Quality

Q lit Control,
C t l Quality
Q lit Assurance,
A
Proficiency Testing I & II
HKU SPACE
Higher Certificate in Medical Laboratory Science
Laboratory Management
Vanessa Lo
Adjunct Lecturer
Clinical Scientist, FFSc(RCPA)
2&9S
September
t b 2022 (6 (6:30
30 – 9:30
9 30 pm))

• Errors are found everywhere

• To ensure test results are fit for clinical
application laboratory need to ensure all
application,
quality related activities are fit for clinical
application
• Inappropriate centrifugal force with respect to
bl d ttube
blood b
1. Speed too high or time prolong
• Cell rupture, in particular fragile tumor cell
• Release intracellular components
• False elevation of intracellular ingredients
e g potassium
e.g. potassium, inorganic phosphate
phosphate, AST
AST,
LDH 2
2 Speed too low or time too short
2.
• Fail to centrifuge down all buffy coat of
anti coagulated blood e
anti-coagulated e.g.
g Lithium
heparinized blood
• Neutrophil
N t hil granules l
• Suspend in plasma
• Result in sporadic and inconsistent
release of ALP from granules
g
• Lead to fluctuate and inconsistent false
elevation of ALP level in patient
samples

Quality
Q lit Management
M t
System (QMS)

Internal

External
E ternal
Quality
Assurance
4
Two Types of Laboratory Error
1. Systematic Error
• Poor
P accuracy / Bias
Bi
• Definite cause
• Reproducible
Reprod cible
• Avoidable through adoption of the best
practice or state of the art technology
2. Random Error
• Poor precision
• Non specific cause
• NOT Reproducible
R d ibl
• Can happen anytime
• NOT avoidable
id bl
5

Quality Control in Bacteriology

Culture Media
Dehydrated
• Order quantities for 6 months but no longer
than 1 year
• Store in a dark, cool, well ventilated place
• Stored
S d quantity
i should
h ld bbe wellll packed
k d and
d
used up in 1–2 months
• Write date of receipt on each container
p
• When a container is opened , write the date of
opening
• Discard all dehydrated media that are either
caked or darkened 6
In-house Prepared
• Distilled or de-mineralized water should be
used for media preparation
• Protect against sunlight and heat
• Media containing heat sensitive ingredient ee.g.
g
blood and antibiotics should be stored in
refrigerator
• Store in cool and dark place
• Shelf
Sh lf life
lif depends
d d on ttype off container
t i used
d
1. Tubes with cotton wool – 3 weeks
2. Petri dishes sealed in plastic bags – 4
weeks
3. Containers with screw caps – 3 months 7

Commercially Prepared
• Clinical and Laboratory Standard Institute
(CLSI) subcommittee on media quality control
collected data over several years regarding
the incidence off QC failure
f off commonly
used microbiology media
• Based on the findings the subcommittee
published a list of media that did not require
retesting in the user's laboratory if purchased
from a manufactures who follow CLSI
guidelines

8
However:
1. Quality control tests should be carried out
by the end-user laboratory to ensure that
• the performance characteristics of the
medium are within specification and
• the methodology of medium preparation
is satisfactory
2. Each lot/batch of prepared medium should
b subjected
be bj t d tto a minimal
i i l testing
t ti program
which will
• ensure that
th t it is
i acceptable
t bl andd
• demonstrate a typical bacterial
performance
f
9

pH Value
• Check that pH of the prepared medium, when
tested in final form at ambient temperature
(25°C) lies within the range given on the
product label
• The medium should be discarded if the pH
value lies outside the specified range

Stability
• Periodically perform ALL checking
procedures on stored prepared media in order
to determine whether the storage conditions
will give optimal results
10
Sterility Check
• Check that pH of the prepared medium, when
tested in final form at ambient temperature
(25°C) lies a representative sample of each
lot/batch of medium should be incubated for 2
to 5 days at 35 to 37°C
• Testingg sample
p number depends
p on lot size
• <=100 units  3 to 5% samples
• > 100 units  10 random samples
• There should be no evidence of microbial
gro th after incubation
growth inc bation
• Discard all samples of the batch which did not
pass the sterility check
11

Growth Performance
• Test the growth support properties of the product
byy inoculating
g the medium with appropriate
pp p
stock cultures and/or fresh isolates
• Use a standard inoculation procedure
p
• Examine the quantitative and qualitative
results
• Test with organisms expecred to:
• grow
g
• give positive / negative reaction
• If testing new lots/batches of media –
inoculate old and new lots in one test and
compare the performance of the two lots side by
side 12
Blood agar
g
Test for growth and haemolysis with
Streptococcus
p Pyogenes
y g

