ASSAY DEVELOPMENT

Some types of sample collection and transport are well established and validated. For example, using a cellulose paper for collecting neonatal blood for testing inherited metabolic diseases has been practiced for 40 years and has well-established guidelines.¹ However, for the majority of assay developers, especially rapid or point-of-care (POC) assays, the mechanism of collecting and preparing samples can be a major development obstacle.

One common problem in diagnostic assays is that the detection target is often present in very low amounts relative to other materials (e.g., viral or cancer cells are present in low concentrations compared with the host or healthy cells). Such low levels of the target cells can cause problems with sensitivity and selectivity. Either the target is obscured by the high quantity of extraneous material present, or the detection system used nonspecifically reacts with the large amounts of other material.

What Sample Preparation Offers

Introducing a sample preparation step can significantly affect how well an assay works. Adding sample preparation can improve assay sensitivity and selectivity, or even reduce the total assay time.

In sample preparation, the amount of target material in a sample is increased by either retaining the materials of interest or removing those of no interest. Historically, sample preparation has been done by techniques such as centrifugation, sedimentation, or differential extraction (for molecular diagnostics).^2–3 Even though all these techniques do work, they require either expensive equipment or operator skill, and a certain amount of time to perform. In many cases, a simple filtration step can perform the cell separation in a more time- and cost-efficient manner.

Figure 1. (click to enlarge) Sizes of typical cells and the filters that can remove them.

Malaria Detection Using Filtration Sample Preparation

Malaria is a tropical disease that affects more than 40% of the world’s population. According to a 2005 World Health Organization report, malaria threatens 3.2 billion people, with 350 million to 500 million cases resulting in 1.5 million to 2.7 million deaths per year.⁶ Currently, the principal method for detecting malaria is a trained operator viewing blood smears. Viewing blood smears is a specialized activity that requires a significant amount of operator training. Using blood smears is also insensitive: a parasite concentration of 20,000 to 30,000 parasites per ml (p/ml) of whole blood is needed for an accurate diagnosis.⁷

A multiwell plate with a double layer of a glass fiber–based red cell enrichment material can isolate red cells from large amounts of human DNA.⁸ Removing the human DNA that is present in white cells results in the ability to detect lower concentrations of parasites with polymerase chain reaction (PCR) rather than using PCR on whole human blood. The minimum detection limit for real-time PCR (RT-PCR) on whole blood is approximately 1000 p/ml.⁹ However, by removing the DNA-containing cells, the sensitivity of RT-PCR is increased to approx­imately 20 p/ml.

By using a filterplate, the parasite-containing red cells are rapidly separated from the cells containing human DNA. The background signal observed is therefore significantly lower than the signal observed in traditional PCR reactions on human blood. Such lower signal allows a more rapid diagnosis of disease with less chance of a false-positive result. The ability to decrease the minimum detection limit by separating cells prior to PCR amplification may be applied in areas other than malaria. In any application in which the target cell is smaller than human white cells, purifying the target cell of interest should be possible. Subsequent cell lysis and PCR should then result in improved sensitivity.

Cell Capture Using TEMs

Using a fibrous media results in cells being trapped in the matrix of the media. However, if cells are to be visualized or recovered, a depth filter is ineffective. Rather, a true surface capture filter (e.g., TEMs) is the best method for such purposes.

Figure 2. (click to enlarge) Comparison of a TEM and a cast membrane by scanning electron microscopy (SEM).

The structure of cast membranes is similar to “open-celled foams (i.e., vacuoles with branched walls), and fastening the cellular network together are long hose- and chain-like ribs which spread out in three dimensions.”¹⁰ It can be seen that “the pore diameters thus have no direct relationship to the size of the particles being filtered because the pores are actually crevice-shaped rather then circular.”¹¹

The situation becomes more complex as the claimed pore size is based on measurements of physical properties (e.g., bubble point). The actual size of particles removed by the filter can therefore be very different from the claimed pore size of a cast membrane.

For several years, the ability to specifically isolate a particular cell type based on size has been exploited for microbiology and cancer testing. However, extending this ability into isolating viral particles from the cellular component of whole blood could have a significant impact, especially in molecular diagnostics.

Solid Phase Sample Collection in IVDs

For many years, the advantages of solid phase materials in remotely collecting and subsequently transporting clinical samples have been known. For example, collecting a sample onto a piece of high-purity cellulose allows the sample to be held in a defined area and transported at room temperature after collection. After a sample is received in a testing lab, extracting the pad allows certain proteins to be tested. The concept is simple, and has been extended to the collection of many other sample types in remote areas.

However, the problem with cellulose as a sample collection matrix is that the sample is not protected in any way. Temperature, humidity, and microbial contamination can destroy a sample before it gets to a testing lab. A sample may also be infectious: plain cellulose paper does not inactivate pathogens, so the potential for infection remains. With a plain paper collection media, neither the sample nor the end-user is protected.

To study viral diseases adequately, the virus should be collected safely, and the viral nucleic acid should be stabilized for further study. Tissue or body fluid samples are often frozen to stabilize the virus immediately after collection. But this can frequently be inconvenient when collecting samples in the field or away from the laboratory. To address such issues, Whatman Inc. (Florham Park, NJ) has developed the FTA technology for collecting, stabilizing, and purifying RNA and DNA viruses from plant and animal sources.

