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![95% Range Mean[12] Mean[11]
Myeloblasts 0–3 1.4 0.4
Promyelocytes 3–12 7.8 13.7[*]
Myelocytes (neutrophil) 2–13 7.6 –
Metamyelocytes 2–6 4.1 –
Neutrophils 22–46 32.1M; 37.4W 35.5
Myelocytes (eosinophil) 0–3 1.3 1.6
Eosinophils 0.3–4 2.2 1.7
Basophils 0–0.5 0.1 0.2
Lymphocytes 5–20 13.1 16.1
Monocytes 0–3 1.3 2.5
Plasma cells 0–3.5 0.6 1.9
Erythroblasts[†] 5–35 28.1M; 22.5W 23.5
Megakaryocytes 0–2 0.5
Macrophages 0–2 0.4 2.0
Normal ranges for differential counts on aspirated bone marrow (500 cells should be counted)](https://image.slidesharecdn.com/bmevaluationpart1pbsbma-231223041249-22f30be9/85/Bone-Marrow-evaluation-EVALUATION-PBS-BMA-pptx-29-320.jpg)
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Bone Marrow evaluation EVALUATION (PBS+BMA).pptx
- 1.
- 3. When? Indications for BMexamination • Haematological abnormalities that cannot be explained by available clinical and laboratory data. • 3 main indications : – Diagnostic purposes – Staging for malignant diseases – Monitoring • A good indication is essential for an accurate diagnosis.
- 4. Indication of marrowexamination: • Investigation of unexplained peripheral blood abnormalities • Diagnosis of suspected primary hematopoietic neoplasm • Infectious disease work-up if other systemic investigations non-contributory • Evaluation of suspected constitutional disorder • Assess for storage disorder • Assess for involvement by metastatic neoplasm • Staging of lymphoma • Ongoing monitoring after therapy
- 5.
- 7. Importance of touch/Imprint smear • Gives cytological details when aspirate is not obtained.(fibrotic or inaspirable) • Shows more neoplastic cells than aspirate. • Can show marrow infiltration , not seen in aspirate • Important for potential ancillary techniques (e.g., cytochemistry, FISH, iron stains) • Particularly if BM aspirate is hemodilute/dry tap • ≥ 3 air-dried imprints Be gentle, be delicate ! Do NOT CRUSH !
- 8. Importance of ClotSection • Assessment of bone marrow cellularity. • For detecting granuloma and tumor infiltrates complementary to biopsy. • No decalcification associated nucleic acid or protein damage. • Immunostains may also be performed.
- 9. BM Aspirate orTrephine Biopsy
- 10. BM aspirate ortrephine biopsy
- 11. How to collectthese specimens ? • Anatomic sites • Collection procedure • Adequacy
- 12. Anatomic sites Posterior iliaccrest aspiration is suitable for children, infants and many neonates. Tibial aspiration is suitable for very small babies but has no advantages over iliac crest aspiration in older infants.
- 13. BM Aspirate Adequacy •Obtain at least 3 particles per slide • Obtain at least 4 slides – 2 for routine staining – 1 for iron (as needed) – 1 or more for potential ancillary tests (e.g.,cytochemistry, FISH) • Not crushed/Not too thick/Not clotted • Allow to dry quickly • Dry tapes represent 2-7% of the cases • If an adequate aspirate has not been possible, considerations should be given to preparing touch imprints of the core biopsy prior to placing it in fixative.
- 14. BM Trephine Biopsy •11-gauge needle AT LEAST • If osteopenic, a 8-gauge needle allows the collection of an intact core biopsy with minimal crush artifact • 13-gauge biopsy needle for pediatric patients Adequate core biopsy: • At least 1.6 cm to 2 cm long • Exclusive of cortical bone, cartilage, or periosteum • Free of crush artifact or interstitial hemorrhage or fragmentation
- 15. BM Evaluation • Clinicalhistory • Clinical impression • Laboratory data • Morphology • Immunophenotype • Genetic features
- 18. • Erythrocytes remainmacrocytic from the first 11 weeks of gestation until day 5 of postnatal life. Peripheral blood film for a normal newborn demonstrating a normal lymphocyte, macrocytes, polychromasia, and one nucleated red blood cell (×1000).
- 19. • Orthochromic normoblastsfrequently are observed in the full-term infant on the first day of life but disappear within postnatal days 3-5. • NRBCs may persist longer than a week in immature infants. • The average number of NRBCs ranges from 3 to 10 per 100 WBCs in a normal full-term infant to 25 NRBCs per 100 WBCs in a premature infant. • The presence of NRBCs for more than 5 days suggests hemolysis, hypoxic stress, or acute infection.
