Anemia is the most common hematological complication in HIV-infected adults [1,2] and is positively associated with disease progression [3–5]. In adults, anemia results primarily from reduced erythropoiesis [6–9]. Information about anemia mechanisms in HIV-infected children is scarce [10–13] and there have been no pediatric studies from sub-Saharan Africa.
We have previously reported that HIV infection was more common among severely anemic Malawian children than in a carefully selected control population (13 vs. 6%, P < 0.001) . The aim of the present study was to determine whether HIV infection was associated with reduced erythroid precursor cells, or increased rates of apoptosis and dyserythropoiesis, and to investigate the role of cytokines, erythropoietin and plasma vitamin A in reducing apoptosis.
This study was part of a large case–control study investigating the causes of severe anemia in southern Malawi . All children aged 6–60 months with a primary diagnosis of severe anemia (hemoglobin concentration <5 g/dl), and no blood transfusion within the previous month, were recruited prospectively between 2002 and 2004. HIV-uninfected children aged 6–60 months with no obvious signs of infection and undergoing elective operations were recruited as controls.
An automated full blood count, including reticulocytes, was performed on peripheral blood samples (Coulter counter, Beckman Coulter, Durban, South Africa). Malaria slides were read by two independent microscopists. Stained bone marrow aspirate smears from all children were used to determine the myeloid: erythroid ratio  and assess dyserythropoiesis, which was defined and scored according to a published protocol .
C-reactive protein and erythropoietin were determined using a Roche p800/e170 system (Roche, Basel, Switzerland). Inflammatory cytokine profiles were measured by Cytometric Bead Array flow cytometry (FACS-Calibur, BD Biosciences, San Jose, California, USA). Serum vitamin A (retinol) was measured using HPLC . HIV testing was performed using two rapid tests (Determine, Abbott-Laboratories, Tokyo, Japan; Unigold, Trinity-Biotech, Dublin, Ireland). Reactive results in children less than 18 months of age and discordant results were resolved by PCR .
Fresh bone marrow aspirates underwent automated cell count (Coulter counter, Beckman Coulter) and four color flow cytometry (FACS-Calibur, BD Biosciences). Bone marrow cells were separated and incubated with different combinations of CD14-PE-Cy5 (Tük4), CD34-FITC/PE (QBEND/10), CD36-PE (CLB-IVC7), CD235a-FITC (CLB-AME-1) (all from Sanquin Reagents, Amsterdam, The Netherlands), laser dye styril-751 (LDS, Applied Laser Technology, Maarheeze, The Netherlands) and Annexin V and propidium iodide (IQ-products, Groningen, The Netherlands) .
Patient characteristics and hematological variables were compared using the χ2-test, Fisher's exact test, Student's t-test and the Mann–Whitney U-test. Correlations were assessed using the Pearson product–moment correlation coefficient or Spearman's rank correlation coefficient. A two-sided significance level was set at P = 0.05.
Complete data (bone marrow samples and HIV tests) for this study were available for 329 of 381 children enrolled in our original case–control study. The original study had shown that bacteremia, malaria, hookworm, HIV, glucose-6-phosphate dehydrogenase (G6PD ) deficiency and vitamin A and B12 deficiency were associated with severe anemia. Iron deficiency was negatively associated with severe anemia. Folate deficiency and sickle cell disease were uncommon .
Forty of the 329 children (12%) were infected with HIV. Their median age was 25 months compared with 16 months for HIV-uninfected children (P < 0.01). No significant differences were found between HIV-infected and uninfected children with regard to other baseline characteristics, mean hemoglobin levels (P = 0.67) or other erythrocytic indices (Table 1) .
HIV-infected children had fewer bone marrow CD34+ hematopoietic progenitors, erythroid progenitor cells and erythroid precursor cells than HIV-uninfected children, but numbers of bone marrow proerythroblasts, basophilic and polychromatic erythroblasts and peripheral blood reticulocytes were similar (Table 1). Correction for age or malaria did not alter the results (data not displayed).
