Regulation of Laboratory Developed Tests

Regulation of Laboratory Developed Tests 

Michael P. Ryan, MT, Ph.D. 

Director, Division of Laboratory Quality Certification (DLQC) 

Wadsworth Center 

New York State Department of Health 

mpr02@health.state.ny.us


Division of Laboratory Quality Certification 

(DLQC) 

DLQC Programs 

•Environmental Laboratory Approval Program (ELAP) 

•Blood and Tissue Resources Program (BTRP) 

•Physicians Office Laboratory Evaluation Program (POLEP) 

•Regulatory Affairs/Laboratory Investigative Unit (RA/LIU) 

•Breath Alcohol Permit Program (BAPP) 

•Regulated Medical Waste Program (RMW) 

•Clinical Laboratory Evaluation Program (CLEP)


Clinical Laboratory Reference System 

• Division of Environmental Health Sciences 

• Division of Genetics 

• Division of Infectious Disease 

• Division of Translational Medicine 

• Division of Laboratory Quality Certification

Clinical Laboratory Reference System 

The Department of Health's Wadsworth Center is charged with 

oversight of clinical laboratories and blood banks operating in New York 

State. 

This oversight is carried out through the Clinical Laboratory Reference 

System. 

Reference system activities include: 

•on-site surveys of facilities 

•proficiency testing of laboratories 

•review of laboratory personnel 

•reviewing and approving laboratory-developed assays

Part 58-1.10(g) of 10 NYCRR 

Assay Approval 

All technical procedures employed in a laboratory shall be of 

proven reliability and generally accepted by leading 

authorities in the specialty of laboratory medicine and/or 

approved by the department.


We review assays in all disciplines of laboratory medicine 

including: 

Bacteriology 

Cellular immunology 

Clinical chemistry 

Cytogenetics 

Cytopathology 

Diagnostic immunology 

Endocrinology 

Fetal defect markers 

Forensic identity 

Genetics 

Hematology 

Histocompatibility 

HIV 

Immunohematology 

Mycobacteriology 

Mycology, oncology 

Parasitology 

Therapeutic substance monitoring 

Toxicology 

Virology


In the past 10 years, over 5,000 packages have been 

submitted for review. 

In 2009, approximately 40% of the 950 laboratories that 

we regulate submitted packages for review.

Scope of Reviews 

What type of assays require review and approval? 

Assays not expressly approved (PMA), cleared (510k), or 

exempted by the FDA, including: 

•Commercialized test kits variously labeled as Research 

Use Only (RUO) or Investigational Use Only (IUO) 

•Laboratory Developed Tests (LDTs) that include the use 

of reagents prepared by the lab and/or analyte-specific 

reagents (ASRs) 

•FDA cleared/approved tests that have been modified 

from their intended use


Comprehensive Test Approval Policy and Guidelines 

Description of Assays/Tests 

Instructions on how to apply 

General Requirements for Assay Validation 

Guidance documents for specific scientific disciplines 

•Cellular Immunology 

•Forensic Identity 

•Genetic Testing-Molecular 

•Molecular Microbiology 

•Oncology-Molecular 

•Toxicology 

•Trace Elements 

•Cytogenetics under development 

(http://www.wadsworth.org/labcert/TestApproval/forms/Submission_Guidelines_Policy.pdf)


Molecular Microbiology-nucleic acid amplification assays 

http://www.wadsworth.org/labcert/TestApproval/forms/M 

icrobiology_NAAT_Checklist.pdf



Information Reviewed: 

•standard operating procedure manual (SOPM) 

•test requisitions and reports 

•pertinent literature references 

•summary of validation studies and supporting data 

•plans for carrying out proficiency testing



Standard Operating Procedure Manual 

Overview of the Assay 

• an overview describing the scientific basis of the test and 

an explanation of the assay 

• target gene or region being amplified with the location of 

primers and probes 

• oligonucleotide list with sequences 

• target population of the assay and clinical validity




Procedure: 

• detailed step-by-step protocol 

•specimen collection, processing, storage, rejection criteria 

•sources of reagents and equipment 

•description of controls 

•interpretation of results 

•technical limitations of the assay 

•steps taken to minimize amplicon contamination




Guidance on use and preparation of controls 

Description of the types of controls that need to be included: 

•positive lysis/extraction control 

•negative lysis/extraction control 

•inhibition control 

•reagent contamination control



Method Validation 

Data needs to be submitted that demonstrates the 

analytical and clinical validity of the assay. 

