Analytical Verification

Before a new or improved LDT is introduced into the laboratory menu, careful evaluation of performance characteristics of the assay under laboratory conditions needs to be done. In addition to evaluating these characteristics, undertaking analytical verification can provide useful information with regard to issues of practicality that will have to be addressed before introducing the LDA. It is important to point out that the performance of analytical validation programs has been challenging because of the lack of standards for a large number of nucleic acid targets. This shortfall has impacted the laboratory's ability to determine assay sensitivity and accuracy. Table 4 describes a number of different suppliers of commercially available reference materials that can be used during analytical verification and as a source of material for quality control purposes.

Analytical verification provides information about the performance characteristics of the assay. This section provides information about the design and execution of analytical verification studies. As part of the analytical verification, laboratories must determine the assay's analytical sensitivity, analytical

Table 4

Commercial Sources for Control Materials


Available material

Boston Biomedica Inc. (

Advanced Biotechnology Inc. (

National Institute of Standards and Technology (

Coriell Institute for Medical Research (

Stratagene ( AcroMetrix ( Promega Corporation ( American Type Culture Collection

Quantitative and qualitative controls, panels for HCV, HIV, HBV, CMV,

Chlamidya trachomatis, Mycobacterium tuberculosis Control DNA, virus and cell lines (HIV, HTLV, EBV, CMV, HSV, VZV, HCV, SIV, rubella, native and recombinant products purified viruses, antigens, proteins, antibodies, research kits) PCR-Based DNA profiling standard for human identity testing

The Coriell Cell Repositories provide essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing cells, cultures, and DNA derived from cell cultures for inherited disorders, microsatellite fingerprinting Universal Human Reference RNA for microarray technology HIV, HCV, and HBV

A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis and mutation detection Bacteria, bacteriophages, cell lines and hybridomas, filamentous fungi and yeast, tissue cultures, viruses, etc.

specificity, accuracy, and precision. In addition, for quantitative assays, the information regarding the linear dynamic range or reportable range of the overall process provides valuable information to determine when a measurement or change in the quantity of the analyte is considered clinically relevant or the result of inherent test error. A number of national and international organizations are taking steps in developing standard reference materials. The National Institute of Standards and Technology (NIST) developed one of the first nucleic acid standard reference materials for human identity testing. More recently, the World Health Organization (WHO) introduced a standard reference material for hepatitis C that has been used for verification of nucleic acid tests (NATs) for screening of blood and blood products and has also provided useful material for other in vitro diagnostics devices. There are currently three registered WHO standards for virologic molecular testing. The WHO HCV (heptatitis C virus) standard (HCV 96/790) consists of a lyophilized material, which contains 50,000 units per 0.5 mL or 100,000IU/mL. This material was assigned a value of 5.14 log genome equivalents/mL when tested by a variety of different molecular methods in different laboratories. Following the HCV standard, WHO introduced an HIV standard, HIV 97/656. Similar to the HCV standard, the HIV standard consist of a lyophilized preparation of HIV-1-genotype B virus-positive plasmapheresis donation. Several laboratories have tested this material with different technologies, obtaining a mean value of 4.79 log genome equivalents/mL, which was assigned a value of 100,000 IU/mL. In addition, the HBV (hepatitis B virus) standard 97/746 has been assigned a value of 1,000,000 IU/mL. Reference panels calibrated to WHO standard reference material are commercially available (Boston Biomedica Inc., Boston, MA).

Because of the lack of standards, laboratories have relied in the use of reference materials of different kinds to validate different tests. Laboratories can develop their own reference material for analytical validation, which can be used subsequently for monitoring daily performance of the assay. Creating an in-house reference material involves the use of an independently established method for determining target nucleic acid concentration. Alternatively, the laboratory can conduct studies to compare with another established assay. For example, samples can be split for a comparison study with another laboratory that performs a similar molecular test. These samples might be available in-house or from an outside source such as a collaborating laboratory, government (CDC, FDA, or National Institutes of Health), or even a commercial supplier (Boston Biomedical, Accrometrix). Natural analytical reference materials are those that consist of a known analyte or known quantity of an analyte as it occurs naturally in the test matrix, purified from the test matrix, and derived by culture or even cell lines considered nonsynthetic reference materials. Some examples used by a number of laboratories are intact virus particles, bacteria naturally containing the target in their genome, cell lines containing a specific genetic change, plasmids, intracellular RNA or DNA, and so forth. Furthermore, in some instances, it has been very difficult to obtain a natural analytical reference material and laboratories have depended on the development of synthetic reference materials. These synthetic reference materials could be in the form of DNA, either single or double stranded, or RNA manufactured in vitro, which can be accurately quantified by several physical and/or biochemical methods. Such synthetic reference materials can include synthesized DNA in the form of oligonucleotides, single-stranded DNA produced by cloning recombinant phage, cloning into vectors such us plasmids, or a DNA fragment produced by chemical or physical method from a larger DNA molecule followed by purification. On the other hand, synthetic RNA reference materials can be generated by in vitro transcription of DNA templates.

