There are two major clinical assay formats widely used in molecular diagnostic laboratories. One of the formats are those tests developed by in vitro manufacturers and the other are assays developed by each laboratory (LDA), also known as home-brew assays.
2.1. TESTS DEVELOPED BY IN VITRO MANUFACTURERS This first category of tests is composed of kits developed by manufacturing companies to provide quality-controlled reagents for the performance of the entire molecular test for a determined clinical condition. For example, kits for monitoring patients diagnosed with human immunodeficiency virus-1 (HIV-1) infection through quantification of plasma HIV-1 RNA. These kits usually include the reagents necessary for nucleic acid isolation, amplification, and detection/quanti-tation. These complete kit assays usually include information about sensitivity, specificity, and tolerance limits for a particular clinical condition. They can be labeled by the manufacturer as FDA-approved, FDA-cleared, for research use only (RUO), or for investigational use only (IUO). Recently, we have seen the development and introduction of automated platforms for molecular diagnostic assays. A large number of these platforms have been developed by different in vitro diagnostic manufacturers, to provide automation for a single step or several steps of the testing procedure. Single-step automated instruments have been incorporated into molecular diagnostic laboratories, such as automated nucleic acid extractors, robotics for reagent preparation, and automated DNA sequencers. More recently, automated instruments that perform more than a single function have been developed and introduced in molecular diagnostic laboratories. An automated platform introduced by Roche Molecular Systems is the COBAS Amplicor® analyzer. This instrument contains a thermocycler for nucleic acid amplification, a 3D Cartesian arm with a gripper, heating blocks, wash stations, and even a colorimeter reader. It is anticipated that fully automated instruments will be developed and implemented in the near future. Analytical and clinical validation of these kits drastically differs depending on the labeling. Validation procedures will be discussed later.
2.2. LABORATORY DEVELOPED TESTS Laboratory developed tests are those tests (LDTs) fully developed and validated by the laboratory that performs them. They are also referred to as "home-brew" tests. Usually, these tests use a combination of reagents that are purchased separately from a variety of manufacturers. Each laboratory determines the performance characteristics of the assay for a specific clinical condition and a particular patient population. There are a number of different commercially available kits that are developed by a manufacturer to provide quality-controlled reagents to perform a particular step in molecular testing. For example, kits are commercially available for nucleic acid extraction, amplification reagents including controls, in vitro amplified nucleic acid detection systems, and so forth. In some instances, the laboratory develops a particular molecular test by combining two or more kits from the same or even different manufacturers for a determined clinical condition in a particular patient population. The analytical and clinical validation of the entire testing process is the responsibility of each laboratory. Analytical and clinical validation of each step and the entire procedure will be discussed in a later section.
2.3. ANALYTE-SPECIFIC REAGENT RULE AND THE ROLE OF THE FDA IN THE DEVELOPMENT OF LDT A new term has entered the lexicon of molecular diagnostics. The term "analyte-specific reagent" (ASR) was devised by the FDA to refer to reagent(s) used in LDT that confers specificity for detecting a particular analyte. The FDA has been involved in the review of in vitro diagnostic devices (IVDs) since the introduction of the Medical Device Amendment of 1976. This amendment expanded the FDA jurisdiction to include all reagents, assays, and equipment used for the diagnosis and/or treatment of diseases. The Safe Medical Devices Act of 1990 further classified medical devices into three categories that require different levels of regulatory stringency. Class I devices are regulated as general controls, such as registration of the manufacturer with the FDA, listing of all medical devices and/or drugs manufactured by a company, manufacture devices and/or reagents under good manufacturing practices, appropriate record keeping and restrictions on the use, sale, and distribution of the device. Class I devices are subject to premarket notification submissions to establish "substantial equivalence" (510K review), even though the FDA can exempt a Class I device from premarket notification. Most Class I in vitro diagnostic devices are considered of low risk and are often exempted from premarket review. Class II devices require special controls in addition to general controls to assure the safety and effectiveness of the IVD. Special controls include performance standards, postmarket surveillance of product problems, and guidelines for product use. Class II devices are considered of moderate risk and subject to premarket notification submission requirements or 510K review. Class III devices are considered high-risk products and are regulated by premarket approval applications or PMA review. Furthermore, devices are classified by generic device types into six categories: (1) generalpurpose reagents, which are Class I devices and mostly exempted from any review; (2) research use only (RUO), where FDA review is expected within 30 mo; (3) investigational use only (IUO), where the IVD is used to establish characteristics for FDA review; (4) kits for analysis (Class II or III requiring FDA review; (5) immunohistochemicals (Class I, II, or III); and, most recently, (6) analyte-specific reagents or ASRs. Most of the ASRs are considered Class I, but depending on their use, they could also be classified as Class II or Class III.
