Page 683 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )
P. 683
CHAPTER 31 ■ Molecular Diagnostic Techniques and Applications 667
■ Other techniques are used to enhance the sensitivity and size, the Southern blot procedure can detect these changes
speci city o ampli cation techniques. T e selection o in DNA sequences.
one technique over another is o en based on actors such
as sensitivity and speci city pro les, cost, turnaround Fluorescent In Situ Hybridization (FISH)
time, and local experience.
■ Tis is a tissue-based molecular diagnostic assay. T e rapid
DNA Sequencing expansion in the availability o polyclonal and monoclo-
nal antibodies has ostered a dramatic increase in light
■ Molecular genetic testing ocuses on the examination o microscopic immunohistochemistry (IHC) and in situ
nucleic acids (DNA or RNA) by special techniques to hybridization.
determine whether a speci c nucleotide base sequence is ■ FISH analysis is used in the diagnosis o hematological
present. malignancies including CML, AML, Burkitt lymphoma,
■ Te applications o nucleic acid testing have expanded, and other lymphomas (e.g., ollicular lymphoma, mantle
despite higher costs associated with testing, in various cell lymphoma, MAL lymphoma, and anaplastic large
areas o the clinical laboratory. cell lymphoma).
■ Molecular testing has the ollowing advantages: aster ■ FISH analysis is generally better or detection o deletions
turnaround time, smaller required sample volumes, and and inversions than PCR.
increased speci city and sensitivity.
■ DNA sequencing is the determination o the precise Next-Generation Sequencing
sequence o nucleotides in a sample o DNA. T e most
popular method or doing this is called the dideoxy ■ NGS is not a method, it is an approach.
method or Sanger method. ■ Tis approach overcomes the limitations o traditional
■ Melting curve analysis (MCA) is a method o assessing the Sanger sequencing by providing highly parallel sequenc-
dissociation characteristics o double-stranded (DS) DNA ing with a separate sequence result or every sequence o
using a f uorophore during heating. interest.
■ Capillary electrophoresis (CE) is a relatively new, power- ■ T is has positioned NGS as the method o choice or tar-
ul separation technique that is ideally suited or handling geted resequencing o regions o the human genome.
small amounts o DNA. ■ NGS has the potential to be more cost-e ective and be
■ Pyrosequencing is an attempt to nd a aster and less able to simultaneously sequence complete genomes o
expensive way o molecular sequencing. patients to deliver personalized medicine.
■ NGS technologies permit analysis o mutation, rearrange-
Southern Blot Technique ment, ampli cations and deletions (DNA sequencing), or
coding and noncoding RNA (RNA sequencing).
■ T e Southern blot procedure is used less commonly than
in the past. Microarray Gene Chips
■ Specimen DNA is denatured and treated with restriction
enzymes to create DNA ragments; then, the ssDNA rag- ■ Microarray gene chip technology represents the merger
ments are separated by electrophoresis. o three elds: the Human Genome Project, abrication o
■ T e Southern blot procedure has clinical diagnostic appli- integrated circuits mounted on a substrate, and sophisti-
cations or disorders associated with signi cant changes cated computer power.
in DNA, a deletion or insertion o at least 50 to 100 bp ■ Te microarray chip technology is becoming a routine
and determination o clonality in lymphomas o or B tool or the high-throughput analysis o gene expression
cell origin. in a wide range o biological systems, including hema-
■ I a single-base mutation changes an enzyme restriction topathology applications such as di use large B-cell
site on the DNA, resulting in an altered band or ragment lymphoma.
REVIEW QUESTIONS
*Indicates ML (optional) and MLS advanced content. 2. T e rst inherited hematologic disorder to be diagnosed
1. Molecular techniques are being used to detect abnor- using molecular biologic assay was
malities o A. hemophilia A
A. erythrocytes B. actor V Leiden
B. leukocytes C. sickle cell anemia
C. some coagulation actors D. CML
D. All o the above
(continued)
