1. Part II Bone Marrow Examination

2. Bone Marrow Examination: Introduction
O bone marrow - a highly vasculized loose connective tissue - volume: ml/kg - hematopoietic compartment hematopoietic cells and stromal cells (supporting cells) red marrow: hematopoietically active, erythroid and myeloid cells yellow marrow: hematopoietically inactive, adipocyte - vascular compartment nutrient artery, central sinus, central vein

3. Bone Marrow Examination
O Indications for bone marrow evaluation

4. Bone Marrow Examination: Bone Marrow Procedure
O sites of hematopoiesis differ by age - at birth: all bones - in adult: skull, scapulae, ribs, sternum, clavicles, vertebrae, the proximal ends of the long bones of the arms and legs -> red marrow - disease state (thalassemia, sickle cell anemia): extend into the long bones O cellularity within red marrow decreases with age - at birth: 90% cellularity - in elderly adult: 50% cellularity O most marrow specimens: taken from the posterior superior iliac crest (Fig. 35-1) O performing the biopsy first and then the aspirating - to avoid distortion of the marrow architecture in the biopsy specimen - disadvantage: clotting O trephine core biopsy: 11-gauge cutting needle aspiration: 2 ml, 18-gauge needle O common complication: localized bleeding, infection

5. Bone Marrow Examination

6. Bone Marrow Examination: Bone Marrow Processing
BONE MARROW ASPIRATE SMEARS, PARTICLE PREPARATION, AND CLOT SECTIONS O about 0.5 ml of the aspirate O the presence of particles: fat, granules, small pieces of bone in the aspirate sample -> evidence for effective aspiration O bone marrow occasionally cannot be aspirated: dry tap - extensive fibrosis (hairy cell leukemia, chronic idiopathic myelofibrosis) - very increased cellularity (leukemia) O direct smears of bone marrow aspirate: at the bedside - the aspirate clots quickly O bone marrow particle crush smears O Wright's stain O aspirate smear: better cell morphology trephine core biopsy and particle preparation: better architecture and cellularity

7. Bone Marrow Examination: Bone Marrow Processing
O clot section O placed in EDTA tube -> filtration -> fixed in 10% buffered formalin O EDTA-anticoagulated marrow aspirate -> placed in Wintrobe tube -> centrifugation -> separated into four layers - fat and perivascular cells - plasma - buffy coat - red cells O ratio between the fat/perivascular and erythroid/myeloid layers - reflects the overall marrow cellularity TOUCH IMPRINTS AND CORE BIOPSY O touch imprint: by gently touching or rolling the tissue several times on glass slides -> some cells form the core specimen will stick to the slide O core biopsy specimen -> fixed in 10% buffered formalin -> decalcification, dehydrating, embedding in paraffin blocks -> sectioning of 2-3 um thick -> staining with hematoxylin and eosin (H and E) O performed for primary diagnosis, disease staging purpose

8. Bone Marrow Examination: Morphologic Interpretation
O cytologic assessment: aspirate smear, touch imprint O histologic assessment: clot, particle preparation, core biopsy O cellularity, cell morphology, quantity of iron and its distribution, tumor cell BONE MARROW ASPIRATE O in conjunction with complete blood count, peripheral blood smear O evaluate the number, morphology, maturation process (Fig. 35-2) O scanned at 100X - to determine cellularity - to select a suitable area for the differential count - area where the cells are destroyed: by the presence of bare nuclei

9. Bone Marrow Examination

10. Bone Marrow Examination: Morphologic Interpretation
O bone marrow cellularity - estimating the percent of bone marrow space occupied by hematopoietic tissue - decrease with age - the number and the distribution of the megakaryocytes O at higher magnification (500X or 1000X) - evaluation of nuclear and cytoplasmic maturation O myelodysplastic syndrome (MDS) - erythroid: multinucleation nuclear/cytoplasmic dysynchrony (nuclear maturation is delayed) - myeloid: abnormal granule development - megakaryocyte: hypolobulation of the nucleus

11. Bone Marrow Examination: Morphologic Interpretation
Bone Marrow Differential Count O 500-1,000 cell differential at 1,000X magnification on the direct smear or the crush particle preparation O concentrate, touch imprint: hypocellularity or dry tap O bone marrow composition changes with age - myeloid precursors are usually increased at birth - within few weeks by a predominance of lymphoid cells hematogenes: normal immature B cells (Fig. 35-3) - in young children, lymphocytes -> about one-third of marrow cellularity decrease gradually after puberty O bone marrow is not as homogeneous as blood - lymphoid follicle O megakaryocytes: not included in the differential count O in adult: composed of granulopoietic and erythropoietic precursors

