Bone Marrow-nonneoplastic

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Last revised 6 February 2007

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See also Bone Marrow tumors (future topic), Leukemia/myelodysplasia,

Lymphoma-B cell, Lymphoma-non B cell, Bone, Hematology (future topic)

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Table of contents

Primary references, images needed, embryonic development

Normal: general, basophils, eosinophils, erythroid maturation, hematogones, lymphocyte maturation, mast cells, megakaryocyte maturation, monocyte maturation, neutrophil maturation, osteoblasts, osteoclasts, plasma cells, age related changes

Bone marrow biopsy and aspirate smear: technique, sample report, routine stains

Alterations in cellularity: cellularity-general, amegakaryocytic thrombocytopenia, aplastic anemia, Diamond-Blackfan anemia, dyskeratosis congenita, Fanconi’s anemia, hypercellular, Schwachman-Diamond syndrome, TAR syndrome, pure red cell aplasia, treatment related

Benign changes: gelatinous transformation, Howell-Jolly bodies, iron, lymphoid aggregates, necrosis, persistent polyclonal lymphocytosis, plasmacytosis, polymorphous reactive lymphoid hyperplasia, systemic polyclonal B-immunoblastic proliferation

Anemias: megaloblastic

Infectious/inflammatory: Candida, CMV, Cryptococcus, Denge fever, granulomatous inflammation, histoplasmosis, HIV/AIDS, human granulocytic anaplasmosis, Leishmania, malaria, mycobacteria, parvovirus B19, Penicilliosis marneffei, Q fever, sarcoidosis, Whipple’s disease

Systemic disorders: Chediak-Higashi syndrome, cystinosis, Fabry’s disease, Gaucher’s disease, mucopolysaccharidosis type VII, Niemann-Pick disease, Pearson's syndrome, sea-blue histiocytosis syndrome, sickle cell

Other toxicity / deposition disorders: alcohol abuse, calcium oxalate, copper, podophyllin

Bone marrow transplantation: general

 

Primary references

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American Journal of Clinical Pathology (AJCP), January 1975 to December 2006

American Journal of Surgical Pathology (AJSP), March 1977 to December 2006

Archives of Pathology and Laboratory Medicine (Archives), January 1976 to December 2006

Human Pathology (Hum Path), March 1970 to December 2006

Journal of Clinical Pathology, January 1966 to December 2006

Modern Pathology (Mod Path), January 1988 to January 2007

Biomed Center, to 19 December 2006

Mills: Sternberg's Diagnostic Surgical Pathology (4th ed), 2004

Rosai: Rosai and Ackerman's Surgical Pathology (9th ed), 2004

Tumors of the Bone Marrow (AFIP Atlas of Tumor Pathology, Series 3, Vol 9)

AFIP images (not copyrighted) courtesy of www.PathologyResources.com

Websites with images: American Society of Hematology, PathoPic, PEIR digital library

Journal search terms: marrow and each disease entity listed

 

Please refer to these primary references for more detailed discussions and photographs

 

Images needed for Bone Marrow

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Embryologic development of bone marrow

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In embryo, hematopoiesis (other than lymphoid) occurs in yolk sac with formation of mesenchymal derived primitive erythroblasts

Aorta also contributes lymphomyeloid stem cells, as do embryonic liver and bone marrow (Development 2002;129:4147, Ann N Y Acad Sci 2005;1044:41)

At weeks 10 to 24, liver is primary hematopoietic organ with production of granulocytes and megakaryocytes in sinusoids

At months 4-5, bone marrow hematopoiesis begins

By birth, liver and spleen have minimal role in myelopoiesis, and bone marrow is major site of hematopoiesis

References: Int J Dev Biol 2005;49:243, Wikipedia (stem cells)

 

 

Normal bone marrow

Normal bone marrow-general

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3-6% of total body weight

Major organ for hematopoiesis at birth; also primary and secondary lymphoid organ

Hematopoiesis sites change from axial and radial skeleton in newborns to flat bones of central skeleton by mid-teens

Pluripotent stem cells develop into myeloid blasts (myeloblasts, monoblasts, erythroblasts and megakaryoblasts) or lymphoblasts

Cells are in storage pools for 5-7 days, then to blood, then to tissues

Hematopoietic stem cells: defined as cells with multilineage hematopoietic differentiation potential and sustained self-renewal activity; detected by their ability to regenerate long-term multilineage hematopoiesis in myeloablated recipients

Vasculature: nutrient (medullary) artery ramifies through marrow space to supply medullary cavity; its arterioles branch into capillaries that are continuous with thin walled sinusoids; sinusoidal walls have inner endothelial cells and outer adventitial reticular cells; outer adventitial reticular cells are phagocytic and can become lipocytes; also synthesize collagen, laminin, fibronectin and proteoglycans

Micro: arterioles, venules, capillaries, sinusoids, adipose tissue, connective tissue and hematopoietic cells; mitotically active cells are usually paratrabecular and perivascular

 

Basophils in bone marrow

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0.5% of all white blood cells

Progresses from myeloid stem cell to basophilic promyelocyte, to basophilic myelocyte, to basophilic metamyelocyte, to basophil

Similar to mast cells, but apparently generated by different CD34+ precursor cells in bone marrow

