Abstract
Alpha-hemoglobin-stabilizing-protein (AHSP) is an abundant erythroid-specific chaperone protein that facilitates incorporation of nascent alpha-globin into hemoglobin A. We characterized AHSP expression by immunohistochemistry in a panel of 100 neoplastic and reactive bone marrow biopsies and splenic tissue with extramedullary hematopoiesis and compared it to established erythroid markers CD71 and CD235a. In all cases, AHSP expression was limited to physiologic nucleated erythroid precursors (EPs) and blasts in erythroid leukemias. While CD71 also stained EPs, it additionally stained non-erythroid malignant cells to varying extents in acute leukemia, diffuse large B cell lymphoma, metastatic carcinomas and small round cell tumors. In contrast, CD235a staining was erythroid-specific but stained non-nucleated RBCs in all specimens, limiting its utility. We conclude that AHSP is superior to CD71 and CD235a for detecting normal and neoplastic nucleated EPs.
Introduction
Identification of erythroid precursors (EPs) in bone marrow biopsies is essential for lineage assignment of immature precursors or blasts, assessment of the myeloid:erythroid ratio, and evaluation of topographic features indicative of myelodysplasia. Morphologic assessment of H&E stained samples can identify erythroid precursors in many circumstances. However, specimens with erythroid dyspoiesis, increased immature forms, topographic disarray, or those involved by non-hematopoietic lesions can be especially challenging and may require the use of immunohistochemical erythroid stains.
Antibodies currently used clinically to identify EPs include CD235, CD71, and hemoglobin A (HgbA). Each of these antibodies has specific limitations. CD235a and HgbA label both EPs and mature, non-nucleated red blood cells (RBCs), creating excessive background staining in specimens with extensive hemorrhage. Furthermore, CD235a may not stain the most immature erythroid precursors.14 CD71 (transferrin receptor 1, TfR1) has recently been shown to label EPs and not non-nucleated RBCs, potentially overcoming the limitations of CD235a.3,10 However, CD71 expression occurs in a wide range of cells such as activated T lymphocytes, T and B lymphocyte precursors, epithelial cells (including keratinocytes), myocytes, and is generally considered to be a marker for rapidly proliferating cells.12 CD71 expression in bone marrow has been reported in acute myeloid and lymphoid leukemias and a variety of lymphomas involving the bone marrow. Thus, the nonspecific nature of CD71 could confound interpretation in diagnostically challenging cases or in limited samples. For these reasons, more sensitive and specific methods to identify EPs in clinical samples are needed to improve the accuracy of hematopathologic diagnoses. This need could be fulfilled by an antibody directed against an erythroid lineage specific antigen that is highly expressed in EPs and downregulated upon their subsequent maturation into anucleate RBCs. We hypothesized that alpha-hemoglobin stabilizing protein (AHSP), a recently discovered erythroid protein, would fulfill these requirements.
AHSP is a 12kDa chaperone protein that binds nascent alpha-globin and facilitates its incorporation into hemoglobin A.8 AHSP binds reversibly with free alpha-globin, preventing its aggregation and stabilizing its structure prior to binding beta globin to form HbA.9 Loss of AHSP expression in a murine model results in globin precipitation with ineffective erythropoiesis.4,9 AHSP is expressed at high levels in lineage-committed EPs that are actively synthesizing hemoglobin. In anucleate reticulocytes and mature RBCs, AHSP synthesis declines and the protein is degraded. These properties make AHSP an ideal candidate for marking nucleated EPs.
We characterized AHSP expression in bone marrow biopsies and splenic specimens from adult and pediatric patients with a variety of hematopoietic neoplasms, metastatic non-hematopoietic cancers and reactive conditions. Comparison to immunohistochemical staining of CD71 and CD235a on the same samples indicates that anti-AHSP staining is the superior approach for detecting EPs.
Materials and Methods
All procedures were reviewed and approved by the Institutional Review Boards at the University of Pennsylvania School of Medicine and the Children’s Hospital of Philadelphia (CHOP).
Polyclonal antibody against full-length recombinant human AHSP was commercially prepared by Covance Research Products (Denver, PA).
