Encyclopedia of Pathology

Living Edition
| Editors: J.H.J.M. van Krieken

Bone Marrow

  • Julie Bruneau
  • Chantal Brouzes
  • Vahid Asnafi
  • Thierry Jo MolinaEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-3-319-28845-1_3875-1

Synonyms

Anatomy

Bone marrow is a semisolid tissue present in all the bones divided schematically into hematopoietically inactive fatty marrow, named yellow marrow, and hematopoietically active marrow, named red marrow. In adults, red marrow is mainly found in the central skeleton such as pelvis, sternum, skull, vertebra, scapulae, as well as epiphyseal areas of long bones such as femur and humerus.

Function

The function of hematopoietic marrow is to regulate myeloid and lymphoid cell trafficking as well as hematopoietic stem and progenitor cell maintenance for normal myelopoiesis and lymphopoiesis. In addition, the marrow contains mesenchymal stem cells that can differentiate into adipocytes, hepatocytes, osteoblasts, chondrocytes, skeletal, and cardiac muscle cells. The nonhematopoietic marrow serves as a large store of reserve lipids. Bone marrow endothelial cells form a barrier preventing entry of red blood cells and platelets from the circulation regulating cell trafficking and osteogenesis. These cells also contribute to specialized perivascular microenvironment where majority of hematopoietic stem cells are present.

Size, Weight

The total mass of an adult bone marrow is estimated between 1600 g and 3700 g.

Macroscopy and Techniques for Studying the Marrow

Evaluation of bone marrow benefits both from thoroughly cytological evaluation by aspirate smear preparations (usually sternal for individuals older than 12 or the pelvic bone) as well as histopathological evaluation by bone marrow trephine biopsy (BMB) (usually posterior superior iliac spine) for evaluation of the architecture, cellularity, focal lesions, fibrosis, or necrosis.

Bone marrow aspirate provides liquid sample with mixed cell population of bone marrow cells. After bone marrow harvest, high quality smears are realized onto glass slide, air-dried and stained using May Grünwald Giemsa. Complementary cytochemistry staining can be done such as Perls staining to see intracellular (erythroblastic) and extracellular iron distribution. Sometimes, myeloperoxidase or esterase staining are useful to better characterize abnormal leukemic cells.

Bone marrow trephine biopsy provides a core of cortical and medullary areas which contain bone trabeculae and hematopoietic tissue. It is optimally a cylinder of 1.5–2 cm length. This biopsy is sent usually in the laboratory of pathology in 10% neutral buffered formalin. European Bone Marrow Working Group promotes slow decalcification using EDTA during 48 h, allowing perfect epitope conservation for immunohistochemistry and fairly good nucleic acid preservation for molecular evaluation (NGS, lymphoid clonality tests, …). Slides of 2–3 μ thickness should be prepared and stained with H&E, Giemsa (crucial for cytological evaluation), Perls for hemosiderin deposits, silver impregnation for fibrosis. When fibrosis is diagnosed on H&E, a trichrome stain is recommended. PAS stain is sometimes used, for example, to better evaluate megakaryocytes as well as immunoglobulin within lymphocytes with plasma cell differentiation.

Microscopy

Cytology on a Marrow Aspirate

Bone marrow aspirate should always be analyzed together with peripheral blood film observation.

At small magnification, cellularity will be evaluated (according to the age) and interpretability will take into account a possible hemodilution (Figs. 1 and 2). This small magnification allows to look for megakaryocytes and to appreciate their morphology (dysmegakaryocytopoiesis will be diagnosed if present on more than 10% of 30 megakaryocytes observed according to the 2016 WHO recommendation). It allows also to detect other cells such as mastocytes, macrophages, or potential extra-hematopoietic cells grouped into a cellular cluster (in case of suspicion of carcinoma metastasis, for example).
Fig. 1

Cytology of normal bone marrow with normal cellularity with one megakaryocyte (arrow) – May-Grünwald Giemsa stain. Low magnification X100

