NORMAL BONE MARROW:
IMMUNOHISTOCHEMICAL IDENTIFICATION OF DIFFERENT CELL COMPONENTS
Normal bone marrow consists of a heterogeneous population of cells proceeding along various differentiation
pathways. Although most cell types can be easily distinguished on bone marrow aspirate smears and biopsy
sections of appropriate thickness, immunohistochemistry is valuable in identifying specific cell subsets and
in assessing their proliferative capability.
Erythropoiesis
The erythroid cells account for 5-38% of the nucleated cells in normal bone marrow. The erythroid cells can
be identified by staining the biopsy sections with a polyclonal anti hemoglobin antibody. Similar results
can also be obtained by using an anti glycophorin A antibody. The earliest identifiable erythroid cell is
the proerythroblast. In the erythroid series, cell division occurs down to the stage of polychromatophilic
erythroblast. Normally, 2/3 of the erythroblasts in the adult bone marrow express the proliferation
associated markers PCNA/PC10 and Ki67/MIB-1.
Granulopoiesis
Myeloid cells account for 23-85% of the nucleated cells in the normal bone marrow. Myeloid cells can be
identified by numerous antibodies. The most specific and most sensitive, however, is myeloperoxidase (MPX).
Myeloperoxidase is expressed in cells belonging to the neutrophilic and eosinophilic series as well as by
some monocytes. CD34 and HLA-DR are expressed by myeloblasts; both markers are lost when myeloblasts mature
into promyelocytes. Elastase, a marker restricted to the primary neutrophilic granules, has also been
proposed as a mean to identify immature myeloid precursors (e.g. promyelocytes). However, it is a less
sensitive marker than MPX. Lactopherrin, a product contained in secondary granules, can identify more mature
precursors (e.g. myelocytes and metamyelocytes). Cells belonging to the later stages of neutrophilic
differentiation can also be recognized by CD15. CD45RO, CD74, BCL-2, and CD43 also stain myeloid cells in
marrow sections but are non-specific. The epitopes of CD68 (PG-M1, HAM-56 and KP-1) react with myeloid
cells as well as macrophages. PG-M1 is the most specific for macrophages/monocytes. The other two
antibodies may be used as secondary markers for myeloid cells. (See section on macrophages). Lysozyme is
also a useful marker for myeloid cells, but less specific than MPX. CD117 (c-KIT) is generally not reactive
with myeloblasts in decalcified material such as marrow biopsies (only stains mast cells).
Proliferation associated antigens are positive in myeloid cells down to the metamyelocyte stage. Basophils,
mast cells, and eosinophils are usually identified by their morphologic and histochemical characteristics.
The normal mast cell shows reactivity with c-KIT, CD68/KP-1, as well as with an immunohistologic TRAP stain
in many cases.
Megakaryopoiesis
Megakaryocytic progenitor cells express CD34, and HLA-DR. Some of these cells express CD4. The maturation
process of the megakaryocytic series is characterized by repeated endomitosis with the generation of large
cells with lobulated nuclei demonstrating 8, 16, or 32 ploidy. Megakaryocytes can be identified in tissue
sections by their positivity with Factor VIII antigen, CD61, and CD31. Although these markers are more
strongly expressed in granular (mature) megakaryocytes they can also be used to identify immature cells of
the megakaryocytic lineage. Staining of the megakaryocytes with PCNA and MIB-1 is variable and not
particularly related to the nuclear segmentation of the cells. However, increased staining can be observed
after treatment with thrombopoietic growth factors.
Lymphoid Cells
Lymphocytes account for 1-5% of the nucleated cells in the normal bone marrow. However, higher values may
be observed in older patients and in children. Usually T-cells outnumber B cells by 4:1. Lymphoid
follicles may also be observed, usually in adult subjects, especially after the age of fifty. The T-cells
can be identified by a large number of T-cell antibodies which are reactive in routinely processed marrow
(e.g. CD45RO, CD2, CD3, CD4, CD8, CD5, CD7). Normally, CD8 T-lymphocytes are more numerous than CD4
T-lymphocytes. The marrow B-cells are stained by CD20, CD45RA and CD79a. CD74 is non specific and should
not be used as a pan B cell reagent. Rare TdT, CD10, and CD79a positive early B-cell precursors (also
termed hematogones) are normally present in the marrow. Those are more common in the pediatric age group
where one may experience problems in separating these reactive cells from lymphoblasts in patients with
acute lymphoblastic leukemia. In ALL, however, the blasts usually consistently express CD34 and TdT,
whereas in normal marrows, the number of TdT positive cells is higher than the number of CD34 positive
cells. In non neoplastic marrows, CD34 positive cells are present singly and usually account for less than
2% of the marrow nucleated cells. NK cells can be recognized in marrow sections by CD56 staining; S-100 can
also be positive in those cells.
Bone Marrow Stroma
The extracellular matrix which can be demonstrated in routine preparations includes reticulin (collagen
III), and collagen IV. Bone marrow reticulum cells can be identified by NGFR positivity. The amount of
staining is correlated with the amount of stainable reticulin observed (Cattoretti et al., 1993). Vascular
endothelial cells can be stained by FVIII, CD34, and CD31. Differences in staining pattern can be observed
with these antibodies (e.g. sinusoids are negative with CD34).
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