Investigation methods in

Hematology

Contents

Cell counts
Blood smear
Bone marrow examination
Flow cytometry
Cytogenetic
Molecular biology

Cell count
Careful assessment of the blood elements first
step in assessment of hematologic function and
diagnosis.
Examination of blood smears and hematologic
parameters often offer an important diagnostic
information, allow differential diagnosis and
indicate additional specific tests.
Blood elements include red cells (erythrocytes ),
white cells (leukocytes) and platelets. Evaluation
of blood requires quantification of each of these
cellular elements by manual or automatic
methods. Automatic methods are usually more
precise than manual procedures.
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Cell count
The blood sample should be obtained in proper
conditions. There are factors that affect the
hematologic measurements : patient activity,
level of hydration, medication, sex, age, race,
smoking and also the age of the sample may
affect the data collected.
Blood is collected by venipuncture into collection
tubes with anticoagulant. The most preferred
anticoagulant is EDTA ( tripotassium of trisodium
salts of ethylenediaminetetraacetic acid)
remove calcium - because produces complete
anticoagulation with minimal morphologic and
physical effects on cells.
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Red blood cells parameters

hemoglobin concentration Hb
hematocrit Ht (volume of packed red cells)
the volume of a blood sample occupied by
red cells
red cell count
mean corpuscular volume (MCV)= the
average volume of the red blood cells
mean corpuscular hemoglobin (MCH)
measure of the average Hb content per red
cell. MCH=Hb (g/l)/ red cell count
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Red blood cells parameters

mean corpuscular hemoglobin concentration
(MCHC) the average concentration of Hb in a
given red cell volume. MCHC=Hb (g/l)/Ht
red cell width (RDW) reflects the heterogeneity
of red cells size in sample
reticulocyte counts provide useful information
about the bone marrow capacity to synthesize
and release red cells in response to a physiologic
challenge ( anemia). Reticulocyte count
performed manually using supravital staining with
metilene blue looks as a dark blue meshwork or
granules ( it is staining precipitated RNA).
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Leukocyte analysis

automated WBC counts may be falsely elevated in

the presence of cryoglobulins , aggregated platelets

Platelets analysis

falsely low PLT caused by the presence of platelets

clumps or platelets agglutinins

falsely high PLT caused by fragments of red and

white blood cells

mean platelet volume (MPV) has been correlated

with some diseases. MPV has an inverse
relationship with PLT number : high MPV in ITP
( low number of PLT)
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Blood smear
Optimal morphology and staining are obtained
from nonanticoagulated blood from a
fingerstick procedure : a drop of blood
not too small, not too large, is placed in the
center of the clean glass slides; a second
spreader slide is placed at a 30 to 45 degree
angle and moved backward to make contact
with the blood drop, which will spread along
the slide edge. This technique creates a film of
blood 3-4 cm long.
Blood smears are usually stained with MayGrunwald-Giemsa stains.
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Blood smear
Examination of the blood smear :
initially under an intermediate power (10 to
20 X objective) in order to asses the
cellular distribution and staining; should
scan over the entire blood smear to ensure
the abnormal populations.
After use an oil immersion lens (50 to
100x objective)
Important to evaluate each cell type
for quantitative and qualitative
abnormalities
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Red cells

evaluated for changes in size, shape, Hb distribution,

presence of cellular inclusions
normal red cell dimeter 7,2-7,9 m
variation in red cell size = anisocytosis
red cell > 9 m macrocyte
red cell < 6 m microcyte
variation in red cell shape = poikilocytosis; examples :
spherocytes , teardrop cell (dacryocyte), drepanocyte (sickle
shaped), schistocyte ( fragmented cells).
hypochomia = poor hemoglobinization , leading to a very thin
rim of Hb or an increased area of central pallor
target cell = cell with a central spot of Hb surrounded by an
area of pallor ( because abnormal distribution of Hb)
red cells may contain inclusions : Howell-Jolly bodies
remnants of nuclear material, Pappenheimer bodies
remnants of mitochondria, infectious agents (malarial
parasites)
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The association between the pathologic red cell and disease

state

Cabot rings ( circular blue inclusion)

postsplenectomy, hemolytic anemia,
megaloblastic anemia
Howell-Jolly bodies - postsplenectomy, hemolytic
anemia, megaloblastic anemia
Ovalocyte ( elliptocyte) hereditary elliptocytosis
Hypochromic red cell iron deficiency anemia,
thalassemia , sideroblastic anemia
Macrocyte - increased erythropoiesis; oval
macrocyte in megaloblastic anemia, round
macrocyte in liver disease
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The association between the pathologic red cell and disease

state

Pappenheimer bodies sideroblastic

anemia, postsplenectomy
Rouleaux paraproteinemia
Schistocyte microangiopathic hemolytic
anemia ( DIC, TTP, severe burns)
Drepanocyte sickle cell disorders
Spherocytes hereditary spherocytosis,
immuno-hemolytic anemia
Target cell liver disease,
postsplenectomy, thalassemia
Teardrop cell myelofibrosis
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Platelets

PLT number may be estimated from the blood film.

