Page 682 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

666 PART 8 ■ Fundamentals of Hematological Analysis

history o the disease; thus, some patients may relapse
CHAPTER SUMMARY
with a clone di erent to that observed at presentation.

The Human Genome Project
Minimal Residual Disease

■ T e goal o the Human Genome Project was to sequence

the exact order o the base pairs in a segment o DNA in ■ Molecular techniques, or example, PCR, real-time quan-

order to establish our genetic database. titative PCR (RQ-PCR), f ow cytometry, and cytogenetic

■ Genetic variations associated with speci c diseases or marker studies, are more sensitive to a low number o cells

increased the risk o speci c diseases are the target o than morphologic appearance in the peripheral blood.

genome investigations. ■ PCR is able to detect one malignant cell in a population

■ It is estimated that about 19,500 genes are present in o 1 million cells.

human beings. ■ umor load, type o leukemia, whether disease speci c

■ Te initial method o analysis used by scientists working on marker is identi able, and technological limits will deter-

the Human Genome Project in 2003 was rst-generation mine the optimum methodology or monitoring MRD.

DNA sequencing, Sanger sequencing. oday, second-gen-

eration sequencing or next-generation sequencing (NGS) Molecular Genetics in Hematology

analyzes millions o ragments o DNA in sequenced uni- ■ echniques in molecular genetics are beginning to be used

son rom a single patient specimen. extensively in hematology.

■ A wide range o abnormalities can be detected with these
Molecular Techniques in Hematology techniques. PCR is an in vitro method that ampli es low

■ Molecular genetic testing ocuses on examination o levels o speci c DNA sequences in a sample to higher
nucleic acids (DNA or RNA) by special techniques to quantities suitable or urther analysis. PCR analysis can

determine i a speci c nucleotide base sequence is present. lead to the detection o gene mutations that signi y the

■ Te distinct advantages o molecular testing include early development o cancer.
greater accuracy in diagnosis, aster turnaround time,

smaller required sample volumes, and increased speci c- Single Nucleotide Polymorphisms

ity and sensitivity in the detection o minimal residual dis- Single nucleotide polymorphisms (SNPs) comprise the

ease a er treatment or cancer. ■
most abundant source o genetic variation in the human

genome.
Hematopathology
■ Since the decoding o the human genome and the result-

■ T e bene ts o molecular techniques in hematopathology ing greater than 3 million SNPs, laboratory techniques
diagnosis and monitoring include aster turnaround time, have been able to associate disease states and pharmaco-

smaller required sample volumes, and increased speci c- logical responses with individual SNPs.

ity and sensitivity.

■ oday, molecular methods are used to identi y changes Polymerase Chain Reaction
ranging rom a single chromosome disorder to alterations

involving the interchange o DNA between chromosomes. ■ An amplicon is a piece o genetic material, such as DNA,

■ Hematological malignancies were the rst orm o human that can be ormed as the product o a natural event or
arti cial ampli cation technique, such as a polymerase
cancer to be studied in depth at the molecular level.

Investigation o the Philadelphia chromosome at the chain reaction (PCR).

molecular level revealed a translocation-induced gene ■ In laboratories in which PCR is per ormed requently,

rearrangement involving the Bcr and Abl genes that results any alse positives are generally caused by amplicon

in activation o the Abl cellular oncogene. contamination.
■ PCR is an in vitro method that ampli es low levels o spe-

ci c DNA sequences in a sample to higher quantities suit-
Gene Rearrangement Studies
able or urther analysis.

■ Gene rearrangement studies are important in diagnostic ■ T e three important applications o PCR are ampli cation
hematopathology as indicators o clonality and as aids in o DNA, identi cation o a target sequence, and synthesis

determining the cellular lineage o a particular malignant o a labeled antisense probe.

proli eration. ■ PCR has become increasingly popular or detecting chro-

■ Immunophenotyping categorizations are aided by the use o mosomal breakpoints, usion genes, and MRD a er che-
cluster designation (CD) or speci c lineages o cells. CDs motherapy or leukemia and lymphoma.

indicate a known cluster o monoclonal antibodies binding ■ One adaptation o PCR uses nested primers.

to a known antigen on the cell sur ace o hematopoietic cells. ■ Real- ime PCR is another method based on PCR.

■ Although it is sensitive, the technology is susceptible to ■ Q-PCR assumes 100% e ciency, variation in e ciency o
alse negatives due to clonal evolution during natural ampli cation, and no ampli cation o some specimens.