Molecular Tests In The Clinical Coagulation Laboratory

4.1. GENERAL ASPECTS The applications of molecular diagnostics in coagulation testing are confined mainly to patients with thrombosis, an area for which laboratory testing has increased steadily over the past years. The availability of molecular diagnostics for hereditary thrombophilic disorders depends on the underlying molecular defect. Based on their unique molecular abnormalities, the FVL mutation, Prothrombin G20210A mutation, and the MTHFR mutation all lend themselves to relatively straightforward molecular analysis. In contrast, other hereditary thrombophilic defects such as antithrombin, protein C, and protein S deficiencies are not typically assessed by molecular methods in the clinical laboratory given their many different mutations.

Molecular testing for the FVL, the Prothrombin G20210A mutation, and the MTHFR mutation have become readily available in many clinical laboratories. Several molecular methods are available and the choice of a particular method is based on the experience of the laboratory and the availability of infrastructure. Similar to other areas of a clinical laboratory, reagent cost and labor costs account for most of the expenses of a molecular pathology laboratory.

By far the most common molecular approach for the hereditary thrombophilias are polymerase chain reaction (PCR)-based assays. PCR is ideal for detection of point mutations or single-nucleotide polymorphisms (SNPs), including those associated with coagulation disorders. A high degree of concordance between laboratories shows that Factor V Leiden can be reliably detected by a variety of PCR-based assays (55).

EDTA-anticoagulated peripheral blood is collected from the patient and DNA is extracted from the white blood cells. Subsequently, in a typical PCR assay, the template DNA is mixed with a pair of oligonucleotide primers of varying length, specific for a target gene sequence. After the numerous cycles of denaturation, annealing, and polymerase extension (synthesis) in a thermocycler, a PCR product is obtained that can then be analyzed by one of several different methods (56). With multiplex PCR, more than one PCR product can be obtained in a single test run. Several manual and automated multiplexed PCR assays have been developed for simultaneous detection of Factor V Leiden, prothrombin 20210A mutation, and the MTHFR mutations (57-59).

The most common method used to analyze PCR products is restriction fragment length polymorphism (RFLP) analysis. Other molecular methods that include PCR-based and non-PCR-based assays, some of which eliminate the use of restriction digestion, have also been incorporated into the molecular diagnostics laboratory with improved cost-efficiency (60-63). Furthermore, the use of hybridization probes have become commonplace for analysis and monitoring of the PCR product (56). In response to a need for rapid, simple, and reliable diagnostic assays, sequence specific primers (PCR-SSP) (60), allele-specific oligonucleotide (ASO) hybridization (55,56), rapid-cycle PCR using the LightCycler instrument (61), and the

Factor Leiden Allele

Fig. 2. Polyacrylamide gel electrophoresis of PCR-RFLP products for the Factor V Leiden (bottom) and prothrombin (FII) G20210A (top) polymorphisms. Lane 1, molecular size markers; lane 2, blank control; lane 3, normal control; lane 4, double heterozygous control; lanes 5, 6 and 8, normal patients; lane 7 heterozygous G2010A and normal FV Leiden patient.

Fig. 2. Polyacrylamide gel electrophoresis of PCR-RFLP products for the Factor V Leiden (bottom) and prothrombin (FII) G20210A (top) polymorphisms. Lane 1, molecular size markers; lane 2, blank control; lane 3, normal control; lane 4, double heterozygous control; lanes 5, 6 and 8, normal patients; lane 7 heterozygous G2010A and normal FV Leiden patient.

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Fig. 3. The Roche LightCycler real-time PCR instrument. For more information, see https://biochem.roche.com/lightcycler.

Nanochip technology based on electronic microarrays (62) have all been incorporated for molecular diagnosis in coagulation disorders.

4.2. RESTRICTION FRAGMENT LENGTH POLYMORPHISM The original molecular approach used for the detection of the FVL was PCR followed by RFLP. The FVL mutation abolishes a normal restriction enzyme site for Mull and creates a new restriction fragment pattern, allowing the mutant allele to be distinguished from the normal allele (36) (Fig. 2). The Mull fragments can be separated on agarose or polyarcylamide gels and visualized as wild-type or mutant bands. Of note, the Mull cleavage site might rarely be affected by other mutations that, although distinct from the FVL, could give a false-positive test with RFLP (55).

lf the inherited mutation does not result in change of the restriction enzyme site, a restriction site can also be introduced through site-directed mutagenesis. For the detection of the

Prothrombin G20210A mutation, a mutagenic primer creates a new Hiudlll restriction site, allowing identification of the mutant allele (42) (Fig. 2). As with FVL testing, typically PCR is used to amplify the 3' untranslated region of the Prothrombin gene surrounding the G20210A polymorphism, followed by restriction endonuclease digestion (64).