Bone marrow biopsy and splenic specimens from reactive and neoplastic conditions were identified by searching the laboratory information systems of the Hospital of the University of Pennsylvania and CHOP. All adult biopsies were fixed in B5 and decalcified for 1–2 hours using RDO rapid decalcifying solution (Darlco, Oradell, NJ). Pediatric bone marrow biopsies (for cases of neuroblastoma, rhabdomyosarcoma, primitive neuroectodermal tumor, and retinoblastoma) were fixed in acetic acid-zinc-formalin (AZF) and decalcified for 30 minutes using RDO rapid decalcifying solution (Darlco, Oradell, NJ). Splenic tissue was fixed in formalin. All samples were routinely embedded in paraffin. Immunohistochemical staining was performed on 4 micron sections for AHSP (rabbit polyclonal 1:8000), CD71 (Invitrogen, Grand Island, NY, H68.4, 1:1600), and CD235a (Dako, Carpineria, CA, JC159, 1:1000). Deparaffinization, epitope retrieval with a pH9 buffer, and detection were performed on a Leica Bond-Max automated stainer. Staining was assessed visually in all specimens and categorized as “positive” or “negative” in each cell lineage, any neoplastic cells present, and non-cellular background material. All staining was performed in the same laboratory with adequate positive and negative controls and all samples were reviewed by two pathologists (PWR, JKC). Images included in this manuscript were obtained by scanning slides at 40X original magnification with an Aperio Scanscope CS; no color enhancement or alteration was performed on any images after scanning.
Results
Immunohistochemical staining for AHSP, CD71, and CD235a was performed on 100 bone marrow samples representing a variety of neoplastic and reactive conditions and splenic tissue with extramedullary hematopoiesis; the results are detailed in Table 1. In summary, AHSP stained EPs in all specimens tested and did not stain non-erythroid cells. AHSP demonstrates a cytoplasmic staining pattern and brightly labels nucleated EPs from early (pronormoblast) through orthochromatic normoblast stages. CD235a stained both non-neoplastic erythroid precursors and mature (anucleate) RBCs, confounding evaluation in specimens with extensive hemorrhage and in splenic tissue with extramedullary hematopoiesis and congestion. Like AHSP, CD71 stained EPs in all specimens tested; however, it also stained neoplastic cells in a subset of non-erythroid acute leukemias, diffuse large B cell lymphoma (DLBCL), metastatic carcinomas, and small round blue cell tumors. Neither AHSP nor CD71 stained non-nucleated RBCs.
Table 1.
Immunohistochemical expression of AHSP, CD71, and CD235a in bone marrow and splenic specimens.
| Diagnosis | n | AHSP | CD71 | CD235a | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Nucleated EPs | Lesional Cells | Megakaryocytes | Nucleated EPs | Lesional Cells | Megakaryocytes | Nucleated EPs | Lesional Cells | Megakaryocytes | ||
| Non-neoplastic | ||||||||||
| Normal | 5 | 5 | NA | 1 | 5 | NA | 0 | 5 | NA | 0 |
| Relative erythroid hyperplasia | 6 | 6 | NA | 0 | 6 | NA | 0 | 6 | NA | 0 |
| Spleen with extramedullary hematopoiesis | 5 | 5 | NA | 0 | 5 | NA | 0 | 5 | NA | 0 |
| Parvoviral infection | 2 | 2 | 2* | 0 | 2 | 2* | 0 | 2 | 0* | 0 |
| AML with recurrent genetic abnormalities | ||||||||||
| Acute promyelocytic leukemia with t(15;17)(g22;12) | 5 | 5 | 0 | 0 | 5 | 1 | 1 | 5 | 0 | 0 |
| AML (megakaryoblastic) with t(1;22)(p13;q13) | 1 | 1 | 0 | NA | 1 | 1 | NA | 1 | 0 | NA |
| AML, NOS | ||||||||||
