Bone Marrow-nonneoplastic
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Last revised 6 February 2007
Copyright © 2001-2007 PathologyOutlines.com, Inc.
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
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
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Embryologic development of bone marrow
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
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
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
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
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 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
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
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
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
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
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)
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
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
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
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
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
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
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)
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
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
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
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