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
Reagents
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
Giemsa solution
Staining
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
Don’t adjust
the pH of the
buffered
water!
Check the pH
of the
Sorensen
buffer!
May-Grünwald STAINING
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 Jolly Bodies Spherocyte
Dacryocyte (Teardrop cells)
Red Cell morphology
Dacryocyte Schistocyte Codocyte
Drepanocyte Jolly Bodies Spherocyte
Schistocyte definition
JF LESESVE (Hématologie)
Red Cell morphology
Dacryocyte Schistocyte Codocyte
Drepanocyte Jolly Bodies Spherocyte
Codocytes
Red Cell morphology
Dacryocyte Schistocyte Codocyte
Drepanocyte Jolly Bodies Spherocyte
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
With courtesy of G.Detry (CH Jolimont)
Lack of detection by automated hematology analyser
22
MYH9-related disease
Giant platelets Döhle bodies
Lack of detection by automated
hematology analyser
Platelet
distribution (MPV)
and count
White Blood Cell morphology
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)
Lymphocytic population
Benign Neoplastic ? Monomorphism
lymphocyte
immunophenotype
Polymorphism
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
1. Villous lymphocytes
2. Plasmocytoid
3. Cleaved cells
4. Clumped chromatin
5. Nucleoli
6. …
!!! pleiomorphic
Marginal zone lymphoma
• Red granules without peroxydase activity
Lymphoplasmocytic Lymphoma
S. Daliphard et al., Hématologie 2006; 12(1): 77-79
Cytoplasmic inclusions in B lymphomas
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
CLINIQUE CMF
EM (Blasts)
ALL AML
Phase Contrast Microscopy: blasts
Belgian EQA march 2010
Belgian EQA march 2010
ALL
PEROXIDASE: negative
Whole blood AML M3 (APL)
Quality Assessment and Education
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
Belgian EQAS
virtual microscopy
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