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Transferring molecular biology to the clinical laboratory
CLINICAL
O v e r v i e w o f R e q u i r e m e n t s for Q u a l i t y C o n t r o l a n d A s s u r a n c e i n M o l e c u l a r D i a g n o s t i c s T r a n s f e r r e d to the Clinical Laboratory and the Role of the FDA a n d U S D A i n L i c e n s i n g T e s t Kits T h a t M a k e T h i s Possible A. Bennett Jenson I and Stanley Geyer 2 IGeorgetown University Medical Center, Washington, DC ; 2Department of Pathology and Laboratory Medicine, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania
olecular diagnostics is defined as the clinical application of nucleic acid techniques to diagnose and monitor disease states, screen for susceptibility genes, and test for parentage and forensic identity. The potential applicalions have evolved rapidly and there are many different local, national, and international groups and societies that are focusing on its emerging roles in clinical and
M
diagnostic medicine. One of these, the College of American Pathology (CAP), Molecular Pathology Committee, was formed in 1989 to develop a laboratory accreditation checklist, and to provide long-range planning for proficiency assays) The CAP committee identified seven areas of interest: 1) molecular oncology testing, 2) molecular genetic disease testing, 3) molecular HLA/histocompatibility testing,
4) parentage testing by DNA polymorphisms, 5) forensic identity testing by DNA polym0rphisms, 6) in situ hybridi7ation testing, and 7) molecular testing for infectious diseases. Recent advances in biotechnology research have provided the basis for the rational use of molecular diagnostic test kits continued on page 138
Transferring Molecular Biology to the Clinical Laboratory Stanley J. Geyer I and A. Bennett Jenson 2 IDepartment of Pathology and Laboratory Medicine, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania; 2Georgetown University Medical Center, Washington, DC
olecular biology techniques in the research laboratory offer improved analytical sensitivity and specificity compared to most other detection methods, thus enabling investigators to identify and study smaller numbers of relevant molecules with greater accuracy in laboratory samples. These powerful methods--such as the polymerase chain reaction (PCR), Southern blotting, gene rear-
M
CIMNDC 13(11) 137-152
Elsevier
rangement analysis, and fluorescence in situ hybridiT~tiow--when applied to the study of genetic, infectious, neoplastic, and other human diseases, offer new insights about the causes and pathogenesis of disease. Molecular biology techniques are useful for identifying and studying genetic disorders, such as cystic fibrosis and Duchenne muscular dystrophy?4 Infeccontinued on page 138 0197-1859/93/$0.00 + 6.00
138 CLINICAL I M M U N O L O G Y Newsletter
R e q u i r e m e n t | for Quality Control a n d Assurance in Molecular Diagnostics continuedfrom page 137
in human and nonhuman animal medicine. Many large laboratories are incorporating both human and nonhuman diagnostic tests into their reference laboratories. Others concentrate on either human or nonhuman diseases. Many of these laboratories will be using molecular diagnostics in the near future. The largest difference between routine diagnostic tests (histopathology, electron microscopy, immunocytochemistry) and molecular diagnostic tests is that the latter will require the use of diagnostic
Transferring Molecular Biology to the Clinical Laboratory continuedfrom page 137
tious diseases, such as tuberculosis, 6 herpes simplex encephalitis, 7 human papilloma virus infection, g cytomegalovirus infection, 9 and others, can be detected using PCR and other molecular analyses, such as plasmid-profile analysis, genomic f'mgerprinting by restriction-endonuclease analysis, and Southern-hybridization fingerprinting. ~° The use of molecular biology assays to detect gene rearrangements and oncogenes assists in the diagnosis of certain cancers. ~:2 Molecular methods also play a key role in histocompatibility typing and tissue transplantation, t3 paternity testing, and forensic medicine. The improved analytical sensitivity and specificity offered by molecular biology appear to make the technology ideally suited for use in clinical laboratories. Because the methods and applications of mo-
Vol. 13, No. 11, 1993
test kits. Diagnostic test kits have multiple advantages, which include statements of their utility, reproducibility, and quality control, both for the kit and the laboratory. In addition, various companies spend years testing and packaging the kits for FDA (human) and USDA (nonhuman) licensing. Eventually, medical economics and the benefit derived from those diagnostic kits will determine their place in screening and diagnosis of human and nonhuman disease. Transferring molecular technology to the hospital laboratory where it can he used for diagnosis and clinical management of different diseases and genetic predispositions is a current subject of great interest. It requires both basic and practical knowledge of how this can be accom-
plished in a well-prepared service laboratory. Of equal importance is knowledge of which tests or test kits can be best used to make testing efficacious and feasible. In this issue, Drs. Geyer and Jenson discuss some of the considerations for transferring molecular technology to the clinical laboratory. Licensing test kits has placed a Iremendous burden on the FDA and USDA. We appreciate the contribution of the USDA to this issue, t:lN
lecular biology have been reviewed extensively elsewhere, this article will not recapitulate the biochemical basis or specific applications of molecular biology procedures, but rather will deal with important aspects of implementing these forms of testing in a hospital clinical laboratory. Table 1 contains a list of some of the issues that must be considered and addressed before implementing patient-care testing using molecular biology techniques. The evaluation of the clinical need to offer molecular biology testing must consider factors such as the advantages of molecular testing over alternative available forms of testing, turnaround time required to produce high-quality patient care, and the sensitivity and specificity of the testing. For example, the laboratory testing for tuberculosis is enhanced by PCR testing because it shortens the time required to identify the presence of organisms in a specimen and offers a substantial improvement in analytical sensitivity in detecting organisms. Using standard laboratory
methods, Mycobacterium tuberculosis requires weeks of growth in culture to become detectable and microscopic examination of specimen smears requires approximately 10,000 organism per ml of sample for detection. PCR testing permits the identification of organisms within hours and offers superior sensitivity. 6 Because of the improved turnaround time and increased analytical sensitivity, PCR testing offers the clinician the opportunity to choose the most appropriate therapy at the earliest time, thus improving therapeutic decision-making. Despite the obvious benefits of increased sensitivity and reduced turnaround time, the implementation of molecular biology testing for tuberculosis is also accompanied by some costs, which may be prohibitive in developing the testing at this time in a standard hospital diagnostic laboratory. Because no kit is yet available for tuberculosis testing, offering this test requires the development of procedures; the manufacture of reagents, such as primers
References 1.