Extra Work for In-house Prepared

Amount prepared Sterilization methods
g
Source of each ingredient Proper
p color
Lot number Depth
Preparation date Smoothness
Expiration date Clarity
Agar
g p plate – 1 month
Tube media – 6 months Hemolysis

Contamination Excessive bubbles

13

Stock Culture
• Laboratory should maintain stock culture
• Source:
1 Commercial
1. C i l
2 Clinical specimens
2.
3. Proficiency testing samples
4. Reference laboratories
5. Official Culture Collection (American Type
Culture Collection (ATCC))
14
Stock Strain
• Appropriate ATCC control strains
• Suggested by CLSI
Strain ATCC
Escherichia coli 25922
P
Pseudomonas
d aeruginosa
i 27853
Staphylococcus aureus 25923
Streptococcus pneumoniae 49619
Enterococcus faecalis 29212
Haemophilus influenzae 49247
P
Pneumoniae
i kl b i ll
klebsiella 70063
15

Media-Antimicrobial Susceptibility
• Verified with approved reference organism
CLSI (NCCLS)-M2-A7
• Variables that can affect results accuracy:
• Antibiotic
t b ot c pote
potency
cy
• Agar depth (Kirby-Bauer Test = Disc
Diffusion Test)
• pH
• Inoculum
• Incubation time
• Incubation
I b ti ttemperature
t
• Moisture
• CO2 concentration 16
 Frequency
• Everyday of testing
• Weekly y – Unless laboratory
y adopted
p in house
verified procedure that
1 the appropriate QC strains were tested for a
1.
minimum of 30 consecutive days
2 have demonstrated acceptable
2.
performance
• Use
U specific ifi strains
t i
1. Haemophilus influenzae – gram negative
coccobacilli
2. Nesisseria gonorrhoeae – gram negative
diplococcus 17

Weekly – Unless
laboratory adopted in
house verified
procedure that
1. the appropriate QC
strains were tested
for a minimum of 30
consecutive days
2. have demonstrated
acceptable
t bl

18
19

20
21

22
23

24
Reagent – Daily
In-use reagent vial is refrigerated at night but
usually left at room temperature during the day
• has the opportunity to degrade while in use
• should
h ld bbe tested
t t d each h day
d off use with
ith b
both
th
positive and negative controls
Reagent Controls
C t l
Catalase C t l
Catalase (+) vs. C
Catalase
t l ((-)) Staph.
St h
Coaglulase Coagulase (+) vs. Coagulase (-) Staph.
Cytochrome C Oxidase (+)
Oxidase Neisseria, Moraxella, Campylobacter
and Pasteurella species vs. (-)
25

Reagent – Weekly
Reagents that are documented to have
consistent and dependable
p results may
y be
tested less frequently
Reagent Controls
Gram stain Gram positive and negative
Acid fast stain Ziehl–Neelsen stain for TB
Bacitracin Cyclic peptides by Bacillus subtilis
Optochin Streptococcus pneumoniae
ONPG (ortho- Lactose fermenter – Escherichia
Nitrophenyl-β-
p y β coli,, Klebsiella spp,
pp, Enterobacter
galactoside) spp 26
Reagent – Weekly
Reagents that are documented to have
consistent and dependable
p results may
y be
tested less frequently

With each new batch of

reagent

With each
h new vial
i l off reagentt

27

Reagent – Commonly Used Test

28
 29

Reagent – Stain

30
Reagent – Monthly and As Required
• Typing sera should be tested with
• Each new lot number and
• Each month of use
• Examples
• Salmonella spp
spp.
• Escherichia Coli
• Vibrio Cholerae

31

Equipment
• Biological safety cabinets
• Autoclave
• Oven
• Incubator
• Refrigerator
• Freezer
• Water bath
• Mi
Microscope
• Anaerobic jar
• Centrifuge
• Thermometer
• MALDI-TOF 32
Autoclave

Refrigerator and Freezer

33

Highlights for Identification of Cultured

Microorganisms with MALDI-TOF-MS
• In April 2017, the Clinical and Laboratory
Standards Institute (CLSI) published a
document – Methods for the Identification of
g
Cultured Microorganisms Using
g Matrix-
Assisted Laser Desorption/Ionization Time-
of-Flight
of Flight (M58
(M58-Ed1)
Ed1)
• Lays out comprehensive recommendations
for MALDI-TOF MS in clinical microbiology
laboratories
34
Need
N d ffor robust
b t internal
i t l and
d external
t l quality
lit
control (QC) designed to account for the unique
performance and limitations of MALDI-TOF MS
1 Laboratories must perform internal QC before
1.
using MALDI-TOF MS to identify
microorganisms
2. Internal Q
QC consists of an automatic
instrument calibration using a
manufacturer-specified
manufacturer specified calibration
standard

