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Investigation Methods in Hematology Power
Investigation Methods in Hematology Power
methods in
Hematology
Contents
Cell counts
Blood smear
Bone marrow examination
Flow cytometry
Cytogenetic
Molecular biology
Cell count
Cell count
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
Leukocyte analysis
Platelets analysis
Blood smear
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
Red cells
Platelets
Diameter 1-2
Leukocytes
Bone marrow
examination
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
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.
Bone marrow
examination
Bone marrow
examination
Immunohystochemical
tests
Cytochemical stains
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.
Cytochemical stains
Terminal deoxynucleotidyl transferase TdT
- intranuclear enzyme found in immature lymphoid cells
- useful marker to identify ALL and lymphomas
Flow cytometry
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
Flow cytometry
Cytogenetics
Chromosome structure study
Cytogenetic analysis became very important
for :
- diagnosis,
- classification,
- management of therapy,
- prognosis ,
- quantification of therapy response
research of hematologic disorders
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.
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.
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 :
In Situ Hybridization
AML1-ETO AML2
Inversion 16 AML4