5.1. PRECISION Every laboratory's measurement has an inherent error or random variation. The evaluation of the precision of an assay allows determining what constitutes a change in the analyte as a result of changes in clinical condition vs expected fluctuations of the laboratory measurement. Precision refers to the agreement of values obtained between replicates of the same material. Evaluation of the precision should be measured for the entire process from nucleic acid extraction, amplification detection, and/or quantitation of the intended analyte. Precision studies should be carried out using test material or reference material that is similar or closely resembles the intended patient specimen. This could be achieved by using a serial dilution of a target positive specimen into a negative specimen. If a characterized patient specimen is not available, a reference material could be created by mixing a cell line, purified virus micro-organism, and so forth into a pool of patient's specimen known to be negative for the specific ana-lyte. In addition, enough material should be prepared in order to last the entire experiment. This material could well be used for daily monitoring of the assay's performance. The concentration of the analyte spiked into the patient's specimen will depend on the type of assay being developed. For qualitative assays, a single reference control with a concentration close to the limit of detection is recommended. On the other hand, for quantitative methods, at least two concentrations should be tested. These two concentrations should span the linear dynamic range of the assays, and when possible, be close to the value used for clinical decision-making. Whenever possible, precision studies should be performed with more than one lot or batch of reagents and/or materials.

5.2. ACCURACY Accuracy of a method refers to its ability to measure the true value of the particular analyte. Determination of the accuracy of molecular methods has been challenging because molecular methods have proven to be more sensitive than a large number of well-established gold standard methods. Generally, when evaluating a new assay, the results obtained should be compared to the results obtained from an established assay, which is considered a "gold standard method." In the absence of a gold standard to compare the results to, a laboratory could purchase reference material to be used for the analytical verification.

5.3. QUANTITATIVE METHODS: DETERMINATION OF LINEARITY AND REPORTABLE RANGE Linearity of an assay is the measure of the degree to which a curve approximates a straight line. Furthermore, the linear range of an assay is the span of analyte concentration for which the final value output is directly proportional to the analyte concentration, with acceptable accuracy and precision. The boundaries of the linear range constitute the upper and lower limit of quantitation, but they are not necessary for the limit of detection. Determination of the linearity of a quantitative assay might be performed by testing at least four different levels of the analyte. Again, test material could be prepared by spiking the analyte into negative patient sample or by performing serial dilution of a patient specimen known to contain a very high level of the analyte.

5.4. ANALYTICAL SENSITIVITY Analytical sensitivity is the lowest amount of a specific analyte that the method can repro-ducibly detect. The analytical sensitivity represents the ability of a test to obtain a positive result in concordance with positive results obtained by a reference method. For quantitative molecular methods, the lowest amount that the method can detect might be different from the lower limit of quantification for a particular nucleic acid or micro-organism. The lower limit of quantification is the lowest amount of a nucleic acid sequence that can be detected with acceptable precision. Analytical sensitivity could be determined by performing serial dilutions of an appropriate number of samples containing different concentrations of the analyte.

5.5. ANALYTICAL SPECIFICITY Analytical specificity is the ability of an analytical method to detect and/or quantify what the analyte is intended to measure. One aspect of specificity that can easily be measured is the lack of crossreactivity with closely related nucleic acid sequences, organisms, and so forth. In addition, for infectious disease testing, it is important to determine lack of crossreactivity with nucleic acids from organism present in the normal flora or that would normally be present in a patient specimen.

In addition to the parameters already discussed, it is important to determine the effect that interfering substances might have on the ability of a test to detect and/or quantitate the anlyte of interest. An interfering substance is a component present in the patient specimens that interferes with the accurate detection and/or quantitation of a specific target. The source of the interfering substance could be endogenous or exogenous. Exogenous interfering substances could be drugs, parenteral nutrition, anticoagulants, and so forth. Furthermore, it has been shown that the use of some anticoagulants such us heparin could interfere with the amplification process. On the other hand, endogenous interfering substances could be result of pathologic conditions, (e.g., lipids, bilirubin, etc.). Several approaches could be used to address the effects of different interfering substances. The addition of nucleic acid targets, either purified nucleic acid, cells, or micro-organisms to a variety of different specimens that contain interfering substances can be a means to address this issue. Furthermore, the specific target could be added to specimens from patients with specific conditions or receiving specific drug treatment.

5.6. PREANALYTICAL CONSIDERATION Appropriate specimen handling, including specimen collection and transport conditions, is critical to the testing process to ensure specimen integrity. Inappropriate specimen handling could result in nucleic acid degradation, which can lead to false negatives and/or inaccurate quantitation of nucleic acid. This is critical for assays that detect and/or measure eukaryote RNA and viral RNA. The best specimen type and quantity should be determined because molecular tests have been applied to a variety of specimen types. Appropriate selection of specimen type will depend on a variety of factors, including the condition being assayed and the type of nucleic acid required for the test. The format of the molecular assay being developed could greatly affect the amount of specimen required; for example, tests that require samples to be run in duplicate would require more specimen. Specimen transport and storage should be evaluated for every assay and type of nucleic acid. Specimen transport and storage conditions could vary significantly between specimen type, analyte (RNA vs DNA), cells, and micro-organism and must be determined by each laboratory. Special determinations of specimen transport and storage is crucial for RNA, because it is highly susceptible to degradation by ubiquitous enzymes. Transport and storage conditions can vary greatly for different specimens, analyte, and assay type. These conditions could vary from specimen storage at room temperature to having to centrifuge samples, remove plasma or serum, and store them at -80°C until tested.

Spiking target-negative specimens with a known amount of purified target, micro-organism, or cells can be used to perform the assessment of the preanalytical variables mentioned earlier. The same approach can also be used to assess the effect on performance by lipids, hemoglobin, bilirubin, therapeutic drugs, and specimen anticoagulants.

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