- 21.
- 22. Evaluating Aspirate Smears Screenall slides at low power (4 x) • Evaluate cellularity, look for abnormal infiltrate • Find good areas of slides for counting • Evaluate number of megakaryocytes Count cells from several areas (100 x) • If spicules are lacking, accurate counts may be obtained at edges of smears • If cells are mainly neutrophils and lymphocytes, smears may represent peripeheral blood rather than marrow The preparation can be considered satisfactory only when marrow particles and free marrow cells can be seen in stained films.
- 23. Normocellular bone marrowon aspirate High cellularity oaf 95-100% Cellularity of 40-50% Hypocellular bone marrow (10-20%)
- 24. Epidemiology • Dramatic age-relatedvariations in BM cellularity -Highest cellularity in neonates: ~ 80-100% -Gradual decline in cellularity with age • BM cellularity in elderly patients: ~ 20-30%
- 25. Mean values (observedrange) for bone marrow cells in healthy infants and children
- 26. • The bonemarrow at birth has major erythroid and myeloid components with few lymphocytes and very few plasma cells. • The percentage of erythroid cells declines steeply in the first weeks. • The percentage of lymphocytes increases during the first month and remains at a high level until 18 months of age (up to 40-50% in young children) • Numbers decline during childhood and in adults they do not generally comprise more than 15–25% of nucleated cells, unless the marrow aspirate has been considerably diluted with peripheral blood. Evaluating Aspirate Smears
- 27. • Megakaryocytes ofhaematologically normal neonates and infants, up to the age of 10 months, are smaller and more homogeneous in size than those of older children and adults. • In children above the age of 2 years, the proportions of different cell types do not differ greatly from those in normal adult bone marrow. • Osteoblasts are uncommon in bone marrow aspirates of healthy adults but, when present, often appear in small clumps. They are much more numerous in the bone marrow of children and adolescents. Evaluating Aspirate Smears
- 28. Osteoclast: note thehighly granular cytoplasm and the multiple nuclei (2- 100)which are uniform in size and have indistinct, medium - sized, single nucleoli. OSTEOBLAST: have basophillic cytoplasm,extruding nucleus and regular chromatin with 1-4 nucleoli. Can be distinguished from plasma cells by their larger size and the position of the Golgi zone, which is not immediately adjacent to the nucleus. Mature megakaryocyte having loblated nucleus and pink granular cytoplasm.platelets are formed by budding of the cytoplasm which are shed in the circulation.
- 29. 95% Range Mean[12]Mean[11] Myeloblasts 0–3 1.4 0.4 Promyelocytes 3–12 7.8 13.7[*] Myelocytes (neutrophil) 2–13 7.6 – Metamyelocytes 2–6 4.1 – Neutrophils 22–46 32.1M; 37.4W 35.5 Myelocytes (eosinophil) 0–3 1.3 1.6 Eosinophils 0.3–4 2.2 1.7 Basophils 0–0.5 0.1 0.2 Lymphocytes 5–20 13.1 16.1 Monocytes 0–3 1.3 2.5 Plasma cells 0–3.5 0.6 1.9 Erythroblasts[†] 5–35 28.1M; 22.5W 23.5 Megakaryocytes 0–2 0.5 Macrophages 0–2 0.4 2.0 Normal ranges for differential counts on aspirated bone marrow (500 cells should be counted)
- 30.
- 31. How To ExamineA Trephine Section 10X Evaluation •Evaluate bone trabeculae •Look for gross abnormalities •Evaluate marrow cellularity •Screen slide for adequacy of sample 20X Evaluation •Marrow architecture 40X Evaluation •Morphology of erythroid, myeloid elements and Megakaryocytes Have an organized approach! 1. Assessment of architecture and cellularity 2. Assessment of accessory structures 3. Distribution of cellular elements (topography) 4. Assessment of cell morphology
- 32. Adequacy: Quality of marrowmore important than length of core -Adequate sampling: 1.5 cm (1.6-2cm) -H&E (at least 2 levels) and reticulin stains •Avoid Crushed areas Subcortical fatty marrow Intertrabecular hemorrhage This BM core biopsy is suboptimal for evaluation with cartilage on both ends of the biopsy flanking minimal subcortical BM. Subcortical marrow is often not representative of the marrow as a whole, and a statement to this effect should be issued in the final report.
