Blood Cell Morphology

Pr François MULLIER, Pr Bernard CHATELAIN BHS course, October 12th, 2013 Blood cell morphology

• Validation of the hematology counter parameters (PLT count, MCV…) • Lack of sensitivity and specificity of the hematology counter alarms (lymphoblasts (FN), Immature granulocytes (FP),… • Answer to questions coming from – Biological results – Clinical situation

Material and Methods

• K2EDTA Sample • Smear – Wedge smear: classical – Spun smear: centrifugation – Automated device – Buffy coat (rare cell detection, diff if low WBC count…) • Staining • Observation (classical, image capture and analysis) • Interpretation BLOOD SMEAR (Wedge) Hemaprep Diff spin

• Higher monocyte percentage • Lack of Smudge cells in CLL May-Grünwald STAINING Check the pH of the Reagents Sorensen buffer! Sorensen Phosphate Buffer pH 6.8 : Na2HPO4.2H2O M/15 (2.56 g/l) and KH2PO4 M/15 (6.63 g/l) Buffered water : dilution 1/20e of Sorensen Phosphate Buffer in distilled water May-Grünwald solution Don’t adjust Giemsa solution the pH of the buffered Staining water! Methanol 10 minutes Pure May-Grünwald solution 5 minutes May-Grünwald (50/50 in buffered water buffer pH 6.8) 2-3 minutes Giemsa (1/10 in buffered water pH 6.8) 15 minutes Rinse with running water Buffered water pH 6.8 30 seconds Let dry smears upright

Guidelines for blood smear preparation and staining procedure for setting up an external quality assessment scheme for blood smear interpretation. Part I: control material (EQALM), J-L Vives Corrons, S. Albarède, G. Flandrin, S. Heller, K. Hovarth, B. Houwen, G. Nordin, E. Sarkani, M. Skitek, M. Van Blerk, J-C. Libeer, Clin Chem Lab Med, 2004, 42(8):922- 926

EQALM : External Quality Assurance Programmes in Laboratory Medecine Microscope

• High resolution objective (highest N.A.) 60X 1.4 N.A. oil i. planapochromatic • Kohler setting with condenser whose N.A. is matched with the objective N.A. • Condenser diaphragm adjustment: full open if N.A. close to 1.4.

Red Cell morphology

Dacryocyte Schistocyte Codocyte

Drepanocyte Spherocyte Jolly Bodies Dacryocyte (Teardrop cells) Red Cell morphology

Dacryocyte Schistocyte Codocyte

Drepanocyte Spherocyte Jolly Bodies Schistocyte definition

JF LESESVE (Hématologie) Red Cell morphology

Dacryocyte Schistocyte Codocyte

Drepanocyte Spherocyte Jolly Bodies Codocytes Red Cell morphology

Dacryocyte Schistocyte Codocyte

Drepanocyte Spherocyte Jolly Bodies SPHEROCYTE Plasmodium falciparum Platelet Morphology Platelet agglutination and aggregation • Variable sensitivity of the automated hematology analyser Platelet satellitism

• Lack of detection by Automated hematology analyser

Grey platelet syndrome Lack of detection by automated hematology analyser

With courtesy of G.Detry (CH Jolimont) MYH9-related disease

Giant platelets Döhle bodies

Platelet Lack of detection by automated distribution (MPV) hematology analyser and count 22 White Blood Cell morphology Lymphocyte morphology (t < 4h)

Cytoplasmic Cytoplasm Nucleus membrane • N/C ratio • smudge cells (lymphocyte • smooth or irregular count + good prognostic factor in CLL • Basophilic cyt, golgi but not specific of CLL) • mobility, fragility, pattern • Granules, inclusions • shape • chromatin structure • nucleolus (i) Lymphocytic population

Benign ? Neoplastic

Polymorphism Monomorphism

lymphocyte immunophenotype Reactive Lymphocytes (infectious mononucleosis)