4.3. PCR-SEQUENCE SPECIFIC PRIMERS A PCR reaction with sequence specific primers (PCR-SSP), without the need for postamplification restriction enzyme analysis, is also available for FVL analysis (60). Using a sense primer complementary to both FV alleles along with either of two antisense allele-specific primers, each complementary to the normal and mutant allele, this simple and rapid PCR assay correlated 100% with the PCR-RFLP assay (60).

4.4. ALLELE-SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION Allele-specific oligonucleotide (ASO) hybridization analysis is also available for FVL analysis. With ASO hybridization, both normal and mutant alleles are detected when the PCR-amplified products are spotted onto two membranes followed by hybridization with normal and mutant labeled probes (55,56). This method is very useful when the SNP does not result in change of restriction enzyme recognition sites (56).

4.5. LIGHTCYCLER RAPID PCR In the LightCycler (Roche) platform (Fig. 3), amplification of DNA in glass capillaries occurs under very rapid cycling conditions (61). During PCR, probes labeled with fluorescein (FLU) and LC-Red640 emit fluorescence, that, depending on the degree of match between the probes and target DNA, will decrease when dissociation of the hybridized probes occurs under temperature control. A single-point mutation resulting in a nonperfect match will produce an earlier dissociation with a given temperature (melting point) than a perfect match will. With the LightCycler, PCR amplification and fluorescence detection can be done in the same tube within 60 min. The LightCycler is a convenient and economic platform for parallel detection of the three mutations (61). Of note, mutations within the Factor V gene can lead to a false-positive result.

4.6. DNA CHIPS With recent advances in gene chip technology, automated platforms are now available in which PCR products are hybridized to an array of probes to screen for multiple mutations. A DNA chip generally consists of DNA sequences representative of a genome of interest bound to a solid support. The target DNA to be tested is fluorescently labeled and, hybridized to the array, and the differential fluorescence is detected by laser scanning with confocal microscopy (65).

The Affymetrix chip is one type of DNA microchip in which short oligonucleotide probes with known nucleotide sequences are bound to a solid support surface (66). Affymetrix designed chips that contain oligonucleotide probes with many known SNPs, so-called "SniP chips," have been developed. These DNA microchips might provide a profile array that can be used to analyze gene expression levels in several different coagulation disorders (65,66).

Although global gene expression through the use of DNA microchips such as the Affymetrix GeneChip can provide valuable information, use of a small panel of genes that can provide rapid diagnosis of specific targets of interests have been emerging as a diagnostic tool. Nanogen lnc. has developed a DNA

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Fig. 4. The Nanogen Nanochip workstation (A), microarray cartridge (B) and electronic pads (C). For more information, see http://www. nanogen.com.

microchip wherein small subsets of target DNA can be transferred, denatured, and hybridized with the use of electric fields (62).

In contrast to DNA chips with a huge array of probes, the NanoChip provides a platform in which a small percentage of expressed genes from different patients can be analyzed. The NanoChip® (Fig. 4) is based on an electronic microarray in which voltage and current are applied for electronic field control. By confining hybridization to a specific site on a microar-ray, multiple genetic analysis can be done on a single microarray by distributing different assays to specific subsets of array sites (62). SNPs for different genes from different samples can all be detected in the single platform provided by the NanoChip. These small electronic chips have become recently available for FVL, Prothrombin G20210A, and MTHFR mutation analysis (67).

In the NanoChip system, biotinylated oligonucleotide capture probes are electronically located on specific sites on the NanoChip cartridge. After multiplexed PCR for FVL and G20210A, the amplified PCR product is transferred to a well plate and addressed electronically to pads on the Nanochip cartridge for hybridization to the capture probes. Results are read with fluorescently labeled wild and mutant reporter oligonu-cleotides; the intensity of the fluorescent signal correlates with the amount of target hybridized on the microarray (62). The use of an electric field permits improved hybridization.

Advantages of the NanoChip over the usual RFLP and LightCycler assay is very high throughput, automation, and a comprehensive results display (67). However, compared to the LightCycler, the NanoChip assay has a longer turnaround time.

