QUALITY CONTROL ○ Reporting

○ Quality Control measures

LECTURER: Michelle Gie C. Obial, RMT ○ Quality Assurance

COURSE OUTLINE
II. SPECIMEN HANDLING
I. Phase of Analytical Testing
II. Specimen Handling
III. Collection Tubes for Molecular Testing ● very important part of the testing in pre-analytical
IV. Precautions phase because error in th pre-analytical phase will
V. Holding and Storage Requirements boomerang and impact the rest of the phases
VI. Test Performance ● In molecular biology, the condition of the specimen
VII. Controls should be noted on the requisition form or logbook
VIII. Quality Assurance ○ Temperature
IX. Proficiency Testing ○ Type of tube
○ Type of specimen present
LEARNING OBJECTIVES ○ Patient Id
● Describe proper specimen acception for Molecular Testing
Patient Identifiers
● Describe the optimal conditions for holding and storage
specimens of Nucleic Acid
● Explain the Basic Components of Molecular Test Performance ● Complete name
including Quality Assurance Controls ● Age
● Discuss instruments maintenance, repair and calibration ● Date of birth
● Describe recommendation, preparation and use of reagents ● Sex
● Explain documentation and reporting of results including Gene ● Date and time of collection
Template results ● Initials of phlebotomist (if blood sample)
● Type of specimen
I. PHASES OF ANALYTICAL TESTING ○ e.g. tissue, blood, csf

● involve complex procedures that can be divided into ● specimen must be properly labeled and must match
three main phases that play a crucial role in ensuring the accompanying requisition
the accuracy and reliability of the results ○ mortal sin of MT “mislabeling”
● are interconnected and essential for the successful
execution of molecular biology experiments A. CHAIN OF CUSTODY (in some cases)
● any errors or inconsistencies in any of these phases
can lead to inaccurate or unreliable results, ● condition of specimen and Chain of Custody (COC) is
potentially affecting patient care and research reviewed on receipt in the laboratory
outcomes ● should include the following:
○ who handles the specimens
Pre-analytical ○ who collected the specimens
○ who packed it
● encompasses all the activities that occur before the ○ who sent and received it
actual testing process ○ who tested it
○ Requisition
○ Patient Identification B. TEST REQUISITION
○ Specimen Collection
○ Transportation Test Requisition should include in the following:
○ Specimen Handling
○ Specimens Storage Type of Specimen Material
○ Accession of specimen ● e.g., blood, tissue, csf

Analytical Ordered Test

● e.g., molecular testing, PCR, genetic testing, parental
● core phase where the actual testing procedures take testing
place
○ Running of Controls and Standards Date and Time of Collection
○ Running of Specimen ● as much as possible, specimens must be fresh
● different laboratory have different criteria on what
Post-analytical they considered as a “fresh specimen”

● ensures that datas are accurate, reliable, and Contact details of the Ordering Physician
communicated effectively ● any arising questions before,during, and after the
○ Releasing of results testing, MT can immediately call the doctor
○ Interpretation of the results
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 ○ Plasma (EDTA): The liquid component of
C. MOLECULAR GENETICS or PARENTAGE TESTING blood that remains after the blood cells
(red, white, and platelets) have been
This testing include the following: removed. It contains proteins, electrolytes,
and other substances.
1. Patient Consent Forms ○ Serum (red): The liquid component of blood
○ Patient should know that his/her specimen that remains after the blood has clotted and
is for genetic testing or parentage testing the clot has been removed. It is essentially
○ Legally, there must be patient consent plasma without the clotting factors.

2. Ethnicity
3. Photo Identification of the Person tested LYSIS OF WHITE BLOOD CELLS (leukocytes)
4. Transfusion history or Pedigree
● heavily avoided in molecular biology
D. FOR FORENSIC SPECIMENS ● when WBCs are lysed, DNA or RNA yield will be
reduced
This testing includes: ○ WBCs are nucleated, inside its nucleus is
1. Documented Chain of Custody where your DNA is located, so if it is lysed, it
2. All other informations mentioned above will be exposed and gives us small yield of
DNA or RNA that we want to see

E. WRITTEN PROCEDURES FOR DOCUMENTATION OF

SPECIMEN ACCESSION SEQUESTRATION OF ANTICOAGULANTS OR HEMOGLOBIN

IF SPECIMEN IS UNACCEPTABLE ● is used for a more rapid nucleic acid isolation

without the inhibitory effects of these substances or
● disposal or retention of the specimen is recorded in without the effects of anticoagulants or hemoglobin
the patient report or laboratory quality assurance ● to do that we use:
record ○ buffers such as BloodDirect (Novagen) and
● record the reason why it is not accepted Extract-N-Amp (Sigma)
○ e.g., severe hemolyzed blood sample ○ resins such as Chelex
● immediately call the physician why the sample got ● these buffers and resins used to inhibit the activity of
rejected the anticoagulants or hemoglobin that is present
already in the sample