Leaves bone marrow as terminally differentiated granulocyte

Named because it stains with basic dyes

Basophils and mast cells are effecter cells in allergen/IgE-mediated immune responses; they induce type 1 immediate immune response in airways and elsewhere, causing bronchial asthma and other allergic diseases (Allergol Int 2006;55:105)

Also play a critical role in host defense against infection with helminthes (Allergol Int 2006;55:99)

Basophil activation test, using CD203c or CD63 as an activation marker, has become a reliable test for in vitro investigations of immediate allergy, complementing other in vitro tests (Clin Mol Allergy 2005;3:9)

Micro:

basophilic myeloblast: difficult to distinguish types of granulocyte blasts; large round cell with basophilic cytoplasm without granules; N/C ratio is 80%; dispersed chromatin with nucleolus

basophilic promyelocyte: intermediate in development between basophilic myeloblast and myelocyte; large round cell with a few undifferentiated cytoplasmic granules; slight chromatin clumping, nucleolus present

basophilic myelocyte: round/oval cell; minimal cytoplasm with slight basophilia, moderate cytoplasmic purple-black granules of varying size and shape; granules are usually larger than neutrophilic granules; N/C ratio is 50%; chromatin moderately condensed, no distinct nucleolus

basophilic metamyelocyte: oval cell with abundant pale cytoplasm with large and fairly uniform specific granules; N/C ratio is 40%; nucleus is small and indented with condensed chromatin, no nucleolus

basophil: smaller than other WBCs (10-15 microns); cytoplasm is homogenous pale-blue but often obscured by purple-blue granules (containing heparin and histamine); N/C is 20%; nucleus is often unsegmented or bilobed, chromatin is coarse

Positive stains: commonly used - CD9, CD25, CD38; also CD11a, CD11b, CD11c, CD13, CD15u, CD17, CD18, CD26, CD31, CD32, CD33, CD35, CD38, CD43, CD44, CD45, CD46, CD47, CD49d, CD50, CD55, CD58, CD59, CD63, CD68, CD71 (dim by flow cytometry), CD85A, CD85H, CD87, CD88, CD99, CD102, CD116, CD121b, CD123, Cd125, CD126, CDw128a, CD203c, HLA-DR (immature basophils, Allergy 2006;61:1063), histidine decarboxylase, 2D7 (J Clin Pathol 2006;59:396), basogranulin (AJCP 2006;125:273)

Positive stains: allergic subjects - CD32, CD122, CD124, CD130, CD154 (J Allergy Clin Immunol 2000;106:1190)

Variable: CD14, CD15, myeloperoxidase

Negative stains: CD2, CD3, CD7, CDw12, CD16, CD19, CD20, CD21, CD22, CD23, CD56, CD57, CD114, CD122, CD124, tryptase

References: Wikipedia, Cytometry 1999;35:249-flow cytometry markers, Allergy 1994;49:861-markers, Blood 1987;70:1872-markers

 

Eosinophils in bone marrow

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1-4% of all white blood cells

Progresses from myeloid stem cell to eosinophilic promyelocyte, to eosinophilic myelocyte, to eosinophilic metamyelocyte, to eosinophil

Named because granules stain deeply with eosin

Have a role in response to parasitic infections and allergic conditions

Degranulation is strictly controlled, which allows it to differentially release its contents in an ordered manner, which prevents tissue injury during migration (Semin Respir Crit Care Med 2006;27:117)

Produces: IL-2, IL-3, IL4, IL-5, IL-7, IL-13, IL-16, tumor necrosis factor-alpha, transforming growth factor-beta, RANTES, eosinophil cationic protein, eosinophil peroxidase, eosinophil derived neurotoxin, major basic protein and Charcot-Leyden crystal lysophospholipase

Micro:

eosinophilic promyelocyte: intermediate in development between a myeloblast and myelocyte; 15 microns in diameter with large nucleus and nucleolus; contains a few undifferentiated (primary, coreless) cytoplasmic granules in intensely basophilic cytoplasm

eosinophilic myelocyte: round/oval large cells with moderate cytoplasm containing prominent primary purple granules and secondary red-orange, refractile granules of similar size; N/C ratio is 50% with moderately condensed chromatin and indistinct nucleolus

eosinophilic metamyelocyte: round/oval cells with abundant cytoplasm containing large blue-orange granules; N/C ratio is 40%; nucleus is indented with moderately condensed chromatin and no nucleolus

eosinophil: 9-15 microns with coarsely granular cytoplasm containing refractile orange granules grouped around a single, horseshoe shaped nucleus with 2-3 lobes

Positive stains: CD9; also CD15, CD16, CD23, CD32, CD35, CD47R (weak), CD49d, CD50, CD52, CD62L, CD69, CD85A, CD85D, CD88, CD89, CD116, CDw125, CD183, myeloperoxidase, Sudan Black, PNL2

Negative stains: CD114, tryptase

References: Wikipedia, eMedicine

 

Erythroid maturation in bone marrow

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Erythroid maturation (erythropoiesis) is characterized by increasing hemoglobin synthesis, decreasing cell size, decreasing cytoplasmic basophilia and extrusion of nucleus outside cell at orthochromatic stage of development