One of the key benefits of FTA technology for clinical samples is that both the sample and the user are protected. FTA is a patented cocktail of chemicals that can be added onto any solid phase. The chemicals in the FTA material will lyse cells and stabilize the nucleic acids that are liberated. While FTA is available in many different formats, the most common are based on the familiar blood stain cards that are made of cellulose papers. Because cells, including pathogenic organisms, are lysed on contact, FTA paper also protects the users from potential infection. Studies have shown that both viral and bacterial cells are lysed when they come into contact with the FTA paper.^12,13

Another key feature of FTA is that it stabilizes viral nucleic acids in a sample. Viral RNA is unstable and can be destroyed in the environment. FTA allows samples to be collected remotely and transported to the lab while stabilizing their RNA at room temperature. FTA can also stabilize RNA for several weeks at room temperature with no sample degradation.

Diagnostic Applications of FTA

In one study, several rabies virus isolates and characterized strains were propagated in in vitro cell cultures or in vivo animal hosts.¹⁴ Serial dilutions of these isolates in a DMEM cell culture medium were spotted on FTA cards. Six-mm punches were taken and eluted overnight in a 300-µl DMEM medium at 4°C. RNA was extracted from approximately 150 µl of the eluate using the Viral RNA Mini Kit by Qiagen NV (Venlo, The Netherlands). Viral RNA specific for the rabies virus was detected by a nested RT-PCR process.

A viral inactivation experiment found that drying the virus onto FTA cards for 2 hours rendered the virus completely inactive, which is important in the field when collecting specimens. The safe handling of specimens for molecular testing and their declassification as biohazardous materials are critical factors. Inactivating a virus facilitates the shipping of samples back to a central testing facility.

When the products of the RT-PCR process were separated on agarose gels, the amplified fragments gave bands of the expected size when compared with a frozen virus. In addition, sequencing the products of the RT-PCR amplification from a virus on FTA gave greater than 99% sequence homology to a frozen virus, which is considered the gold standard.

Viral RNA was completely stable at an ambient temperature for 43 days. Such stability and the easy collection of samples allow small amounts of biohazardous materials from live animals to be collected for epidemiology studies and for determining a disease’s transmission routes.

A more recent study of the porcine reproductive and respiratory syndrome virus (PRRSV) used FTA cards to detect viral levels in whole blood using a simplified procedure.¹⁵ In this study, a live virus was diluted in sterile saline or whole blood to determine the detection parameters. In a real-world test, blood samples from piglets were examined for the presence of PRRSV.

The study outlined a protocol for the RT-PCR analysis of PRRSV on FTA without extracting the viral DNA from the FTA disk. The method used fixed RNA to the FTA matrix by treatment with a phenolic solution.¹⁶ PCR inhibitors and cellular debris were washed away, and RNA remained bound to the FTA matrix for in situ first-strand cDNA synthesis. An aliquot of the cDNA was used in an RT-PCR to detect PRRSV. The RT-PCR profiles of viral RNA captured on FTA cards was identical to positive control viral RNA purified by a conven­tional manner.

In collaboration with others in the field, Ambion Inc. (Austin, TX) used FTA cards to capture West Nile virus (WNV) from brain tis­sue and oral swabs from birds, and to detect bovine vial diarrhea virus (BVDV) in whole bovine blood. Such viruses were captured and stabilized on FTA cards for transport back to central veterinary diagnostic labs where the viral RNA was extracted from the cards using the MagMax system by Ambion.

In basic cancer and clinical monitoring research, detecting disease biomarkers is invaluable for determining the targets for drug discovery and the severity and stage of cancer progression. The presence and levels of genetic biomarkers could also be linked to the prognosis of the course of the disease. For example, if particular biomarkers (e.g., circulating mutant DNA or cells containing active oncogenes) could be linked to a particular disease phenotype (e.g., development of metastatic cancer from a localized tumor), molecular diagnostic testing could aid in the treatment decisions for a particular patient.

With experiments and population studies of patients who have cancers with high metastatic potential, such correlations could be developed and become statistically significant. Such studies could identify subsets of patients whose treatment regimen is aggressive, and spare others the unnecessary exposure to chemo­therapeutic agents.

FTA has been used in several clinical studies examining genetic biomarkers for tumor progression in melanoma breast cancer lympho­proliferative disorders and upper aerodigestive tract cancer.^17–22 In these studies, microsatellites served as the biomarkers. Microsatellite alterations included loss of heterozygosity (LOH), allelic imbalance, and mutation. In one study, alcoholic patients and healthy volunteers were genotyped for the alcohol dehydrogenase 1C*1 (ADH1C*1) allele to determine the role of this gene in tumor formation.

In addition, whole blood samples are ideal for FTA-based DNA purification since intact cells are the best sample type for the FTA process. When in contact with the FTA chemical-coated matrix, intact cells quickly lyse, and the DNA becomes trapped (physically, not chemically) in the fibers in the matrix. Proteins, nucleases, and microbial organisms are inactivated by the FTA chemicals, and free radical traps protect DNA from damage at room temperature for extended periods of time. Collecting biological samples on FTA offers three advantages. First, the archiving of samples allows for look-back studies as new disease biomarkers are developed. Second, room-temperature storage has space-saving efficiency. Third, samples can be collected at the point of care and shipped to a central molecular diagnostic laboratory through the mail with no special handling required.

Conclusion

Sample preparation and collection are becoming increasingly important for IVD assays. A filtration step either to concentrate the target or remove the nonspecific materials gives developers of diagnostic assays a simple way to improve their assays’ performance.

Kevin D. Jones, PhD, is chief scientific officer at EDP Biotech (Knoxville, TN). He can be reached at kevinjones@edpbiotech.com.	Betsy Moran, PhD, is technical marketing manager at Whatman Inc. (Florham Park, NJ). She can be reached at betsy.moran@whatman.com.