Fig. 2

Hemodiluted bone marrow. Low magnification X100

Then, bone marrow smear will be observed at higher magnification after putting a drop of oil in a reading area where cell spreading is appropriate in density and quality (Fig. 3a, b, c). The first step consists to ensure the correct distribution of cells, the presence of immature precursors, and to look for the presence of abnormal cells or morphological abnormalities.
Fig. 3

(a, b, c) Cytology of normal polymorph bone marrow at medium magnification (X500) – May-Grünwald Giemsa stain

Cytologic analysis of normal bone marrow aspirate smears allows to recognize each hematopoietic lineage (Table 1). Considering granulopoiesis, myeloblasts, promyelocytes, myelocytes, metamyelocytes, and polynuclear (banded and segmented) could be identified and counted (Fig. 4). It represents approximatively 60–70% of all hematopoietic marrow cells. These are granular cells, with basophilic cytoplasm, azurophilic, and neutrophilic granulations. With the maturation, the granulations of these cells become neutrophilic, the nucleus, initially round, central, with immature chromatin, becomes elongated and segmented with compacted chromatin, and basophilia of the cytoplasm gradually disappears. Erythroblastic lineage cells (around 20–30% of hematopoietic cells) appear as round central nucleated cells (Fig. 5); immature erythroblasts such as proerythroblasts (or pronormoblast) and basophilic erythroblasts have a round immature nucleus with fine chromatin and a large basophilic crowned cytoplasm around the nucleus. With the maturation, the cells become smaller, with condensed chromatin and polychromatophilic then acidophilic cytoplasm due to iron enrichment. Lymphocytes, plasma cells, mastocytes, and monocytes/macrophages are also present in bone marrow, and cytological examination allows to quantify these different subpopulations (Fig. 6) and to detect morphological abnormalities.
Table 1

2014 Reference values of normal adult bone marrow according to the French Society of Hematology

Lineage

Cell type

Reference range (%)

Progenitors

Blasts/stem cell/undifferentiated cell

1–2

Granulocytic

Myeloblasts

2–3

Promyelocytes

4–8

Neutrophilic myelocytes

10–15

Eosinophilic myelocytes

 

Basophilic myelocytes

 

Neutrophilic metamyelocytes

15–20

Eosinophilic metamyelocytes

 

Basophilic metamyelocytes

 

(Segmented and band) neutrophils

20–30

(Segmented and band) eosinophils

1–4

(Segmented and band) basophils

0.5–1

Monocyte

Monocytes

2–3

Macrophages

 

Erythroblastic

Proerythroblasts

1–2

Basophilic erythroblasts

4–8

Polychromatic erythroblasts

6–10

Acidophilic (orthochromatic) erythroblast

4–10

Lymphocyte

Lymphocytes

5–15

Plasma cells

1–3

Fig. 4

Cytology of normal bone marrow – granulocytic lineage: myeloblast (a), promyelocyte (b, arrow), band neutrophil (b), metamyelocyte (c, arrow), segmented neutrophil (c), and eosinophilic myelocyte (d). May-Grünwald Giemsa stain. High magnification (X1000)

Fig. 5

Cytology of normal bone marrow – erythroblastic lineage: proerythroblast (a), basophilic erythroblast (b), polychromatic erythroblast (c), acidophilic erythroblast (d). May-Grünwald Giemsa stain. High magnification (X1000)

Fig. 6

Cytology of normal bone marrow – other marrow cells: macrophage (“nurse cell” with erythroblasts) (a), mastocyte (b), lymphocyte (c), plasma cell (d). May-Grünwald Giemsa stain. High magnification (X1000)