Progresses from myeloid stem cell to pronormoblast, to basophilic normoblast, to polychromatophilic normoblast, to orthochromatic normoblast (then extrusion of nucleus), to reticulocyte (young erythrocyte), to erythrocyte (red blood cell)

Early erythroid precursors cluster in islands randomly distributed throughout marrow but related to vascular structures

Erythroblast islands may be specialized niches where intercellular interconnections and cytokines regulate erythropoiesis (Curr Opin Hematol 2006;13:137)

Note: normoblast is also called erythroblast

Micro:

pronormoblast: 13-18 microns, round/ovoid with thin rim of basophilic cytoplasm, large spherical nucleus with fine chromatin and 1-2 nucleoli; usually perinuclear halo; N/C ratio is 90%

basophilic normoblast: 12-17 microns; increase in deeply basophilic cytoplasm compared to pronormoblast and slightly smaller nucleus with slight chromatin condensation; often perinuclear halo; no granules, no nucleolus; N/C ratio is 75%-85%

polychromatophilic normoblast: 12-15 microns; round/ovoid with abundant, dull gray to gray-green, variegated cytoplasm due to polyribosomes (basophilic) and hemoglobin (eosinophilic); round, condensed and basophilic nucleus has coarse granules that give it a checkboard (cart-wheel) appearance; perinuclear halo present; no nucleolus; N/C ratio is 60-80%

orthochromatophilic normoblast: 8-12 microns; round/ovoid cells with pink-orange uniformly staining cytoplasm, dark and opaque nucleus that may be pyknotic or in the process of being extruded, no nucleolus

reticulocyte: 7-10 microns; cannot identify without supravital stain (new methylene blue) that colors RNA deep blue and granular; must have at least 2 granules to classify as reticulocyte; cytoplasm is red to pale blue due to RNA, no nucleus is present; larger than mature erythrocyte and lacks central pallor

erythrocyte: 7-8 microns; round/ovoid biconcave disc with orange-red cytoplasm, no RNA, no nucleus

Positive stains: red blood cells - GLUT1, CD35, CD36 (early precursors), CD38, CD41, CD43, CD44, CD47, CD49d (erythrocyte precursors only), CD58, CD71 (precursors through reticulocytes), CD75, CD105 (erythrocyte precursors only), CD108, CD111, CD139 (weak), CD147, CD233, CD235a, CD235b, CD235ab, CD236, CD236R, CD238, CD239, CD240 CE, CD240 D, CD240 DCE, CD241

Negative stains: red blood cells - CD9, CD10, CD15, CD24, CD37, CD42a, CD45, CD46, CD47R, CD49d, CD53, CD57, CD71, CD81, CD82, CD114, CD226

References: Wikipedia (reticulocyte)

 

Hematogones in bone marrow

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Hematogones are lymphoid progenitor cells

Often found in young children as normal finding, and may be most common lymphoid population in neonates (Biol Neonate 2004;86:247)

Also associated with childhood cytopenias, neoplasms, ITP (Egypt J Immunol 2005;12:9), and regenerative marrow after chemotherapy or bone marrow transplantation

May distort analysis of acute lymphoblastic leukemia in flow cytometry since markers are similar

Have been separated into three types (Neoplasma 2005;52:502)

Case reports: excessive hematogones in CMV+ neonate with immune thrombocytopenia (Leuk Res 2003;27:193), CD5+ hematogones in 5 year old girl with Shwachman-Diamond syndrome (Pediatr Dev Pathol 2001;4:505), sisters with Schwachman-Diamond syndrome who died as neonates (Archives 2000;124:1379)

Micro:

lymphoblast: resembles lymphoblasts in ALL; 10-20 microns (small/medium size), round/oval with sparse deeply basophilic cytoplasm without granules but may have vacuoles; indented nucleus with homogeneous fine, lacy and smudged chromatin; variable nucleoli

prolymphocyte: same size as lymphoblasts (10-20 microns) but more cytoplasm than lymphoblasts or mature lymphocytes, usually homogeneously blue cytoplasm; central round nucleus with single prominent nucleolus; coarser chromatin than lymphoblasts; N/C ratio is 75-85%

lymphocytes: 7-15 microns, round/ovoid but may have notches or indentations; variable light blue cytoplasm (often sparse); dense chromatin, usually no nucleolus; N/C ratio is 35-85%

Positive stains: commonly CD10+, CD38 (bright), CD19+ by flow cytometry; heterogeneous expression of CD19, CD20, CD22, CD10, CD34, TdT; also CD38, CD43 (Br J Haematol 2005;128:820); more CD20+ cells than CD34/TdT+ cells (AJCP 2000;114:66)

Negative staining: surface immunoglobulin

DD: ALL (staining is homogeneous for various markers, not heterogeneous; deviates from normal B cell maturation spectrum with maturation arrest, aberrant expression of myeloid antigens and asynchronous expression of B cell precursors, Blood 2001;98:2498, Leuk Lymphoma 2004;45:277)

References: AJCP 1994;102:202 (adults)

 

Lymphocyte maturation in bone marrow

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Lymphocyte precursors originate in bone marrow

B cells complete most of their development within the bone marrow, but T cells are generated in the thymus from precursor cells that migrate from the bone marrow

B cell development in marrow is dependent on CD10+ stromal cells (J Pathol 2005;205:311), which form specific adhesive contacts with developing B lineage cells, and also provide growth factors (stem cell factor, IL-7, stromal cell derived factor 1)