Dyserythropoiesis occurred in 2.8% and 3.8% of erythroid precursors in HIV-infected and uninfected children, respectively (P = 0.12, Table 1). The proportions of viable erythroid precursor cells and those at various stages of apoptosis were similar between the two groups (Table 1). The proportions of dyserythropoietic cells and red cells undergoing early apoptosis were positively correlated (range r = 0.34, P = 0.01). There were no correlations (r = −0.14–0.15) between the proportion of either dyserythropoietic or apoptotic cells and the peripheral blood concentrations of cytokines tumor necrosis factor-α (P = 0.90 and 0.28), interferon-γ (P = 0.15 and 0.36), interleukin-10 (P = 0.74 and 0.19), erythropoietin (P = 0.22 and 0.83) or vitamin A (P = 0.83 and 0.22).
This study is the first detailed prospective analysis of erythropoiesis using bone marrow samples and flow cytometry in HIV-infected children. HIV-infected children with severe anemia had 33% fewer CD34+ hematopoietic progenitors and 35% less erythroid progenitors in their bone marrow than uninfected children. This supports the hypothesis that red cell production failure is an important cause of severe anemia in HIV-infected children and may be caused by a reduced stem cell capacity . However, the proportion of more mature erythroid precursor cells in bone marrow or peripheral blood (reticulocytes) did not differ between the two groups, suggesting that HIV-uninfected children had less efficient later stages of erythropoiesis than HIV-infected children. This is supported by the trend toward less dyserythropoiesis and apoptosis in HIV-infected children, but is in contrast to previous reports suggesting that anemia due to dyserythropoiesis is more common in later stages of HIV disease [2,10]. Alternatively, the lost CD34 cells in HIV-infected children may have been precursors that were not committed to erythropoiesis.
HIV infection affects hematopoietic processes possibly through abnormal expression of cellular genes and cytokines . The African HIV-1 subtype C can directly infect CD34+ hematopoietic progenitors . Unlike previous studies [24,25], we found no association between dyserythropoiesis or apoptosis and altered cytokine levels or vitamin A deficiency [26,27], despite 90% of children having vitamin A deficiency . More intensive investigations might identify cytokines that affect regulatory signals and could potentially be therapeutic targets to reduce hemopoietic inhibition in HIV patients.
In common with previous studies, we did not find any differences in peripheral blood erythrocytic indices or bone marrow microscopy in HIV-infected compared with uninfected children [6–8,10,28], possibly because of the multifactorial cause of anemia in African children [14,29].
None of the children were on antiretroviral therapy, which can exacerbate blood and bone marrow abnormalities . Although not all tests were done on all children, the large sample size increases confidence that the study sample was representative.
The findings in these severely anemic Malawian children indicate that despite an HIV-associated reduction in early red cell precursors, subsequent erythropoiesis appears to proceed similarly in HIV-infected and HIV-uninfected children with severe anemia.
This study was funded by the Wellcome Trust and supported by independent grants of the Nutricia Research Foundation and the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences.
We thank the parents and guardians of the children admitted to the study, the SevAna study team, the staff of the Queen Elizabeth Central Hospital, Chikwawa District Hospital and Wellcome Trust Research Laboratories, and particularly, S.M. Graham, E.M. Molyneux, M. Cornelissen, M. Beld, L. van Lieshout, F.A. Wijnberg and W.J. van Lüling for their contributions to the study.
1. Spivak JL, Bender BS, Quinn TC. Hematologic abnormalities in the acquired immune deficiency syndrome. Am J Med 1984; 77:224–228.
2. Zon LI, Arkin C, Groopman JE. Haematologic manifestations of the human immune deficiency virus (HIV). Br J Haematol 1987; 66:251–256.
3. Mocroft A, Kirk O, Barton SE, Dietrich M, Proenca R, Colebunders R, et al
. Anaemia is an independent predictive marker for clinical prognosis in HIV-infected patients from across Europe. EuroSIDA study group. AIDS 1999; 13:943–950.
4. Moore RD, Keruly JC, Chaisson RE. Anemia and survival in HIV infection. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 19:29–33.
5. Sullivan PS, Hanson DL, Chu SY, Jones JL, Ward JW. Epidemiology of anemia in human immunodeficiency virus (HIV)-infected persons: results from the multistate adult and adolescent spectrum of HIV disease surveillance project. Blood 1998; 91:301–308.
6. Bain BJ. The haematological features of HIV infection. Br J Haematol 1997; 99:1–8.
7. Bain BJ. Pathogenesis and pathophysiology of anemia in HIV infection. Curr Opin Hematol 1999; 6:89–93.
8. Moses A, Nelson J, Bagby GC Jr. The influence of human immunodeficiency virus-1 on hematopoiesis. Blood 1998; 91:1479–1495.