35

3. Depending on the system, calibrators /

calibration strain include
• a manufactured
f t d extract
t t off Escherichia
E h i hi colili
(E. coli) or
• a specific E. coli
4 Laboratories should ensure full compliance
4.
and follow manufacturers’ specifications
while handling calibrator strain:
• Preparing
• Using
• Storing
36
Calibration
• Laboratories must perform calibration before
every run
• The calibrator generates and automatically
analyzes
l a mass spectrum
t t
to
1. check the spectrum baseline and
2. ensure the expected calibration peaks are
p
present
• Laboratories use these parameters to confirm
their instrument settings are appropriate and
their instruments will automatically adjust if
necessary
37

• The calibrator spectrum

p is run against
g the
reference database to ensure the correct
identification is ggiven with a level of
confidence that meets the manufacturer’s
specifications
p
• To ensure a successful calibration, the
College of American Pathologists (CAP)
Microbiology Checklist requires laboratories
run a calibrator control each day of patient
testing
1 when a new target is used,
1. used or
2. more often according the manufacturer’s
recommendation
d ti
38
• The CAP checklist requires laboratories to
maintain a written procedure for
• instruments operation and calibration
• all calibration records
• It is important for laboratories not only
d
document t calibration
lib ti resultslt bbutt also
l promptly
tl
investigate calibration failures
• Spectral acquisition is not allowed until
successful calibration

39

C lib ti failures
Calibration f il
1. Improper application of the calibrator –
laboratories can assess potential user error by
reapplying and re-analyzing the calibrator
2. Improper preparation of the calibration
3 P
3. Problems
bl crop up with
i h the
h
• Matrix
• Reagents
• Target
• Instrument

40
 Total Testing Process and Associated
Laboratoryy Errors (%)

41

Histopathology and Cytopathology

Pre-analytical
• The accuracy of the histopathological and
cytological diagnosis of tissue specimens
depends on adequate and good quality
samples
• Processes involve
1. Sample taking
• Surgical removal
• Biopsy
• Fine needle aspiration etc
etc..
2. Sample transport
3 Sample receipt in the laboratory
3.
42
Analytical
y
Accurate histopathological and cytological
diagnosis
g depends
p on:
1. Pathologist [Result Interpretation]
• Appropriate
pp p macroscopic
p description
p
• Microscopic interpretation
• Correlatingg cytological
y g and histological
g
diagnosis
2. Cytotechnologist
y g / Medical Technologist g
[Result Interpretation]
• Microscopic
p interpretation
p
3. Medical Technologist [Sample Processing]
• Technical processing g
• Staining 43

Post analytical
Post-analytical
• There is subjective element(s) in the report
content
• The interpretative reports are opinion of the
reporting pathologists
• Some diagnoses require the combined input
off a cytologist
t l i t andd histopathologist
hi t th l i t
• There are a variety of reasons why clinical
appearances, cytology, biopsy and excision
results may appear discrepant
• Multi-disciplinary team (MDT) meetings can
often resolve p
perceived discrepancies
p
44
Highlights for Histopathology
1. Sample ‘Chain of Custody’
• A robust ‘chain of custody’ across the
specimen pathway
• These involve cross-checking of a minimum
of three patient identifiers at each stage,
to typically include name, hospital number
and accession number
• Slide labels will include patient surname in
addition to the accession number

45

2. Sample Processing and Embedding

• Dedicated facilities will be provided for
sample embedding
• A record will be kept of any tissue that does
not survive the tissue processing schedule
3. Sample Sectioning
• Health and safety procedures have to be
followed at all times to prevent cuts from
microtome blades

46
4. Slide Staining
• Stains
• Haematoxylin and Eosin
• Special stains and immunohistochemical
stains
t i
• Prepare and use according to
manufacturer’s instructions with appropriate
regard to both positive and negative
control slides
• Internal technical q qualityy checks should
be carried out routinely including quality of
stainingg and q
quality
y of p
preparation
p
47

5 Mi
5. Microscopici diagnosis
di i depends
d d
crucially on quality control
• Methods used for quality assessment have
to:
1. Incorporate a process of continuous
dialogue within the laboratory
2. Improve individual histopathology
reporting accuracy
• Internal quality control of reporting can be
monitored by a variety of methods include:

