Page 674 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

658 PART 8 ■ Fundamentals of Hematological Analysis

PCR assay has been run. DNA and other biomolecules can be

separated based on charge, size, and shape (see Figs. 31.3 and

31.4). DNA has a net negative charge and will migrate toward

the anode (positive pole). PCR products are loaded into an aga-

rose gel and electrophoresed. Ethidium bromide is a dye that

intercalates into nucleic acids and will f uoresce with an orange

color under ultraviolet (UV) irradiation. An image analyzer

uses UV light to capture computer images o the PCR products.

Melting curve analysis (MCA) is a method o assessing

the dissociation characteristics o double-stranded (DS)

DNA using a f uorophore during heating. When the tem-

perature is raised, the DS begins to dissociate, which leads

to a rise in the absorbance intensity, hyperchromicity. Factor

V Leiden coagulopathies are a good example, i a particular

mutation is known.

T e principle o MCA is that slightly mismatched DNA

will denature at a lower temperature than per ectly matched FIGURE 31.3 PCR. Molecular gene structure o chromosomes 14

DNA. T e temperature is gradually increased, and the change and 18. Example o bcl-2 translocation detection by PCR. Lane 1, a
reactive lymphoid tissue showing no evidence o bcl-2 translocation
in f uorescence is measured, such as Factor V Leiden.
while in lanes 2 and 3 show 2 patients with ollicular lymphoma

DNA Sequencing having bcl-2 rearrangement. mw, molecular size markers. (From
McClatchey KD. Clinical Laboratory Medicine, 2nd ed, Philadelphia,
In the past decade, DNA sequencing has enabled the system- PA: Lippincott Williams & Wilkins, 2002.)

atic sequencing o more than 10,000 cancer exomes and 2,500

whole cancer genomes. T is has revolutionized the under- ■ Faster turnaround time

standing o the genetics o cancer and has lead to previously ■ Smaller required sample volumes

unrecognized cancer genes and new mutation signatures. ■ Increased speci city and sensitivity

Mutations arise rom replication errors or rom DNA

damage that is either repaired incorrectly or le unrepaired. Capillary electrophoresis (CE) (Figs. 31.5 and 31.6) is a

Leukemias, such as AML, typically have the lowest numbers o relatively new, power ul separation technique that is ideally

mutations compared to tumors, such as lung cancer. Di erent

mutational processes lead to idiosyncratic patterns o muta-

tions, termed “mutational signatures.” T ese patterns allow

identi cation o known and novel mutations. Features that can

characterize the action o a given mutation process include the

type o mutations observed, local sequence context, distri-

bution across the genome, and evidence o repair. Since the

discovery o the Philadelphia chromosome, mutation reoccur-

rence has proven to be a power ul tool or the identi cation o

new cancer genes. Estimates o the mutation rate in human

and B lymphocytes are thought to be on the order o 2 to 10

mutations per diploid genome per cell division. Sequencing

studies in normal blood has revealed insights into patterns

o clonal expansion associated with driver mutations. In the

blood, mutations ref ect about the relationship between aging,

particularly individuals older than 65 years o age, and the

typical epidemiological pattern seen in leukemias.

Many o the revolutionary changes that have occurred in

research in the biological sciences, particularly the Human FIGURE 31.4 PCR detection o actor V Leiden. Exon 10 o Factor

Genome Project, can be directly attributed to the ability to V gene is PCR ampli ed and cut with (D) resection enzyme MnlI.

manipulate DNA in de ned ways. Molecular genetic testing DNA rom normal (N) individuals contains MnlI, recognition site

ocuses on the examination o nucleic acids (DNA or RNA) (GAGG), which is lost in individuals carrying mutation. Undigested

by special techniques to determine whether a speci c nucle- DNA in normal (N) individuals shows 267 bp (upper bands) DNA

otide base sequence is present. ragment. A er resection enzyme treatment, normal individuals show
163 bp ragment (dashed arrow). In homozygous (Hom) patients,
T e applications o nucleic acid testing have expanded, digestion shows 200 bp ragment (solid arrow). In heterozygous (Het)

despite higher costs associated with testing, in various areas patients, both 163 bp and 200 bp ragments are seen. Internal control

o the clinical laboratory. T ese include genetic testing or o 67 bp in digested samples is seen in the bottom. U 5, undigested;

diagnosis and monitoring. Molecular testing has the ollow- D 5, digested. (From McClatchey KD. Clinical Laboratory Medicine,

ing advantages: 2nd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2002.)