- 33. • A biopsyspecimen containing at least five or six intertrabecular spaces is desirable, not only for an adequate assessment of cellularity but also to give a reasonable probability of detecting focal bone marrow lesions. • Ideally this requires a core of 2–3 cm in length. A core length of at least 0.5 cm has been advised in children but one study found 1.0 cm was necessary to avoid a high rate of non‐interpretable specimens. Adequacy:
- 34. • A sectionof a trephine biopsy specimen of adequate size from a patient with Hodgkin lymphoma showing only a small area of infiltration at one end of the specimen, illustrating how a small biopsy may miss focal lesions. at least 16 mm Adequacy:
- 35. Common artifacts inmarrow trephine biopsy • Washed off of marrow space- technical artifact leading to inconclusive findings • Aspiration artifact • Crushed artifact
- 36. 1. ASSESMENT OFARCHITECTURE AND CELLULARITY HOW TO EXAMINE A TREPHINE SECTION
- 37. • Cellularity variesaccording to location and age • Hypocellular in subcortical marrow (three intertrabecular space) Evaluation of cellularity
- 38. Evaluation of cellularity •Average cellularity in the bone marrow of children, assessed on core biopsy or clot sections: • 80% at 2 years • 69% at 2–4 years • 59% at 5–9 years • around 60% thereafter • 20-40% in elderly
- 40. Architecture Myeloid cells Paratrabecular Immature myeloidelements are adjacent to bone trabeculae Mature cells towards centre Erythroid cells Centre in colonies Megakaryocytes Centre around sinusoids are singletons (<3 together)
- 42. 2. ASSESMENT OFACCESSORY STRUCTURES HOW TO EXAMINE A TREPHINE SECTION
- 43. Accessory structures • Bothosteoclasts and osteoblasts normally evident along bony trabeculae in specimens from pediatric patients. • Incomplete ossification typical in infants/young children • Evidence of bony remodeling normal in children, abnormal in middle- aged to elderly adults.
- 44. Trabeculae consists oflamellar bone Osteoblasts along endosteal surface Ossifying cartilage. Osteocytes within lacuna (arrow head). Osteoclast
- 45. BM trephine biopsysection from a child showing endochondrial ossification; a bony spicule with a core of cartilage is lined by osteoblasts. Cartilage cells are dispersed singly or in small groups and are aligned into columns. BM trephine biopsy section from an adult showing cartilage adjacent to the cortex. By contrast with childhood appearances, a well‐defined layer of cortical bone separates this cartilage from the bone marrow.
- 46. HOW TO EXAMINEA TREPHINE SECTION 3. DISTRIBUTION OF CELLULAR ELEMENTS (TOPOGRAPHY) 4. ASSESMENT OF CELL MORPHOLOGY
- 47. Diagrammaticrepresentation of thetopography of normal bonemarrow. Osteoclasts, osteoblasts, myeloblasts, and promyelocytes are adjacent to the spicule of bone. Deeper in the intertrabecular space are maturing cells of neutrophil lineage, erythroid islands with a central macrophage, and interstitial lymphocytes. Eosinophils and their precursorsare apparently randomly scattered, plasma cells are interstitial or form a sheath around capillaries, and megakaryocytes abut on a sinusoidat one extremity of the cell.
- 48. Marrow elements 1-Hematopoietic component • Erythroidseries • Myeloid Series • Megakaryocyticseries • Lymphoid cells t 2-Stromal componen • Macrophage • Fat cells • Fibroblast • Delicate fiber network • Blood vessels(mostly sinusoid) • Nerve bundles • Trabecular bone • Extracellular matrix • Osteoblast and Osteoclast Stromal component facilitate maintenance of HSC and support differentiation and maturation of the progenitors These two components not only coexist but closely interact with each other
- 49. Special Stains onMarrow -Giemsa(routine) • Differentiates cytoplasm of erythroid elements and myeloid elements • Plasma cells, mast cells more obvious -Reticulin (special) • Evaluates bone marrow fibrosis • clues: crush artifact, dry tap, enlarged vascular spaces -PAS stains highlighting megakaryocytes, fungal organism in institutions with large populations of immunosuppressed patients -Silver stain for assessment of reticulin fibrosisIncases of myeloproliferative neoplasms -Not recommend the routine Perl stain of the core biopsyif a satisfactory marrow aspirate cannot be obtained, iron stains of the clot or biopsysections>>>potential false-negative results.
