Bone Marrow evaluation EVALUATION (PBS+BMA).pptx

Bone marrow pathology

1. BONE MARROW EXAMINATION

2. When? Indications for BM examination 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 .

3. Indication of marrow examination : 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

4. What specimens to collect ?

5. 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 !

6. Importance of Clot Section 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.

7. BM Aspirate or Trephine Biopsy

8. BM aspirate or trephine biopsy

9. How to collect these specimens ? Anatomic sites Collection procedure Adequacy

10. Anatomic sites Posterior iliac crest 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.

11. 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.

12. 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

13. BM Evaluation Clinical history Clinical impression Laboratory data Morphology Immunophenotype Genetic features

14. Erythrocytes remain macrocytic 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).

15. Orthochromic normoblasts  frequently 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.

16. Embryonic/fetal HP sites Yolk sac: Earliest site Dorsal aorta: Early gestation Liver/spleen : Mid gestation BM : Late gestation Postnatal HP - Long bones, vertebrae, pelvis early in life - Pelvis , vertebrae: Late childhood, adults

17. Bone Marrow Aspiration

18. Evaluating Aspirate Smears Screen all 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.

19. Normocellular bone marrow on aspirate High cellularity o a f 95-100% Cellularity of 40-50% Hypocellular bone marrow (10-20%)

20. Epidemiology Dramatic age-related variations in BM cellularity - Highest cellularity in neonates: ~ 80-100% - Gradual decline in cellularity with age BM cellularity in elderly patients: ~ 20-30 %

21. Mean values (observed range) for bone marrow cells in healthy infants and children

22. The bone marrow 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

23. Megakaryocytes of haematologically 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

24. O steoclast : note the highly 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.

25. 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 32.1 M ; 37.4 W 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 28.1 M ; 22.5 W 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 )

26. BM Evaluation Bone Marrow Biopsy

27. How To Examine A Trephine Section  10X Evaluation Evaluate bone trabeculae Look for gross abnormalities Evaluate marrow cellularity Screen slide for adequacy of sample  20 X Evaluation Marrow architecture  40 X 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

28. Adequacy : Quality of marrow more 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.

29. A biopsy specimen 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 :

30. A section of 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 :

31. Common art i facts in marrow trephine biopsy Washed off of marrow space- technical art i fact leading to inconclusive findings Aspiration art i fact Crushed art i fact

32. 1. ASSESMENT OF ARCHITECTURE AND CELLULARITY HOW TO EXAMINE A TREPHINE SECTION

33. Cellularity varies according to location and age Hypocellular in subcortical marrow (three intertrabecular space) Evaluation of cellularity

34. 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

35. Architecture Myeloid cells Paratrabecular Immature myeloid elements are adjacent to bone trabeculae Mature cells towards centre Erythroid cells Centre in colonies Megakaryocytes Centre around sinusoids are singletons (<3 together)

36. 2. ASSESMENT OF ACCESSORY STRUCTURES HOW TO EXAMINE A TREPHINE SECTION

37. Accessory structures Both osteoclasts 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.

38. Trabeculae consists of lamellar bone Osteoblasts along endosteal surface Ossifying cartilage. Osteocytes within lacuna (arrow head). O steoclast

39. BM trephine biopsy section 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.

40. HOW TO EXAMINE A TREPHINE SECTION 3. DISTRIBUTION OF CELLULAR ELEMENTS (TOPOGRAPHY) 4. ASSESMENT OF CELL MORPHOLOGY

41. Diagrammatic representation of the topography of normal bone marrow. 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 precursors are apparently randomly scattered, plasma cells are interstitial or form a sheath around capillaries, and megakaryocytes abut on a sinusoid at one extremity of the cell.

42. Marrow elements 1-Hematopoietic component Erythroid series Myeloid Series Megakaryocytic series 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

43. Special Stains on Marrow - 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 fibrosis In cases of myeloproliferative neoplasms - N ot recommend the routine Perl stain of the core biopsy if a satisfactory marrow aspirate cannot be obtained, iron stains of the clot or biopsy sections>>>potential false-negative results.

44. Reticulin fibrosis Reticulin is 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

45. grade grade 1 grade 2 , grade 3 grade 4

46. Iron Assessment Typically assess 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 .

47. 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)

48. Grading of bone marrow 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

49. Perl’s stain s i d e r oblast Perl’s stain Macrophage with iron Ringed sideroblast

50. ,CD41

51. Major patterns of the IHC staining in formalin-fixed and paraffin-embedded tissue sections

52. MPO showing distribution of normal myeloid cells CD79a showing distribution of normal B cells CD20 CD3 showing distribution of normal T cells CD34 showing positivity of endothelial cells CD34 showing very occasional positive cells

53. Pathologic Key Features Of 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.

54. The evaluation of 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

55. Congenital syndromes and 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

56. Two features are 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

57. Finally, certain types 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

58. Pitfalls Of Pediatric Bone 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.

59. Pitfalls Of Pediatric Bone 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.

60. Pitfalls Of Pediatric Bone 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

61. Pitfalls Of Pediatric Bone 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.

62. Pitfalls Of Pediatric Bone 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.

63. Pitfalls Of Pediatric Bone Marrow Interpretation Parvovirus B19 infections occurring in patients with constitutional red cell disorders may cause erythroid hyperplasia and significant dyserythropoiesis , resembling congenital dyserythropoietic anemia.

64. Acute lymphoblastic leukaemia 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

65. Pitfalls Of Pediatric Bone 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

66. Small cell tumors 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

67. Pitfalls Of Pediatric Bone Marrow Interpretation It should be noted that, in children, apparent aplastic anemia may represent an aplastic presentation of ALL.

68. Pitfalls Of Pediatric Bone 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.

69. Pitfalls Of Pediatric Bone 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

70. CONCLUSION Integration of clinical, 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.