Immunophenotype : CD3+/ CD8+/ CD38+/ HLA-DR+ ou CD19+ activés Binucleated polyclonal B lymphocytosis Chronic Lymphocytic Leukaemia B Prolymphocytic Leukaemia Mantle cell lymphoma Mantle cell lymphoma Follicular lymphoma Lymphocyte mobility Hairy-cell leukaemia (1) Hairy-cell leukaemia (2) Splenic Lymphoma with villous lymphocytes Lymphoplasmocytic lymphoma Marginal zone lymphoma

1. Villous lymphocytes 2. Plasmocytoid 3. Cleaved cells 4. Clumped chromatin 5. Nucleoli 6. …

!!! pleiomorphic Lymphoplasmocytic Lymphoma

• Red granules without peroxydase activity Cytoplasmic inclusions in B lymphomas

S. Daliphard et al., Hématologie 2006; 12(1): 77-79 Acute Lymphoblastic Leukaemia Keep in mind… • Granules may also be present in B lineage • Neoplastic population may some time be present in low percentage • Smudge cells are not restricted to CLL

• Consider always the DD with Acute Leukaemia

MORPHO

CMF CLINIQUE EM (Blasts)

ALL AML Phase Contrast Microscopy: blasts Belgian EQA march 2010 Belgian EQA march 2010

PEROXIDASE: negative ALL Whole blood AML M3 (APL) Quality Assessment and Education Belgian EQAS virtual microscopy 2004 November Acute Lymphoblastic Leukemia 2005 March Normal, B Prolymphocytic Leukemia 2005 June Normal, Hereditary Spherocytosis 2005 November Normal, Secondary leukemia 2006 March Normal, B12 deficiency 2006 June Normal, Drepanocytosis, May Hegglin (didactic) 2006 November Myeloma, Chediak-Higashi (didactic) 2007 March Normal, CML 2007 June Normal, Reactive Lymphocytosis 2007 November Normal, Hairy Cell Leukemia 2008 March Normal, TTP 2008 May B12 deficiency, Secondary leukemia (didactic) (bone marrow aspirate) 2008 June Secondary ALL, BiN Polyclonal Hyperlymphocytosis (didactic) 2008 November T Prolymphocytic leukaemia, MDS Ider20Q (didactic) 2009 March Hairy Cell Leukaemia, t(8;21) AML (didactic) 2009 June Normal, T cell large Granular Lymphocytic leukaemia 2009 November Normal, Leukemoid reaction 2009 November Myeloma (bone marrow aspirate) 2010 March CLL, Mucopolysaccharidosis Maroteaux-Lamy syndrome (didactic) Belgian EQAS virtual microscopy 2010 June Normal, Plasmodium Falciparum, TTP 2010 November Normal, Sezary Lymphoma 2010 November Mantle Cell Lymphoma 2011 March Normal, Acute Lymphoblastic Leukemia 2011 June Normal, Acute Myeloblastic Leukemia 2011 November Normal, Oat cell carcinoma (I.G. + erythroblastosis) 2011 November Gaucher disease 2012 March Grey platelet syndrome, Reactive Lymphocytosis (didactic) t+6h 2012 June Normal pediatric sample, Normal, 2012 November Hereditary spherocytosis (band III), G6PD deficiency (didactic) 2012 November Cup like Acute Myeloblastic Leukemia

2013 March Acute Monoblastic Leukaemia, Chronic Myelogenous Leukemia (UKNEQAS EQALM) 2013 June T (7;11) Acute Myeloblastic Leukemia, Normal www.e-hematimage.eu

References: • Repères en diagnostic de laboratoire, X. BOSSUYT, J.-M. BOEYNAEMS Ed: Garant ISBN 90-441-1022-5 • Wegwijzer in Laboratorium-diagnose, X. BOSSUYT, J.-M. BOEYNAEMS Ed: Garant ISBN 90-441-1021-7 • Blood Cells A Practical Guide, Barbara J. BAIN, ISBN 1-4051-4265-0 • Bone Marrow Pathology, Barbara J. BAIN, David M. CLARK, Bridget S. WILKINS, ISBN 978- 1-4051-6825-0