples had a mean reticulocyte count of 62,828/mm³ and a relative IRF of 3/47 (6.4%), whereas in the study group, the mean reticulocyte count was of 100,628/mm³ and IRF of 5/47 (10.8%). Of these, 34/47 (72.3%) samples had a reticulocyte count and an IRF higher than the reference values. Conclusion: The results suggest that the increase in the reticulocyte count and its IRF in the study group may be important early risk markers for medullary intoxication by benzene.

Key words: reticulocyte count; benzene; hydrocarbons; occupational exposure; anemia, aplastic.




Introdução: A exposição ocupacional prolongada a componentes tóxicos da gasolina, como o benzeno, há muito tempo é reconhecida como um importante fator de alterações hematológicas. Objetivo: Avaliar a contagem de reticulócitos e suas frações imaturas (IRF) em amostras de trabalhadores de postos de combustíveis (“frentistas”) atendidos em um laboratório particular de Belém, Pará, no período de janeiro a dezembro de 2021. Método: Foram analisadas 47 amostras de sangue periférico de trabalhadores de postos de combustíveis do sexo masculino (grupo do estudo), independentemente do tempo de atividade laboral, e que realizam exames laboratoriais periódicos em um laboratório particular de Belém, e 47 amostras de clientes, também do sexo masculino, que realizaram hemograma de rotina no mesmo laboratório e não apresentavam doença hematológica prévia (grupo controle). Resultados: No grupo controle, 46/47 (97,9%) das amostras apresentavam média de contagem de reticulócitos de 62.828/mm³ e de IRF relativas de 3/47 (6,4%), já nos trabalhadores do grupo de estudo a média de contagem de reticulócitos foi de 100.628/mm³ e de IRF de 5/47 (10,8%). Destas, 34/47 (72,3%) amostras apresentavam contagem de reticulócitos e de IRF superiores aos valores de referência. Conclusão: Os resultados sugerem que o aumento da contagem de reticulócitos e suas IRF em frentistas de postos de combustíveis podem ser importantes marcadores de risco precoce de intoxicação medular pelo benzeno.

Palavras-chave: contagem de reticulócitos; benzeno; hidrocarbonetos; exposição ocupacional; anemia aplástica.




Introducción: La exposición ocupacional prolongada a los componentes tóxicos de la gasolina, como el benceno, se ha reconocido durante mucho tiempo como un factor importante en los cambios hematológicos. Objetivo: Evaluar conteos de reticulocitos y sus fracciones inmaduras (IRF) en muestras de trabajadores en gasolineras atendidos en un laboratorio privado en Belém, Pará, de enero a diciembre de 2021. Método: Se analizaron 47 muestras de sangre periférica de individuos de sexo masculino, independientemente del tiempo de actividad laboral, que son trabajadores de gasolineras y que realizan exámenes de laboratorio periódicos en un laboratorio privado en Belém (grupo estudio), así como 47 muestras de clientes, también de sexo masculino, a quienes se les realizaron hemogramas de rutina en el mismo laboratorio y no presentaban enfermedad hematológica previa (grupo control). Resultados: En el grupo control, 46/47 (97,9%) de las muestras tenían un recuento promedio de reticulocitos de 62.828/mm³ y un IRF relativo de 3/47 (6,4%), mientras que en los trabajadores del grupo estudio el recuento promedio de reticulocitos fue de 100.628/mm³ y un IRF de 5/47 (10,8%). De estas, 34/47 (72,3%) muestras tenían recuentos de reticulocitos e IRF superiores a los valores de referencia. Conclusión: Los resultados sugieren que el aumento de los recuentos de reticulocitos y sus IRF en los trabajadores de las gasolineras pueden ser marcadores importantes del riesgo temprano de intoxicación medular por benceno.

Palabras clave: recuento de reticulocitos; benceno; hidrocarburos; exposición profesional; anemia aplásica.




Exposure to hydrocarbons and their derivatives, such as benzene, present in detergents, plastics and gasoline has generated several alerts by the International Agency for Research on Cancer (IARC) about their potential carcinogenic risk1-8.


Many of these substances are absorbed by the human body through inhalation, dermal contact or even by ingestion. Especially in relation to benzene, after its absorption, its toxic effects can be detected in the liver (primary metabolism), bone marrow (secondary metabolism) and kidneys (excretion)2,4,5,7,9,10.


The primary hematological changes due to this harmful exposure to benzene are often observed only when the blood count already shows anemia, leukopenia, thrombocytopenia or pancytopenia associated with bone marrow aplasia or, in more severe cases, when leukemias appear6-8,10,11.


Naoum et al.12 and Ruiz et al.11 already suggested that the functionality and integrity of the bone marrow in these individuals could be estimated through the quantification, of the concentration of reticulocytes13-16 in peripheral blood. More recently, with the automated counting of reticulocytes13,16,17 and immature reticulocytes fractions (IRF), these parameters have gained even greater accuracy for the evaluation of erythropoiesis activity14-16.


This study aimed to evaluate the count of reticulocytes and IRF in gas station workers as a possible method of identifying early bone marrow injury.



Peripheral blood samples from 47 male workers (study group) of a company that manages gas stations in the metropolitan region of Belém, Pará were included, regardless of the time in the work function and who performed blood counts as routine exams at a private laboratory in the same city (study group) from January to December 2021. The control group was randomly selected and consisted of 47 male clients who performed routine blood counts in the same laboratory and period. As a criterion for inclusion in the study, all individuals in both groups should be non-smokers, did not present quali-quantitative alterations or previous disease of the red series.