Earliest stem cells are in subendosteum, adjacent to inner bone surface; with maturation, B lineage cells move towards central axis of marrow; final stages of development of immature B cells occur in peripheral lymphoid organs (spleen, lymph nodes)

Micro: diffusely scattered throughout interstitium; 10% of marrow cells in adults; aggregates often present

References: Immunobiology online textbook

 

Mast cells in bone marrow

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Part of immune system

Similar to basophils, but generated by different CD34+ precursor cells in bone marrow

Mast cells leave bone marrow and circulate in immature form, and only mature at tissue site

Play a central role in allergic reactions through IgE receptor mediated responses

Stem cell factor is crucial for their development, proliferation and maturation (Immunol Res 2006;34:97)

Micro: rare in normal marrow; larger (up to 100%) than basophils with irregular elongated spindle shapes and cytoplasmic extensions; cytoplasm is packed with basophilic granules that may obscure nuclear margin; nucleus is round and single

Positive stains: Giemsa stain, Leder stain (chloroacetate esterase), methylene blue (granules stain purple), microphthalmia transcription factor, CD13 (immature and neoplastic mast cells), CD29, CD33, CD34, CD41, CD43, CD45, CD50, CD52, CD61, CD63, CD68, CD88 (J Allergy Clin Immunol 2005;115:1162), CD117, CD172a, CD203c

Negative stains: CD1-CD8, CD10-CD17, CD19-CD24, CD25 (non-neoplastic mast cells), CD38

References: Wikipedia, Blood 1989;73:1778

 

Megakaryocyte maturation in bone marrow

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Progresses from myeloid stem cell to megakaryoblast to promegakaryocyte to megakaryocyte to proplatelets (released into circulation) to platelets (J Thromb Haemost 2003;1:1580, Front Biosci 2007;12:2050)

Maturation is characterized by an increase in size and lobulation of nuclei, and is controlled by thrombopoietin (J Clin Invest 2005;115:3339)

Megakaryocytes form demarcation membrane within cytosol, which leads to production of platelets

Micro:

megakaryoblast: variable size (7-35 microns); may be designated micromegakaryoblasts if less than 15 microns; round/ovoid cells with scanty blue agranular cytoplasm that often forms a rim around nucleus and may have a few small budding protrusions at periphery; nuclei are round/oval with coarse granular chromatin, one or more nucleoli

megakaryocyte: randomly disbursed throughout bone marrow; 50-150 microns (largest normal nucleated cell in marrow); micromegakaryocytes measure 15-30 microns; abundant light blue to pink cytoplasm with numerous purple-red or pink granules; nucleus has 8, 16 or 32 overlapping lobes; no nucleolus; megakaryocytes producing platelets may have demarcated granular clumps of platelets streaming from the margins

Positive stains: CD41, CD61; also CD9, CD31, CD34, CD36, CD42a, CD42b, CD42c, CD42d, CD43, CD49b, CD49f, CD51, CD62P, CD110, CD111, CD112, CD141, CD151, CD226 (Eur J Haematol 2005;74:228)

Negative stains: CD45, CD68

References: J Clin Invest 2005;115:3348, J Clin Invest 2005;115:3332

 

Monocyte maturation in bone marrow

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Progresses from myeloid stem cell to monoblast to promonocyte to monocyte (bone marrow) to monocyte (peripheral blood) to macrophage (in tissues)

Difficult to identify monoblasts and promonocytes in normal bone marrow

Contain granules similar to those in neutrophils, but fewer and smaller

Monocytes are also precursors of dendritic cells

Micro:

monoblast - 12-20 microns, moderate basophilic cytoplasm without granules, often intense staining on periphery and with perinuclear zone, round/oval nuclei with fine chromatin and 1-4 nucleoli; nucleus may show indentations or folding

promonocyte - features intermediate between monoblasts and monocytes

monocyte - largest of leukocytes (12-20 microns); round with smooth margins or pseudopod-like cytoplasmic extensions; abundant agranular light-blue cytoplasm with fine pink azurophilic granules; may have vacuoles or phagocytized material; large bilobed, kidney shaped or U shaped nucleus with moderately clumped chromatin; no nucleolus; N/C ratio is 65-80%;

Positive stains: mainly CD14; also CD7, CD11a, CD11b, CD11c, CD11d, CD12, CD13, CD15 (variable), CD15u, CD17, CD18, CD23 (activated), CD29, CD30, CD32, CD33, CD36, CD37, CD38, CD39, CD40, CD43, CD44R, CD45, CD45RB, CD45RC, CD45RO, CD48, CD49a, CD49b, CD49d, CD49e, CD49f, CD51, CD52, CD54, CD61, CD62L, CD64, CD65, CD65s, CD68, CD83 (transient), CD84, CD85A, CD85B, CD85D, CD85E, CD85F, CD85I, CD85J, CD85K, CD85M, CD86, CD87, CD88, CD89, CD91, CD92, CD93, CD97, CD101, CD102, CD105 (activated), CD111, CD112, CD114, CD116, CD122, CD123 (plasmacytoid), CD126, CD127, CD128, CD132, CD137, CD139, CD141, CD148, CD156, CD157, CD163, CD165, CD166 (activated), CD171, CD180, CD210, CD226, CD227