SPECIMEN WITH CELLULAR CONTENT

G. SOLID TISSUES
● e.g., buccal specimens
● these samples are centrifuged to collect cells before FRESH or FROZEN SAMPLES
extracting DNA or RNA
● best analyzed especially for SOUTHERN BLOT or
LONG-RANGE PCR methods that require relatively
CROSS-CONTAMINATION OF SPECIMENS high-quality DNA
○ not normal way of freezing
● must be avoided ○ the technique is snap-freezing in liquid
○ improper PPE nitrogen
○ using the same pipette tips

ALIQUOT SNAP-FREEZING IN LIQUID NITROGEN

● small volume of specimens from a bigger volume ● when specimen is collected, automatically freeze it
● aliquot removed from a specimen is never returned ● used to preserve nucleic acid and gene expression
to the original tube or vessel patterns that may change upon tissue storage
○ can be a source of contamination ○ usually found in histopath section
● if you're going to have a sample with large volume, ○ usually done in pathology laboratory, as it is
you must make aliquot the common process to preserve your
tissue specimen

F. BLOOD SPECIMENS
FIXED, PARAFFIN-EMBEDDED TISSUE
● should be inspected for visual of hemolysis
○ Hemolysis - cause release of hemoglobin in ● also used as specimen aside from fresh or frozen
the sample samples, however, it yield lower-quality DNA and
○ Hemoglobin - tends to interfere with some RNA “bati lantawon”
of the reagents in other molecular
techniques

Signs of Hemolysis in Blood Sample

● after centrifugation, the color of the plasma is

reddish and the serum is reddish or pinkish
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 III. COLLECTION TUBES FOR B. REGULAR COLLECTION TUBES
MOLECULAR TESTING
● e.g. EDTA, Blue Top, Heparin (commonly used in the
HEPARIN IS SELDOM USED laboratory
○ treat with Trizol or TriReagent (by Sigma)
● it tends to inhibit enzyme used in molecular analysis
● not all techniques in molecular biology can use Trizol or TriReagent by Sigma
sodium heparin as their sample because of its
inhibitory properties ● can lyse separated white blood cells from standard
collection tubes
TRIPOTASSIUM EDTA (lavender) & ● lysate can be stored at -70°C to stabilize RNA for
ACID CITRATE DEXTROSE SOLUTION (yellow)
several days
○ this is for the actual extraction steps
● mostly used
Serial Analysis in the Laboratory
BLUE TOP
● requires analysis that the specimens received in the
● can still be use also aside from T-EDTA and ACDS
laboratory at different times be handled as
CONSISTENTLY AS POSSIBLE
● immediate stabilization of the RNA is tantamount to
having COMPARABLE RESULTS

● serial analysis refers to the repeated testing of a

sample or patient over time
○ three samples will be consecutively sent to
the lab for the analysis
○ same sample = same test performed
○ done to monitor changes, evaluate
treatment effectiveness, identify trends
○ to make sure that the results are
comparable with each other (no outside or
external variable that affects the results)

IV. PRECAUTIONS

● the same with the precautions applied to other

departments
A. SPECIAL COLLECTION TUBES
A. STANDARD PRECAUTIONS
PLASMA PREPARATION TUBES
● all specimens are considered potentially infectious
● VACUTAINER PPT, BECTON DICKINSON ○ e.g. all body fluids, patient samples
● contains a polymer gel that separates granulocytes ● proper PPE is a must to prevent disease
and some lymphocytes from erythrocytes upon transmission
centrifugation
○ granulocytes (type of WBC with small
Standards Precautions in Labs:
granules inside them)
○ lymphocytes (type of WBC that is part of
● Hand Hygiene: Frequent handwashing or
immune system)
use of sanitizer.
○ erythrocytes (RBC)
● PPE: Gloves, gowns, masks, eye protection.
● Respiratory Hygiene: Cover coughs and
● used for HIV and HCV analysis
sneezes.
○ HIV (Human Immunodeficiency Virus)
● Environmental Cleaning: Regular cleaning
○ HCV (Hepatitis C Virus)
and disinfection.
● Safe Injection Practices: Use single-use
● used for separation of white cells for genetics and
needles and syringes.
chimerism testing
● Patient Placement: Isolate highly
contagious patients.
● Eating is prohibited: food will be
TEMPUS RNA BLOOD TUBE (by Applied Biosystem) or
contaminated with agents
PAXgene BLOOD RNA TUBE (by PreAnalytix)

● contains proprietary RNA stabilization agents that

maintain the integrity of the RNA from collection
through isolation
○ basically, if your main target or you want to
amplify RNA, then you can use these tubes