48
 1. Performance evaluations
2 Periodic audit of histopathology outcomes
2.
3. Non-conformities monitoring
4. Slides review by MDT (Multi-Disciplinary
Team)
5. Histopathology
p gy detection and reporting
p g
rates monitoring
6 Correlation of cytology with
6.
clinical/histological outcome

49

Highlights for Cytopathology

1. Regulations as to the number of specimens a
cytotechnologist may evaluate in a 24-hour
period are currently set at 100 slides / 8 hour
day
2. The vaginal/ectocervical/endocervical
cytology sample should be interpreted
preferably by using the Bethesda System
• Developed
D l d iin 1988
• For reporting cervical or vaginal cytologic
diagnoses
p smear result
• Pap’s
50
Highlights
g g ts for
o Immunohistochemistry
u o stoc e st y
(IHC)
Six controls commonly used to:
1. Determine specificity of the observed antibody
staining
t i i
2. Confirm the observed staining pattern is true,
accurate and reliable
p
3. Exclude experimental artefacts
Positive Negative
Endogenous tissue background Isotype
No primary antibody Absorption
51

1. Positive
• Reveal specific binding and true positive
results
• Specimens contain biomarker of interest
with specific characteristics in known
location
• Histomorphology and cytomorphology can
be visualized by fluorescent or
chromogenic stain
2. Negative
• Reveal non-specific
p bindingg and false
positive results
• Specimens do not express the biomarker
or protein of interest 52
3 N
3. No P
Primary
i Antibody
A tib d
• Reveal non-specific binding and false
positive results
• Samples are incubated with NO primary y
antibody but ONLY the antibody diluent
• To determine if secondary antibody binds
non-specifically to cellular components
that
a do noto co
contain
a theebbiomarker
o a e o or p
protein
oe
of interest

53

4. Endogenous Tissue Background

• Rule out endogenous
g background
g
• Before applying primary antibodies, cells
and tissues are inspected under the
microscope with either fluorescence or
bright-field
bright field illumination
• To ensure there is no signal inherent to
the tissue itself

54
5. Isotype
yp – Particularlyy crucial for monoclonal
antibodies
• Antibody
• of the same isotype (IgG2 / IgM / IgY),
clonality conjugate,
clonality, conjugate and host species
as the primary antibody
• can target a protein or biomaker that is
not present in the sample
• To
T validate
lid t the
th observed
b d staining
t i i is i
1. specific to the protein of interest
2. not produced by non-specific
interactions of the antibody y with the
other components of the tissue samples 55

6. Adsorption
• To demonstrate that an antibody y is binding
g
specifically to the protein of interest
• Pre
Pre-incubate
incubate antibodies with the
immunogen / purified antigen
• inactivate the antibody
• the tissue show little or no staining

56
Regulation in Blood Bank
• In the years before the HIV epidemic, blood
banks were perceived as organizations that
provided a community service
• Increased occurrence of HIV and increased
public scrutiny resulted in stricter FDA
regulations
• FDA regulatory oversight has resulted in an
increased effort to provide a safe, high-
quality product at low cost

57

• Primaryyggoal is transfusion of a SAFE unit of

blood
• To achieve quality must have:
• Well constructed SOPs
• Well
W ll ttrained
i d personnell full
f ll compliance
li t
to
SOP
• Comprehensive guidelines in compliance
with Joint Commission, FDA, AABB, CAP
• Fatal consequences can result from failure in:
• QQualityy of blood collected
• Screening of collected blood
• Non-compliance
Non compliance of SOPs
58
 Facilities and Equipments
Temperature Monitoring is critical for refrigerators,
freezers incubators and water baths
freezers,
• Must be manually or electronically recorded daily
• Refrigerators and freezers must have a device to
record the temperature round the clock
• When temperature is out of range must be:
• Immediately attended
• Documented of reason and corrective action
taken if any
• Alarms on refrigerators and freezers must be
tested periodically to make sure they will sound
as required
i d
59

Supplies and Reagents

Daily (Potency and Reactivity)
Antihuman globulin serum Antibody screening cells
Blood grouping anti-serums Reverse grouping cells
Lectins Enzymes

N
New Kit S
Sensitivity
iti it & S
Specificity
ifi it

Every Run (Donor Collection Center)

Hepatitis testing reagents HIV testing reagents
HTLV I/II reagents
HTLV-I/II t S hili serology
Syphilis l reagents
t
Liver enzyme, alanine transaminase (ALT) testing
reagents
60
Transfusion Committee
• Medical staff responsible
p for
1. assessing adequacy of transfusion
services and
2. proper use of blood components
• Reviews
1. Usage of all components for
appropriateness
i t
2. Records of all transfusion reactions
3. Order practices – Minimize wastage

