Blood Cell Morphology. Pr François MULLIER, Pr Bernard CHATELAIN BHS course, October 12th, 2013

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1 Blood Cell Morphology Pr François MULLIER, Pr Bernard CHATELAIN BHS course, October 12th, 2013

2 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

3 K 2 EDTA Sample Smear Material and Methods 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

4 BLOOD SMEAR (Wedge)

5 Hemaprep

6 Diff spin Higher monocyte percentage Lack of Smudge cells in CLL

7 Reagents Sorensen Phosphate Buffer ph 6.8 : Na 2 HPO 4.2H2O M/15 (2.56 g/l) and KH 2 PO 4 M/15 (6.63 g/l) Buffered water : dilution 1/20 e of Sorensen Phosphate Buffer in distilled water May-Grünwald solution Giemsa solution Staining Methanol Pure May-Grünwald solution May-Grünwald (50/50 in buffered water buffer ph 6.8) Giemsa (1/10 in buffered water ph 6.8) Rinse with running water Buffered water ph 6.8 Let dry smears upright May-Grünwald STAINING 10 minutes 5 minutes 2-3 minutes 15 minutes 30 seconds 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): EQALM : External Quality Assurance Programmes in Laboratory Medecine Check the ph of the Sorensen buffer! Don t adjust the ph of the buffered water!

8 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.

9 Red Cell morphology Dacryocyte Schistocyte Codocyte Drepanocyte Spherocyte Jolly Bodies

10 Dacryocyte (Teardrop cells)

11 Red Cell morphology Dacryocyte Schistocyte Codocyte Drepanocyte Spherocyte Jolly Bodies

12 JF LESESVE (Hématologie) Schistocyte definition

13 Red Cell morphology Dacryocyte Schistocyte Codocyte Drepanocyte Spherocyte Jolly Bodies

14 Codocytes

15 Red Cell morphology Dacryocyte Schistocyte Codocyte Drepanocyte Spherocyte Jolly Bodies

16 SPHEROCYTE

17 Plasmodium falciparum

18 Platelet Morphology

19 Platelet agglutination and aggregation Variable sensitivity of the automated hematology analyser

20 Platelet satellitism Lack of detection by Automated hematology analyser

21 Grey platelet syndrome Lack of detection by automated hematology analyser With courtesy of G.Detry (CH Jolimont)

22 MYH9-related disease Giant platelets Döhle bodies Platelet distribution (MPV) and count Lack of detection by automated hematology analyser 22

23 White Blood Cell morphology

24 Lymphocyte morphology (t < 4h) Cytoplasmic membrane smooth or irregular mobility, fragility, pattern Cytoplasm N/C ratio Basophilic cyt, golgi Granules, inclusions Nucleus smudge cells (lymphocyte count + good prognostic factor in CLL but not specific of CLL) shape chromatin structure nucleolus (i)

25 Lymphocytic population Benign? Neoplastic Polymorphism Monomorphism lymphocyte immunophenotype

26 Reactive Lymphocytes (infectious mononucleosis) Immunophenotype : CD3+/ CD8+/ CD38+/ HLA-DR+ ou CD19+ activés

27 Binucleated polyclonal B lymphocytosis

28 Chronic Lymphocytic Leukaemia

29 B Prolymphocytic Leukaemia

30 Mantle cell lymphoma

31 Mantle cell lymphoma

32 Follicular lymphoma

33 Lymphocyte mobility

34 Hairy-cell leukaemia (1)

35 Hairy-cell leukaemia (2)

36 Splenic Lymphoma with villous lymphocytes

37 Lymphoplasmocytic lymphoma

38 Marginal zone lymphoma 1. Villous lymphocytes 2. Plasmocytoid 3. Cleaved cells 4. Clumped chromatin 5. Nucleoli 6.!!! pleiomorphic

39 Lymphoplasmocytic Lymphoma Red granules without peroxydase activity

40 Cytoplasmic inclusions in B lymphomas S. Daliphard et al., Hématologie 2006; 12(1): 77-79

41 Acute Lymphoblastic Leukaemia

42 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

43 EM (Blasts) ALL AML

44 Phase Contrast Microscopy: blasts

45 Belgian EQA march 2010

46 Belgian EQA march 2010 PEROXIDASE: negative ALL

47 Whole blood AML M3 (APL)

48 Quality Assessment and Education

49 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) Belgian EQAS virtual microscopy 2010 March CLL, Mucopolysaccharidosis Maroteaux-Lamy syndrome (didactic)

50 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 Belgian EQAS virtual microscopy

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53 References: Repères en diagnostic de laboratoire, X. BOSSUYT, J.-M. BOEYNAEMS Ed: Garant ISBN Wegwijzer in Laboratorium-diagnose, X. BOSSUYT, J.-M. BOEYNAEMS Ed: Garant ISBN Blood Cells A Practical Guide, Barbara J. BAIN, ISBN Bone Marrow Pathology, Barbara J. BAIN, David M. CLARK, Bridget S. WILKINS, ISBN