Overall, an excess of one cell category or a maturation block could easily be identified by cytologic observation. It is the case in leukemia: excess of lymphoblasts in lymphoblastic leukemia (Figs. 7 and 8) or excess of myeloblasts in acute myeloid leukemia (Figs. 9 and 10a, b). Morphological abnormalities of hematopoietic precursors could also be observed: degranulation or chromatin abnormalities of the granulocytic lineage, nucleocytoplasmic asynchronism of the erythroblastic lineage, dehemoglobinization of erythroblast’s cytoplasm, abnormal cytoplasmic inclusions such as Auer rods in myeloblast. If there is suspicion of myelodysplastic syndrome with dyserythropoiesis, a Perls staining will be performed to look for ring sideroblasts. In addition, bone marrow could be infiltrated, for example, by abnormal lymphoplasmocytic cells in Waldenström’s macroglobulinemia, lymphoid cells in mantle cell lymphoma (Figs. 11 and 12), or by dysmorphic plasma cells in myeloma.
Fig. 7

Lymphoblasts in B-acute lymphoblastic leukemia (B-ALL) – May-Grünwald Giemsa stain. High magnification (X1000)

Fig. 8

Flow cytometry analysis: dot plots of B-ALL marrow sample: expression of CD34 +and TdT+ immaturity markers and low CD45 expression; expression of B lymphoid markers: CD19 +, CD22 +, CD79a +, CD20+, and CD10+

Fig. 9

Myeloblasts in acute myeloid leukemia (AML) with abnormal eosinophilic granular precursor (arrow) (AML with inv (16) (p13.1q22); AML4Eo FAB subtype) – May-Grünwald Giemsa stain. High magnification (X1000)

Fig. 10

(a, b) Flow cytometry analysis: dot plots of AML marrow sample: expression of CD34 + immaturity markers and low CD45 expression; expression of myeloid markers: CD117 +, CD33 +, CD13 +, MPO +, partial CD15 +; expression of HLA-DR; partial expression of CD2 frequently documented although nonspecific in the context of inv (16) (p13.1q22)/AML4Eo; no expression of B-cell and T-cell markers

Fig. 11

Marrow infiltration by mantle cell lymphoma: May-Grünwald Giemsa stain. High magnification (X1000)

Fig. 12

Flow cytometry analysis: dot plots of bone marrow infiltration by mantle cell lymphoma: expression of B lymphoid markers: CD19 +, CD22 +, CD79b +; aberrant expression of CD5 antigen, and absence of expression of CD23 antigen; kappa monotypic light chain expression

In conclusion, bone marrow cytological examination is the best way to study the quantitative and qualitative constitution of hematopoietic system but must be associated in some cases to histological examination depending on the context (to look for presence of fibrosis, for a diagnosis of Aplastic anemia, in case of focal lesions or unexplained fever….).

Histopathology on BMB

Before any histological evaluation, the size and interpretability of BMB should be evaluated. A small biopsy will prevent the detection of a focal lesions and crushing artifact or dislocation of medullary spaces during the biopsies will prevent any interpretation (Fig. 13). At least three medullary spaces should be evaluated for a minimal interpretation. However, more than 10 partially preserved intertrabecular areas should be preserved for an optimal interpretation.
Fig. 13

Bone marrow biopsy, H&E: normal bone marrow biopsy with numerous preserved intertrabecular areas (a); focal lesions (circle) such as a granuloma in a bone marrow biopsy (b); Noninterpretable marrow due to crushing artifact (c); or dislocation of medullary spaces becoming empty (d)

The analysis of a BMB should systematically evaluate bone trabeculae and medullary spaces. Within medullary spaces, one should evaluate adipocytes, hematopoietic cells (granulocytic lineage, erythroblastic lineage, megakaryocytic lineage), lymphocytes, plasma cells, mastocytes, histiocytes, as well as vascular and reticulin network (Fig. 14).
Fig. 14

Bone marrow biopsy, Giemsa stain: normal bone marrow, polymorphic with all marrow lineages

Cellularity should be evaluated and is defined by the area of a histological section occupied by hematopoietic cells compared to the area occupied by fat cells (excluding bone trabeculae). In neonates, the cellularity approaches 100%. In the adult, the cellularity is around 60–70% (Fig. 15), whereas in the elderly, cellularity decreases around 20%. One should be aware of the presence of subcortical physiological areas devoid of hematopoietic cells that do not correspond to aplastic areas.
Fig. 15