| AML with minimal differentiation, with maturation, and without maturation | 5 | 5 | 0 | 0 | 5 | 3 | 1 | 5 | 0 | 0 |
| Acute myelomonocytic, monocytic, and monoblastic leukemia | 4 | 4 | 0 | 0 | 4 | 4 | 0 | 4 | 0 | 0 |
| Erythroleukemia | 5 | 5 | 5 | 0 | 5 | 5 | 0 | 5 | 0 | 0 |
| Pure erythroid leukemia | 2 | 2 | 2 | 0 | 2 | 2 | 0 | 2 | 0 | 0 |
| Acute megakaryoblastic leukemia | 2 | 2 | 0 | NA | 2 | 2 | NA | 2 | 0 | NA |
| AML with myelodysplasia-related changes | 5 | 5 | 0 | 0 | 5 | 0 | 0 | 5 | 0 | 0 |
| Myelodysplastic syndromes | ||||||||||
| Refractory cytopenia with multilineage dysplasia | 5 | 5 | NA | 0 | 5 | NA | 0 | 5 | NA | 0 |
| Refractory anemia with excess blasts | 5 | 5 | NA | 1 | 5 | NA | 2 | 5 | NA | 0 |
| Precursor lymphoid neoplasms | ||||||||||
| B lymphoblastic leukemia, NOS | 4 | 4 | 0 | 0 | 4 | 1 | 1 | 4 | 0 | 0 |
| T lymphoblastic leukemia, NOS | 4 | 4 | 0 | 0 | 4 | 1 | 0 | 4 | 0 | 0 |
| Myelodysplastic/myeloproliferative neoplasms | ||||||||||
| Chronic myelomonocytic leukemia | 5 | 5 | 0 | 2 | 5 | 1 | 1 | 5 | 0 | 0 |
| Myeloproliferative neoplasms | ||||||||||
| Chronic myelogenous leukemia | 5 | 5 | 0 | 2 | 5 | NA | 2 | 5 | NA | 0 |
| Primary myelofibrosis | 5 | 5 | 0 | 4 | 5 | NA | 4 | 5 | NA | 0 |
| Metastatic neoplasms | ||||||||||
| Diffuse large B cell lymphoma | 4 | 4 | 0 | 0 | 4 | 4 | 0 | 4 | 0 | 0 |
| Carcinoma | 4 | 4 | 0 | 0 | 4 | 2 | 0 | 4 | 0 | 0 |
| Neuroblastoma | 5 | 5 | 0 | 1 | 5 | 5 | 0 | 5 | 0 | 0 |
| Rhabdomyosarcoma | 3 | 3 | 0 | 0 | 3 | 1 | 0 | 3 | 0 | 0 |
| Primitive neuroectodermal tumor | 3 | 3 | 0 | 1 | 3 | 0 | 0 | 3 | 0 | 0 |
| Retinoblastoma | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 |
Legend: The number in each column refers to the number of specimens with positive staining for each category of cells. AML = acute myeloid leukemia; NA = not applicable; NOS = not otherwise specified;
= giant pronormoblasts are considered lesional cells for the purposes of this table.
AHSP specifically stained EPs in all normal bone marrow biopsies, all bone marrow biopsies with relative erythroid hyperplasia, and in all adult splenic samples with extramedullary hematopoiesis (Figure 1). CD71 and CD235a also stained erythroid precursors in all of these specimens. In addition, acute erythroid leukemia blasts (both erythroleukemic and pure erythroid leukemic blasts) were positive for AHSP staining (Figure 2). CD71 and CD235a also stained erythroid blasts in all cases of acute erythroid leukemia. However, CD71 also stained blasts to varying degrees in every morphologic subset of acute myeloid leukemia (Figure 3) and both acute T- and B-lymphoblastic leukemia. In general, non-M6 AMLs stained less intensely for CD71 than EPs. However, a minority of myeloid blasts stained strongly for this antigen at an intensity approximating that exhibited by EPs.
Figure 1. AHSP and CD71 stain nucleated EPs.
A. Normal bone marrow, H&E. B. CD235a stains both nucleated EPs and mature, anucleate RBCs. C. AHSP stains nucleated EPs, but not mature, anucleate RBCs. D. CD71 stains nucleated EPs, but not mature, anucleate RBCs. E. Spleen with extramedullary hematopoiesis, H&E. F. AHSP stains nucleated EPs in splenic extramedullary hematopoiesis.
Figure 2. AHSP and CD71 stain erythroid blasts in acute erythroleukemia.
A. Acute erythroleukemia, H&E. B. CD235a stains erythroid blasts and mature, anucleate RBCs. C. AHSP stains erythroid blasts. D. CD71 stains erythroid blasts.
Figure 3. CD71 stains myeloid blasts in acute myeloid leukemia, whereas AHSP does not.