Normal PLT counts should have several platelets ( 515) per oil immersion field or 1 platetet for 10 to 20
red blood cells

Diameter 1-2
Leukocytes

are analyzed last

neutrophils, eosinophils, basophils, lymphocytes,

monocytes normally seen on the blood smear

immature myeloid cells : myelocytes, metamyelocytes,

promyelocytes , blasts abnormal

should be counted at least 100 elements

should screen for morphologic abnormalities of the

cytoplasm and nucleus : toxic granulation ( azurophilic
granules) increased in infection or after growth factor
therapy; hypogranularity of neutrophils in
myelodysplastic disorder
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Bone marrow examination

Importance : diagnosis and
management of hematologic diseases
Examination of BM include 2
specimens : cytologic preparation of
BM cells obtained by aspiration of
BM and smear of the cells; the second
needle biopsy of the bone and
associated marrow, allowing optimal
evaluation of marrow cellularity,
fibrosis, infections, infiltrative diseases
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Bone marrow examination

Indications :
evaluation of primary bone marrow
tumors
staging for bone marrow involvement by
metastatic tumors
assessment of infectious diseases
fever of unknown origin
evaluation of metabolic storage diseases
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Bone marrow examination

Methods :
younger children may have marrow examination from the
anterior medial tibial area ( at birth, hematopoietic marrow is
found in all bones, by early childhood fat cells replace the bone
marrow hematopoietic cells in the extremities).
in adults aspiration from sternum at the second intercostal
space or anterior/posterior iliac crest aria. For biopsy posterior iliac crest area
The skin, subcutaneous tissue and periosteum in the area of
biopsy are anesthetized with a local anesthetic ( lidocaine).
From aspirated material we make smears; if additional
material is needed for flow cytometry , cytogenetics, culture
it is needed additional aspiration, collected into tubes with
anticoagulant.
The BM biopsy is made with a larger needle; touch preparations
of the bone biopsy should be made, particularly if no aspirate
was obtained.
The BM aspirate or touch preparations are stained with Wright
or May-Grunwald-Giemsa stains.
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Bone marrow examination

The aspirate smear allows cytologic examination of
BM cells . Should be evaluated minimum 500
nucleated cells under oil immersion magnification.
Erythroid cells represents 10-40% of marrow cells;
erythroid cells should be examined for abnormalities
in morphology and iron content. The myeloid cells
are usually predominant within BM and mature
forms are more numerous. If immature forms are
predominant reveals a pathologic process.
Megakaryocytes represent < 1% of BM cells. Some
other non-hematopoietic cells may be seen within
the BM : macrophages, mast cells, stromal cells, fat
cells , representing < 1% from total marrow
cellularity.
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Bone marrow examination

Bone marrow core biopsies and the clot are
fixed in formalin; they are stained with
hematoxylin and eosin or Giemsa stain for
morphologic examination.The BM biopsy
section provides the best representation of
BM and its anatomic relationships normal
localization of immature myeloid cells
adjacent to bony trabeculae; useful for
evaluation of infiltrative processes
( carcinoma, lymphoma), fibrosis. When
aspiration of BM is not possible the biopsy
remains the only method for diagnosis.
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Immunohystochemical tests
Performed in Pathology Department
On solid samples : bone marrow biopsy,
lymph node biopsy or any other tissue
Using monoclonal antibodies
Very important for a positive diagnosis
of acute leukemias, chronic
lymphoproliferative disorders,
metastatic tumors with bone marrow
involvement
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Cytochemical stains
useful in diagnostic and classification
of acute leukemias , differential
diagnosis between myeloid and
lymphoid acute leukemia
made on peripheral blood films, bone
marrow aspirates or touch
preparations from BM, lymph node or
other tissue biopsies
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Cytochemical stains
Myeloperoxidase
primary granules of neutrophils and secondary
granules of eosinophils contain MPO. Lymphocytes
and nucleated red blood cells lack the enzyme.
Positive in AML
Sudan Black B
the pattern of staining is parallel with MPO positive
staining for granulocytic cells and eosinophils
Specific esterase
used to identify cells of granulocytic lineage
Nonspecific esterase
used to identify monocytic cells, but do not stain
granulocytes and eosinophils.
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Cytochemical stains
Terminal deoxynucleotidyl transferase TdT
- intranuclear enzyme found in immature lymphoid cells
- useful marker to identify ALL and lymphomas