Bone marrow biopsy, H&E: normal cellularity around 50–60%

Cellularity of the myelomonocytic lineage is around 50–70%. In marrow sections, early granulopoietic cells (myeloblasts, promyelocytes) are mainly found near the endosteum of bone trabeculae whereas maturing granulocytes are found in the center of medullary spaces, adjacent to sinusoids (Fig. 16). However, few promyelocytes or myelocytes are present in small clusters at sites away from bone trabeculae. Promyelocytes are difficult to differentiate from myelocyte and normal myeloblasts are difficult to identify when not in excess. More mature granulocytes are easily identified. Giemsa stain allows to differentiate early granulocytic cells due to their azurophilic granules. Eosinophil lineage is easily identified based on bright red, refractile granules whereas basophilic granules are extracted during fixation and cannot be identified on BMB. In the absence of immunostain, monocytes cannot be identified with certainty on BMB.
Fig. 16

Bone marrow biopsy, Giemsa stain: normal granulocytic lineage with predominance of promyelocytes and myelocytes close to endosteum whereas neutrophils in the center (a). Neutrophil granules within cells from granulocytic lineage (b, c, d)

Erythroblastic lineage (20–40%) is easily identified due to the presence of erythroblasts of varying degrees of maturity in aggregates. Giemsa stain allows easily to differentiate basophilic immature erythroblasts (pronormoblast or basophilic normoblast) from more mature erythroblast with a smaller nucleus and a poorly stained cytoplasm (Fig. 17). In thick slides, these cells can be difficult to differentiate from lymphocytes.
Fig. 17

Bone marrow biopsy, Giemsa stain: Erythroblastic lineage with mature erythroblast (a, circle) and basophilic erythroblast (b, circle)

Megakaryocytes are easy to recognize by their large size, pink cytoplasm, and lobulated nuclei on H&E. In normal marrow, they are singly scattered or rarely in loose clusters (other cells between megakaryocytes). Around 4–8 are found by medullary spaces (Fig. 18). A small number of megakaryocytes with hyperchromatic nuclei, minimal or no cytoplasm can be seen physiologically.
Fig. 18

Bone marrow biopsy, Giemsa stain: 3 Megakaryocytes with normally lobulated nuclei (arrows) not far from the lumen of a venous sinus (star)

Mature lymphocytes are distributed within the interstitium with few aggregates increasing with age. T lymphocytes predominate over B lymphocytes (Fig. 19).
Fig. 19

Bone marrow biopsy, Giemsa stain: normal lymphocyte distribution with rare interstitial CD3+ T cells (a) predominating over CD20+ B cells (b); rare lymphoid aggregates (c); or lymphoid follicle with germinal center (d)

Plasma cells are often located on a perivascular topography in intimate association with small blood vessels lacking smooth muscle layer. Scattered mast cells are present as single cells rarely spindle and easily identified on Giemsa stain (Fig. 20). Macrophages are numerous throughout the BMB and are better appreciated using immunostaining.
Fig. 20

Bone marrow biopsy, Giemsa stain: plasma cells (circle) are normally present preferentially in a pericapillary distribution (star within a capillary lumen) (a). Mastocytes (arrow) are normally rare (b). Plasma cells are normally polytypic expressing either kappa or lambda light chains (c, d)

Blood supply comprises arterioles, capillaries, and large sinusoids draining through a system of collecting venules. Normal marrow contains an incomplete network of type III collagen (reticulin) fibers (Fig. 21). A higher concentration is found around the walls of larger arteries and near the endosteum.
Fig. 21

Bone marrow biopsy, silver impregnation stain: normal pattern with incomplete network of reticulin fibers

Immunophenotype

Multiparametric flow cytometry evaluation of bone marrow is a very useful complement to quantify and characterize normal or pathologic hematopoietic cells.

All hematopoietic cells express the panleucocyte antigen CD45, except erythroblastic cells that are positive for CD36 and Glycophorine A.