- 50. Reticulin fibrosis Reticulinis normally present in BM ‘Normal’ reticulin • Around blood vessels • Short wisps • Day-to-day variation in silver • Underdone reticulin stain • - No staining around blood vessels -> repeat • Overdone reticulin stain • - Staining of cell membranes, cell nuceli stained darkly - > repeat or discount • Collagen fibrosis Most notably seen in association with myeloproliferative neoplasms Detected with trichrome stain Not normally present in BM Unlikely to be reversible 2 types of BM fibrosis: - Reticulin fibrosis - Collagen fibrosis
- 52. grade 0 grade1 grade 2, grade 3 grade 4
- 53. Iron Assessment • Typicallyassess 2 types of iron stores • Sideroblastic iron (a.k.a. erythroid iron) • Macrophage iron (a.k.a. storage iron) • Assessment of storage iron requires that an adequate number of fragments are obtained • A minimum of seven fragments in one or more bone marrow films need to be examined in order to state with reasonably reliability that bone marrow iron is absent .
- 54. Erythroid iron Preferred specimen:Spicular, unstained, air-dried BM aspirate smear/touch preparation containing adequate red blood cell precursors Normal: 20-50% of red blood cell precursors demonstrate 1-3 small cytoplasmic granules Abnormal – Abnormal size (large), shape (chunky), or location (perinuclear, ring) of granules – Ring sideroblast is defined by WHO as 1/3 of red blood cell nucleus tightly surrounded by 5 or more iron granules. Storage iron Preferred specimen: Spicular, unstained, air-dried BMA smear Grading may be semiquantitatively assessed (normal, none, increased, decreased) or assessed by grading scale (0-6) In routine practice, grading as absent, scanty, reduced, normal or increased is also a practical approach. Need adequate positive control for accuracy May have heterogeneous iron deposition in BMA Formalin fixation/histologic processing/decalcification may interfere with staining of iron stores (ferritin)
- 55. Grading of bonemarrow storage iron 0 No stainable iron 1+ Small iron particles just visible in reticulum cells using an oil objective 2+ Small, sparse iron particles in reticulum cells, visible at lower power 3+ Numerous small particles in reticulum cells 4+ Larger particles with a tendency to aggregate into clumps 5+ Dense, large clumps 6+ Very large clumps and extracellular iron
- 56. Perl’s stain sideroblast Perl’s stain Macrophagewith iron Ringed sideroblast
- 57.
- 58. Majorpatternsof theIHCstainingin formalin-fixedandparaffin-embedded tissuesections
- 59. MPO showing distributionof normal myeloid cells CD79a showing distribution of normalB cells CD20 CD3 showing distribution of normalT cells CD34 showing positivity of endothelial cells CD34showing very occasional positive cells
- 60. Pathologic Key FeaturesOf Pediatric Bone Marrow Interpretation • Interpretation of pediatric bone marrow samples requires that the pathologist integrate clinical, morphologic, and ancillary study results to arrive at the correct diagnosis. • The morphology of normal pediatric bone marrow overlaps significantly with that of adults.
- 61. • The evaluationof pediatric bone marrow poses specific challenges when compared with the general adult population. • These challenges stem in part from the higher likelihood of congenital disorders with hematopoietic manifestations, some of which may give rise to hematologic malignancies. Pathologic Key Features Of Pediatric Bone Marrow Interpretation
- 62. • Congenital syndromesand florid reactive processes must always be considered in the differential diagnosis of hematologic malignancies in the pediatric population • Recognition of the general pathologic pattern (hypercellular, hypocellular, or single-lineage defect) aids in narrowing the differential diagnosis when interpreting bone marrow samples from pediatric patients. Pathologic Key Features Of Pediatric Bone Marrow Interpretation
- 63. • Two featuresare significantly different and may affect bone marrow interpretation: the overall cellularity and the presence of benign lymphoid precursors (hematogones), which may be markedly increased in some patients. • Also it is important to realize that a large proportion of the benign and malignant disorders diagnosed during childhood are likely to be related to underlying genetic abnormalities, sometimes as the first manifestation of these defects Pathologic Key Features Of Pediatric Bone Marrow Interpretation
- 64. • Finally, certaintypes of malignant neoplasms with partially overlapping blastic morphologic features (so called “small blue-cell tumors”), but with vastly different biology requiring distinct therapeutic approaches, occur relatively frequently in young patients. Pathologic Key Features Of Pediatric Bone Marrow Interpretation
- 65. Pitfalls Of PediatricBone Marrow Interpretation • Benign B-cell precursors (hematogones) can occur in significant numbers in the bone marrow of children with cytopenias of nonneoplastic causes and may be mistaken for involvement by B- lymphoblastic leukemia.