9. Volberding PA, Baker KR, Levine AM. Human immunodeficiency virus hematology
. Hematology Am Soc Hematol Educ Program
10. Ellaurie M, Burns ER, Rubinstein A. Hematologic manifestations in pediatric HIV infection: severe anemia as a prognostic factor. Am J Pediatr Hematol Oncol 1990; 12:449–453.
11. Meira DG, Lorand-Metze I, Toro ADC, Silva MTN, Vilela MMDS. Bone marrow features in children with HIV infection and peripheral blood cytopenias. J Trop Pediatr 2005; 51:114–119.
12. Mueller BU, Tannenbaum S, Pizzo PA. Bone marrow aspirates and biopsies in children with human immunodeficiency virus infection. J Pediatr Hematol Oncol 1996; 18:266–271.
13. Sandhaus LM, Scudder R. Hematologic and bone marrow abnormalities in pediatric patients with human immunodeficiency virus (HIV) infection. Pediatr Pathol 1989; 9:277–288.
14. Calis JCJ, Phiri KS, Faragher EB, Brabin BJ, Bates I, Cuevas LE, et al
. Factors associated with severe anemia in Malawian children. N Engl J Med 2008; 358:888–899.
15. Bain BJ, Clark DM, Lampert IA, Wilkins ES. Bone Marrow Pathology. Oxford: Blackwell Science; 2007.
16. Newton CR, Warn PA, Winstanley PA, Peshu N, Snow RW, Pasvol G, et al
. Severe anaemia in children living in a malaria endemic area of Kenya. Trop Med Int Health 1997; 2:165–178.
17. Bieri JG, Tolliver TJ, Catignani GL. Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr 1979; 32:2143–2149.
18. Molyneux EM, Walsh AL, Malenga G, Rogerson S, Molyneux ME. Salmonella meningitis in children in Blantyre, Malawi, 1996–1999. Ann Trop Paediatr 2000; 20:41–44.
19. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84:1415–1420.
20. Dibley MJ, Goldsby JB, Staehling NW, Trowbridge FL. Development of normalized curves for the international growth reference: historical and technical considerations. Am J Clin Nutr 1987; 46:736–748.
21. Redd AD, Avalos A, Phiri K, Essex M. Effects of HIV type 1 infection on hematopoiesis in Botswana. AIDS Res Hum Retroviruses 2007; 23:996–1003.
22. Koka PS, Reddy ST. Cytopenias in HIV infection: mechanisms and alleviation of hematopoietic inhibition. Curr HIV Res 2004; 2:275–282.
23. Redd AD, Avalos A, Essex M. Infection of hematopoietic progenitor cells by HIV-1 subtype C, and its association with anemia in southern Africa. Blood 2007; 110:3143–3149.
24. Ellaurie M, Rubinstein A. Elevated tumor necrosis factor-alpha in association with severe anemia in human immunodeficiency virus infection and Mycobacterium avium
intracellulare infection. Pediatr Hematol Oncol 1995; 12:221–230.
25. Testa U. Apoptotic mechanisms in the control of erythropoiesis. Leukemia 2004; 18:1176–1199.
26. Herault O, Domenech J, Georget MT, Clement N, Colombat P, Binet C. All-trans retinoic acid prevents apoptosis of human marrow CD34+ cells deprived of haematopoietic growth factors. Br J Haematol 2002; 118:289–295.
27. Zauli G, Visani G, Vitale M, Gibellini D, Bertolaso L, Capitani S. All-trans retinoic acid shows multiple effects on the survival, proliferation and differentiation of human fetal CD34+ haemopoietic progenitor cells. Br J Haematol 1995; 90:274–282.
28. Galli L, de Martino M, Rossi ME, Panza B, Farina S, Vierucci A. Hemochrome parameters during the first two years of life in children with perinatal HIV-1 infection. Pediatr AIDS HIV Infect 1995; 6:340–345.
29. Brabin BJ, Premji Z, Verhoeff F. An analysis of anemia and child mortality. J Nutr 2001; 131:636S–645S.
30. Harris CE, Biggs JC, Concanon AJ, Dodds A. Peripheral blood and bone marrow findings in patients with acquired immune deficiency syndrome. Pathology 1990; 22:206–211.