AHSP stains residual EPs and not myeloid blasts in acute myeloid leukemia with minimal differentiation (A), whereas CD71 stains both myeloid blasts and EPs (B). AHSP does not stain myeloid blasts in acute myelomonocytic leukemia (D), whereas CD71 does (E). C and F are corresponding H&Es, respectively.
AHSP and CD71 antibodies performed comparably in specimens with myeloproliferative neoplasms and myelodysplastic syndromes, with both antibodies staining erythroid precursors in all bone marrow biopsies examined. All cases of primary myelofibrosis exhibited mildly increased staining of megakaryocytes by both AHSP and CD71; CD235a staining did not stain megakaryocytes (Figure 4). AHSP staining in megakaryocytes was variable between cases and between individual megakaryocytes within the same case.
Figure 4. AHSP and CD71 stain megakaryocytes in primary myelofibrosis.
A. Primary myelofibrosis, H&E. B. CD235a stains both nucleated EPs and mature, anucleate RBCs. AHSP (C) and CD71 (D) variably stain megakaryocytes and also stain nucleated EPs.
CD71 expression was also noted in many bone marrow biopsies with metastatic malignancies, including DLBCL (4 of 5 cases, Figure 5 A–C) and carcinoma (2 of 4 cases, Figure 5 D–F) in adults and neuroblastoma (5 of 5 cases) and rhabdomyosarcoma (1 of 3 cases) in children. CD71 staining ranged in intensity from dim to bright in metastatic lesions, but was equal to that observed in EPs in multiple cases. CD235a stained both EPs and non-nucleated RBCs and did not stain metastatic lesions.
Figure 5.
AHSP (A) stains residual EPs and not lymphoma cells in DLBCL, whereas CD71 (B) stains both lymphoma cells and EPs. C. Corresponding H&E. AHSP (D) does not metastatic carcinoma, whereas CD71 (E) does. F. Corresponding H&E.
Both AHSP and CD71 stained giant pronormoblasts in bone marrow biopsies from patients with parvovirus infection (Figure 6), whereas CD235a was negative in these cells, consistent with CD235a expression relatively late in erythroid development.
Figure 6.
Giant pronormoblasts are evident in parvoviral infection (H&E, A). B. CD235a does not stain giant pronormoblasts. AHSP (C) and CD71 (D) stain giant pronormoblasts.
AHSP and CD71 both stained less than 1% of non-nucleated RBCs in all tested specimens, likely marking young reticulocytes that had recently extruded their nucleus. No increase in the frequency of positively staining non-nucleated RBCs was detected in biopsies with myelodysplasia stained with AHSP or CD71. In rare instances, AHSP-stained slides exhibited increased levels of background signal that was localized to non-cellular proteinaceous fluid present in the specimens. This artifact was infrequent and was not over-represented in any individual class of diagnoses. This finding is attributed to non-specific binding of the polyclonal antibody to serum proteins and did not affect interpretation of cellular staining patterns.
Discussion
Our results demonstrate that AHSP is a lineage-specific marker of nucleated EPs with improved specificity and equivalent sensitivity as compared to CD71 and CD235a. AHSP staining characteristics did not differ after various fixation protocols (B5, AZF, and formalin) and were not affected by routine decalcification procedures. In virtually every case, AHSP antibody stained only EPs and erythroid malignancies, whereas CD71 stained malignant cells in a subset of non-erythroid acute leukemias, DLBCL, and metastatic non-hematopoietic malignancies. This result is expected in light of the biological restriction of AHSP to the erythroid lineage as a necessary factor for the effective production of hemoglobin.
In contrast, CD71 is a widely expressed protein involved in iron acquisition for most cell types. CD71 expression levels are particularly high in EPs to supply adequate levels of iron for Hgb production. However, many rapidly dividing cells, including malignant ones, also exhibit relatively high levels of CD71 in order to supply iron to meet metabolic demands.12 While CD71 and AHSP stain nucleated EPs, analysis of CD235a staining patterns is confounded by staining of anucleate RBCs that frequently contaminate pathological specimens. This effect is most pronounced in samples of splenic extramedullary hematopoiesis and in bone marrow biopsies with extensive hemorrhage. Thus, among current antigens used for detecting EPs in pathological samples, only AHSP meets two important criteria: high specificity for the erythroid lineage and lack of staining in anucleate RBCs. These properties reflect the biological functions of AHSP. As a molecular chaperone for alpha globin, AHSP expression coincides with Hgb synthesis, which occurs specifically in erythroid tissues.8,16 As Hgb synthesis declines during the reticulocyte stage of erythropoiesis, AHSP is no longer needed and the protein is degraded.
