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Blasts In The Bone Marrow: Information FromAspirate Smears, Biopsy Sections and Flow Cytometry
Importance of Accurate and PreciseIdentification of Bone Marrow Blasts
• Diagnosis
• Classification
• Prognosis
• Treatment decisions
Confounding Issues for Accurate andPrecise Assessment of Blasts
• Technical– Specimen collection and quality of specimen
– Quality of the preparation
• Other factors– Drug induced changes
• G‐CSF, chemotherapy, etc
• Limitations of method– Aspirate smears, biopsy sections, flowcytometry
Modalities for Assessing Blasts in theBone Marrow
1. Bone Marrow Aspirate Smears
Myeloblasts Types 1 and 2 and a Promyelocyte
MPO
NSE
CD41
Aspirate Smears For Assessment Of BlastsIn Bone Marrow
Advantages
• Historical standard
• Rapid and ease
• Allows for identification ofabnormal features
• Lineage identification
• Assessment of backgroundfeatures‐‐myelodysplasia
• Cytochemical evaluation
– MPO, NSE
Issues
• Hemodilution—myelofibrosis
• Technical quality of smear
• Limited cell count subject tostatistical error
• Variation in morphologiccriteria for a blast (subjectivity)
– Distinction of Type 2 & 3blasts from promyelocytes
– Normal from neoplastic
– Myeloblast and lymphoblast
Modalities for Assessing Blasts in theBone Marrow
2. Bone Marrow Trephine BiopsySections
ALIP
CD34
CD20 CD79a
TdT CD10
CD34 CD61
CD34 Glycophorin A
Biopsy Sections For Assessment Of BlastsIn Bone Marrow
Advantages
• Truest visualization ofintact marrow architecture
• Recognition of abnormalrelationships‐‐‐ALIP
• Blast counts not altered bymyelofibrosis
• Applicability ofimmunohistochemistry
• Identification of lineage
Issues
Modalities for Assessing Blasts in theBone Marrow
3. Flow Cytometry
Enumeration of Blasts in Multiple Aspirate Specimens
Aspirate Pull
1 2 3
1. HLA‐DR+/CD11b‐ 3.2 2.0 1.8
CD34+/HLA‐DR+ 2.9 1.9 1.4
2. HLA‐DR+/CD11b‐ 1.3 0.6 0.4
CD34+/HLA‐DR+ 1.5 0.5 0.5
3. HLA‐DR+/CD11b‐ 2.1 1.6 1.1
CD34+/HLA‐DR+ 1.1 0.9 0.8
4. HLA‐DR+/CD11b‐ 1.1 1.2 1.2
CD34+/HLA‐DR+ 1.3 1.0 0.8
Loken MR, etal. Normalization of Bone Marrow Aspirates for Hemodilution inFlow Cytometric Analysis. Cytometry B (2007) 76B: 27‐36
Criteria to Define a Phenotypic Myeloblast
• Combining CD45 and side scatter with differentreagent combinations:
• CD34 and HLA‐Dr/CD11b or CD117
• Correct for lymphoblasts
• Redundancy covers for aberrant loss of any oneantigen on the myeloblast population
• Expressing the proportion of myeloblasts / non‐erythroid cells
• Circumvents the problem of variable erythroidcell lysis in the preparation
Normal blasts
MPO
Hematogones
• Bone marrow B‐cellprecursors
• Size and morphology bridgemature lymphocytes andneoplastic lymphoblasts
• Large percentages seen inhealthy infants and youngchildren
• Increased in– Regenerating marrows– Autoimmune or congenital
cytopenias– Lymphoma, neuroblastoma– AIDS
Mckenna Leuk and Lymph 2004
TdT
CD10
CD20
Clinical Necessity Of IdentifyingNeoplastic Blasts In low percentage
• Distinction of normal from low levelneoplastic blasts at diagnosis of somemyeloid neoplasms (MDS) and in post‐therapy marrow
• Minimal residual disease (MRD) assessment
Sensitivity of Methods for Detection ofNeoplastic Blasts in Bone Marrow
• Sensitivity– Bone marrow smears ‐‐‐‐ 1% to 5%
– Bone marrow biopsies ‐‐‐ ?
– Cytogenetic banding ‐‐‐‐‐ 1% to 5%
– FISH ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 0.1% to 5%
– Flow Cytometry ‐‐‐‐‐‐‐‐‐‐‐‐ 0.01% to 0.005%
– Molecular (PCR) ‐‐‐‐‐‐‐‐‐‐‐‐ 0.01% to 0.0001%
Prognostic Significance of End of Consolidation MRD in ALL
Borowitz MJ, etal. (2008)Blood; 111: 5477‐5485
Minimal Residual Disease Detection
CD38
CD45
CD10
CD19
CD34
CD45
Flow Cytometry Assessment For BoneMarrow Blasts
Advantages
• Best sensitivity– MRD assessment
• Large number of cellscounted– Dramatically reducescounting statistical error
• Objective criteria can beused to define a blast
• Lineage identification
Issues
• Specimen hemodilution– Aspirate procedure– Preparation
• Frequent lack ofcorrelation with blastcounts on aspirates andsections
• Lack of consensuscriteria to define aphenotypic myeloblast
• No visualization of cells
Summary‐‐Generalizations
• Aspirate smears typically provide the mostaccurate quantification of blasts in the marrowand frequently identify lineage
• Trephine biopsies are superior for quantificationof blasts in fibrotic bone marrow and providematerial for immunophenotyping
• Flow cytometry is most sensitive for detection oflow numbers of blasts and MRD and best forlineage characterization
Summary
• Bone marrow smears, biopsysections and flow cytometry are alleffective methods for assessment ofblasts in the bone marrow
• Each may independently contributediagnostic information
Summary
• One of the three methods may provemost suitable in a given clinical setting
• Often it is the integration of data fromthese different modalities that providesdiagnostic information
• For optimal practice of bone marrowhematopathology all 3 methodologiesshould be used