Bone Marrow Examination and Techniques



Bone Marrow Examination and Techniques


Kaaren K. Reichard, MD








One important component of a thorough BME is a spicular, well-spread, and well-stained aspirate smear. Here one sees progressive maturation of the erythroid and granulocytic lineages.






Ancillary techniques play a significant role in the work-up and diagnosis of bone marrow specimens. Flow cytometric analysis is one of these specialized techniques that is routinely utilized.


TERMINOLOGY


Abbreviations



  • Peripheral blood (PB)


  • Bone marrow (BM)


  • Bone marrow examination (BME)


  • Flow cytometry (FC)


  • Immunohistochemistry (IHC)


  • Fluorescence in situ hybridization (FISH)


  • Conventional cytogenetics (CC)


MICROSCOPIC FINDINGS


Morphology



  • Interpretation



    • Proper acquisition, preparation, and staining of BME components


    • See overview chapter on hematopoiesis for in-depth morphology review


    • Certain components of BME are optimal for certain tests (e.g., morphologic review, special studies)



      • PB: Dysplasia, increased blasts, abnormal lymphocytes, cytochemistry, FC, CC, FISH


      • BM aspirate: Assess trilineage hematopoiesis, dysplasia, increased blasts, lymphoid cells, histiocytic proliferations, cytochemistry, FC, CC, FISH


      • BM biopsy: Bone, stroma, cell composition and architecture, lymphoid aggregates, granulomas, IHC, molecular studies


      • Core biopsy can be disaggregated for FC, CC, FISH if BM inaspirable


    • Perform ancillary testing as indicated based on PB/BM review or protocol requirements



      • Based on review of PB and BM and clinical scenario or protocol requirements


    • Perform step sectioning to ensure adequate review of BM core biopsy



      • Particularly when assessing for involvement by focal/metastatic lesion


CLINICAL ISSUES


Indications



  • Investigation of unexplained PB abnormalities


  • Diagnosis of suspected primary hematopoietic neoplasm (e.g., leukemia, myeloproliferative neoplasm, myelodysplasia, myeloma)


  • Infectious disease work-up if other systemic investigations noncontributory


  • Evaluation of suspected constitutional disorder


  • Assess for storage disorder


  • Assess for involvement by metastatic neoplasm


  • Staging of lymphoma


  • Investigate radiologic bone or BM abnormality


  • Ongoing monitoring after therapy



    • After BM transplant to assess for BM recovery


    • Protocol requirement


Contraindications



  • Severe coagulopathy


  • Severe bleeding disorder


  • Overlying skin/soft tissue infection


Complications of BM Biopsy



  • Rare but well recognized


  • Incidence not rigorously documented


  • In UK survey, 0.07% incidence of adverse events after BM aspiration &/or biopsy


  • Hemorrhage most common


  • Other: Ongoing pain, fracture, anaphylaxis, and infection


Prebiopsy Preparations



  • Discussions regarding



    • Indications for procedure


    • Potential complications



    • Steps in procedure


    • Duration of procedure


    • Postprocedure expected discomfort and instructions


    • Time interval for test results


  • Appropriate materials should be available as needed (e.g., media for ancillary studies)



    • Ample slides for touch preparation and bone marrow aspirate smears


    • Sodium heparin-coated syringes for cytogenetics, flow cytometry


TECHNICAL ISSUES


Sites of BME



  • Posterior iliac crest



    • Standard site


    • Unilateral specimen adequate for work-up of most conditions


    • Bilateral specimens uncommon



      • Used for staging of BM involvement in past


  • Anterior iliac crest



    • Rare


    • Used when posterior iliac crest is not an option


  • Sternum



    • Exceedingly rare; high risk of sternal plate puncture


    • Aspiration specimen only; guard required on needle


Components of BME



  • CBC data


  • Peripheral blood smear


  • BM aspirate


  • BM touch preparation


  • BM clot section


  • BM trephine biopsy


Adequacy of BM Aspiration and Biopsy



  • BM aspiration



    • Obtain at least 3 particles per slide


    • Obtain at least 4 slides



      • 2 for routine staining


      • 1 for iron (as needed)


      • 1 for potential ancillary tests (e.g., cytochemistry, FISH)


    • Acquire additional material for ancillary tests (as needed)


    • Optimal Wright (or equivalent) stain for satisfactory cytologic assessment


    • Heparin-coated syringe for FC, CC, molecular genetic studies


  • BM trephine biopsy



    • 1.5-3 cm in length


    • No significant aspiration artifact



      • Perform biopsy prior to aspiration


    • Perform touch preparation (see BM touch preparation section)



      • Particularly if BM aspirate is suboptimal


    • Ensure adequate and proper decalcification, formalin fixation and processing, sectioning, and staining


    • Lengthy decalcification results in



      • Suboptimal morphology


      • Suboptimal immunohistochemistry


      • Suboptimal special stains (reticulin)


  • BM touch preparation



    • Adequate stain for cytologic assessment



      • Particularly in event of no/suboptimal aspirate


    • Important for potential ancillary techniques (e.g., cytochemistry, FISH, iron stains)



      • Particularly if BM aspirate is hemodilute secondary to tumor infiltration, fibrosis


    • ≥ 3 air-dried imprints


REPORTING CRITERIA


Individual BM Reports



  • Should be completed on average within 3 days


  • Integrate additional ancillary testing information



    • IHC


    • FC


    • Cytochemistry


  • Utilize standardized template &/or synoptic reporting


  • Organize report such that key diagnostic and prognostic information is readily available to clinician