College of American Pathology Conference XXIV, Molecular Pathology, August 19-22. 1922, Vancouver, BC
CLINICAL I M M U N O L O G Y NEWSLETTER (ISSN 0197-I 859) is issuedmonthly in one indexed volume per year by ElsevierScience PublishingCo., Inc.,655 Avenue of the Americas, N e w York, N Y 10010. Subscriptionpriceper year:.$145.00. For surfaceairliftadd $43.00. Second-classpostage paid at N e w York, NY, and at additionalmailing offices.Postmaster: Send address changes to ClinicalImmunologyNewsletter,ElsevierScience PublishingCo., Inc.,655 Avenue of the Americas, N e w York, N Y 10010. NOTE: No responsibilityis assumed by the Publisherfor any injuryand/or damage to pe~ons or property as a matter of p~ducts liability,negligence or otherwise,or from any use or operationof any m e , otis,producls,instructionsor ideas contained in the materialherein.N o suggestedtestor procedure should be carriedout unless,in the reader'sjudgment, itsriskisjustified.Because of rapid advances in the medical sciences,we n~rtmend thatthe independent verificationof diagnoses and drug doses should be made. Diso.~sious,views and recommendations as to medical procedures, choice of drugs and drog dosages are the respousibilityof the authors.
I 0197-1859/93/$0.00 + 6.00
I © 1993 Elsevier Science Publishing Co., Inc.
C L I N I C A L I M M U N O L O G Y Newsletter 139
Vol. 13, No. 11, 1993
and probes; the acquisition of new equipment; the remodelling of space to separate reagent preparation, amplification, and detection procedures to minimize carryover, contamination, and false-positive results; the development of quality control and quality assurance procedures; and possibly the payment of licensing fees for the use of the PCR technology. Many of these matters are unfamiliar to clinical laboratory technologists and pathologists and create considerable obstacles to the development of this technology. In a hospital with a low volume of tuberculosis testing, there may be little justification to considering the development of molecular testing, regardless of its clinical advantages. If sufficient clinical justification does exist for developing a particular molecular biology test in a clinical laboratory, the cost of the testing and the cost of the alternative testing should be compared. The cost analysis should include an evaluation of the cost of labor based on the time and the salaries of the individuals who are developing, performing, or evaluating the testing, the cost of equipment, the cost of reagents and supplies, the costs of quality control material, and the cost of licensing fees or user fees associated with the use of patented technology, such as PCR. The evaluation of personnel costs must include an accounting for the time spent to develop a test. Also, the professional time required to supervise the individuals performing the tests and the professional time needed for test interpretation must be included in the cost of the testing. Some aspects of molecular biology testing may require remodelling of space. For PCR testing, some laboratories have chosen to isolate the reagent preparation areas, the amplification or thermal cycling area, and the detection area to reduce the likelihood of contaminating a specimen by carryover from a previously amplified sample. For a laboratory that chooses to use isolated areas for the various steps of PCR, the availability of space and the cost of remodelling must be considered and accounted for in the developmental costs of adding molecular biology testing to the repertoire of the laboratory. Manufacturers are attempting to devise test methods and re-
agents that eliminate false-positive results and obviate the need for specially designed space and its associated costs. The Roche Amplicor kit substitutes deoxyuridine triphosphate (dUTP) for thymidine triphosphate (TIP) and uses uracil-N-glycosylase to catalyze the destruction of uracil-containing DNA strands before thermal cycling to break apart contaminating amplicons from previously amplified specimens. Eastman-Kodak is developing an automated closed-vessel system for in vitro PCR diagnostics that attempts to overcome special space requirements, t4 However, commercial PCR kits are not available for widespread application and the addition of tests developed within the hospital's laboratory may still require specially designed space to accomplish the analytical goals of the laboratory. The evaluation of cost must be accompanied by a similar appraisal of revenues. The net revenues depend on a successful billing and collection system and the willingness of payers to provide reimbursement for molecular biology testing. The charges associated with the testing must be related to the costs of testing, which are determined by a thorough cost-accounting system. Bills must be properly coded to identify the method involved in the testing and to receive the proper attention and reimbursement from payers. Because of the sophisticated nature of the testing and the need for a professional component of supervision and interpretation of the testing, the revenue plan should consider a mechanism for receiving payment for the professional aspects of interpreting the molecular diagnostic results. If the clinical needs, the benefits, the anticipated costs, and the projected revenues associated with a particular clinical test justify proceeding with the development of the test, the laboratory then must construct a plan for implementing the testing. In most hospital clinical laboratories, the availability of a test in kit form offers considerable advantages to developing a test de novo. Most kit-based tests contain instructions and supplies, pre-measured quantities of reagents that minimize technologist preparation time and maximize
© 1993 Elsevier Science Publishing Co., Inc.