61

Utilization Review
Agreed by Joint Commission – Assess
1 Blood ordering and transfusion
1. f practices
of medical staff
2. Cross match / Transfusion ratio
• Number of units cross matched divided by y
the actual number transfused
• Used as an indicator that too much blood
is being requested to be on hold
• Could result in high outdate or wastage
3. Number of autologous transfusions
62
4. Number of emergency releases
5. Justification of transfusion
6. Define and audit transfusion criteria
• Hematocrit < 24%
• Hemoglobin < 8 g/dL
• Symptoms due to anemia
• Recent estimated blood loss of > 10% of
total blood volume
7. Record of actions taken for physicians who
violated the transfusion criteria

63

Haematology
gy
Daily Internal Quality Control
Routine
1. Start up status and background count check
• Monitor for acceptable
p background
g
• If required take corrective action
2. QC monitoring g using
g multilevel controls
• Commercial – Low, normal, high level –
Monitor L-J chart and control rules
• Retained patient samples – Monitor %CV
• Dailyy routine patient
p samples
p – Monitor
moving averages
• If required
q take corrective action
64
Establish Commercial Multilevel Controls
Range (Mean and SD) – Commercial
• Far before adoption of new lot control material
material,
it should be analyzed in parallel with current lot
• This may be accomplished by running the new
controls twice a day for five days
• Determine
D t i mean and d SD off th
the tten runs as
tentative mean and SD
• Verify validity of the tentative mean and SD by
collecting more data
• Establish a running mean and SD for the lot

65

Establish Commercial Multilevel Controls

Range (Mean and SD) – Retained Patient
Samples
• Store patient samples with normal counts at 2 –
8oC = Retained patient samples
• Run retained patient samples as scheduled:
1. At the start of the day after running the
commercial controls
2. Every hour or after 30 patient samples
3. As the last sample before analyzer shut
down
• Calculate mean and SD
66
Multi-QC Rules

67

Multi-QC Rules – Warningg Rule – Observe

and no action to be taken

68
Multi-QC Rules – Rejection
j Rule

Bias

69

Multi-QC Rules – Rejection

j Rule

Imprecision

70
Other Internal Quality
y Control Rules

71

Peripheral Blood Smear (PBS)

Frosted Area Zone of
Body
Morphology

Head Tail

• Ensure g
good drying
y g of the smear
• Do not apply overused stain
• Maintain required buffer pH
• Compile with staining time
72
Highlights
g g ts for
o Flow
o Cyto
Cytometry
et y
1. Clinical flow cytometry has evolved from two-
parameter quantitative assessment of
peripheral blood lymphocytes to six-
parameter qualitative evaluation of bone
marrow for hematopathology
2. Leukemia
eu e a a and
d lymphoma
y p o a immuno-u o
phenotyping represent an extremely
important
p complement
p to morphology
p gy in the
diagnosis and monitoring of hematopoietic
malignancies
g
3. The complexity of five- and six-parameter
analyses
y and corresponding
p g data
interpretation rely on: 73

a. Instrument standardization and validation

b. Reagents
c. Procedure
• Laboratories are required to document:
a. Sample
p p preparation
p
b. Method performance parameters:
• Accuracy y
• Precision
• Specificity
p y
• Sensitivity
c. Proficiencyy testing
g results
• NCCLS and the U.S.-Canadian Consensus
Conference have provided recommendations
74
 Molecular Diagnostic
Performance Characteristics
1 Reportable range / Linearity
1.
study for quantitative assay
2 Precision / Replication
2.
experiment
3 Accuracy
3. A / Trueness
T /
Comparison of method study
4. Analytical sensitivity / Limit of
detection study
5. Analytical specificity /
Interference study
6. Reference interval 75

Laboratory
y Developed
p Genetic Test
1. Conduct a review of available scientific studies
and pertinent references
2. Define appropriate patient populations for which
the test should be performed
3 Select
3. S l t the
th appropriate
i t test
t t methodology
th d l f the
for th
disease or condition being evaluated
4 Establish analytic performance specifications
4.
and determine quality-control procedures using
the appropriate number
number, type,
type and variety of
samples
5. Ensure that test results can be interpreted for an
individual patient or family
6. Ensure that the limitations of the test are well
defined and reported 76
Specimen
p Selection for Validation
1. Evaluate specimen adequacy of the
specimens for the prevalence of the disease
and the mutations or variants
2 Specimen types may include blood
2. blood, buccal
swabs, dried blood spots, fresh or frozen tissue,
paraffin-embedded tissue,
tissue or prenatal
specimens
3 For a multiplex genetic test,
3. test all the mutations
or variants to be detected should be included in
performance establishment
4. For rare genotypes/mutations, alternative
samples
l acceptable
t bl
77