12. Bone Marrow Examination

13. Bone Marrow Examination: Morphologic Interpretation
Myeloid to Erythroid Ratio O M:E ratio: ratio between all granulocytes and their precursors and all nucleated red cell precursors - low: erythroid hyperplasia or myeloid hypoplasia - reference range: between 2:1 and 4:1 (1.5:1 to 3:1) - chronic myelogenous leukemia: increased ratio myelodysplastic syndrome: decreased ratio TOUCH IMPRINTS O Fig O when bone marrow: dry tap (myelofibrosis, hairy cell leukemia)

14. Bone Marrow Examination

15. Bone Marrow Examination: Morphologic Interpretation
BONE MARROW PARTICLE PREPARATION, CLOT AND CORE BIOPSY O for histologic examination O core biopsy: not good to study marrow iron stores due to decalcification O reticulin and trichrome stain: evaluate fibrosis O granuloma: a distinctive pattern of chronic reaction in which the predominant cell type is an activated macrophage with an epithelial-like appearance O specific tumor marker: immunohistochemical stain O disadvantage: lose fine cellular details in the processing O PCR: B cell or T cell gene rearrangement, various leukemia, minimal residual disease, diagnosis of infectious disease

16. Bone Marrow Examination: Morphologic Interpretation
Bone Marrow Cellularity
O bone marrow biopsy is the optimal sample
O estimated by comparing the amount of hematopoietic tissue with the amount of
adipose tissue
O normal expected cellularity: subtract the patient's age from 100% and +/- 10%
O subcortical and paratrabecular area are more hypocellular than
the deeper medullary area
O reported as decreased, normal, increased
O Fig. 35-5

17. Bone Marrow Examination

18. Bone Marrow Examination: Morphologic Interpretation
BENIGN LYMPHOID AGGREGATES VERSUS MALIGNANT LYMPHOMA O benign (reactive) aggregates (Fig. 35-6) - nonparatrabecular - single, small, well-defined, - polymorphic populations of lymphocyte with plasma cells at the periphery - presence of blood vessels within the aggregates O lymphoid aggregates of lymphoma (Fig. 35-7) - paratrabecular, interstitial, diffuse, patchy - small to large, ill-defined - monomorphic cells

19. Bone Marrow Examination

20. Bone Marrow Examination: Morphologic Interpretation
BONE MARROW IRON STORES O iron - stored as ferritin and hemosiderin in reticuloendotheilial cells and erythroblast - storage iron in the bone marrow: in the form of hemosiderin Prussian Blue Stain for Iron O EDTA chelating method: used to decalcify the bone marrow biopsy for iron study - rapid acid decalcifying solution: extract iron O procedure - weak acid solution: free the iron form the loose protein bonds - free iron combine with potassium ferrocyanide -> ferric ferrocyanide - greenish blue (Fig. 35-8)

21. Bone Marrow Examination

22. Bone Marrow Examination: Morphologic Interpretation
O intracellular ferritin: not stain with Prussian blue O serum ferritin: parallel the concentration of storage iron O nonspecific increase of ferritin: - chronic infection, inflammation, liver disease, malignancy -> hemosiderin: accurate indicator of iron sufficiency Use of Bone Marrow Iron Evaluation O useful in the classifying anemias associated with defective hemoglobin synthesis - decrease: iron-deficiency anemia - normal or increase: anemia of chronic disease and thalassemias - increase: sideroblastic anemia ringed sideroblast: erythroid precursor that have a deposition of excess iron around the nucleus arranged like a necklace. This iron is located within the mitochondria that surround the nucleus

23. Bone Marrow Examination: Special studies
FLOW CYTOMETRY
O simultaneous analysis of multiple characteristics of cells in suspension as they pass through
a laser light beam
- size, internal complexity, presence of specific cell markers
O useful in - the presence of clonality
- diagnose and classify malignant lymphomas and leukemias
- detect minimal residual disease after chemotherapy
O performed only on fresh samples (Table35-2)
CYTOGENETICS
O used in the primary diagnosis of acute leukemia, malignant lymphomas,
myelodysplastic syndromes and myeloproliferative disorders
O lymphoma cells are more difficult to grow
O FISH (fluorescent in situ hybridization)

24. Bone Marrow Examination

25. Bone Marrow Examination: Special studies
MOLECULAR GENETICS O PCR - demonstrate cell clonality and lineage - identify specific genetic rearrangements - detect minimal residual disease O fresh and frozen sample, samples fixed in formalin or embedded in paraffin O Table 35-3 CYTOCHEMICAL STAINS O cytochemistry: in vitro staining of cells to allow microscopic examination of the cells' chemical composition O differentiating the lineage of immature cells O some cellular constituents are sensitive to heat, light, storage, and processing technique

26. Bone Marrow Examination