TABLE 1. FACTORS TO BE CONSIDERED IN IMPLEME1WHNGMOLECULAR BIOLOGY TESTING IN A HOSPITAL CLINICAL LABORATORY Clinical
need
Improved sensitivity or specificity Improved turnaround time No suitable alternative form of testing available Cost of testing Personnel Equipment Reagents and supplies Controls and standards Cost of alternative forms of testing Billing and revenue Availability and training of testing personnel New test development versus use of kits Development of quality control Development of quality assurance Reporting Interpretations Quantitative results and reference ranges Administrative and regulatory issues
the cost-effective use of reagents, and quality control materials. Furthermore, the manufacturers of kits usually develop procedures that optimize the technology to produce best results. Kits offer convenience and standardization, without associated developmental costs and, thus, are ideally suited for use in a hospital laboratory. However, only a limited number of molecular biology tests are available from manufacturers in kit form. The development of a new test, without the availability of a kit or other manufacturer support, requires the production of reagents and quality control materials, writing about the technical procedures, and testing the new procedure against existing methods to determine the suitability of the test for clinical purposes. Whenever a new molecular biology test is added in the clinical laboratory, whether in the form of a manufacture's product or by the use of a newly developed method within the laboratory, the testing must be 0197-1859D3/$O.00 + 6.00
140 C L I N I C A L I M M U N O L O G Y Newsletter
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TABLE 2. REGULATORYQUESTIONS PERTAINING TO THE IMPLEMENTATIONOF MOLECULAR DIAGNOSTIC TESTING What role will the FDA play in regulating molecular diagnostic testing? What are the joint and separate responsibilities of the Health Care Financing Administration (HCFA) in regulating molecular biology testing? What does the FDA require of manufacturers of molecular diagnostic kits approved for clinical use? Can a molecular diagnostic kit that states "For research use ordy--not for in vitro diagnostic procedures" be used legally for patient-care purposes? What is the Compliance Policy Guide and where can a copy be obtained? What does the FDA require from a clinical laboratory for it to use a molecular diagnostic kit that has received FDA approval? Can molecular infectious disease kits that are labeled "Confirmation use only" be used for making the primaD, diagnosis? What are the FDA regulations concerning the use of "home brew" for patient care? Who is eligible to use "home brew" for patient testing? Who is legally responsible for "home brew" tests? What qualifications make someone eligible to use "home brew" materials? Under what circumstances can "home brew" tests become part of a patient record? Can a clinical laboratory charge a patient or insurance carder for "home brew" testing? Can a laboratory that performs "home brew" testing be a referral laboratory? If so, can the testing be offered on an interstate basis?
accompanied by quality control and quality assurance practices, similar to those applied to other laboratory tests. The quality control practices must include the use of positive and negative controls to ascertain proper performance of the method, equipment, reagents, and technical personnel. The quality control procedures should include methods for detecting false positives and false negatives and for minimizing their occurrences. Because of the exquisite analytical sensitivity of PCR, analytical false-positive reactions occur because of sample carryover, from improper pipetting techniques, and because of aerosol dispersion of previously amplified products. Safeguards can be developed to avoid false-positive PCR reactions and must be meticulously incorporated into all methods using this form of testing. Positive and negative control samples should be assayed each time molecular biology testing is performed on patient sampies. To reduce the cost per reportable patient test, a batching strategy should be employed that permits testing the largest number of clinical samples per control specimen. When testing of the control specimens fails to yield the proper result, patient testing results should not be reported until the cause of the control failure is determined and corrected. I
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If a molecular biology test produces quantitative results, high quality, standardized calibrating materials must be used to assure the accuracy and linearity of the quantitative methods. Likewise, quantitative assays must be accompanied by a reference range that enables the user to interpret the numerical results for diagnostic purposes or for selection of therapy. Likewise, some understanding of the distinctions between analytical and clinical sensitivity and analytical and clinical specificity is useful prior to implementing the testing and offering interpretations of the results, whether through the use of kits or by methods developed within a clinical laboratory. Analytical sensitivity refers to the ability of a test to detect small amounts of an analyte, whereas the clinical sensitivity of a test is def'med as the percent of positive test results in a population known to have the clinical condition for which the testing is done (true positives divided by the sum of true positives plus false negatives, times 100%). Analytical specificity refers to the ability of a test to identify only the substance under investigation, while clinical specificity is defined as the percent of true negative test results that occur in a population that does not have the condition for the which the test is used (true negatives divided by the sum of true
© 1993 Elsevier Science Publishing Co., Inc.
negatives plus false positives, times 100%). The strength of most molecular biology tests resides in their superior analytical sensitivity and specificity. However, this analytical power can create some clinical dilemmas. For example, using PCR to amplify the DNA of clinical samples, very small numbers of leukemia and lymphoma cells containing clonal gene rearrangements can be detected because of the sensitivity of the assay. But it is also possible to detect clonal rearrangements in several clinically benign disorders, ~-~7thereby producing a possible clinical false-positive result despite an analytical true positive test. If gene rearrangement studies are to be performed for the detection of malignant lymphoproliferative disorders, it is important for the person interpreting the test to understand that analytically positive results do not always translate into the clinical presence of malignancy. The same situation applies to other molecular biology tests and clinical conditions, where the presence of a positive test does not always correspond to the presence of disease and an analytical true negative does not always indicate with certainty the absence of a clinical condition. In addition to standard quality control methods, the clinical laboratory performing molecular biology testing should develop quality assurance procedures to guarantee the success of the testing program. Once testing is offered for patient care, a plan must exist to assure the continuous availability of the testing. If a particular method fails, a backup method with equal performance characteristics---turnaround time, sensitivity and specificity, cost--should be available. For example, for PCR testing, if the test fails because of reagent contamination with the production of analytically false-positive results, a suitable alternative PCR method, with a different set of primers, may be employed to replace the primary method, thus assuring continuity of the service to maintain the confidence of the physicians who order the testing. Quality assurance activities should be developed in the clinical laboratory to assess the success and proper utilization of
