Quality Assurance

and Quality Control

in the Molecular
Laboratory

Nusrat Hossain
 What is quality assurance in the Molecular
laboratory?
In the molecular laboratory, quality assurance involves the entire testing
process: pre-analytical, analytical (testing), and post-analytical processes.

ü Evaluate the effectiveness of the lab's policies and procedures.

ü Identify and correct problems.
ü Assure the accurate, reliable, and prompt reporting of test results.
ü Assure the adequacy and competency of the staff.

Quality control (QC) is one of the most important impacts

on laboratory testing, it ensures both precision and accuracy of patient
sample results. The integrity of quality control samples is important to
both management of overall quality as well as to meeting requirements of
proficiency testing.

The lab must also initiate corrective action when problems occur and
document all quality assurance activities. As the saying goes, if it is not
written down, it did not happen.
Molecular diagnostic testing has an ever-expanding role in clinical laboratory
assessment. The frequently used molecular techniques in a molecular diagnostic lab are
nucleic acid isolation, gel electrophoresis, different types of PCR, RT-PCR and real time
PCR, microarray, sequencing and restriction enzyme analysis. Recently, NGS is also
becoming an indispensable part of a molecular lab. A false genetic test results can have
serious repercussions for patients and their families.

Presently, compared with other laboratory disciplines, the quality control (QC) practices
for molecular diagnostic tests have fallen behind. QC in the molecular diagnostic lab
includes QC at different steps-assay validation, pre-analytical, analytical and post
analytical.

QC for molecular diagnostic tests encounters the following challenges: New and rapidly
evolving technologies, high expectations of accuracy for once in a lifetime genetic tests,
lack of quality control materials, lack of quantitative test system outputs and the almost
daily appearance of new genetic test targets. In the face of such issues, clinical
laboratories are struggling to develop appropriate quality assurance programs for the
molecular diagnostic tests they conduct. The clinical laboratory improvement
amendments (CLIA) address the issues related to laboratory quality. FDA also plays a
role in regulating molecular diagnostics, including the authority to regulate laboratory
developed tests (LDT). Due to uniquely difficult challenges, good QC practices for
molecular diagnostics have taken longer to evolve than other laboratory disciplines.
However, by continuing to work together, the in vitro diagnostics (IVD) industry and the
laboratory community can improve molecular QC practices to promote good medicine
and avoid burdensome legislation.
 Quality Assurance Standards:

Assessment required for:

1.Patient test management

2.Quality control
3.Proficiency testing
4.Test comparisons
5.Relate results to clinical data
6.Personnel
7.Communications
8.Complaints
9.Staff review
10.Records
 Clinical Laboratory Improvement Amendments (CLIA)
Standards for Quality Assurance

The regulations specify ten different standards to be included in a laboratory’s quality

assurance program.

The first standard emphasizes having a system in place for monitoring and
evaluating the procedures for Patient Test Management, including:
•Patient preparation
•Specimen collection
•Labeling
•Preservation and transportation
•Test requisition completeness
•Relevance and necessity for testing
•Use of appropriate criteria for specimen rejection
•Test report completeness
•Relevance and accuracy
•Timely reporting of results
•Accuracy and reliability of test reporting systems
•Storage and retrieval of result
The second standard is quality control. The lab is to have systems in place to evaluate the
effectiveness of corrective actions in regard to the QC program, including:

•Problems identified during the evaluation of calibration and QC

•Problems identified during the evaluation of patient test results (to verify the reference
range of a test method)
•Errors detected in results

The third standard is proficiency testing. The lab is to assess the effectiveness of
corrective action taken to address any unacceptable, unsatisfactory, or unsuccessful PT
results.

The fourth standard is a comparison of test results. If a laboratory has more than one
method of performing the same test, the lab must (twice a year) evaluate and define the
relationship between the two methods (i.e., run the same specimen by each method and
check for comparable results). If the lab performs testing on non-regulated analytes, the lab
must have a method for verifying the accuracy of its test results. Proficiency testing or split
sampling may be used.

The fifth standard involves the relationship of patient information to patient test results.
This is an internal quality assurance function. The lab must have a system in place to
identify and evaluate patient test results when they appear inconsistent such criteria as the
patient’s age, sex, diagnosis, and the relationship with other test results.
The sixth standard involves personnel assessment. The laboratory must have a system in
place to evaluate the effectiveness of its policies and procedures for assuring employee
competence. If the lab has an outside consultant, the lab should have a method for evaluating
his/her effectiveness, also.