Several other antibodies or antibody panels have been characterized as potential markers of EPs, highlighting the clinical need for such a stain. To our knowledge, all previously described markers other than CD235a stain non-EPs in bone marrow biopsies. A panel of three immunohistochemical markers targeting proteins whose expression is not limited to the erythroid lineage has also recently been described.13 Although this panel of markers sensitively stains for erythroid precursors, it also marked blasts in a subset of non-erythroid acute leukemias. E-cadherin has also been shown to be expressed in erythroid precursors, although its expression is present in many epithelial cell types and also is not expressed in erythroleukemia.1,2 CD36, the thrombospondin receptor, is expressed in EPs, but is also expressed in other cell types including platelets, macrophages, and endothelial cells.5,6
CD71 has been previously reported to label blasts in a subset of non-erythroid acute leukemias but not by others.3,10 Our results confirm the former findings. While CD71 expression is variable in non-erythroid malignancies, certain cases demonstrated CD71 expression in blasts that approximated levels exhibited by EPs. AHSP did not stain leukemic blasts in any cases of non-erythroid acute leukemias. AHSP staining was also limited to erythroid precursors in all cases of marrow involvement by DLBCL or non-hematopoietic malignancies, whereas CD71 stained the neoplastic cells in many of these cases. CD71 expression has been reported in a wide variety of activated or proliferating cell types, concordant with its biological role as the transferrin receptor. CD71 expression is likely upregulated in these cases secondary to increased iron demand in these rapidly proliferating cells.
Detection of AHSP in megakaryocytes associated with primary myelofibrosis is of uncertain etiology. Non-specific staining is unlikely, as the antibody does not stain normal megakaryocytes in control specimens or bone marrow affected by B-cell lymphoma or parvovirus. It is possible that the pathological megakaryocytes in primary myelofibrosis aberrantly express AHSP and occasionally CD71 as well, reflecting dysmegakaryopoiesis with de-repression of an erythroid gene expression program. Erythroid and megakaryocytic lineages derive from a common bipotential progenitor and express overlapping sets of hematopoietic transcription factors.11 Megakaryocytes in primary myelofibrosis have been demonstrated to aberrantly express multiple genes and miRNAs; however, AHSP has never been specifically studied.7,15 Importantly, AHSP did not stain blasts in acute megakaryocytic leukemia, whereas CD71 marked them brightly.
AHSP and CD71 both demonstrated intense staining of giant pronormoblasts in cases of parvovirus infection, whereas CD235a is negative. Negative CD235a expression and CD71 positivity in infected giant pronormoblasts has previously been reported.3,14 AHSP positivity in giant pronormoblasts confirms the ability of AHSP to mark EPs that are in the earliest stages of the erythroid lineage.
In addition to its potential clinical utility as a specific immunohistochemical marker of erythroid precursors, AHSP expression may be useful in other clinical and research settings. For example, further characterization of AHSP as an intracellular flow cytometric marker to identify acute erythroid leukemias and potentially help differentiate between morphologic subtypes (erythroleukemia vs. acute erythroid leukemia) is one area of potential study.
In summary, AHSP is a novel marker of erythroid precursors whose biologic role as a chaperone protein necessary for hemoglobin formation confers lineage-specificity and whose expression is limited to nucleated erythroid precursors. AHSP may be useful in many clinical scenarios including assigning lineage to neoplastic or reactive immature cells and identifying erythroid precursors in dyspoietic marrows, and assessing topographic disruption in bone marrow biopsies.
Acknowledgements
We would like to thank Lynita Thomas, Joanne Mauger, and the histotechnology staff at the Hospital of the University of Pennsylvania and Children’s Hospital of Philadelphia for their expert technical assistance. This work was supported in part by the National Institutes of Health (NIH) under grants R01 DK061692, R01 HL087427 and P30DK090969 (MJW).
