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