Final Integrated BM Reports



  • Should be promptly completed upon finalization of last piece of testing data (e.g., CC, FISH, &/or molecular studies)


  • Report significance of ancillary studies


  • Report prognostic implications


  • Suggest any additional testing as needed


  • Utilize standard classification systems if possible


  • Synoptic (or similar) reporting allows for standardization and reproducible location of key information


ANCILLARY TECHNIQUES


Cytochemistry



  • Myeloperoxidase (MPO) and nonspecific esterase (NSE) (a.k.a. α-naphthyl butyrate esterase) are most common


  • Requires unfixed/unstained specimen



    • PB


    • BM aspirate/touch preparation


    • Cytospin preparation from FC/CC aspirate


  • MPO



    • Stains granules within neutrophilic lineage


    • Intensity of staining greatly increases at promyelocyte stage of maturation


    • Blasts of myeloid lineage show scattered positive granules


    • May rarely see sparse positive granules in blasts of monocytic &/or lymphoid lineage


    • All normal myeloblasts should exhibit scattered MPO positivity


    • MPO positivity does not discriminate neoplastic from nonneoplastic blasts


    • Not all myeloblasts are MPO positive



      • Need immunophenotyping and genetic studies


    • Fast



      • Technique usually takes ˜10 minutes to perform


  • NSE




    • Stains cells within monocytic lineage



      • Monoblasts


      • Promonocytes


      • Monocytes and histiocytes


    • Diffuse cytoplasmic brown staining


    • Assay is technically tricky



      • Need reliable external &/or internal positive control


    • Positivity does not discriminate neoplastic from nonneoplastic monocytes



      • Requires morphologic integration


Special Stains for Iron



  • Iron studies



    • Nonenzymatic stain; Prussian blue


    • Highlights iron particles/stores as blue-green cytoplasmic granules &/or clumps


  • Assess 2 features



    • Erythroid iron


    • Macrophage stores


  • Erythroid iron



    • Need adequate, unstained, air-dried BM aspirate smear/touch preparation


    • Normal red cell iron



      • ˜ 20-50% of red blood cell precursors demonstrate 1-3 small cytoplasmic granules


    • Pathologic red cell iron



      • Abnormal size (large), shape (chunky), or location (ring) of granules


      • Ring sideroblast is defined by World Health Organization as 1/3 of red blood cell nucleus tightly surrounded by 5 or more iron granules


    • Storage iron



      • Grading may be semiquantitatively assessed (normal, none, increased, decreased) or assessed by grading scale (0-6)


      • Need good-quality BM aspirate with sufficient particles


      • Need adequate positive control to insure accuracy


      • May have heterogeneous iron deposition in BM aspirate


      • Formalin fixation may interfere with staining of iron stores (ferritin)


Special Stains for Fibrosis



  • 2 types of BM fibrosis: Reticulin and collagen


  • Reticulin fibrosis



    • May be seen in various neoplastic and nonneoplastic disorders


    • Detected by silver stain


    • Reticulin is present normally in bone marrow



      • Seen as scant thin fibers around small vessels and rare sinusoids


    • Often reversible with eradication of underlying pathology


    • Variability occurs within staining; ensure reproducibility


    • Preanalytical factors may affect quality: Fixation, decalcification, thick sections, manual vs. automated procedure


  • Collagen fibrosis



    • Detected with trichrome stain


    • Not normally present in bone marrow


    • Unlikely to be reversible


  • Grading scale



    • Recent proposal of 0-3 scale


Immunohistochemistry



  • Performed on fixed specimen (BM clot or core biopsy)


  • Numerous IHC stains available



    • Lymphoid cells



      • B; CD19, CD20, CD79a, CD22


      • T; CD1a, CD2, CD3, CD4, CD5, CD7, CD8


      • NK; CD2(+), sCD3(-), CD16, CD56


    • Cells in neutrophil lineage



      • CD13, CD33 (not routinely available by IHC), MPO


    • Monocytic cells



      • CD163, CD68, CD4 (weak)


    • Blasts (not lineage-specific)



      • CD34, TdT, CD117


    • Erythroid cells



      • CD117 (weak +) in pronormoblasts, hemoglobin A, glycophorin A, CD71


    • Megakaryocytic lineage



      • CD31, CD41, CD42b, CD61


    • Mast cells



      • CD117, tryptase


    • Plasma cells



      • CD138, CD38, cytoplasmic κ and λ


  • Indications for IHC assessment of BM biopsy specimens



    • Evaluate for morphologically occult process (use CD3, CD20, CD34)


    • Evaluation of unexplained hypocellular BM (e.g., hairy cell leukemia, increased blasts)


    • Evaluation of unexplained hypercellular marrow (e.g., increased blasts, increase in mast cells)


    • Evaluation of lymphoid aggregates if suspicious (e.g., paratrabecular aggregates) &/or flow suboptimal or negative


  • Caveats of IHC



    • Variable IHC results depending on fixation, decalcification, antibody clones/dilution


  • Comparative features of IHC vs. FC



    • Slower turnaround time (TAT) for IHC


    • Generally uniparametric; occasionally dual parametric IHC


    • Preserved architecture in tissue section


    • No significant risk of loss of antigens if properly preserved (IHC)


    • Assess infectious agents, proliferation rate, aberrant oncoproteins



      • e.g., EBER, Ki-67, p53, cyclin D1, ALK1


Flow Cytometry

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Jun 13, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Bone Marrow Examination and Techniques

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