The seventh standard involves communications. The lab must have a mechanism for
documenting problems arising as a result of a breakdown in communication. Corrective
actions must be taken to both resolve the problem and minimize future communication
breakdowns.

The eighth standard addresses complaint investigations. The lab must have a system to
assure that all complaints and problems are documented. Investigations must be made and
corrective action taken.

The ninth standard is quality assurance review with staff. In addition to documenting and
assessing problems identified in QA reviews, the lab personnel must discuss the issues and
take corrective action to prevent recurrences.

The tenth standard addresses QA records. Documentation of all QA activities must be

made available to the Department of Health and Human Services.
SPECIMEN ACCESSION
ØPre-analytical error is the consequence of erroneous or
misleading results caused by events that occur prior to
sample analysis.
ØThe condition of the specimen and, if necessary, the
chain of supervision is reviewed upon receipt in the
laboratory.
ØNo specimen is accepted without proper labeling and
identification.
ØIf a specimen is unacceptable, the disposal or
retention of the specimen is recorded.
PRECAUTIONS
All specimens are potentially infectious and should be
handled with standard precautions using proper
personal protective equipment (PPE).
­ Gloves are highly recommended not only as part of standard
precautions, but also to protect nucleic acids from nuclease
degradation. Gloves are absolutely required for handling of
RNA.
Transmission-based precautions, including respirators,
are used with airborne or contact transmissible agents.
Contact precautions are designed for direct patient
care.
 SPECIMENS FOR MOLECULAR TESTING
Cross-contamination must be avoided.
The specimen is inspected for hemolysis.
If white blood cell lysis has occurred, DNA and RNA
yield will be reduced.
Solid tissues are best analyzed from fresh or frozen
tissues.
The quality of nucleic acid from fixed tissue depends on
the fixing process and the fixative used.
 TEST VALIDATION
Commercially developed and FDA-approved molecular
methods are verified by using the purchased reagent
sets to test validation specimens in the laboratory.
If the commercial test is modified, validation is required
to show equal or superior performance of the modified
procedure.
Once a procedure has been established, the method is
documented in the laboratory according to Clinical and
Laboratory Standards Institute (CLSI) guidelines.
QUALITY ASSURANCE

Periodic review and documentation of test results is

required.
Molecular quantitative methods should have a defined
dynamic range, sensitivity level, and accuracy.
Assay levels that distinguish positive from negative results
(cut-off values) must also be well defined and verified at
regular intervals.
 INSTRUMENT MAINTENANCE
Manufacturers supply recommendations for routine
maintenance.
The laboratory maintains a schedule and instructions for all
routine maintenance.
Technologists should be aware of the limits of user-
recommended repairs and when service calls are indicated.
Regular calibration, or fitting an instrument or test system
output with the actual concentration of a reference analyte,
is required for detection systems.
HAZARDOUS CHEMICALS: TRANSPORT AND
STORAGE
Secondary or reinforced containers are required for transport and
handling of dangerous chemicals such as concentrated acids or phenol.
Volatile and flammable reagents are stored in properly vented and
explosion- proof cabinets or refrigeration units.
HAZARDOUS CHEMICALS: RADIOACTIVE
MATERIAL
The Nuclear Regulatory Commission (NRC) requires
that laboratories working with radioactive reagents
maintain a radiation safety manual providing
procedures for the safe handling of radioactive
substances.
Use and storage of radioactive reagents are in
designated areas.
 REPORTING TEST RESULTS
The test report must convey the method or
manufactured kit used, the locus, mutation or organism
tested, the analytical interpretation of the raw data,
and the clinical interpretation of the analytical result.
The likelihood of false-positive or false-negative results
are also included on a report.
DOCUMENTATION OF TEST RESULTS
Test results in the form of electropherograms, gel images, or
autoradiograms should be of sufficiently high quality that
results are unequivocal.
Documentation of assay conditions, reagent lot numbers and
quality and quantity of the isolated DNA or RNA is required.
In situ results such as FISH are correlated with histological
findings (stained sections of tissue specimens under a
microscope) of tissue morphology.
Raw data is retained with the final report and clinical
interpretation of the test results.