78
Analytical
y Testing
g Phase
CLIA requires laboratories having procedures to:
1 monitor and minimize contamination during
1.
the testing process
2 ensure a unidirectional
2. idi ti l workflow
kfl f
for
amplification procedures that are not contained
i closed
in l d systems
t (42 CFR §493.1101)
§493 1101)

Separate Areas for: Use no

no-template
template
 Reagent preparation control to detect
 Sample
S l preparation
ti contamination
 Amplification area (negative pressure if PCR)
 Post-PCR area (if amplicons are manipulated)79

Internal Quality Control Samples

• Quality control samples should be taken through
1. Extraction phase when appropriate and
practical
2. Amplification phase, and
3. Detection phase of the assay
• When possible
possible, quality control samples should
be similar to patient specimens in order to
monitor the quality of all analytical steps of
the testing process

80
1. Commercial
1
2. In-house – remainder patient specimens or
synthesized
3. Rare genetic diseases – positive controls are
h d tto obtain
hard bt i
4. CLSI guideline Verification and Validation of
Multiplex Nucleic Acid Assays (MM-17A; CLSI,
2008)
5. CDC’s Genetic Testing Reference Materials
Coordination Program (GeT-RM)
6. Positive and negative controls should be
tested each time p
patient samples
p are assayed
y
81

Quantitative Assays
• Quality control statistics are performed
monthly to
1 define analytic imprecision and
1.
2. monitor trends over time
• The laboratory must use statistical methods
1 Calculate SD and CV monthly t
1.
2. Evaluate variance in numeric QC data
• Multi-QC rules are applied if appropriate

82
Multi-QC Rules

83

Qualitative Assays
y
1. Cut-off value distinguishes positive from
negative result
• Establish the cut-off value initially
• Verify with every lot change or at least
every 6 months
2 Limit
2. i i off detection
d i (LoD)
( D) differentiate
diff i a
positive from negative result
• Establish the LoD when the test is initially
placed in service
• Verify with every change in lot (e.g. new
master mix),
), instrument maintenance,, or
at least every six months thereafter 84
• A low-positive
p control that is close to the
LoD is required to:
1 Identify weak-positive
1. weak positive patient sample
2. Verify reference material was prepared in
appropriate matrix

85

Post Analytical Testing Phase – Report

• Should be understandable by
• Non-geneticist health professionals and
p
• Other specific users of the test results
• The reports should contain:
1. Unique patient identifier e.g. name and
identification number e.g. HKID number,
passport number, hospital number
2. Name and address of laboratory
y where the
test was performed
3 Indication for testing
3.
86
 4
4. Specimen source (when appropriate)
5. Test performed
6. Units of measurement (if applicable)
7
7. Test results
8. Result interpretation
9. Recommendation
10 Guidance (if applicable)
10.
11. Genetic counseling (if applicable) –
Particularly important for positive test
result(s)
87

Post Analytical Testing Phase – Record

Retention
1 L
1. Laboratory
b t iis required
i d tto retain
t i or be
b ablebl to
t
retrieve copies of original test reports for at
l
leastt 2 years after
ft the
th date
d t off reporting
ti
• Final
• Preliminary
• Corrected
C t d
2. Retention of molecular g
genetic test results for
25 years / an approximate entire generation,
is recommended
88
TWO Tools For Overcoming Clinical Lab's
Toughest Quality Control Challenges to Ensure
NGS-Based Tests for Personalized Medicine Are
Safe and Effective
• ... Today, there are many different offerings on
the market across all components of the NGS
workflow …
• … Different tests use different:
• Extraction methods
• Target enrichment chemistries
• Library
Lib preparation
ti techniques
t h i
• Sequencing platforms
• Bioinformatic analysis pipelines 89

• … Each of these variables can have a profound

impact on performance. Furthermore, external
factors such as specimen characteristics,
sample handling and stability, and the
presence of interfering substances can also
affect
ff results.
• … All this variations can lead to different types of
failures:
1. Random variation may y reduce data quality
q y
so that results are not interpretable, leading to
increased cost and delayedy reports.
p
2. Systematic bias can cause incorrect results,
the most catastrophic outcome of which is
errors in patient treatment. … 90
Reliable Reference Materials,, the Foundation of
Effective NGS Quality Control
1
1. Challenge NGS assays across a broad range
of variant types
2. Highly multiplexed because NGS-based
assays are highly multiplexed
3. Specific and sensitive
4
4. Result of a robust,
robust highly-sensitive
highly sensitive
comparator method
5. Remain constant over time to allow well-
controlled evaluation of change
91