The NanoChip is currently recommended for laboratories with medium to large test volume (67).

4.7. NON-PCR-BASED HYBRIDIZATION ASSAY

Direct hybridization methods without prior amplification with PCR are also available for DNA analysis. A signal amplification system known as the Invader assay (Third Wave Technologies, Madison, WI) can detect mutations or SNPs using a combination of hybridization technique with enzyme recognition (63). The assay is based on the formation of a three-stranded product comprised of a downstream signal probe, the target DNA template with normal or mutant nucleotide sequence, and the upstream Invader probe; this product is then cleaved by an enzyme, resulting in a measurable product (63).

The enzyme used in the Invader assay is referred to as Cleavase. This enzyme cleaves DNA molecules within a given temperature range and at specific recognizable structures generated by the hybridization of the two oligonucleotide probes to the target DNA. Detection of the Invader reaction products can be achieved by gel electrophoresis, or enzyme-linked immunoassay (63).

The ability of the Invader assay to detect a mutation is based on the substrate specificity of the cleavage enzyme used. Probe design is simpler because specificity does not rely entirely on the hybridization step but also on enzyme substrate recognition. Sample processing is simplified because the need for target amplification is eliminated. However, methods based on probe hybridization alone, such as the Invader assay, are generally considered less sensitive than PCR (56).

4.8. COMPARING MOLECULAR VS NONMOLECULAR TESTING FOR THE FACTOR V LEIDEN MUTATION Two different diagnostic approaches—a clot-based assay and a molecular assay—can be used for detection of Factor V Leiden mutation (68). The functional clot-based assays for evaluation of the phenotypic abnormality associated with the FV Leiden genetic defect (APC resistance) have been available as first-and second-generation assays.

The first-generation tests are not as sensitive as the second-generation assays or DNA-based analysis; however, APC resistance caused by mutations other than the FVL mutation can be detected by this first-generation assay. This has raised the question of the significance of cases being missed by not performing first-generation clot-based assays. The clinical importance of detecting these non-FVL cases is uncertain. A recent study concluded that measurement of APC resistance without dilution in FV-deficient plasma (a typical first-generation clot-based assay) was necessary to evaluate for potentially important risk factors associated with thrombosis other than FVL (69).

On the other hand, Anticoagulation treatment, coagulation inhibitors, and acute thrombosis all can variably interfere with the first-generation clot-based assays for APC resistance; in contrast, the current second-generation APC assays and molecular-based testing can both be performed in these circumstances. In addition, the second-generation assay and molecular genetic testing can distinguish between heterozygotes and homozygotes (68,70). Distinguishing between the heterozygote and homozygote state is important because of the risk associated with each FVL carrier state: an overall 3- to 7-fold increased risk of venous thrombosis for heterozygotes and a 50- to 100-fold increased risk for homozygotes (71).

Although a FVL functional clot-based assay is proposed by some as an appropriate starting point for the evaluation of APC resistance phenotype, the best approach to FVL analysis remains controversial (68). At our institution, testing for the FVL is done by DNA-based testing without performing a functional first or second-generation clot-based assay.

4.9. ADVANTAGES/DISADVANTAGES OF MOLECULAR TESTING The major advantages of molecular genetic testing are increased specificity and sensitivity. To some degree, the challenges posed on conventional clot-based coagulation assays given the dynamic nature of the coagulation process is circumvented with direct genetic analysis. DNA-based assays can be performed while the patient is on anticoagulation treatment, in the presence of specific or nonspecific inhibitors, including lupus anticoagulant, and in the setting of a recent thrombosis where expected consumption of coagulation proteins occurs regardless of the presence of a hereditary defect. In addition, as mentioned earlier, molecular testing can distinguish between heterozygotes and homozygotes.

Limitations of molecular testing include those generally known for all PCR-based assays, including the need to know the specific sequence of the DNA of interest, PCR contamination, and specificity resulting from nonspecific DNA products (56). In theory, all mutation detection methods not based on sequencing could give misleading results if there are other base exchanges in the vicinity of the mutation of interest. False positives caused by mutations or polymorphisms that alter a different basepair at the same restriction site when using RFLP have been described (56). False-negative PCR assays can occur in patients with the APC resistance phenotype because of mutations other than Factor V Leiden, albeit the significance of these mutations is controversial.

As mentioned previously, molecular genetic testing for antithrombin, protein C, and protein S deficiency is currently impractical given the inability to screen for all the mutations associated with these defects.

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