Vol. 13, No. 11, 1993
molecular biology testing by measuring the actual number of clinically reported tests performed and by comparing the results of the molecular biology methods with the results obtained by other types of testing. A good quality assurance program should be able to determine the diagnostic sensitivity and specificity and the predictive value of the new testing when compared to the existing methods. Also, the quality assurance program should assess the proper clinical use of the test to minimize unnecessary utilization. Likewise, a quality assurance program associated with clinical molecular biology testing should include proficiency testing or external quality control, in which the personnel performing and interpreting the tests are responsible for analyzing control specimens obtained from a source external to the department. The College of American Pathologists (CAP) has not yet developed a proficiency testing program for PCR. However, the CAP offers an external quality assurance program for Southern blot analysis of antigen receptor (B and T cell) gene rearrangement. If molecular biology is to gain a niche in routine clinical laboratory testing, it will need to be subjected to the same standards of quality assessment and quality assurance as other forms of testing. Until commercially available proficiency testing material becomes available, a laboratory may consider producing its own program by exchanging samples with other laboratories performing similar tests for the same target analytes. While this development of external quality control may add to the cost of testing, it offers additional evidence and assurance that the laboratory is performing the testing proficiently. Regulatory matters will affect the implementation of certain forms of molecular testing, particularly PCR testing. Table 2 contains a list of questions that need to be addressed by Regulatory Agencies--particularly the U.S. Food and Drug Administration---in order to achieve uniform development and competent applications of molecular diagnostic testing in clinical laboratories. Despite the authors' inquiries to the FDA, the questions remain unan-
CLINICAL I M M U N O L O G Y Newsletter 141
swered. The uncertainty of the regulations pertaining to molecular diagnostics presents a significant obstacle to the development of new tests within a hospital laboratory. In summary, molecular biology testing performed in a hospital clinical laboratory for patient-care purposes holds great promise for improving sensitivity and specificity of testing, for decreasing turnaround time of testing required for critical diagnoses, for adding to the understanding of disease mechanisms, for improving tissue typing results, for improving paternity testing, and for adding valuable tools to forensic investigations. However, serious limitations--developmental and operational costs, special space requirements, the creation of quality control and quality assurance practices, and regulatory matters---still preclude widespread application of this type of testing in a routine clinical laboratory. Commercial kits will hasten the introduction of molecular biology testing in clinical laboratories by obviating the need for costly development within a hospital laboratory. Most of the methods used in the kit-based testing are already familiar to most clinical laboratory technologists, and the kits also contain appropriate control material, thus avoiding the necessity for making costly control samples within the laboratory. Finally, in the case of kits marketed by Roche Diagnostic Systems, the cost of purchasing the kit includes the licensing fee associated with using the Roche patented PCR-based testing. Transferring molecular biology to the diagnostic laboratory requires collaboration between basic scientists, clinical pathologists and technologists, manufacturers, and regulatory agencies to assure competent and economical clinical use of molecular technology. The challenge is to have the wisdom to manage health care resources effectively and to use tests that elevate the quality of patient care. ClN References 1.
Killeen AA, Orr I-IT: Molecular genetics of human disease: Clinical applications of genetic linkage analysis. In: Fenoglio-Preiser CM, Willman CL (eds): Molecular Diagnostics in Pathology. Baltimore, Williams & Wilkins, 1991, pp 67-80.
© 1993 Elsevier Science Publishing Co., Inc.
2.
Koenig M, Hoffman EP, Bertelson CJ, et al.: Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50:509-517, 1987. 3. Tsui L-C, Buchwald M, Barker D, et aL: Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 230:10541057, 1985. 4. Wainwright BJ, Scrambler P J, Schmidtke J, et al.: Localization of cystic fibrosis Incus to human chromosome 7cen-q22. Nature 318:384-385, 1985. 5. White R, Woodward S, Leppe~ M, et at.: A closely linked genetic marker for cystic fibrosis. Nature 318:382-384, 1985. 6. Manjunath N, Shankar P, Rajan L, et at.: Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis. Tubercle 72:21-27, 1991. 7. Puchhammer-Stockl E, Popow-Kraupp T, Heinz FX, et al.: Establishment of PCR for the early diagnosis of herpes simplex encephalitis. J Med Virol 32:77-82, 1990. 8. Nuovo GJ, Becker J, MargioUa M, et al.: Histologic distribution of polymerase chain reactionamplified human papillomavirus 6 and 11 in penile lesions. Am J Surg Pathol 16:269-275, 1992. 9. Buffone GJ, Demmler GJ, Schimbor CM, Greet J: Improved amplification of cytomegalovims DNA from urine after porification of DNA with glass beads. Clin Chem 37:1945--1949, 1991. 10. Tompkins LS: The use of molecular methods in infectious diseases. N Engl J Med 327:1290-1297, 1992. 11. Reid AH, Omningham RE, Frizzera G, O'Leary TJ: hel-2 Rearrangement in Hodgkin's disease. Am J Pathol 142:395-402, 1993. 12. Lonn U, Lonn S, Nylen U, et al.: Gene amplification detected in carcinoma from human urinary bladder washings by the polymerase chain reaction method. Cancer 71:3605--3610, 1993. 13. KuKuruga D, Eisenbrey AB: Role of molecular tools in tissue transplantation. Lab Med 24:589595, 1993. 14. Findlay JB, Atwood SM, Bergneyer L, et al.: Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction. Clin Chem 39:1927-1933, 1993. 15. Fishleder A, Tubbs R, Hesse DO, Levine H: Uniform detection of immunoglobulin-gene rearrangement in benign lymphoepithelial lesions. New Engl J Med 316:1118-1121, 1987. 16. Weiss LM, Wood GS, Ellisen LW, et al.: Clonal T-cell populations in pityriasis lichenoides et variolaforma acuta (Mucha-Haherman disease). Am J Pathol 126:417-421, 1987. 17. LeBoit PE, Abel EA, Clear), ML, et al.: CIonal rearrangement of the T cell beta gene in the circulating blood of lymphocytes of erythrodermic mucinosis. Blood 71:1329-1333, 1988.
