Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow and then circulate for about a day in the bloodstream before developing into mature red blood cells. In mammals, a reticulocyte does not have a cell nucleus. The reticulocyte stage is the non-nucleated stage in the development of the red blood cell that occurs just before it matures to an erythrocyte. The final steps of erythropoiesis involve enucleation and remodeling of both the cytoskeleton of the developing RBC and its membrane proteins to form a biconcave erythrocyte [1]. The young cells are called reticulocytes because of a reticular (mesh-like) network of ribosomal RNA that appears as dark blue dots and curved linear structures which becomes visible under a microscope with certain stains such as new methylene blue and brilliant cresyl blue. During staining with supravital stains, the RNA is precipitated and is then seen as the characteristic scattered granules and the reticular structure that differentiates reticulocytes from other erythrocytes [2]. They can also be distinguished from other RBCs in Romanowsky stains by their characteristic polychromatic staining appearance. Another distinguishing feature is that reticulocytes are relatively larger than normal RBCs, a characteristic that is defined by the mean corpuscular volume (MCV).

To accurately measure reticulocyte counts, automated counters use a combination of laser excitation, detectors, and a fluorescent dye that marks RNA and DNA (such as titan yellow or polymethine). Reticulocytes can be distinguished from other circulating cells because they emit a signal that is neither strong (like lymphocytes) nor weak (like red blood cells) [3] with fluorescent dyes. In the recent past, acridine orange, a fluorescent dye, has also served as a better alternative to supravital stains, being more sensitive in the manual microscopic count of reticulocytes [2].

The normal fraction of reticulocytes in the blood depends on the clinical situation and the state of the bone marrow but is usually 0.5% to 2.5% of the circulating red blood cells in adults and 2% to 6% in infants [4]. The relative number of reticulocytes is a reflection of bone marrow activity because it represents recent production. A higher than normal count may indicate anemia, depending on the bone marrow health status. The calculation of the reticulocyte production index (RPI) is an additional step in the proper interpretation of the reticulocyte count and determining its appropriateness to the situation. This serves a better purpose than just knowing that the percentage is within the normal range. A RI <2% with anemia indicates decreased production of reticulocytes (i.e. inadequate response to correct the anemia) and therefore RBCs, whereas a RI >3% with anemia indicates loss of RBCs [4]. Thus, a person who is severely anemic with a reticulocyte percentage of only 1% indicates that the bone marrow is likely not producing new blood cells at a rate that will appropriately compensate for the anemia. The combination of the reticulocyte count and the reticulocyte production index is useful in determining whether the anemia is caused by a production problem or otherwise, while also being relevant in monitoring the progress of treatment for anemia. Ordinarily, the compensatory erythropoiesis in cases of chronic or severe loss of erythrocytes, such as may be found in severe hemolysis and acute hemorrhage, results in an accelerated erythropoiesis (or stress erythropoiesis) with markedly high number and percentage of reticulocytes. Otherwise, abnormally low numbers of reticulocytes are usually due to chemotherapy, aplastic anemia, pernicious anemia, bone marrow malignancies, problems of erythropoietin production, various vitamin, or mineral deficiencies (iron, vitamin B12, folic acid), disease states (anemia of chronic disease), bone marrow infiltration by pathogens and other causes of anemia due to poor RBC production.

A number of authors have asserted that anemia is prevalent in HIV infections [5-7]. Many workers have also claimed that reticulocyte count is not usually increased in HIV infections. These outcomes have been attributed to the indirect effect of the virus on the bone marrow, leading to defective erythropoiesis [8]. The hematological status of naive HIV patients, however, is not the same as that of those on active antiretroviral therapy (ART). Highly active antiretroviral therapy (HAART) is a customized combination of different classes of antiretroviral medication recommended to aggressively suppress viral replication and halt progress of HIV to acquired immunodeficiency syndrome (AIDS) [9]. The goal is to prolong survival in people infected with HIV and improve the quality of life. A physician may prescribe HAART based on such factors as the patient’s viral load, the particular strain of the virus, the CD4+ cell count, age and other considerations (e.g. disease symptoms) [10]. In affected individuals, HAART has to be taken daily for life, since HAART does not completely eliminate the virus. Availability of HAART has raised the life expectancy of victims and brought great relief to the medical world, turning what hitherto used to be a death sentence into a manageable chronic disease. Currently, the term HAART is no longer commonly used, as there are newer treatment options that are easier to take, longer lasting and efficacious, with far less resistance and less side effects [11]. These drugs are therefore now more appropriately called Anti-retroviral treatment. However, the HAART regimen is still commonly used in low-resource settings, since most of them are given to nationals as donations. A person is considered to be HAART naïve, if he/she is HIV positive and has never taken any ART for the infection.