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 B. TRANSMISSION-BASED PRECAUTIONS V. HOLDING AND STORAGE REQUIREMENTS

● used with airborne or contact-transmissible agents INTERPHASE AND METAPHASE FLOURESECENT IN SITU
● must used or have: HYBRIDIZATION (FISH) and karyotyping

RESPIRATORY MASK / RESPIRATORS ● are methods that requires intact cellular structures or
culture cells so that only fresh specimens are
● Surgical masks: used for general protection against acceptable
large-particle airborne contaminants ○ if you are going to perform FISH, the doctor
● N95 respirators: provide higher levels of protection must send a fresh specimen
against airborne particles, including those that can ○ only fresh specimen are acceptable
be inhaled and cause disease
A. GENERAL RULE OF HOLDING SPECIMEN FOR
BIOSAFETY CABINET PROECCESING

● Class I biosafety cabinets: Provide protection for the ● differ depending on the analyte and it's stability in
operator from exposure to hazardous materials. the cell
● Class II biosafety cabinets: Provide protection for
both the operator and the environment from BLOOD and BONE MARROW
exposure to hazardous materials.
● Class III biosafety cabinets: Provide the highest level ● if your laboratory are not capable of testing the
of protection for both the operator and the sample, specimens are sent to outside laboratories
environment,typically used for work with highly for molecular analysis can be shipped overnight at
infectious agents. room temperature or with ice packs
○ depending with the SOP of the laboratory,
must follow their SOP so that the sample
C. CONTACT PRECAUTIONS will not be rejected and will be processed
○ SOP = Standard Operating Procedure
● designed for direct patient care where there is
potential for direct exposure to infectious agents on TISSUES
or from the patient
○ in direct contact with patients ● best shipped frozen on dry ice
○ very evident during COVID-19 pandemic ● best when fresh or snap-frozen
● prevent the spread of infections agents, and other ● [ -70°C ] last at least 2 years
pathogens

WEAR COMPLETE PPE

● avoid possible contacting with virus

MOLECULAR LABORATORY TECHNOLOGIST

● who has no direct contact with patients, used gloves

and gowns as PPE

EYES PROTECTION OR MASKS

● required in cases where frozen tissue is being

processed or where spraying or splashing of a
sample may occur

GLOVES

● are important, not only as part of standard

precautions but also to protect nucleic acids from
nuclease degradation
● absolutely required for handling of RNA

● DNA — less susceptible to degradation from

contaminating DNases, however, repeated handling
of samples without gloves will adversely affect the
integrity of the DNA over time

● separate areas for DNA and RNA isolation were

recommended
○ to avoid contamination of the two

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Buffer → maintain the pH of the specimen

VI. TEST PERFORMANCE

SENSITIVITY
PRECISION
● ability of the test to designate a person with the
actual disease as POSITIVE ● closeness of the test results regardless if it's in the
● the higher the sensitivity of the test, the lesser the range or outside the range
false negative results ● a measure of reproducibility of test results
● ability of a test to correctly identify individuals with a ● degree to which repeated measurements of the
particular condition same quantity give similar results
● Formula: True Positives / (True Positives + False ○ e.g., a precise scale would consistently give
Negatives) the same weight for the same object
○ e.g., in a blood test for a disease, a high
sensitivity means it's good at detecting the ACCURACY
disease in people who actually have it
● test results are close to the true value, means it hits
ANALYTE SENSITIVITY → considered as the lower limit of the target/mark
detection the analyte ● a measure of correctness or production of correct
results
CLINICAL SENSITIVITY → ability of test results to predict a ● degree to which a measurement or observation
clinical condition conforms to the true value of the quantity being
measured
○ e.g., a thermometer that consistently reads
SPECIFICITY the correct temperature is accurate

● ability of the test to designate the person without the

disease as NEGATIVE
● the more specific the test, the lesser the false
positive results
● ability of a test to correctly identify individuals
without a particular condition
● Formula: True Negatives / (True Negatives + False
Positives)
○ e.g., a high specificity means the test is
good at ruling out the disease in people
who don't have it

ANALYTE SPECIFICITY → ability to detect only the analyte

and not non-specific targets

CLINICAL SPECIFICITY → disease-associated results only in

patients who actually have the disease conditions

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 VII. CONTROLS
A. TEST VALIDATION
● interpretation of test results always includes
● performed on specimens of the types that will be inspection of controls and standards to verify
encountered in the routine use of the test, such as; acceptable test performance
○ frozen tissue ● controls are sample with known concentration
○ paraffin-embedded tissue ● you can't proceed to the actual testing of the sample
○ body fluids if your controls are not in range
○ cultured cells

