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654 PART 8 ■ Fundamentals of Hematological Analysis
Hematopathology the diagnosis o -cell malignancies. T e Ig and CR gene
rearrangements during normal B and -lymphocyte devel-
T e bene ts o molecular techniques in diagnosis and moni- opment, respectively, generate unique usions o variable,
toring include diversity, and joining (VDJ) segments, interspersed by ran-
■ Faster turnaround time dom nucleotide (N) insertion and/or deletion. T ese B and
■ Smaller required sample volumes -clonal recombinations generate patient-speci c DNA
■ Increased speci city and sensitivity length and sequences, which represent ideal molecular
markers or detection and quanti cation o leukemic cells
CML was the rst human malignancy to be consis- among normal lymphocytes in remission samples. Although
tently associated with a chromosome abnormality, the it is sensitive, the technology is susceptible to alse negatives
Philadelphia (Ph) chromosome. oday, molecular methods due to clonal evolution during natural history o the disease;
are used to identi y changes ranging rom a single chromo- thus, some patients may relapse with a clone di erent to that
some disorder to alterations involving the interchange o observed at presentation.
DNA between chromosomes. Abnormalities o erythrocytes
(sickle cell disease, and α- and β-thalassemias), leukocytes Minimal Residual Disease
(acute myelogenous leukemia [AML], acute lymphoblastic
leukemia [ALL], chronic myelogenous leukemia [CML], Molecular techniques, or example, PCR, real-time quan-
and lymphoma), and coagulation actors (hemophilia A, titative PCR (RQ-PCR), f ow cytometry, and cytogenetic
hemophilia B, and actor V Leiden de ect) can be detected marker studies, are more sensitive to a low number o cells
by molecular methods. than morphologic appearance in the peripheral blood. PCR
T e study o hematological malignancies, hematopathol- is able to detect one malignant cell in a population o 1 mil-
ogy, was the rst orm o human cancer to be studied in lion cells.
depth at the molecular level. Investigation o the Philadelphia Molecular techniques permit early detection o leukemia
chromosome at the molecular level revealed a translocation- relapse at subclinical levels, allow or early clinical interven-
induced gene rearrangement involving the Bcr and Abl tion, perhaps be ore early progenitor cells, including CD34+
genes that results in activation o the Abl cellular oncogene. cells, acquire genetic lesions that increase the aggressiveness
Fluorescence In Situ Hybridization (FISH) analysis is com- o the clone.
monly per ormed in the search or translocation (9;22) In the past, molecular detection and monitoring o
(BCR/ABL), which is diagnostic o CML. patients with chronic myeloid leukemia patients have been
Cytogenetic, FISH, and other molecular genetic tech- success ul.
niques can aid in establishing a diagnosis o a malignancy, Now, the current state o the art and development o
or example, ALL, detecting blast trans ormation emerging molecular techniques in other leukemias, or example, child-
rom CML, or determining a patient’s prognosis. In addition, hood ALL, is o growing interest. umor load, type o leu-
molecular techniques provide a diagnostic tool or clinicians kemia, whether disease speci c marker is identi able, and
in order to technological limits will determine the optimum methodol-
ogy or monitoring MRD.
■ Detect minimal residual disease (MRD) in hematological
malignancies
■ Purge malignant cells (e.g., bcr-positive cells) rom autolo- MOLECULAR TECHNIQUES
gous bone marrow be ore in usion IN HEMATOLOGY
■ Monitor patients ollowing bone marrow transplantation
■ Discover an early relapse in patients treated or a hemato- Since the inception o research on the Human Genome
logical malignancy Project, molecular biology has a high pro le in the eld or
medicine. Molecular techniques have enabled more accurate
Gene Rearrangement Studies classi cation o various disorders and advanced progress on
treatment o these disorders.
Gene rearrangement studies are important in diagnostic Characteristics o nucleic acids are discussed in Chapter 3.
hematopathology as indicators o clonality and as aids in Molecular genetic testing ocuses on examination o nucleic
determining the cellular lineage o a particular malignant acids (DNA or RNA) by special techniques to determine i
proli eration. Immunophenotyping categorizations are aided a speci c nucleotide base sequence is present. Applications
by the use o cluster designation (CD) or speci c lineages o o nucleic acid testing have expanded, despite higher costs
cells. CDs indicate a known cluster o monoclonal antibodies associated with testing, in various areas o the clinical labora-
binding to a known antigen on the cell sur ace o hematopoi- tory, including hematology (hematopathology). T e distinct
etic cells. advantages o molecular testing include greater accuracy in
Molecular diagnostic assays to detect heavy chain or diagnosis, aster turnaround time, smaller required sample
kappa chain rearrangements are use ul or establishing the volumes, and increased speci city and sensitivity in the
diagnosis o B-cell neoplasms. -cell receptor ( CR) beta, detection o minimal residual disease a er treatment or
gamma, and delta rearrangements are use ul in establishing cancer.