Several observational studies have reported a higher mortality in HIV infected patients from low hemoglobin levels even after adjusting for CD4 cell count and viral load. The etiology of anemia in HIV infection is multifactorial and typically, the anemia may result from low production of red blood cells, increased RBC destruction, or ineffective RBC production [7]. Since HIV infection is challenging for the sufferers, social and family support is required to maintain a relatively good quality of life [9] which are rarely available in poor resource settings. Poor nutrition is commonly an associated factor which may further complicate the condition, resulting in anemia [6]. Frequently, the laboratory features are compatible with anemia of chronic disease with a low reticulocyte count, normocytic and normochromic red blood cells with normal iron stores and cytokine-mediated poor erythropoietin response [6]. It has also been observed that HIV-infected patients with anemia have a greater risk for mortality than patients without anemia [7].

Moreover, there is a wide variation in the prevalence of anemia among HIV/AIDS patients in different studies all over the world. In patients without HIV infections and bone marrow complications, anemia elicits production of reticulocytes [2]. Expectedly, HIV infection introduces an alteration to this known pattern by diminishing bone marrow activity [8]. One of the most common causes of anemia in HIV patients and their associated laboratory findings is decreased RBC production. Ineffective RBC production may be seen due to folic acid and vitamin B12 deficiency [12]. In this case laboratory analysis of blood shows low reticulocyte counts, high indirect bilirubin, and high mean cell volume (MCV). Anemia also occurs in HIV patients due to the following other reasons: HIV-induced iron deficiency, which is due to blood loss, most commonly from gastrointestinal bleeding or menstrual hemorrhage in pre-menopausal women, neoplasm infiltrating the bone marrow, infection in the marrow, certain drugs such as zidovudine, interferon-alfa, gancyclovir, pyrimethamine, amphotericin and cancer therapies, among others. Laboratory investigations reveal low reticulocyte counts, normal levels of indirect bilirubin, low mean cell volume (MCV) in iron deficiency and anemia of chronic disease, while MCV becomes high with zidovudine treatment [12,13]. Another mechanism by which HIV induces anemia is by red blood cell destruction. Blood tests usually reveal high reticulocyte counts, high indirect bilirubin, and high lactate dehydrogenase (LDH), and low haptoglobin, while the peripheral blood smear may present with fragmented RBCs, spherocytes, and schistocytes [13,14].

When the cause is due to bone marrow infiltration by virus, treatment of HIV patients with HAART regimen reduces the viral load, thus restoring activity to the bone marrow [15]. This study therefore sought to assess the reticulocyte count among HAART-HIV positive persons within the Ilorin environ, with a view to determining the effect of HAART treatment on the bone marrow erythropoietic response in HIV infected persons.

A cross sectional design was used for this study. Approval for this study was obtained from the Kwara State Ministry of Health Research Ethical Review Committee, Ministry of Health, Ilorin. The sample collection was explained to the patient using the information sheet prepared by the researcher. Each patient was required to give a written informed consent before being recruited to the study. This study comprises 100 confirmed HIV-positive patients undergoing antiretroviral treatment which include children, teenagers, adults, and the elderly, recruited from within Ilorin who were HIV patients attending Sobi Specialist Hospital Ilorin and 40 control subjects recruited from some schools and the organized labor workforce. All test subjects were already on ART.

Sample collection and analysis

Three ml of venous blood was collected from the patient into a sterile EDTA anticoagulant tube and transported to the hematology laboratory. Two ml was used for screening for HIV status while 1 ml was used for the reticulocyte count. Both the test subjects and the controls were screened again for their HIV status using the Determine HIV 1 and 2 immunochromatographic screening kit. Invalid or borderline samples from the control subjects were confirmed with the western blot technique.

Reticulocytes staining

The young red cells that have lost their nuclei but not the cytoplasmic RNA called reticulocytes are demonstrated with a supra vital stain, when the RNA appears as a network of strands or granules. Two drops of blood were placed in a sterile glass Khan tube and an equal volume of new methylene blue was added and then incubated at 37^oC for 30 minutes in a water bath after which it was removed from the incubator and mixed thoroughly. A thin film was then made and allowed to dry before being examined under the microscope using x100 objective.

Manual counting

An area on the smear where the red cells do not overlap was selected for the count. The number of red cells and reticulocytes in each field were counted. Ten successive fields were counted without overlapping count, making a total of 1,000 red cells. Classification of results was based on the WHO classification criteria. Briefly, a normal value of reticulocyte count for a healthy individual who is not anemic is 0.5% - 2.5%.