Negative stains: CD24, CD56, CD57, CD60 (usually), CD231

 

Neutrophil maturation in bone marrow

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Progresses from myeloid stem cell to myeloblast type I to myeloblast type II to promyelocyte to neutrophilic myelocyte to neutrophilic metamyelocyte to neutrophilic band to segmented neutrophil (polymorphonuclear neutrophil-PMN)

Maturation is characterized by decreasing N/C ratio and increasing granule production and nuclear segmentation

Immature forms are usually paratrabecular or perivascular; exceptions are after bone marrow transplantation, cytokine administration or chemotherapy

Primary granules: large, round, red-pink, electron dense; contain myeloperoxidase, elastase, lysozyme, cathepsin G, acid hydrolases; also called azurophilic (stains sky blue with azure stains used in the past); these granules are formed in promyelocytes (Blood 1979;53:179 , free full text)

Secondary (specific) granules: smaller, electron lucent (clear), cause characteristic cytoplasmic color in Wright stained preparations; contain lactoferrin and lysozyme; these granules are formed in myelocytes

Micro:

myeloblast: 15-20 microns, round/oval; usually scanty basophil cytoplasm with no perinuclear halo; may contain Auer rods (due to fusion of azurophilic granules) or delicate azurophilic granules; round/oval nuclei with occasional indentations or clefts; one or more nucleoli; finely reticulated chromatin; N/C ratio is 80-85%

type I myeloblast: no granules in cytoplasm

type II myeloblast: up to 15-20 delicate granules in cytoplasm

type III myeloblast: more than 15-20 cytoplasmic granules, but otherwise has features of a blast cell

promyelocyte: 10-20 microns; increased basophilic cytoplasm (compared to blasts) with primary coarse red-purple, azurophilic granules; large, round/oval nucleus with red-blue and fine to slightly condensed chromatin; 1-2 nucleoli; N/C ratio is 75-85%

myelocyte: 10-18 microns; round/oval with abundant pink cytoplasm with prominent red-purple azurophilic (primary) granules and numerous fine, lilac, specific (neutrophilic) secondary granules; round/oval to slightly indented nucleus with red-blue and slightly condensed chromatin; usually no nucleolus; N/C ratio is 50-65%

metamyelocyte: 10-16 microns; moderate pink or colorless cytoplasm with occasional red-purple azurophilic (primary) granules and variable fine, lilac, specific (neutrophilic) secondary granules; indented nucleus with light blue-purple and granular chromatin; no nucleolus; N/C ratio is 40-50%

band: 10-15 microns; abundant pink cytoplasm with many fine, lilac, neutrophilic (secondary) granules and possibly a few red-purple azurophilic (primary) granules; nucleus is indented to more than half the distance from the farthest nuclear margin; elongated and horseshoe-shaped nucleus; if lobulated, the bridge or isthmus between the lobes must be wide enough to have two distinct parallel dark margins with light nuclear chromatin between; has blue-purple and clumped granular chromatin; no nucleolus; N/C ratio is 33-40%

neutrophil: 10-15 microns; abundant pink cytoplasm with many fine, lilac, neutrophilic (specific or secondary) granules; lobulated (segmented) nucleus with 2-5 lobes, connected by a thin filament of chromatin; the filament is so narrow that there is no visible chromatin between the two sides; filaments may be difficult to visualize due to folding or twisting of nucleus; in other areas, the chromatin is dense with no nucleolus; N/C ratio is 33%

classify cell with folded nucleus as neutrophil if: (1) margins of two adjacent lobes are completely separated; (2) width of either of the two adjacent lobes markedly narrows or converges towards the junction of the lobes (making it possible for there to be a hidden filament), or (3) the nucleus is so extensively folded that one cannot determine if a filament is present

classify cell with folded nucleus as band if: (1) elongated band form crosses over itself without any evidence of constriction to a filament; (2) only the distal tip of the nucleus is slightly bent upon itself, and (3) the hidden area in the fold between two adjacent lobes is so small and the lobe width is so thick that it is unlikely that a thin filament is present

Positive stains: neutrophils (may also stain other precursors) - CD10, CD11b, CD11c, CD12, CD13, CD14 (weak-30%), CD15, CD15s, CD15u, CD16a, CD16b, CD17, CD18, CD24, CD29 (low), CD30, CD31, CD32, CD33 (low), CD35, CD37 (low), CD43, CD45RO, CD47, CD47R (weak), CD48 (weak), CD49e, CD62L, CD63 (weak), CD64, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD68, CD69, CD83, CD84, CD85F, CD85M, CD87, CD88, CD89, CD92, CD93, CD97, CD101, CD107a, CD107b (weak), CD114, CD116, CD128a, CD128b, CD132, CD139, CD141, CD148, CD156a, CD157, CD170

Negative stains: neutrophils - CD7, CD49d, CD52 (or weak), CD56, CD60, CD81, CD102, CD226

References: CAP proficiency testing handbook, Blood Cells Mol Dis 2002;28:260 (antigenic changes during maturation)

 

Osteoblasts in bone marrow

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Along endosteal surface of bony trabeculae or along margins in marrow smears

Common in children; in adults associated with various diseases and healing biopsy sites