Periodic Acid-Schiff (PAS)

detects intracellular glycogen found in variable quantities in
most hematopoietic cells
AML6 PAS intense positive
Perls stain
used to identify iron in nucleated red blood cells
normally red cell precursors contain one or more blue
granules in 20-50% of the cells. When these granules
surround more than 2/3 of the nucleus of the red cell
precursor, the cell is called ringed sideroblast.

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Flow cytometry
Flow cytometry is the measurement
of cell properties (cytometry) as cells
move in single file ( flow) in a fluid
column and interrupt a beam of laser
light. The method allows the
quantitative and qualitative analysis
of many properties ( multiparameter)
of cell population from body fluids.
Using monoclonal antibodies.
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Flow cytometry
Very important for :
determination of lineage ( myeloid or
lymphoid ), if morphology and
cytochemistry are not enough for diagnosis
distinction between B- and T-cell acute
leukemias
detection of mixed lineage acute leukemias
detection of monoclonality in B-cell
lymphoproliferative disorders
important for prognosis
evaluation of minimal residual disease
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Flow cytometry
Precursor B-cell acute lymphocytic leukemia :
CD34, CD19, HLA-DR, CD10
T-cell acute lymphocytic leukemia : CD34, CD2,
CD7 ,TdT, some are CD10 positive
Acute myeloid leukemia : CD34, CD 117, CD33,
CD13, CD15, CD11b, cMPO.
B-cell chronic lymphoproliferative disorders : CD5,
CD23, CD19, CD20, FMC7, CD38; important to
differentiate types of lymphoproliferative disorders
( mantle lymphoma, CLL , follicular lymphoma,
hairy cell leukemia).
Peripheral T-cell lymphoma clonality may be
suspected by phenotypic abnormalities of major Tcell markers Cd4, CD8
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Cytogenetics
Chromosome structure study
Cytogenetic analysis became very important
for :
- diagnosis,
- classification,
- management of therapy,
- prognosis ,
- quantification of therapy response
- research of hematologic disorders
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Cytogenetics
Diagnosis
the presence of a clonal chromosomal abnormality establish the
presence of a clonal bone marrow disorder
very important for myelodysplastic syndrome diagnosis in
patients with mild cytopenias and bone marrow with minimal or no
dysplasia.
The presence of Philadelphia chromosome=t(9,22) establish
the diagnosis of chronic myeloid leukemia
Classification
the more recent World Health Organization (WHO) classification of
neoplastic diseases of hematopoietic and lymphoid tissues uses
cytogenetic finding into a number of subtypes
Examples :
t (8,21); inv (16); t (15,17) define distinct subsets of AML
del (5q) define a distinct subset of MDS
t (9,22) (q34:q11) present in the bone marrow and peripheral
blood in 95% of patients with CML

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Cytogenetics
Prognosis
AML, ALL, MDS with normal karyotypes
have an intermediate response to treatment
AML therapy related with abnormalities of
chromosome 5 and 7 poor prognosis
De novo AML better prognosis
Stratification of treatment cytogenetic
abnormalities are used in guiding patient
management, especially the choice of
postremission therapy.
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Molecular biology
The molecular biology techniques have an important role for diagnosis and enable a
large-scale synthesis of a number of recombinant proteins for therapy.
Techniques :

Southern Blot analysis

Northern Blot analysis

In Situ Hybridization

Polymerase Chain Reaction Analysis

Qualitative tests RT-PCR :

BCR-ABL major CML

BCR-ABL minor ALL

FIP1L1-PDGFRA primary eosinophilic syndrome

JAK2 policittemia vera, essential thrombocytemia, idiopathic myelofibrosis

Quantitative tests for diagnosis and monitoring the treatment

AML1-ETO AML2

PML-RARa (bcr1) APL

PML-RARa (bcr2) APL

Inversion 16 AML4

BCR-ABL major CML

BCR-ABL minor - ALL

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