XI International Conference Hematopoiesis Immunology Budapest, June 6-7, 2014 FLO CYTOMETRIC ANALYSIS OF NORMAL BONE MARRO Bruno Brando and Arianna Gatti Hematology Laboratory and Transfusion Center Legnano General Hospital (MI) e-mail: bruno.brando@ao-legnano.it
BONE MARRO MORPHOLOGY VS FLO Peripheral blood contamination: how to manage it? Evaluation of Bone Marrow cell representation. Some Authors proposed formulas to calculate the actual Bone Marrow cellularity.
CD13 PE --> CD13 PE --> CD13 PE --> 10 1 10 2 10 3 10 4 CD13 PE CD13 PE CD13 PE 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 A 10 1 10 2 10 3 10 1 10 2 10 3 10 4 CD16- CD16 FITC FITC --> B 10 1 10 2 10 3 10 1 10 2 10 3 10 4 CD16- CD16 FITC FITC --> C N u m b e r 10 50 90 1 3 0 1 8 0 N u m b e r 10 50 90 1 3 0 1 8 0 N u m b e r 10 50 90 1 3 0 1 8 0 10 1 10 2 10 3 10 1 10 2 10 3 10 4 CD16- CD16 FITC FITC --> 10 1 10 2 10 3 10 1 10 2 10 3 10 4 CD16- CD16 FITC FITC --> 10 1 10 2 10 3 10 1 10 2 10 3 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 CD16- CD16 FITC FITC --> CD16- CD16 FITC FITC --> A B C 78% 4% 56% 22% 96% 44% Immature Neutrophil proportion can indicate the degree of peripheral blood contamination Dissociated Marrow Biopsy Peripheral blood Bone Marrow Aspirate Courtesy of Mike Loken, 2009
The percentage of Immature Granulocytes (CD16 dim/neg) reflects the peripheral blood contamination of Bone Marrow Aspirate suspensions 64% 45% 33% 23% Loken MR. Clinical Cytometry 2009; 76B: 27-36.
It is assumed that immature granulocytes (CD16 neg/dim) represent ~ 80% of the whole granular myeloid population. Therefore any dilution by peripheral blood mature PMN reduces this proportion accordingly. The correct BM Blast percentage (and maybe the % of any other BM cell population) may be calculated according to this formula: Normalized BM Blast Count = 80% % CD16+ neg/dim x Blast % Loken MR. Clinical Cytometry 2009; 76B: 27-36.
The Proportion of Erythroid precursors can also give an idea about the overall cell representation in the BM aspirate suspension. Always keep in mind that particle smears and BM suspensions for FCM do contain DIFFERENT PROPORTIONS OF CELL POPULATIONS! Poorly representative BM aspirate Reasonably representative BM aspirate
Expected Cell Levels in Normal Bone Marrow with 80% Purity Immature Cells % Lymphocytes % Monocytes % CD34+ Cells % Brooimans RA. Clinical Cytometry 2009; 76B: 18-26.
SSC CD34+ Precursor Cells in Normal Bone Marrow Granulocyte Line Eosinophils CD34+ Myeloid Precursors Monocyte Lineage Nucleated Erythroid Lineage Cells CD34+ Lymphoblasts CD45 Hematogones (CD34-) CD34+ HPC Lymphocytes Courtesy of Alberto Orfao, 2008
Enumeration of Progenitor Cell Subsets in Sequential BM Aspirate Specimens Aspirate Pull (5mL) n. 1 2 3 HLA-DR+/CD11b- % 3.2 2.0 1.8 CD 34+/HLA-DR+ % 2.9 1.9 1.4 HLA-DR+/CD11b- % 1.3 0.6 0.4 CD 34+/HLA-DR+ % 1.5 0.5 0.5 HLA-DR+/CD11b- % 2.1 1.6 1.1 CD 34+/HLA-DR+ % 1.1 0.9 0.8 HLA-DR+/CD11b- % 1.1 1.2 1.2 CD 34+/HLA-DR+ % 1.3 1.0 0.8 Courtesy of Mike Loken, 2009
CD34 Alone CD34+CD117+ CD34+CD117+DR+ Counting BM Blasts by FCM BM Blast enumeration by FCM using CD34+ CD117+ HLADR+ displays the BEST CORRELATION with morphological blast count as performed by expert readers. Cell denominator is made by CD45+ cells, excluding erythroid precursors. Sandes AF. Clinical Cytometry 2013; 84B: 157-166.
Counting BM Blasts by FCM (MDS Patients) Best counting accuracy using CD45+ nucleated cells as denominator Counting accuracy ORSENS if erythroid precursors are taken into account Sandes AF. Clinical Cytometry 2013; 84B: 157-166.
BM ANALYSIS: PROBLEMS ITH MORPHOLOGY Morphologic analysis may be difficult when samples are hypocellular (500 Cells required!). Blast count and enumeration of other disease-defining cell levels requires experienced readers and displays the highest interobserver variability. Age-related changes and chronic infections can mimic morphological features of MDS, and dyserythropoiesis or dysgranulopoiesis can be observed in relatively healthy elderly individuals. Fibrosis can be evaluated by BM threphine analysis only.