Bone forming cell that arises from marrow mesenchymal cells

Synthesize angiopoietin and osteopontin, which inhibit hematopoietic stem cell proliferation (Br J Haematol 2006;134:467, J Clin Invest 2006;116:1195)

When active, are plump and present on bone surface; eventually are encased within the collagen they produce and get flattened (and become osteocytes)

Synthesize and transport collagenous matrix, initiate and regulate mineralization, control removal of bone via osteoclasts, express Vitamin D receptors; activity is promoted by physical activity (Wolf’s law); express parathormone receptors (mediates the activation of osteoclasts)

Control osteoclast activity via parathyroid hormone (parathormone), PHRP (Parathyroid hormone related protein), IL-1, TNF alpha; digestion of bone by osteoclasts releases cytokines and growth factors for osteoblasts

Micro: large (25-50 microns), often oval, with abundant blue-gray cytoplasm and perinuclear hof; nucleus is round/ovoid with one or more nucleoli

Positive stains: alkaline phosphatase, estrogen receptor, parathyroid hormone, RANKL; also CD10, CD44, CD53, CD56, IL-12, IL-18, IFNgamma (Biosci Rep 2006;26:39); cells in culture express CD11b, CD13, CD16, CD20, CD23, CD25, CD34, CD44, CD54, CD80, CD86, HLA-DR (Cell Physiol Biochem 2002;12:359)

Negative stains: cells in culture are negative for CD3, CD14, CD15, CD45, CD68

EM: resemble fibroblasts due to well developed rough endoplasmic reticulum and Golgi

 

Osteoclasts in bone marrow

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Along endosteal surface of bony trabeculae or along margins in marrow smears

Common in children; in adults associated with various diseases

Involved in bone resorption due primarily to remodeling and not calcium homeostasis

Derived from monocyte fusion

Activated by parathyroid hormone and by cytokines RANKL and macrophage colony-stimulating factor (Arthritis Res Ther 2006;8:201)

Osteoclasts use their ruffled borders (with villous extensions) to bind to matrix adhesion proteins, produce resorption pits/bays (shallow concavities) called Howship’s lacunae; plasma membrane forms a seal with bone; osteoclast acidifies extracellular area, which solubilizes the mineral and releases enzymes which dissolve the matrix

Micro: very large (up to 100 microns), multinucleated (2-12 nuclei) giant cells associated with bone surface; have abundant, blue-purple-pale pink cytoplasm containing many fine, red-purple granules; have multiple, relatively uniform but widely separate nuclei, each with one nucleolus and dense chromatin

Positive stains: CD13, CD31, CD51 (Histochemistry 1991;96:169), CD53, CD54, CD61, CD63, CD68, CD115, acid phosphatase, microphthalmia transcription factor, TRAP

EM: numerous mitochondria, rare lysosomes; ruffled edge in area of cell membrane is associated with bone resorption

References: Keio J Med 2003;52:1 (differentiation), Wikipedia

 

Plasma cells in bone marrow

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Usually less than 1% of marrow cells

Often perivascular and in particle crush specimens

Produces and secretes antibodies

Plasmablast: precursor to plasma cell

Micro: ovoid cells with abundant deep blue cytoplasm and perinuclear hof, eccentric nucleus with coarse chromatin and clockface (cartwheel) pattern; may have occasional binucleated cells; no nucleoli

Positive staining: CD38, CD138, CD19; also CD9, CD27, CD28, CD30 (some), CD31, CD32 (some), CD43, CD45, CD79a, CD79b, hc2, MNDA, PCA-1

Negative staining: CD20, CD21, CD22, CD24, CD37, CD40, CD56 (positive in myeloma, Am J Path 2002;160:1293), CD72 (Am J Hematol 1992;41:151)

EM: prominent Golgi and rough endoplasmic reticulum

 

Age related changes in bone marrow

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Cellularity averages 79% at ages 0-9 years vs. 50% at ages 30-69 vs. 29% at ages 70-79

With aging, hematopoietic tissue is replaced by fat

Deeper medullary areas are typically more cellular than subcortical areas

B cell production declines with age (Curr Opin Immunol 2005;17:463), although the relative abundance of pro-B, pre-B, immature, naive, and mature B cells usually does not change appreciably between ages 24 and 88 years; occasional patients have exceptionally low numbers of lymphocyte precursors  (Blood 2003;101:576)

Hypocellularity in elderly marrow may be due to increased apoptosis (Mech Ageing Dev 2000;117:57)

 

 

Bone marrow biopsy and aspirate smear

Technique

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Trephine: A surgical instrument having circular, sawlike edges, used to cut out disks of bone, usually from the skull

Trephine biopsy: biopsy of a portion of bone containing marrow

Jamshidi-type biopsy needle: recommended, usually 11 gauge

Other needles: single use needle (Biomed Instrum Technol 2005;39:391), for neonates (Br J Haematol 1999;107:458)

Recommended to use separate needles for trephine biopsy and aspiration (J Clin Pathol 2007;60:212); can use same skin incision but sites a few millimeters apart

Bilateral biopsies useful if disease is likely focal (lymphoma or metastatic tumors, Cancer 2002;94:1522)