Reliable Reference Materials,, the Foundation of

Effective NGS Quality Control (Continue)
6
6. Unchanging to allow generation of an assay
assay-
specific baseline
7. Flexible and customizable to ensure the
laboratory has access to the right materials for
the right tests
8. Able to establish meaningful metrics
9. Facilitate inter-laboratory comparisons

92
Total Testing Process and Associated
Laboratory Errors

93

Periodic / Preventive
Maintenance

Follow Manufacturer
Manufacturer’s s
Instruction of Speed and Time
(w.r.t. different blood collection
tube)
94
 Upgrade
D b
Debug

Information
gy (IT)
technology ( )
department /
Outsourced IT
service provider
95

Balance Performance Verification

96
Pipette Performance Verification
Gravimetric Method

Three /
Four
Volumes

97

98
 99

AssureTraceability
Measurement Traceability
• Result of a measurement or the value of a
standard whereby it can be related to stated
references
1 Traceability
1.
2. Standardization
• Usually national or international standards
• Th
Through h an unbroken
b k chain
h i off comparisons
i
all having stated uncertainties
100
Specimen
p Traceability
y
• Important concept in quality assurance
programmes
p g – Chain of custody y
• An effective qualitative assurance procedure
should allow an audit trail to be followed back
from the laboratory report / result
• This should allow access to a complete
documented history
• FROM receipt of the specimen TO the
issue of the report
• Major Objectives:
• Identify the root cause of error
• Apply remedial measures
101

Good Clinical Laboratory

y Practice

102
103

104
 105

Components of Internal Quality

Assurance Program
1
1. Personnel withith adeq
adequate
ate training and
competence
2. Proper specimen collection, storage and
transport
3. Use of techniques with high precision and
accuracy
4. Proper performance of tests
5
5. Efficient processing of results
6. Good quality reagents and equipment
7
7. M th d ffor detecting
Methods d t ti errors
106
 8. Corrective steps when analyses go out of
control
9.
9 Preventive maintenance of equipment
10. Continuous training of staff
11. Documentation
12. Coordination
13. Timely feedback

107

Good Clinical Laboratory Practice – Summary

External quality assessment 108

External Quality Assurance (EQA)
SYSTEM, by means of an EXTERNAL
AGENT designed
AGENT, d i d to
t assess the
th QUALITY off
performance and results obtained by
l b
laboratories
t i in i an OBJECTIVE manner

109

110
 EQA

Proficiency Rechecking On-site

Testing
g Retesting
g Evaluation

Analytical
111

Proficiency Testing (PT)

• Proficiency Testing Provider, a professional
b d organize
body, i and d manage th
the program
• Organize inter-laboratory comparisons
regularly to assess the
1. Performance of analytical laboratories
2. Competence of the analytical personnel
• Procedures
1. Send multiple samples, preferably
human base,
base in regular period to
members of a group of laboratories for
analysis and/or identification
112
2 Compare result(s) of each laboratory with
2.
• Target value or
• Results of other laboratories in the group
e g median
e.g.
3. Assess pre-analytical process of the
l b t
laboratory iin hhandling
dli sample l e.g. recognize
i
identity discrepancy between sample label
andd iinformation
f ti / resultlt entry
t sheet
h t
4. Evaluate post-analytical process of the
laboratory in result reporting e.g. late
reporting and transcription error
113

5 Di
5. Dispatch
t h performance
f reports
t (in
(i
cumulative manner) to the participating
l b t i after
laboratories ft every time
ti reportt
submission
6. Assess the laboratory periodic
performance on regular
p g basis e.g.
g
frequency of late submission, wrong sample
identification, bias, imprecision
p etc..
7. Issue periodic reports with statistical
analysis to quantify the laboratory
performance e.g. quarter / biannual / annual
+/- professional advice / comment
+/
114
PT Provider
PT Organization / Laboratory
Provider

115

Laboratory

116
To receive
T i FULL VALUE off the th proficiency
fi i testing
t ti
program / external quality assurance program,
l b t
laboratory shall
h ll consider
id allll iinformation
f ti obtained
bt i d
from the proficiency testing program directed
t
towardd IMPROVEMENT.
IMPROVEMENT

117

Summary of Jobs Taken by Proficiency

g Providers
Testing
1. Evaluate laboratory performance objectively
and scientifically
2. Compare performance among different test
sites statistically
3. Warn for systemic problems early
4. Identify areas need improvement
5. Provide resources for ongoing training