019%i859/93/$0.00 + 6.00
O v e r v i e w o f R e q u i r e m e n t s for Q u a l i t y C o n t r o l a n d A s s u r a n c e i n M o l e c u l a r D i a g n o s t i c s T r a n s f e r r e d to the Clinical Laboratory and the Role of the FDA a n d U S D A i n L i c e n s i n g T e s t Kits T h a t M a k e T h i s Possible A. Bennett Jenson I and Stanley Geyer 2 IGeorgetown University Medical Center, Washington, DC ; 2Department of Pathology and Laboratory Medicine, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania
olecular diagnostics is defined as the clinical application of nucleic acid techniques to diagnose and monitor disease states, screen for susceptibility genes, and test for parentage and forensic identity. The potential applicalions have evolved rapidly and there are many different local, national, and international groups and societies that are focusing on its emerging roles in clinical and
M
diagnostic medicine. One of these, the College of American Pathology (CAP), Molecular Pathology Committee, was formed in 1989 to develop a laboratory accreditation checklist, and to provide long-range planning for proficiency assays) The CAP committee identified seven areas of interest: 1) molecular oncology testing, 2) molecular genetic disease testing, 3) molecular HLA/histocompatibility testing,
4) parentage testing by DNA polymorphisms, 5) forensic identity testing by DNA polym0rphisms, 6) in situ hybridi7ation testing, and 7) molecular testing for infectious diseases. Recent advances in biotechnology research have provided the basis for the rational use of molecular diagnostic test kits continued on page 138
Transferring Molecular Biology to the Clinical Laboratory Stanley J. Geyer I and A. Bennett Jenson 2 IDepartment of Pathology and Laboratory Medicine, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania; 2Georgetown University Medical Center, Washington, DC
olecular biology techniques in the research laboratory offer improved analytical sensitivity and specificity compared to most other detection methods, thus enabling investigators to identify and study smaller numbers of relevant molecules with greater accuracy in laboratory samples. These powerful methods--such as the polymerase chain reaction (PCR), Southern blotting, gene rear-
M
CIMNDC 13(11) 137-152
Elsevier
rangement analysis, and fluorescence in situ hybridiT~tiow--when applied to the study of genetic, infectious, neoplastic, and other human diseases, offer new insights about the causes and pathogenesis of disease. Molecular biology techniques are useful for identifying and studying genetic disorders, such as cystic fibrosis and Duchenne muscular dystrophy?4 Infeccontinued on page 138 0197-1859/93/$0.00 + 6.00
138 CLINICAL I M M U N O L O G Y Newsletter
R e q u i r e m e n t | for Quality Control a n d Assurance in Molecular Diagnostics continuedfrom page 137
in human and nonhuman animal medicine. Many large laboratories are incorporating both human and nonhuman diagnostic tests into their reference laboratories. Others concentrate on either human or nonhuman diseases. Many of these laboratories will be using molecular diagnostics in the near future. The largest difference between routine diagnostic tests (histopathology, electron microscopy, immunocytochemistry) and molecular diagnostic tests is that the latter will require the use of diagnostic
Transferring Molecular Biology to the Clinical Laboratory continuedfrom page 137
tious diseases, such as tuberculosis, 6 herpes simplex encephalitis, 7 human papilloma virus infection, g cytomegalovirus infection, 9 and others, can be detected using PCR and other molecular analyses, such as plasmid-profile analysis, genomic f'mgerprinting by restriction-endonuclease analysis, and Southern-hybridization fingerprinting. ~° The use of molecular biology assays to detect gene rearrangements and oncogenes assists in the diagnosis of certain cancers. ~:2 Molecular methods also play a key role in histocompatibility typing and tissue transplantation, t3 paternity testing, and forensic medicine. The improved analytical sensitivity and specificity offered by molecular biology appear to make the technology ideally suited for use in clinical laboratories. Because the methods and applications of mo-
Vol. 13, No. 11, 1993
test kits. Diagnostic test kits have multiple advantages, which include statements of their utility, reproducibility, and quality control, both for the kit and the laboratory. In addition, various companies spend years testing and packaging the kits for FDA (human) and USDA (nonhuman) licensing. Eventually, medical economics and the benefit derived from those diagnostic kits will determine their place in screening and diagnosis of human and nonhuman disease. Transferring molecular technology to the hospital laboratory where it can he used for diagnosis and clinical management of different diseases and genetic predispositions is a current subject of great interest. It requires both basic and practical knowledge of how this can be accom-
plished in a well-prepared service laboratory. Of equal importance is knowledge of which tests or test kits can be best used to make testing efficacious and feasible. In this issue, Drs. Geyer and Jenson discuss some of the considerations for transferring molecular technology to the clinical laboratory. Licensing test kits has placed a Iremendous burden on the FDA and USDA. We appreciate the contribution of the USDA to this issue, t:lN
lecular biology have been reviewed extensively elsewhere, this article will not recapitulate the biochemical basis or specific applications of molecular biology procedures, but rather will deal with important aspects of implementing these forms of testing in a hospital clinical laboratory. Table 1 contains a list of some of the issues that must be considered and addressed before implementing patient-care testing using molecular biology techniques. The evaluation of the clinical need to offer molecular biology testing must consider factors such as the advantages of molecular testing over alternative available forms of testing, turnaround time required to produce high-quality patient care, and the sensitivity and specificity of the testing. For example, the laboratory testing for tuberculosis is enhanced by PCR testing because it shortens the time required to identify the presence of organisms in a specimen and offers a substantial improvement in analytical sensitivity in detecting organisms. Using standard laboratory
methods, Mycobacterium tuberculosis requires weeks of growth in culture to become detectable and microscopic examination of specimen smears requires approximately 10,000 organism per ml of sample for detection. PCR testing permits the identification of organisms within hours and offers superior sensitivity. 6 Because of the improved turnaround time and increased analytical sensitivity, PCR testing offers the clinician the opportunity to choose the most appropriate therapy at the earliest time, thus improving therapeutic decision-making. Despite the obvious benefits of increased sensitivity and reduced turnaround time, the implementation of molecular biology testing for tuberculosis is also accompanied by some costs, which may be prohibitive in developing the testing at this time in a standard hospital diagnostic laboratory. Because no kit is yet available for tuberculosis testing, offering this test requires the development of procedures; the manufacture of reagents, such as primers
References 1.