Sites: posterior superior iliac spine

Uses: to evaluate leukemia, lymphoma and lymphoproliferative disorders, myeloproliferative or myelodysplastic disorders, metastatic disease, aplastic anemia and other hematologic conditions, infectious and metabolic disorders; also to evaluate post-chemotherapy cellularity and post bone marrow transplant engraftment

Should be accompanied by aspirated marrow smears and particle crush preparations, and by touch imprints from trephine biopsy

Complications: adverse events in 0.8% (J Clin Pathol 2005;58:406); major complication is hemorrhage / hematoma (apply pressure bandage to biopsy site to prevent; obtain coagulation consultation if patient has bleeding disorder or is on anticoagulants)

Risk factors for hemorrhage are myeloproliferative disorder, aspirin, other putative platelet dysfunction and thrombocytopenia

Processing of trephine biopsy: (1) make imprints from trephine biopsy by gently touching glass slide to specimen; (2) possibly freeze part of trephine biopsy for molecular studies (J Clin Pathol 2006;59:1111), (3) fix tissue in formalin, B5 or Zenker’s; (4) decalcify for 45-60 minutes; (5) embed in paraffin; (6) section at 3-4 micron intervals, saving tissue for possible special stains and molecular studies

Some laboratories prefer plastic embedding, which may provide superior cytologic detail

Processing of aspirate: place some aspirate in EDTA, make smears at bedside with remainder; make smears from buffy coat (nucleated cell layer) and particles

Techniques: ultrasound decalcification may allow more successful FISH, PCR and RT-PCR (AJSP 2006;30:892)

Note: FISH can be performed on tissue imprints, cytopreps, or bone marrow aspirate smears (J Clin Pathol 2005;58:629)

Note: trephine biopsy may also reveal bone disorders not the reason for the biopsy

 

Steps

1. position patient properly

2. prepare and sterilize the site with iodine solution

3. anesthetize skin and bone with lidocaine

4. nick the skin with a blade to facilitate needle insertion

5. insert bone marrow aspiration needle

6. aspiration needle in place with trochar (white) partially withdrawn

7. syringe for aspirating bone marrow is in place

8. part of aspirate is put into EDTA tube to prevent clotting

9. part of aspirate is put on slide to pick particles

10. aspirate smear is made using another slide

11. biopsy needle is inserted at same site

12. feel the give of the needle as it enters the cortex

13. withdraw the trochar from the needle

14. after another centimeter push, rotate the needle to cut the end of the specimen

15. withdraw the needle with the specimen inside the needle

16. push the biopsy specimen from the narrow end to the hub end with the trochar

17. place the biopsy specimen in fixative (such as Zenker’s) for decalcification and processing

 

Micro: bone marrow biopsies are helpful to determine cellularity and presence of fibrosis; purple granular deposits that impair evaluation of touch preparations are due to cartilage in biopsy, and are more common in children (J Clin Pathol 2003;56:883)

References: Medical College of Virginia illustrated guide, eMedicine, J Clin Pathol 2006;59:903 (Hammersmith protocol), J Clin Pathol 2005;58:897 (epoxy resin embedding) , Wikipedia

 

Bone marrow - sample report

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Clinical history: indicate hematologic and other diagnoses, and indication for bone marrow

 

Peripheral blood smear:

List laboratory data (WBC, Hemoglobin, Hematocrit, MCV, Platelet count, RDW, other)

Give microscopic description (number, size, shape, other abnormalities) of red blood cells, white blood cells and platelets

Peripheral smear interpretation

 

Bone marrow biopsy and aspirate:

Biopsy or aspiration site

Cellularity (% and whether hyper-, normo- or hypocellular for age)

Presence or not of trilinear maturation

Adequacy of specimen

Indicate myeloid:erythroid ratio (M:E ratio), and whether normal, increased or decreased

Myeloid cells - normal or abnormal maturation; indicate if excess blasts, abnormal localization of immature precursors

Erythroid cell - normal or abnormal maturation

Provide differential of myeloid and erythroid elements, based on counting 200-500 cells in aspirate smear

Megakaryocytes - normal or abnormal numbers and morphology

Lymphocytes - lymphoid aggregates, hematogones, other abnormalities

Plasma cells - normal or increased, list abnormalities

Histiocytes - normal or increased, list hemophagocytosis or other abnormalities

Iron stores - quantitate (none, 1+, 2+, 3+ or 4+; indicate if normal, increased or decreased); indicate if ringed sideroblasts are present and how many

Fibrosis - present or not; normal or abnormal

Bone - list any abnormalities present

Cytogenetic findings

Molecular findings

Immunohistochemistry findings

Other - presence of metastatic tumor or not, other findings

Bone marrow interpretation / diagnosis - correlate with clinical history, cytogenetics, molecular or immunohistochemistry findings, peripheral blood smear

 

Mandatory features to report for accreditation purposes by the American College of Surgeons Committee on Cancer

Specimen type

Adequacy (adequate, limited, unsatisfactory)

Results of special stains or other studies, if performed

Results of cytogenetics, if performed

Diagnosis (WHO classification)

 

References: College of American Pathologist protocols, Archives 2006;130:1825 (synoptic reporting), how to examine aspirate smears, J Clin Pathol 2001;54:737 (trephine biopsy-Dr. Bain’s protocol), J Clin Pathol 2001;54:657 (aspirate smear-Dr. Bain’s protocol), AFIP Third Series-Bone Marrow-Appendix (specimen processing)