•Analytical validity means the ability of the assay to meet 

technical performance specifications 

•Clinical validity means the proven ability of an assay to 

reliably identify a specific condition in a test subject.




Summary of validation studies and results 

Data: 

•specificity 

•sensitivity (limit of detection) 

•reproducibility (inter- and intra-assay reproducibility) 

•accuracy verification 

Guidance is provided describing approaches that can be 

used to carry out these studies.




Specificity 

Must determine that assay does not detect related organisms, 

organisms producing similar illness, or organisms that may be present in 

specimen types to be tested. 

S. aureus Enterotoxin Type B 

Tested 69 different organisms including: 

• Organisms causing similar enteric illness 

• Genetic near neighbors 

• Other toxin producing S. aureus and Clostridium sp. 

• Select Agents 

* If cross-specificity is found, must be included on the patient report




Sensitivity (limit of detection) 

Data from a dilution series of a known concentration of the target 

(preferably whole organism) that is spiked into negative clinical samples 

for each matrix being validated. 

Perform 3 separate extractions of this dilution series and test in 

duplicate. 

The dilution series must encompass six dilutions including one below the 

limit of detection of the assay. 

If the LOD is determined to be >100 gene copies, linearity data must 

be provided.

Inter-assay reproducibility 




At least 3 authentic clinical samples or spiked clinical samples 

should be run on three different days. 

If spiked, the three samples should include one concentration at 

or near the limit of detection. 

Alternatively, a single positive control run repeatedly over many 

days (15 or more) may be used.

Intra-assay reproducibility 




At least 3 authentic clinical samples or spiked clinical samples 

should be run in triplicate. 

If spiked, the three samples should include at least one 

concentration at or near the limit of detection. 

Also, if different instruments, platforms, models or technicians 

will be used to perform the assay, demonstrate the assay’s 

consistency across these variables.

Assay Verification 




Accuracy should be verified by conducting a randomized, blinded validation 

study where the assay results are compared to those of a gold standard or 

FDA or CLEP approved assay. 

Provide data from at least 30 positive samples and 10 negative samples. The 

samples should be authentic clinical specimens, but spiked samples are 

acceptable when clinical specimens are not available. 

If spiked samples are used, at least 10 samples should be close to the LOD 

(maximum, 10-fold above that level). 

If authentic clinical samples are used, at least 10 of the 30 positives should 

be weak positives, if the methods used allow for this determination. Include 

multiple different strains and specimens.




Assay Verification (cont.) 

For quantitative assays, provide data across the full range of 

concentrations likely to be encountered in clinical samples. 

Multiplex assays (especially those with multiple sample types) can 

present a difficult situation for the design of a validation study, 

since it can be a large and expensive endeavor to assay 40 samples 

(30positive and 10 negative) for each analyte in each specimen type. 

Samples can be spiked with multiple organisms, so as to reduce the 

number of samples required. We may be contacted for guidance 

prior to the submission of a package with multiplex analysis having 

four or more simultaneous targets.




Additional Guidance Provided 

•Broad range sequencing to identify bacteria, mycobacteria, or fungi 

•Subtyping using nucleic acid sequencing and comparisons to sequence 

databases 

•Prognostic viral typing assays

Acknowledgements 

Division of Infectious Diseases 

•Ron Limberger 

•Christina Egan 

•Kim Musser 

Clinical Laboratory Evaluation Program 

•Stephanie Shulman 

•Michael Neal 

Division of Laboratory Quality Certification 

•Kathleen Davis

Regulation of Laboratory Developed Tests

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