Footnotes
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Disclosures:
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REFERENCES
- 1.Acs G, LiVolsi VA. Loss of membrane expression of E-cadherin in leukemic erythroblasts. Arch Pathol Lab Med. 2001;125:198–201. doi: 10.5858/2001-125-0198-LOMEOE. [DOI] [PubMed] [Google Scholar]
- 2.Armeanu S, Buhring HJ, Reuss-Borst M, et al. E-cadherin is functionally involved in the maturation of the erythroid lineage. J Cell Biol. 1995;131:243–249. doi: 10.1083/jcb.131.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Dong HY, Wilkes S, Yang H. CD71 is selectively and ubiquitously expressed at high levels in erythroid precursors of all maturation stages: a comparative immunochemical study with glycophorin A and hemoglobin A. Am J Surg Pathol. 35:723–732. doi: 10.1097/PAS.0b013e31821247a8. [DOI] [PubMed] [Google Scholar]
- 4.Favero ME, Costa FF. Alpha-hemoglobin-stabilizing protein: an erythroid molecular chaperone. Biochem Res Int. 2011:373859. doi: 10.1155/2011/373859. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Febbraio M, Hajjar DP, Silverstein RL. CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest. 2001;108:785–791. doi: 10.1172/JCI14006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Filippone C, Franssila R, Kumar A, et al. Erythroid progenitor cells expanded from peripheral blood without mobilization or preselection: molecular characteristics and functional competence. PLoS One. 5:e9496. doi: 10.1371/journal.pone.0009496. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Hussein K, Theophile K, Dralle W, et al. MicroRNA expression profiling of megakaryocytes in primary myelofibrosis and essential thrombocythemia. Platelets. 2009;20:391–400. doi: 10.1080/09537100903114537. [DOI] [PubMed] [Google Scholar]
- 8.Kihm AJ, Kong Y, Hong W, et al. An abundant erythroid protein that stabilizes free alpha-haemoglobin. Nature. 2002;417:758–763. doi: 10.1038/nature00803. [DOI] [PubMed] [Google Scholar]
- 9.Kong Y, Zhou S, Kihm AJ, et al. Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia. J Clin Invest. 2004;114:1457–1466. doi: 10.1172/JCI21982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Marsee DK, Pinkus GS, Yu H. CD71 (transferrin receptor): an effective marker for erythroid precursors in bone marrow biopsy specimens. Am J Clin Pathol. 134:429–435. doi: 10.1309/AJCPCRK3MOAOJ6AT. [DOI] [PubMed] [Google Scholar]
- 11.Pang L, Weiss MJ, Poncz M. Megakaryocyte biology and related disorders. J Clin Invest. 2005;115:3332–3338. doi: 10.1172/JCI26720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ponka P, Lok CN. The transferrin receptor: role in health and disease. Int J Biochem Cell Biol. 1999;31:1111–1137. doi: 10.1016/s1357-2725(99)00070-9. [DOI] [PubMed] [Google Scholar]
- 13.Rollins-Raval MA, Fuhrer K, Marafioti T, et al. ALDH, CA I, and CD2AP: novel, diagnostically useful immunohistochemical markers to identify erythroid precursors in bone marrow biopsy specimens. Am J Clin Pathol. 137:30–38. doi: 10.1309/AJCP0QFQ0FTSZCPW. [DOI] [PubMed] [Google Scholar]
- 14.Sadahira Y, Sugihara T, Yawata Y. Expression of p53 and Ki-67 antigen in bone marrow giant proerythroblasts associated with human parvovirus B19 infection. Int J Hematol. 2001;74:147–152. doi: 10.1007/BF02981997. [DOI] [PubMed] [Google Scholar]
- 15.Theophile K, Hussein K, Kreipe H, et al. Expression profiling of apoptosis-related genes in megakaryocytes: BNIP3 is downregulated in primary myelofibrosis. Exp Hematol. 2008;36:1728–1738. doi: 10.1016/j.exphem.2008.07.011. [DOI] [PubMed] [Google Scholar]
- 16.Weiss MJ, Zhou S, Feng L, et al. Role of alpha-hemoglobin-stabilizing protein in normal erythropoiesis and beta-thalassemia. Ann N Y Acad Sci. 2005;1054:103–117. doi: 10.1196/annals.1345.013. [DOI] [PubMed] [Google Scholar]