 

Bone marrow - routine stains

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Note: reactivity varies by type of fixation

 

CD45/LCA - lymphocytes

Kappa and lambda light chains - to determine clonality or not of lymphocytes and plasma cells

Myeloperoxidase and lysozyme (for AML subtyping)

B cell markers CD20, CD79a and possibly Pax5

T cell marker CD3

TdT (for ALL)

Iron stains (Prussian blue) on aspirate smears and trephine biopsies (J Clin Pathol 2005;58:269)

   0=absent, 1=trace; 2=present (sparse), 3=present (moderate), 4=abundant (abnormal)

Possibly reticulin stain or Masson trichrome stain

 

Also CD34 (blasts and blood vessels), Factor VIII (blood vessels), CD61 (megakaryocytes), CD68 (macrophages), Hemoglobin A (for AML-M6) and glycophorin (for erythroid cells)

 

Note: to assess iron stores, should examine at least 7-9 particles (J Clin Pathol 2004;57:1038); absence of staining iron is not diagnostic of iron deficiency anemia (Ann Hematol 2001;80:166)

 

 

Alterations in cellularity of bone marrow

Bone marrow cellularity-general

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Best evaluated on biopsy sections or imprints; particle sections are next best choice; aspirate smears may be difficult to evaluate

Theoretically could use automated hematology analyzer or flow cytometry (Ann Clin Lab Sci 2004;34:307)

Iliac crest may not be representative if radiotherapy or other local insults

Varies from 80% (children) to 55% (age 30) to <50% (age 60+)

References: biopsy sections, imprints and aspirate smears are equally reliable - Am J Hematol 1986;22:381, Indian J Pathol Microbiol 1989;32:186

 

Amegakaryocytic thrombocytopenia and bone marrow
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Also called Amega or CAMT (congenital amegakaryocytic thrombocytopenia)

Rare; autosomal recessive; usually diagnosed in early childhood

Presents with isolated nonimmune thrombocytopenia with decreased marrow megakaryocytes and high serum thrombopoietin levels

May have physical anomalies (Br J Haematol 2005;131:636)

Often evolves to aplastic anemia, myelodysplastic syndrome or leukemia

Diagnosis: biallelic mutations in c-mpl (thrombopoietin receptor) at 1p34 (Blood 2001;97:139)

Prognosis: missense mutations are associated with milder or delayed course vs. loss of function mutations (Hum Mutat 2006;27:296)

Case reports: due to anti-c-mpl antibody in woman with systemic sclerosis (Arthritis Rheum 2003;48:1647)

Treatment: stem cell transplantation

References: OMIM 604498

 

Aplastic anemia in bone marrow

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Also called hypoplastic marrow

Rare acquired or congenital state of bone marrow failure with cytopenias and bone marrow hypocellularity

May be due to immune mediated destruction of marrow hematopoietic progenitor/stem cells, mediated via Th1 T cells (BMC Genomics 2006;7:263)

Children may acquire monosomy 7 (AJCP 2006;126:925)

Presence of substance P in B cells may predict progression to acute leukemia (J Clin Pathol 2006;59:935)

Acquired causes: idiopathic or due to drugs (carbamazapine and valproic acid, Epilepsia 2006;47:1232), chemicals, viruses, ionizing radiation; may be associated with thymoma, lupus (J Clin Rheumatol 2001;7:377); in children, cases are considered acquired only after excluding other causes; in adults, aplastic anemia is assumed to be acquired

Congenital causes (see below): dyskeratosis congenital, Fanconi’s anemia, Schwachman-Diamond syndrome, TAR syndrome

Laboratory: variable pancytopenia

Treatment: immunosuppressive therapy, bone marrow transplantation

Case reports: progressing to AML-M0 (J Clin Pathol 2005;58:670), 17 year old man with severe marrow aplasia, associated with trisomy 1q (Cancer Genet Cytogenet 2006;169:73)

Micro:

most severe - marrow space filled with adipose tissue with occasional lymphocytes, plasma cells, mast cells and hemosiderin-laden macrophages (reflecting increased iron stores from repeat transfusions); prominent sinuses and capillaries; no/rare hematopoietic cells

less severe - marrow space has increased adipose tissue and scattered small clusters of erythroblasts, granulocytes and megakaryocytes

rarely has polymorphous, non-clonal lymphoid aggregates; may also be associated with malignant thymoma

DD: newly diagnosed acute leukemia or myelodysplasia (CD34+ blasts and immature myeloperoxidase positive granulocytes are present, AJCP 1997;107:268, Leukemia 2006;20:458)

References: Hematology Am Soc Hematol Educ Program 2004:318, Wikipedia, OMIM 609135

 

Diamond-Blackfan anemia and bone marrow

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Rare; pure red blood cell aplasia

90% are diagnosed in first year of life

May have short stature, abnormal thumbs

May progress to pancytopenia

Laboratory: macrocytic anemia with elevated fetal hemoglobin and increased red blood cell adenosine deaminase

Treatment: 15-25% undergo remission; also corticosteroids; if don’t respond, use red blood cell transfusions and iron chelation; also metoclopramide (Blood 2002;100:2687