Immature cells (blast cells) underexpress CD45 and may be also positive for CD34, TdT, and/or CD117 antigen.

Lymphocytes cells of B lineage are characterized by CD19, CD20, CD22, CD79a, CD79b, and CD10 expression. Lymphocytes cells of T lineage are characterized by intracytoplasmic and membrane CD3 expression and CD5, CD7, CD2, CD4, or CD8 expression.

Myeloid cells are positive for CD13, CD33, CD117, and CD15 antigens. Monocytic cells show a weak expression of CD4 and are CD14 and CD36 positive.

Megakaryoblasts can be characterized by CD61 and CD42 antigen expression.

Immunohistochemistry on Paraffin-Embedded Tissues

To better identify myeloid, lymphoid, and blastic cells in bone marrow, immunohistochemistry is often performed; below are the most used antibodies on BMB.
  • CD34: blasts and endothelial cells

  • Myeloperoxidase, CD15, CD33: maturing granulocytes

  • CD68 (KP1) with some staining of granulocyte lineage, CD68 (PGEM1), CD163: macrophages

  • CD14: monocytes (subpopulation)

  • Glycophorin A, Glycophorin C, CD71: erythroblastic lineage

  • Fact VIII, CD61: megakaryocytes

  • CD117: mastocytes, proerythroblast, early promyelocyte, blasts

  • TdT: lymphoid blasts, hematogones

  • CD20, PAX5: B lymphocytes

  • CD79a: B lymphocytes and plasma cells

  • CD138: plasma cells, (metastatic carcinoma may be CD138+)

  • CD3, CD4, CD5, CD7, CD8; T lymphocytes

  • IgA, G, M, K, L: immunoglobulin heavy and light chains

Table with Important Diseases (Links)

Specific organ

Affected by diseases like

Bone marrow

Acute leukemia

Aplastic anemia

Hemophagocytic syndrome

Infection (tuberculosis, atypical mycobacterial infection, leishmaniosis, …) and inflammatory diseases

Lymphomas

Mastocytosis

Metastasis

Myeloproliferative neoplasms: chronic myeloid leukemia BCR-ABL1 positive, essential thrombocythemia, polycythemia vera, primary myelofibrosis

Myelodysplastic syndromes

Myelodysplastic syndromes/myeloproliferative neoplasms

Plasma cell myeloma

Sea-blue histiocytosis

T-cell large granular lymphocytic leukemia

References and Further Reading

  1. Itkin, T., Gur-Cohen, S., Spencer, J. A., Schajnovitz, A., Ramasamy, S. K., Kusumbe, A. P., Ledergor, G., Jung, Y., Milo, I., Poulos, M. G., Kalinkovich, A., Ludin, A., Kollet, O., Shakhar, G., Butler, J. M., Rafii, S., Adams, R. H., Scadden, D. T., Lin, C. P., & Lapidot, T. (2016). Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature, 532, 323–328.CrossRefGoogle Scholar
  2. Kroft, S. H. (2012). Bone marrow. In S. E. Mills (Ed.), Histology for pathologists (4th ed., pp. 849–887). Philadelphia: Lippincot Williams and Wilkins.Google Scholar
  3. Swerdlow, S. H., Campo, E., Harris, N. L., Jaffe, E. S., Pileri, S. A., Stein, H., & Thiele, J. (2017). WHO classification of tumours of haematopoietic and lymphoid tissues (4th ed.). Lyon: International Agency for Research in Cancer.Google Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Julie Bruneau
    • 1
    • 2
  • Chantal Brouzes
    • 1
    • 2
  • Vahid Asnafi
    • 1
    • 2
  • Thierry Jo Molina
    • 1
    • 2
    Email author
  1. 1.Department of PathologyHôpital Universitaire Necker-Enfants Malades, AP-HP, Université Paris Descartes, Université de ParisParisFrance
  2. 2.Laboratory of Onco-HematologyHôpital Universitaire Necker-Enfants Malades, AP-HP, Université Paris Descartes, Université de ParisParisFrance