College of American Pathology Conference XXIV, Molecular Pathology, August 19-22. 1922, Vancouver, BC
CLINICAL I M M U N O L O G Y NEWSLETTER (ISSN 0197-I 859) is issuedmonthly in one indexed volume per year by ElsevierScience PublishingCo., Inc.,655 Avenue of the Americas, N e w York, N Y 10010. Subscriptionpriceper year:.$145.00. For surfaceairliftadd $43.00. Second-classpostage paid at N e w York, NY, and at additionalmailing offices.Postmaster: Send address changes to ClinicalImmunologyNewsletter,ElsevierScience PublishingCo., Inc.,655 Avenue of the Americas, N e w York, N Y 10010. NOTE: No responsibilityis assumed by the Publisherfor any injuryand/or damage to pe~ons or property as a matter of p~ducts liability,negligence or otherwise,or from any use or operationof any m e , otis,producls,instructionsor ideas contained in the materialherein.N o suggestedtestor procedure should be carriedout unless,in the reader'sjudgment, itsriskisjustified.Because of rapid advances in the medical sciences,we n~rtmend thatthe independent verificationof diagnoses and drug doses should be made. Diso.~sious,views and recommendations as to medical procedures, choice of drugs and drog dosages are the respousibilityof the authors.
I 0197-1859/93/$0.00 + 6.00
I © 1993 Elsevier Science Publishing Co., Inc.
C L I N I C A L I M M U N O L O G Y Newsletter 139
Vol. 13, No. 11, 1993
and probes; the acquisition of new equipment; the remodelling of space to separate reagent preparation, amplification, and detection procedures to minimize carryover, contamination, and false-positive results; the development of quality control and quality assurance procedures; and possibly the payment of licensing fees for the use of the PCR technology. Many of these matters are unfamiliar to clinical laboratory technologists and pathologists and create considerable obstacles to the development of this technology. In a hospital with a low volume of tuberculosis testing, there may be little justification to considering the development of molecular testing, regardless of its clinical advantages. If sufficient clinical justification does exist for developing a particular molecular biology test in a clinical laboratory, the cost of the testing and the cost of the alternative testing should be compared. The cost analysis should include an evaluation of the cost of labor based on the time and the salaries of the individuals who are developing, performing, or evaluating the testing, the cost of equipment, the cost of reagents and supplies, the costs of quality control material, and the cost of licensing fees or user fees associated with the use of patented technology, such as PCR. The evaluation of personnel costs must include an accounting for the time spent to develop a test. Also, the professional time required to supervise the individuals performing the tests and the professional time needed for test interpretation must be included in the cost of the testing. Some aspects of molecular biology testing may require remodelling of space. For PCR testing, some laboratories have chosen to isolate the reagent preparation areas, the amplification or thermal cycling area, and the detection area to reduce the likelihood of contaminating a specimen by carryover from a previously amplified sample. For a laboratory that chooses to use isolated areas for the various steps of PCR, the availability of space and the cost of remodelling must be considered and accounted for in the developmental costs of adding molecular biology testing to the repertoire of the laboratory. Manufacturers are attempting to devise test methods and re-
agents that eliminate false-positive results and obviate the need for specially designed space and its associated costs. The Roche Amplicor kit substitutes deoxyuridine triphosphate (dUTP) for thymidine triphosphate (TIP) and uses uracil-N-glycosylase to catalyze the destruction of uracil-containing DNA strands before thermal cycling to break apart contaminating amplicons from previously amplified specimens. Eastman-Kodak is developing an automated closed-vessel system for in vitro PCR diagnostics that attempts to overcome special space requirements, t4 However, commercial PCR kits are not available for widespread application and the addition of tests developed within the hospital's laboratory may still require specially designed space to accomplish the analytical goals of the laboratory. The evaluation of cost must be accompanied by a similar appraisal of revenues. The net revenues depend on a successful billing and collection system and the willingness of payers to provide reimbursement for molecular biology testing. The charges associated with the testing must be related to the costs of testing, which are determined by a thorough cost-accounting system. Bills must be properly coded to identify the method involved in the testing and to receive the proper attention and reimbursement from payers. Because of the sophisticated nature of the testing and the need for a professional component of supervision and interpretation of the testing, the revenue plan should consider a mechanism for receiving payment for the professional aspects of interpreting the molecular diagnostic results. If the clinical needs, the benefits, the anticipated costs, and the projected revenues associated with a particular clinical test justify proceeding with the development of the test, the laboratory then must construct a plan for implementing the testing. In most hospital clinical laboratories, the availability of a test in kit form offers considerable advantages to developing a test de novo. Most kit-based tests contain instructions and supplies, pre-measured quantities of reagents that minimize technologist preparation time and maximize
© 1993 Elsevier Science Publishing Co., Inc.