Courtesy of Alberto Orfao, 2008
QUANTITATIVE ANALYSIS OF BONE MARRO COMPOSITION: Is it still useful? YES: e live in a world of NUMBERS, and quantitation of certain BM cell populations is mandatory to define disease states ( i.e. Blasts, Ringed Sideroblasts, Plasmacells, Monoblasts+Promonocytes, Dysplastic cell fractions... ). MORPHOLOGIC QUANTITATION is still considered the accepted standard for the enumeration of Plasmacells, Ringed Sideroblasts and Dysplastic cells. FLO CYTOMETRIC QUANTITATION can be used to reliably enumerate Blasts (i.e. CD34+ cells plus CD117+/HLADR+) and other rare cell populations. BUT FLO IS UNBEATABLE HEN THE QUALITY OF BM CELLS HAS TO BE EVALUATED (i.e. Plasmacells, Blast Lineage)
Courtesy of Alberto Orfao, 2008
Aldehyde Dehydrogenase + CD34 - Cells are Erythroid Precursors ALDH + CD34 + ALDH + CD34 - ALDH + CD34 - are CD105+ CD71+ Erythroid Precursors and are Larger than CD34+ Bone Marrow progenitors Mirabelli P. BMC Physiology 2008; 8, 13
CD 71 FITC GLICO A PE Maturing Erythroid Compartment in the Bone Marrow CD 45 / CD 71 / CD 105 / Glycoforin-A NORMAL PATTERN Proerythroblasts Erythroblasts Nucleated Poly- Ortho 10 0 10 1 10 2 10 3 10 4 GLICO A PE 10 0 10 1 10 2 10 3 10 4 CD105 FITC
Maturing Erythroid Compartment in the Bone Marrow Normal Maturation Pattern Variable proportion of cells falling into the different Erythroid maturational steps (shift to the left or to the right). Phenotypic changes through the maturation pathway seem to occur in a DISCRETE fashion rather than as continuous pattern.
Maturing Erythroid Compartment in the Bone Marrow Erythroid Maturation Blockade in Myelodisplasia (RA) R1 Altered Erythroblasts in RA fail to proceed to CD71- Gly-A+ step and Down-regulate CD71
Granulocyte maturation in the normal Bone Marrow Van Lochem EG. Clinical Cytometry 2004; 60B: 1-13.
Myelocyte and Monocyte maturation in the normal Bone Marrow Promyelocytes Myeloblasts Lymphoblasts Monoblasts Mature Monocytes Immature Monocytes Van Lochem EG. Clinical Cytometry 2004; 60B: 1-13.
Monocyte maturation in the normal Bone Marrow Monoblasts Promonocytes Mature Monocytes Courtesy of Alberto Orfao, 2008
Phenotype changes with Age, Exposure & Environment
B Lymphocyte maturation in the normal Bone Marrow Mature B Cells (Pre-B ) Immature B (Pre-B II) Hematogones CD34- (Pre-B I) Lymphoblasts CD34+ TdT+ Van Lochem EG. Clinical Cytometry 2004; 60B: 1-13.
B Lymphocyte maturation in the normal Bone Marrow: Hematogones Hematogones Chantepie SP. Leukemia Research 2013; 37: 1404-1411.
Age-related changes in the normal Bone Marrow (B Cell Lineage) 69 years old 3 years old Van Lochem EG. Clinical Cytometry 2004; 60B: 1-13.
Transitional B Cells in the normal Bone Marrow Transitional B Cells: The first B that migrate into blood. ~ 3% of B cells, CD24++ CD38++ Agrawal S. Clin Experim Immunol 2013; 174: 53-59.
Courtesy of Alberto Orfao, 2005 0 25 50 75 100 Proportion of Plasma Cells by morphology 0 25 50 75 0 25 50 75 100 0 25 50 75 100 In > 50% of cases the FCM enumeration of BM Plasmacells is lower or much lower (usually 1 Log) than by Morphology. So, do not use FCM to count Plasmacells, but rather to define their QUALITY. Proportion of Plasma Cells by FCM Discrepancy between morphologic and FCM counting of Bone Marrow Plasmacells
Normal and Abnormal PLASMACELLS Normal PC Myeloma PC Myeloma PC Normal PC
Normal BM Plasmacells (NPC) may express antigen patterns similar to Abnormal Plasma Cells (APC) Tembhare PR. Leukemia Research 2014; 38: 371-376.
FCM can easily discriminate between CLONAL Plasmacells and normal, Polyclonal Plasmacells according to cyt Ig expression Clonal PC Polyclonal PC Cyt Cyt
A warning: CD56+ Monocytes can be found also in NON-MDS elderly people. Courtesy of Alberto Orfao, 2008
Immunophenotypic Changes of the Myeloid Lineage in MDS Normal Normal MDS MDS MDS Normal MDS MDS Della Porta MG. Clinical Cytometry 2011; 80B: 201-211.
FLO CYTOMETRIC DIAGNOSIS OF MDS: Four Cardinal Parameters in a Tube (CD45 / CD34 / SSC) CD34+ Myeloblasts B Cell Precursors Lymphs SSC Lymphs CD45 MFI PMNs SSC Myeloblasts CD45 MFI Della Porta MG. Clinical Cytometry 2011; 80B: 201-211.
Normal MDS Hemodilution-Independent Parameters to Establish MDS Myeloblast-related cluster size (n.v. > 2%) and B-cell Progenitors cluster size (n.v. > 5%) Ratio between Median Myeloid Lineage Side Scatter and Lymphocyte Side Scatter (n.v. > 6) Ratio between Median Lymphocyte CD45 MFI and Median Myeloblast CD45 MFI (n.v. > 4) Della Porta MG. Haematologica 2012; 97: 1209
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Errors, like straws, upon the surface flow; He who would search for pearls must dive below. John Dryden, from All for Love (1678) prologue Pictures of Epithelial Neoplasm Micrometastases in Bone Marrow
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