● results from the new test are compared with those of WITH QUALITATIVE TESTS
established procedures that may have been
performed on these specimens or with the clinical ● a positive, negative, and in some cases, a sensitive
diagnosis control is required
○ DELTA CHECK = cross-checking
● SENSITIVITY CONTROL → defines the lower limit of
● do validation to ensure that the results that we are detection for a more meaningful interpretation of
releasing are accurate and comparable with each negative results
other ○ e.g., lower limit is 350 um concentration of
● always run: positive control, sensitivity control, and analyte, if it didn't reach the lower limit then
negative control, before doing the actual specimen it is considered negative
testing
○ record everything how much you use to ● AMPLIFICATION CONTROL → is a target that should
ensure the accuracy of serial testing always amplify
○ it is a sensitivity control that is applied in
MOL BIO
○ if it is not amplified, then something is
wrong with reagent or techniques

WITH QUANTITATIVE METHODS

● a high positive, low positive, and negative control is

required
○ can determine if the patient results is in the
borderline
B. INTERPRETATION
○ e.g., 800 is the upper limit, the 600 result of
patient is also considered as high result
● interpretation of qualitative data, acceptable ranges
are required information, such as;
● the high and low levels should be similar to critical
○ band patterns
points in the assay, such as the lowest detectable
○ product sizes
level of analyte
○ melting temperatures
○ reasons for rejecting results
INTERNAL CONTROLS
● useful to incorporate pictures of gel patterns or
instrument output data showing positive, negative,
● are run in the same reaction mix as the test
heterozygous, or other reportable results
specimens for methods requiring the detection of a
TARGET-SPECIFIC PRODUCT or relative amounts of
the target
IN THE COURSE OF VALIDATION
○ e.g., HOUSEKEEPING GENES are used as
internal controls in methods quantifying
● accuracy of a test will determine it's correlation with
infectious agents or detecting tumor cells
disease, as performed in the laboratory in the testing
by TUMOR-SPECIFIC TRANSLOCATION
laboratory
○ CENTROMERE-SPECIFIC PROBES serve as
internal controls in FISH ANALYSIS
C. PROCEDURE MANUAL / SOP
● it is designed to not interfere with the amplification
process of your specimen
● maintained in the laboratory and reviewed at least
● the presence of internal controls will be bases of the
annually
normalization of your results
○ e.g., changing suppliers must be noted
○ SOP must be adjusted according to your
new reagents used in laboratory
PCR
● ensures consistency, accuracy, compliance,
● in PCR, internal control distinguishes
efficiency, and safety in laboratory operations
FALSE-NEGATIVE RESULTS from failed
● provide detailed guidelines from sample handling to
amplifications
data analysis

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CONTROLS are best prepared in larger quantities, aliquoted, ○ e.g., for CC, you will be given a set of vials,
and stored in conditions where they are most stable each vials coresspond to one month of the
year, so you will test it every month and
report it every month as well
VIII. QUALITY ASSURANCE ○ e.g., for MOL BIO, you will be given ten
samples and identify if it is positive or
● more on the paperwork negative, it is one-time testing then send
● review might be in the form of rates of positive and the results
negative results compared with expected rates from
independent sources
NATIONAL REFERENCE LABORATORIES
MOLECULAR QUANTITATIVE METHOD
● Hematology = National Kidney and Transplant
● should have a defined dynamic range, sensitivity Institute
level, and accuracy ● Clinical Chemistry = Lung Center of the Philippines
● HIV, HEPATITIS B & C etc = San Lazaro Hospital or
ASSAY LEVELS SACCL
● Blood Banks = Research Institute for Tropical
● assay levels that distinguish positive results from Medicine (RITM)
negative results (cut-off values) must be well defined ● Bacteriology/Drug Testing = East Avenue Medical
and verified at regular intervals Center
○ this is the reason why, although we
established normal values, since different
laboratories are catering different types of
population, that's why they'll adjust their
normal values, depending on who they are
catering and depending on the machine and
the supplier of their reagents

IX. PROFICIENCY TESTING

● refers to analyzing external specimens from a

reference source supplied to independent
laboratories
○ NEQAS = National External Quality
Assessment Service
○ performed every year and all laboratories
that were given License To Operate by DOH
should participate in this proficiency testing

● Purpose:
○ to assess the skills of laboratory personnel
performing molecular assays and the
performance of the assay itself

○ it also asses the principles of the machine

you are using, because your results will be
compared to other laboratories having or
using the same machine as yours

● it is performed at least TWICE A YEAR, with the

proficiency samples tested within routine patient
runs

NATIONAL EXTERNAL QUALITY ASSESSMENT SERVICE

(NEQAS)

● All laboratories are required to participate in NEQAS

● Laboratories need the certification from NEQAS to
renew license
● Renewal of license in LTO is every year

● Samples will be sent one time to the laboratory

○ e.g., for PARA, you will be given ten slides,
you need to identify the parasites present or
you will be given blood smears and
determine if the specimen is positive for
malaria and specify what specie is present