TABLE 1. FACTORS TO BE CONSIDERED IN IMPLEME1WHNGMOLECULAR BIOLOGY TESTING IN A HOSPITAL CLINICAL LABORATORY Clinical
need
Improved sensitivity or specificity Improved turnaround time No suitable alternative form of testing available Cost of testing Personnel Equipment Reagents and supplies Controls and standards Cost of alternative forms of testing Billing and revenue Availability and training of testing personnel New test development versus use of kits Development of quality control Development of quality assurance Reporting Interpretations Quantitative results and reference ranges Administrative and regulatory issues
the cost-effective use of reagents, and quality control materials. Furthermore, the manufacturers of kits usually develop procedures that optimize the technology to produce best results. Kits offer convenience and standardization, without associated developmental costs and, thus, are ideally suited for use in a hospital laboratory. However, only a limited number of molecular biology tests are available from manufacturers in kit form. The development of a new test, without the availability of a kit or other manufacturer support, requires the production of reagents and quality control materials, writing about the technical procedures, and testing the new procedure against existing methods to determine the suitability of the test for clinical purposes. Whenever a new molecular biology test is added in the clinical laboratory, whether in the form of a manufacture's product or by the use of a newly developed method within the laboratory, the testing must be 0197-1859D3/$O.00 + 6.00
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TABLE 2. REGULATORYQUESTIONS PERTAINING TO THE IMPLEMENTATIONOF MOLECULAR DIAGNOSTIC TESTING What role will the FDA play in regulating molecular diagnostic testing? What are the joint and separate responsibilities of the Health Care Financing Administration (HCFA) in regulating molecular biology testing? What does the FDA require of manufacturers of molecular diagnostic kits approved for clinical use? Can a molecular diagnostic kit that states "For research use ordy--not for in vitro diagnostic procedures" be used legally for patient-care purposes? What is the Compliance Policy Guide and where can a copy be obtained? What does the FDA require from a clinical laboratory for it to use a molecular diagnostic kit that has received FDA approval? Can molecular infectious disease kits that are labeled "Confirmation use only" be used for making the primaD, diagnosis? What are the FDA regulations concerning the use of "home brew" for patient care? Who is eligible to use "home brew" for patient testing? Who is legally responsible for "home brew" tests? What qualifications make someone eligible to use "home brew" materials? Under what circumstances can "home brew" tests become part of a patient record? Can a clinical laboratory charge a patient or insurance carder for "home brew" testing? Can a laboratory that performs "home brew" testing be a referral laboratory? If so, can the testing be offered on an interstate basis?
accompanied by quality control and quality assurance practices, similar to those applied to other laboratory tests. The quality control practices must include the use of positive and negative controls to ascertain proper performance of the method, equipment, reagents, and technical personnel. The quality control procedures should include methods for detecting false positives and false negatives and for minimizing their occurrences. Because of the exquisite analytical sensitivity of PCR, analytical false-positive reactions occur because of sample carryover, from improper pipetting techniques, and because of aerosol dispersion of previously amplified products. Safeguards can be developed to avoid false-positive PCR reactions and must be meticulously incorporated into all methods using this form of testing. Positive and negative control samples should be assayed each time molecular biology testing is performed on patient sampies. To reduce the cost per reportable patient test, a batching strategy should be employed that permits testing the largest number of clinical samples per control specimen. When testing of the control specimens fails to yield the proper result, patient testing results should not be reported until the cause of the control failure is determined and corrected. I
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If a molecular biology test produces quantitative results, high quality, standardized calibrating materials must be used to assure the accuracy and linearity of the quantitative methods. Likewise, quantitative assays must be accompanied by a reference range that enables the user to interpret the numerical results for diagnostic purposes or for selection of therapy. Likewise, some understanding of the distinctions between analytical and clinical sensitivity and analytical and clinical specificity is useful prior to implementing the testing and offering interpretations of the results, whether through the use of kits or by methods developed within a clinical laboratory. Analytical sensitivity refers to the ability of a test to detect small amounts of an analyte, whereas the clinical sensitivity of a test is def'med as the percent of positive test results in a population known to have the clinical condition for which the testing is done (true positives divided by the sum of true positives plus false negatives, times 100%). Analytical specificity refers to the ability of a test to identify only the substance under investigation, while clinical specificity is defined as the percent of true negative test results that occur in a population that does not have the condition for the which the test is used (true negatives divided by the sum of true
© 1993 Elsevier Science Publishing Co., Inc.
negatives plus false positives, times 100%). The strength of most molecular biology tests resides in their superior analytical sensitivity and specificity. However, this analytical power can create some clinical dilemmas. For example, using PCR to amplify the DNA of clinical samples, very small numbers of leukemia and lymphoma cells containing clonal gene rearrangements can be detected because of the sensitivity of the assay. But it is also possible to detect clonal rearrangements in several clinically benign disorders, ~-~7thereby producing a possible clinical false-positive result despite an analytical true positive test. If gene rearrangement studies are to be performed for the detection of malignant lymphoproliferative disorders, it is important for the person interpreting the test to understand that analytically positive results do not always translate into the clinical presence of malignancy. The same situation applies to other molecular biology tests and clinical conditions, where the presence of a positive test does not always correspond to the presence of disease and an analytical true negative does not always indicate with certainty the absence of a clinical condition. In addition to standard quality control methods, the clinical laboratory performing molecular biology testing should develop quality assurance procedures to guarantee the success of the testing program. Once testing is offered for patient care, a plan must exist to assure the continuous availability of the testing. If a particular method fails, a backup method with equal performance characteristics---turnaround time, sensitivity and specificity, cost--should be available. For example, for PCR testing, if the test fails because of reagent contamination with the production of analytically false-positive results, a suitable alternative PCR method, with a different set of primers, may be employed to replace the primary method, thus assuring continuity of the service to maintain the confidence of the physicians who order the testing. Quality assurance activities should be developed in the clinical laboratory to assess the success and proper utilization of