- 66. Pitfalls Of PediatricBone Marrow Interpretation • Benign B-cell precursors (hematogones) often represent a significant proportion of the cells in hypocellular marrow aspirates from acute megakaryoblastic leukemia (with marrow fibrosis) potentially leading to a misdiagnosis of B lymphoblastic leukemia.
- 67. Pitfalls Of PediatricBone Marrow Interpretation • B-lymphoblastic leukemias with a CD45-,CD20-, CD99+ immunophenotype may be mistaken for Ewing sarcoma, especially in small bone or bone marrow biopsies
- 68. Pitfalls Of PediatricBone Marrow Interpretation • Hemophagocytic syndromes may be associated with peripheral T-cell lymphomas; diagnostic lymphoma cells are sometimes present in small numbers in the bone marrow aspirates and may be missed.
- 69. Pitfalls Of PediatricBone Marrow Interpretation • Variably prominent populations of polyclonal CD8+ T lymphocytes with immunophenotypic aberrancies may be present in familial hemophagocytic histiocytosis; they may be mistaken for peripheral T- cell lymphoma.
- 70. Pitfalls Of PediatricBone Marrow Interpretation • Parvovirus B19 infections occurring in patients with constitutional red cell disorders may cause erythroid hyperplasia and significant dyserythropoiesis, resembling congenital dyserythropoietic anemia.
- 71. • Acute lymphoblasticleukaemia can be confused with small cell tumors of childhood, e.g. neuroblastoma, Ewing’s tumour and other primitive neuroectodermal tumours (PNET), rhabdomyosarcoma, medulloblastoma and retinoblastoma. • It should be noted that leukaemic lymphoblasts may fail to express CD45, that neuroendocrine tumors can express PAX5 and that blast cells in a large proportion of ALL patients express CD99, an antigen commonly expressed in Ewing’s tumour/PNET. Pitfalls Of Pediatric Bone Marrow Interpretation
- 72. Pitfalls Of PediatricBone Marrow Interpretation • Terminal deoxynucleotidyl transferase (TDT) in addition to ALL, positive in up to 15% of AML cases. • It has been reported to be positive in a proportion of cases of medulloblastoma and occasionally in other small cell tumors of childhood
- 73. • Small celltumors of childhood, e.g. those with heavily vacuolated cells such as alveolar rhabdomyosarcoma, have been misdiagnosed and even treated as Burkitt lymphoma Pitfalls Of Pediatric Bone Marrow Interpretation
- 74. Pitfalls Of PediatricBone Marrow Interpretation • It should be noted that, in children, apparent aplastic anemia may represent an aplastic presentation of ALL.
- 75. Pitfalls Of PediatricBone Marrow Interpretation • Acute megakaryoblastic leukemia (AMKL), which may yield clusters of cohesive cells mimicking metastatic solid tumors in some cases • Some lymphoblastic leukemias with near-tetraploid DNA content may also show very large blasts reminiscent of solid tumor cells.
- 76. Pitfalls Of PediatricBone Marrow Interpretation • Some leukemias (especially hyperdiploid B-ALL and AMKL) may be accompanied by significant marrow fibrosis, leading to a paucity of neoplastic cells available for ancillary studies in the aspirate samples
- 85. CONCLUSION • Integration ofclinical, morphologic, immunophenotypic, genetic, and other biologic features is mandatory to define specific disease entities • The relative contribution of each feature varies, depending on the case. • Make your cytologists/pathologists/geneticians good ! by providing them relevant clinical information's and optimal samples.
Editor's Notes
- #5 32% of carcinomas and 23% of lymphomas were positive on only one side
- #20 4% to 6% at birth. Reticulocytosis persists for about 3 days after birth, then declines abruptly to 0.8% reticulocytes on postnatal day 4 to 7. At 2 months, the number of reticulocytes increases slightly, followed by a slight decline from 3 months to 2 years, when adult levels of 0.5% to 1.5% are attained.
- #21 Low numbers of blast cells may be present (less than 5% at term, slightly higher earlier in gestation).
- #55 It is usual to base assessment of iron stores mainly on intracellular iron since iron stains are prone to artefactual deposits and it can be difficult to distinguish between extracellular iron and artefact.