The samples were collected as usual and submitted to automated processing in the Sysmex XN-1000 equipment and, after analyzing the results, selected for counting reticulocytes and their IRF in the same equipment, which uses fluorescent dyes as the technology that bind to the RNA of reticulocytes and IRF16,17.


Values between 25,000 and 85,000/mm³ were considered as reference values for the absolute reticulocyte count in people aged over 30 days of life. However, as there are no absolute reference values for the IRF in the literature, these were analyzed only as research parameters17.


As the study was based only on secondary databases, the investigators signed a Term of Commitment to Use Data (TCUD) of the institution responsible for providing the data, in compliance with Resolution number 46618 of December 12, 2012, and supplemented by Article 17, item VII of Resolution 510/201019.


Descriptive and parametric analyzes were performed using the ANOVA test through the StatPlus software version 8.0 and considering p<0.05.



The mean age of the workers in the study group was 35 years, while the means of reticulocyte count was 100,628/mm³ and the IRF was 10.8%. For this group, it was observed that, even with blood cell counts (erythrocytes, leukocytes and platelets) within normal limits according to criteria of the World Health Organization (WHO)20, reticulocyte counts and IRF in 34/47 (72.3%) of the samples analyzed were higher than the reference values established in this study.


Table 1. Concentration of reticulocytes and IRF in the study group and control group who were submitted to routine blood count at a private laboratory in Belém - Pará, from January to December 2021




Concentration of reticulocytes (mm3)



Study Group

Max - Min

20 - 56

45.765 - 223.630

4.7 ± 25.3

X ± SD

35 ± 8.9

100.628 ± 31.124

10.8 ± 4.5

Control Group

Max - Min

18 - 62

34.086 - 85.158

3.2 ± 17.4

X ± SD

37 ± 12.3

62.828 ± 11.640

7.4 ± 2.9

Captions: IRF = immature reticulocytes fractions; Max-Min = maximum and minimum values; X ± SD = mean and standard deviation.



On the other hand, the analysis of the results of the samples of the 47 individuals of the control group (Table 1), who underwent routine hemogram and did not present previous alterations in the erythrocytes, revealed that the average age was 37 years, the reticulocyte count was 62,828/mm³ and the IRF was 7.4%. Of these, 46/47 (97.9%) samples showed reticulocytes count values within the reference values established in this study and also blood counts with erythrogram, leukogram and platelet count indicators within normal limits.


Parametric tests were performed to compare the results between the control and study groups, with a statistical difference (p<0.05) found in the results of reticulocytes (p=0.000001) counts and IRF (p=0.00308) being observed between groups, suggesting that the samples of gas station attendants present higher reticulocytes and IRF counts than those observed in individuals who did not have this occupation.



Figueiredo et al.6, studying the concentration of substances utilized to produce gasoline at gas stations in the city of Rio de Janeiro, Brazil, noticed that benzene concentrations were above the levels recommended by the current legislation and that these places were potentially dangerous for gas station workers who refuel on daily bases due to the carcinogenic potential of this substance21.


Poça et al.7, in their studies on chronic exposure to benzene from gasoline, showed that genotoxic effects (DNA damage assessed by the comet assay) and immunotoxic effects (assessed by low levels of helper T lymphocytes and naive T lymphocytes) were greater among individuals who worked directly in the supply of gasoline compared to what was observed among office workers at the same filling stations.


It is also clear that the chronic effects of exposure to benzene, even in small concentrations and for a long period, may be related to hematological changes ranging from depression of blood cell production, mild (anemia, leukopenia or thrombocytopenia) and even severe conditions (pancytopenia), which can lead to irreversible medullary aplasia10,22 and even alterations in the appearance of leukemias and lymphomas8.


The investigation revealed that the gas station attendants of the gas stations had indicators of erythrogram, leukogram and blood platelet count within the normal limits, however, with reticulocyte and IRF counts higher than those found in individuals who did not have this occupation (control group).


These findings suggest that the continuous and daily exposure of these workers to benzene can be evaluated apparently through the determination of the absolute concentrations of reticulocytes and IRF, which seem to reflect the early bone marrow injury.


The study data, however, could not be compared with other studies of the literature, since it is the first one to address this relationship between changes in reticulocyte and IRF concentration with exposure to benzene. Similar studies carried out by Naoum et al.12 and Ruiz et al.11, over a decade ago, also associated reticulocytosis as an important finding in individuals with benzene intoxication.



The results suggest that the increase in the reticulocytes count and its immature fractions in the study group may be important early risk markers for bone marrow injury by benzene.




To the Directors of the Laboratório Amaral Costa – Medicina Diagnóstica for authorizing the utilization of the data for this study.




Alain Ferreira Vilhena contributed to the study design, acquisition and analysis of the data. Jamille Douahy Rebelo and Rubens Correa de Souza Neto contributed to the acquisition and analysis of the data. Ana Maria Almeida Souza and L. C. Brito Junior contributed to the study design, analysis and interpretation of the data, wording and critical review. All the authors approved the final version for publication.




There is no conflict of interests to declare.




Universidade Federal do Pará. Programa de Pós-Graduação em Análise Clínica (PPGAC).




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Recebido em 8/12/2022

Aprovado em 8/3/2023


Scientific-Editor: Anke Bergmann. Orcid iD:




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