Vol. 13, No. 11, 1993
molecular biology testing by measuring the actual number of clinically reported tests performed and by comparing the results of the molecular biology methods with the results obtained by other types of testing. A good quality assurance program should be able to determine the diagnostic sensitivity and specificity and the predictive value of the new testing when compared to the existing methods. Also, the quality assurance program should assess the proper clinical use of the test to minimize unnecessary utilization. Likewise, a quality assurance program associated with clinical molecular biology testing should include proficiency testing or external quality control, in which the personnel performing and interpreting the tests are responsible for analyzing control specimens obtained from a source external to the department. The College of American Pathologists (CAP) has not yet developed a proficiency testing program for PCR. However, the CAP offers an external quality assurance program for Southern blot analysis of antigen receptor (B and T cell) gene rearrangement. If molecular biology is to gain a niche in routine clinical laboratory testing, it will need to be subjected to the same standards of quality assessment and quality assurance as other forms of testing. Until commercially available proficiency testing material becomes available, a laboratory may consider producing its own program by exchanging samples with other laboratories performing similar tests for the same target analytes. While this development of external quality control may add to the cost of testing, it offers additional evidence and assurance that the laboratory is performing the testing proficiently. Regulatory matters will affect the implementation of certain forms of molecular testing, particularly PCR testing. Table 2 contains a list of questions that need to be addressed by Regulatory Agencies--particularly the U.S. Food and Drug Administration---in order to achieve uniform development and competent applications of molecular diagnostic testing in clinical laboratories. Despite the authors' inquiries to the FDA, the questions remain unan-
CLINICAL I M M U N O L O G Y Newsletter 141
swered. The uncertainty of the regulations pertaining to molecular diagnostics presents a significant obstacle to the development of new tests within a hospital laboratory. In summary, molecular biology testing performed in a hospital clinical laboratory for patient-care purposes holds great promise for improving sensitivity and specificity of testing, for decreasing turnaround time of testing required for critical diagnoses, for adding to the understanding of disease mechanisms, for improving tissue typing results, for improving paternity testing, and for adding valuable tools to forensic investigations. However, serious limitations--developmental and operational costs, special space requirements, the creation of quality control and quality assurance practices, and regulatory matters---still preclude widespread application of this type of testing in a routine clinical laboratory. Commercial kits will hasten the introduction of molecular biology testing in clinical laboratories by obviating the need for costly development within a hospital laboratory. Most of the methods used in the kit-based testing are already familiar to most clinical laboratory technologists, and the kits also contain appropriate control material, thus avoiding the necessity for making costly control samples within the laboratory. Finally, in the case of kits marketed by Roche Diagnostic Systems, the cost of purchasing the kit includes the licensing fee associated with using the Roche patented PCR-based testing. Transferring molecular biology to the diagnostic laboratory requires collaboration between basic scientists, clinical pathologists and technologists, manufacturers, and regulatory agencies to assure competent and economical clinical use of molecular technology. The challenge is to have the wisdom to manage health care resources effectively and to use tests that elevate the quality of patient care. ClN References 1.
Killeen AA, Orr I-IT: Molecular genetics of human disease: Clinical applications of genetic linkage analysis. In: Fenoglio-Preiser CM, Willman CL (eds): Molecular Diagnostics in Pathology. Baltimore, Williams & Wilkins, 1991, pp 67-80.
© 1993 Elsevier Science Publishing Co., Inc.
2.
Koenig M, Hoffman EP, Bertelson CJ, et al.: Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50:509-517, 1987. 3. Tsui L-C, Buchwald M, Barker D, et aL: Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 230:10541057, 1985. 4. Wainwright BJ, Scrambler P J, Schmidtke J, et al.: Localization of cystic fibrosis Incus to human chromosome 7cen-q22. Nature 318:384-385, 1985. 5. White R, Woodward S, Leppe~ M, et at.: A closely linked genetic marker for cystic fibrosis. Nature 318:382-384, 1985. 6. Manjunath N, Shankar P, Rajan L, et at.: Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis. Tubercle 72:21-27, 1991. 7. Puchhammer-Stockl E, Popow-Kraupp T, Heinz FX, et al.: Establishment of PCR for the early diagnosis of herpes simplex encephalitis. J Med Virol 32:77-82, 1990. 8. Nuovo GJ, Becker J, MargioUa M, et al.: Histologic distribution of polymerase chain reactionamplified human papillomavirus 6 and 11 in penile lesions. Am J Surg Pathol 16:269-275, 1992. 9. Buffone GJ, Demmler GJ, Schimbor CM, Greet J: Improved amplification of cytomegalovims DNA from urine after porification of DNA with glass beads. Clin Chem 37:1945--1949, 1991. 10. Tompkins LS: The use of molecular methods in infectious diseases. N Engl J Med 327:1290-1297, 1992. 11. Reid AH, Omningham RE, Frizzera G, O'Leary TJ: hel-2 Rearrangement in Hodgkin's disease. Am J Pathol 142:395-402, 1993. 12. Lonn U, Lonn S, Nylen U, et al.: Gene amplification detected in carcinoma from human urinary bladder washings by the polymerase chain reaction method. Cancer 71:3605--3610, 1993. 13. KuKuruga D, Eisenbrey AB: Role of molecular tools in tissue transplantation. Lab Med 24:589595, 1993. 14. Findlay JB, Atwood SM, Bergneyer L, et al.: Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction. Clin Chem 39:1927-1933, 1993. 15. Fishleder A, Tubbs R, Hesse DO, Levine H: Uniform detection of immunoglobulin-gene rearrangement in benign lymphoepithelial lesions. New Engl J Med 316:1118-1121, 1987. 16. Weiss LM, Wood GS, Ellisen LW, et al.: Clonal T-cell populations in pityriasis lichenoides et variolaforma acuta (Mucha-Haherman disease). Am J Pathol 126:417-421, 1987. 17. LeBoit PE, Abel EA, Clear), ML, et al.: CIonal rearrangement of the T cell beta gene in the circulating blood of lymphocytes of erythrodermic mucinosis. Blood 71:1329-1333, 1988.
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