Calculation

% Reticulocytes = Number of reticulocytes counted x 100/Number of red cells counted + reticulocytes count.

Viral load test

The viral load data for each of the test (HIV-positive) subjects was obtained from the HIV laboratory (Viral load unit) on the day of sample collection, since this test is one of the routine investigations carried out during the patient’s visits to the hospital.

Data analysis

The data was analyzed using IBM statistical Package for Social Sciences (SPSS) version 25.0 and descriptive statistics (frequencies, percentage) was used to describe the data. ANOVA was used to test for the hypothesis. In this study, we assumed 5% significance levels i.e alpha = 0.05, implying that when p<0.05, the null hypothesis is rejected.

A total of 100 HIV positive patients and 40 non-HIV positive subjects as control were recruited for this study. The mean age of the participants was approximately 41 years, and most subjects were between 31 and 40 years. All participants were of black ethnicity. The HIV status of the participants together with their retroviral therapy were determined using a standard questionnaire. The values of the reticulocyte count and viral load were compared between the HIV negative control and the HIV positive patients and results were presented in tables. Furthermore, the subjects were categorized into groups based on the duration of treatment. The basic characteristics of enrolled participants are presented in Table 1.

Table 2 shows the reticulocyte count of HIV positive patients on ART for both genders of various age groups. There was no significant change in the reticulocyte count in the male subjects (p<0.005). However, in the female subjects, there was significant change especially with the duration of infection.

Table 3 shows comparison between HIV positive patients and control subjects. There was no significant difference between the values of RC between the two groups.

Table 4 shows the assessment of the viral load in HIV positive patients across the various age groups and gender. There was no significant difference in the viral load between both genders.

In Table 5, the value of reticulocyte count (RC) and viral load were correlated. The results showed a significant correlation between the reticulocyte count and viral load (R= -0.077, p=0.000 and R= 0.224, p=0.000, respectively). The result has shown that negative significant correlation exists between the reticulocyte count and viral load.

The study population consisted of HIV positive patients (aged 11-70 years) and a control group which consisted of apparently healthy individuals. In this study, the test subjects have already been placed on HAART management for varying lengths of time. The reticulocyte count, viral load of the study group and controls were compared with respect to age, gender, and viral load. Hematological abnormalities are among the most common complications of HIV infection and involve all lineages of blood cells; thus anemia, thrombocytopenia and leukopenia are common findings [8,16]. Reticulocyte count is a simple, easily accessible tool for the assessment of bone marrow function in individuals.

This study shows that there were more HIV positive cases within the age range of 31-40 with females being in the majority. This age range belongs to the youthful age in which most individuals are still sexually active, thus increasing the exposure rate. It is also possible that some of the respondents had contracted the disease some years back and before getting to this age. The dominance of the female gender over their male counterparts in contracting HIV has been documented previously. Some scholars have observed that women are disproportionately affected by HIV around the world as a result of the female gender being subjected to increased risk of HIV infection through economic dependency, intimate partner violence, and lack of power to negotiate safe sex with their male counterparts [17]. Anwuluorah and Asike [18], also found that female sexual passivity, harmful widowhood practices and the patriarchal cultural system are factors that promote exposure to HIV infection on the part of females. Beside this, only females are known worldwide to engage in commercial sex work [19-20]. The female subjects showed a higher count of reticulocytes compared to the male subjects, although the mean values still fell within the normal reference range. Moreover, there was a significant difference in the reticulocyte count of women as the disease advanced. The rate of reticulocyte production in persons with active bone marrow is determined by adequacy of normal red blood cells in the circulation and since the trigger for erythropoiesis occurs by means of negative feedback, most of the female subjects must have been afflicted with low red blood cell levels. Women, unlike the menfolk, lose blood periodically, therefore the increased reticulocyte count is an indication of a normal physiological compensatory mechanism to make up for the loss, hence the greater erythropoietic response among the females. On the contrary, there was no significant difference in the mean values of reticulocyte counts from samples obtained from the menfolk. Our findings here show that HIV infection does not significantly affect the erythropoietic activity of men as it does that of women.

There was also a comparatively higher viral load count among the females across the ages than among the males. Since the number of women in the study are more, various counts of their viral load should be the norm. The degree of viral load in patients is a function of how long they have been under treatment and how each individual is responding to the regimen prescribed. It is important to mention that variations in this count can be influenced by various factors, including the individual's overall health, HIV progression, and response to ART.