There are other mechanisms to offer testing to clinicians and health care providers, such as LDTs. The FDA considers LDTs as medical devices, and such products are subject to FDA regulatory oversight. However, with very few exceptions, the agency has decided not to execute its authority in this area. This decision was based not only on the limited resources available to the FDA but also on the understanding that laboratories developing LDTs for clinical use are regulated by Center for Medicare Services (CMS) under the provisions of CLIA'88. Even with this understanding, the FDA decided in 1998 to propose a modification to its regulations of oversight by introducing the new IVD category, named Analyte-Specific Reagent (ASR). The purpose of the FDA authorities was to introduce a mechanism by which they could clarify their role in the oversight of these devices and to use their regulatory authority to improve general controls and labeling applied to these devices without imposing a new burden for premarket review. In other words, under the ASR rule, the FDA has shifted the regulatory focus away from the assays developed in the laboratory in favor of regulating the reagents purchased by laboratories to develop LDTs.
As part of this new rule, the FDA defines ASRs as "antibodies, both monoclonal and polyclonal, specific receptor proteins, ligands, nucleic acid sequences, and similar reagents, which, through specific binding or chemical reactions with substances in a specimen, are intended for use in a diagnostic application for identification and quantification of an individual chemical substance or ligand in biological specimens." The FDA recognized ASR as the active ingredient of LDTs, that when used in combination with commonly used reagents and general laboratory instrumentations could be the basis for a LDT. In addition to defining ASR, the FDA proposed a series of controls and restrictions that apply to ASRs in order to ensure their quality and consistency and to clarify that laboratories that set up these tests are responsible for the test performance. Interestingly, these new controls applied to both manufacturers and laboratories developing LDTs that use ASRs. Another part of this rule mentions that manufacturers are required to register and list their ASR products with the FDA and follow good manufacturing practices. They are also expected to report adverse events resulting from product failure. Most of these products are exempted from premarket review but are subject to restricted labeling and use. The FDA also limited the labeling of these reagents to "description of the identity and purity including source and method of acquisition of the ASR in addition to standard information already required for general purpose reagents," with the specific disclaimer reading "Analyte specific reagent, analytic and performance characteristics are not established." Specifically, ASRs must be labeled to reflect that these reagents are a component used in LDTs and can only be sold to CLIA-certified high-complexity laboratories. Not every ASR that falls within this definition will be regulated under this rule. ASRs are excluded from the provisions of the rule when they are sold to IVD manufacturers or organizations that use reagents to develop tests for purposes other than providing diagnostic information to patients and practitioners (i.e., academic, research, forensic settings)
Moreover, laboratories developing LDTs are required to meet high-complexity testing requirements, to establish, maintain, and monitor the performance characteristics of the LDT, as specified in the CLIA regulations, and to label these LDTs with a specific statement. The FDA statement reads: "This test was developed and its performance characteristics determined by (Laboratory Name)." It has not been cleared or approved by the U.S. Food and Drug Administration." The rule became effective November 23, 1998. This clearly shifts the burden of analysis of performance characteristics from the manufacturer to the user. Hence, the expectation under the ASR rule is that the laboratory will develop, evaluate, and document the efficacy of diagnostic tests using ASRs and will determine whether there is a need to invest more resources in the proper evaluation of the ASR performance in a laboratory setting.
The restrictions on ASRs are not intended to apply to products developed by laboratories for in-house use. The FDA's preface notes that "the focus of this rule is the classification and regulation of ASRs that move in commerce, not tests developed in-house by clinical laboratories or ASRs created in-house and used exclusively by that laboratory for testing services." In other words, the ASR rule does not state that a laboratory needs to have ASR primers and probes. If primers and probes are made commercially and sold to clinical laboratories, the company must comply with the ASR rule and label the products accordingly. These tests also need to be accompanied by a disclaimer regarding the lack of FDA approval.
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