The age range of 31-40 years showed the highest value for reticulocytes compared to other age groups. This implies that the subjects in this age range responded more to the ART compared to the other age groups. What is responsible for this pattern is not known. However, the most plausible reason would be their compliance with the administration of the HAART regimen, thus leading to a more functional and productive bone marrow. This group of HIV patients cannot afford to be complacent with treatment as this is a very critical period in their lives and most of them would be trying to build a home and a career. It is pertinent to mention though that there was no significant difference in the mean value of viral load of the combined subjects and that of the control.

A generalized effect of HIV/AIDS on hematopoietic and mature blood cells has been reported with a possibility of the virus infecting the hematopoietic precursor cell and inhibiting their differentiation and development to mature blood cells [8,21]. This inhibitory effect of the virus on the bone marrow of the sufferers seems to have been mitigated by treatment with HAART in our study, as there was no significant difference between the bone marrow reticulocyte response of HAART-treated HIV infected individuals and the response in normal persons. It is not known by what mechanism HAART affected the bone marrow of our subjects, because surprisingly, the elderly group (>60 years) who were managed with HAART still produced reticulocytes, whereas the apparently normal elderly (>60 years) control subjects did not. Circulating reticulocytes are decreased in patients with impairment of bone marrow function and are increased in patients with blood loss or destruction, with bone marrow activity. Although HIV infected patients may have a peripheral blood cytopenia which can be due to direct infection of the hematopoietic cells with cytokine dysregulation or ineffective hematopoiesis [21], this feature is more common in patients with advanced disease and low CD4 count. It is pertinent to note that some drugs used in treatment are also myelotoxic. However, in this study, patients on TLD which is a generic combination (Tenofovihoweverroxil, lamivudine and dolutegravir) with lower risk of side effects and which is considered the preferred first line antiretroviral (ARV) regimen for adults and adolescents, showed adequate bone marrow response with reticulocyte count being in the normal range. However, in the current study, HIV patients with high viral loads had slightly lower reticulocyte counts but the difference was not significant.

Our study nonetheless shows a negative correlation between viral load and reticulocyte count. In other words, the higher the viral load, the lower the bone marrow production of reticulocytes, and conversely, a low viral load results in a greater bone marrow response for reticulocyte production and release, that is, given a normal functioning bone marrow. This finding is in tandem with the notion that cytopenia occurs in the advanced stage of infection in which the viral load is usually very high.

We also observed that generally, the duration of the infection did not affect the reticulocyte count obtained. Reticulocytosis usually occurs when there is an increase in erythropoietic activity, but in this study, there was no significant change in reticulocyte count between the HAART subjects and HIV negative subjects (control), as irrespective of the duration of HIV infection, reticulocyte count was not increased. It appears that once HAART treatment gets the viral load to a certain low threshold in patients and provided this threshold is maintained, its effect on the bone marrow remains unchanged.

Our study has shown that more females are exposed to HIV infection in our study population. Likewise, females are more likely to suffer anemia in infection with HIV, if HAART is not administered. The administration of specific HAART in HIV patients in our study had a notable improvement in the CD4+ cell count and viral load with significant age and gender disparity. Moreover, the administration of HAART has mitigated the bone marrow aplasia that could have been caused by the HIV infection in the patients. We also found that the higher the viral load, the lower the reticulocyte count. There may however be a viral load threshold below which bone marrow activity is optimal and unchanged in individuals with HIV infection.

Since hemopoietic deficiencies are a hallmark of HIV infection, reticulocyte count should be included in routine tests to be carried out by HIV positive patients as it provides useful information regarding the degree of regeneration that takes place following antiretroviral therapy/treatment. In addition, the viral load threshold for optimum bone marrow activity in HAART treated HIV patients should be investigated by researchers. For patients acutely ill with a low CD4 count (< 200 cells/microL) there may be a need to consider underlying opportunistic infections, malignancies, and other rare causes. More importantly, patients may also have other cytopenias in addition to anemia.

Ethical approval was obtained via a letter from the Research Ethical Review Committee, Kwara State Ministry of Health, Ilorin.

This research project was wholly funded by the authors

All authors declare no conflict of interest.

The patients' informed consent for use of their samples for research was obtained at the point of sample collection.

Conception and design: P.O.O., A.O.I., Acquisition, analysis, and interpretation of data: P.O.O., L.N.A, Drafting the work or revising: A.O.I., P.O.O. Final Approval of the manuscript: A.O.I., P.O.O., L.N.A.

The authors declare that data supporting the findings of this study are available within the article.