118
Limitations of Proficiency Testing
Program
1. Results can be affected by variables not
related to patient samples e.g. Matrix Effect
2. Cannot detect all problems incurred in the
laboratory
y e.g.
g LIS
3. Cannot detect ALL problems with pre and
post analytical procedures
• Pre-analytical – Bed side order etc..
• Post-analytical – Delta check, phone
clinically
c ca y ccritical
t ca result
esu t etc
etc..
119

4. Reports
p are available 2 – 4 weeks after result
submission – cannot reflect real time
deficiency
y
5. Might not cover all laboratory tests –
laboratory cannot apply those tests for
accreditation

120
Rechecking
g / Retesting
g
Participating
Initiating Laborator
Laboratory
Laboratory
Sample
S l
Exchange

Result
Exchange
Report
p
Exchange
121

Procedure
1. One testing laboratory initiates
2 Other testing laboratory(ies) participates
2.
3. Testing samples
• Most of the time are patient samples
• Can also use proficiency testing samples
4. Evaluate mutual performance with simple
statistical analysis
y
5. Effective feedback
6 Primarily used to assess analytical
6.
performance of analyte(s) not available by
proficiency testing providers
122
 Limitations of Rechecking / Retesting
1. Not blinded
2. Rechecking / retesting cannot detect all
problems incurred in the laboratory
p y e.g.
g LIS
3. Rechecking / retesting cannot detect ALL
problems with pre and post analytical
procedures
• Pre-analytical
P l ti l – Bed
B d side
id order
d etc..
t
• Post-analytical
y – Delta check, p
phone clinically
y
critical result etc..

123

Proficiency
y Testing
g vs. Rechecking/Retesting
g g
Rechecking /
Characteristic Proficiency Testing
Retesting
Inter-laboratory
Yes Yes
Comparison
Simulated Sample Yes No
Patient-matrix Sample Yes / No Yes
Analysis Evaluated Many Few
Laboratoryy Manpower
p
L
Less M
More
Needed
Monetary Spent More Less
Report Turn Around Time Short Long
Comprehensive both
Performance Evaluation Minimal
accuracy & precision 124
Choices of External Quality Assurance
Program
1. Scientific Validity y
• Reliable testing materials
• Valid assigned
g values
• Valid statistical analysis
2. Reliabilityy
• Confidentiality
• Frequent
q distributions
• Short cycle
• Intelligible
g reports
p
• Strictly execution of the schedule
• Scoring g system
y
• Participant number 125

Make the MOST Value Use of External

Quality Assurance
1 Handle and analyze EQA samples same as
1.
patient
2 Monitor and maintain records
2.
3. Investigate deficiencies
4. Communicate outcomes with laboratory staff
y root causes if required
5. Identify q
6. Manage corrective action effectively
7 Establish continuous improvement plan
7.
8. Make good use of EQA resources for
continuous
ti education
d ti andd training
t i i
126
• Laboratory medicine is backbone in the
medical treatment, diagnosis and disease
prevention
• Medical testing laboratory diagnostics
• Influences 70–80% of hospital health care
decisions
• Costs between 3–5% of total health care
costs

GOOD Quality HIGH

Management Quality
System Patient Care
127

Internal quality control – past, present

and future trends. Adv Lab Med 2022.
[Published online May 23
23, 2022]
Carmen Ricós*, Pilar Fernandez-Calle, Carmen
P i h and
Perich d James
J O
O. W
Westgard
t d
This paper
1. Offers an historical view, through a summary
of the internal q
quality
y control ((IQC)) models used
from second half of twentieth century to those
performed today.
p y
2. Wants to give a projection on how the future
should be addressed.
128
The projections for future are:
1. Use third-party control materials.
2. Implement IQC based on patient samples
when no stable control material is available.
3. Use analytical performance specifications
based on biological variation.
variation
4. Encourage external quality assessment
organizers
i t provide
to id commutable
t bl controls
t l
5. Push laboratories to have freezers to maintain
external quality assessment controls
at −80 °C.
129

130
The projections for future are:
1. Use third-party control materials.
2. Implement IQC based on patient samples
when no stable control material is available.
3. Use analytical performance specifications
based on biological variation.
variation
4. Encourage external quality assessment
organizers
i t provide
to id commutable
t bl controls
t l
5. Push laboratories to have freezers to maintain
external quality assessment controls
at −80 °C.
131

Appropriate SD/ CV
Low false rejection
and acceptance rate

132
False Acceptance
• Type II error
• The system accepts a wrong result
• High false acceptance rate
• LJ chart
h t with
ith iinappropriately
i t l ‘LARGE’ SD /
CV
• Present with ‘GOOD’ analytical performance
BUT actually not
False Rejection
• Type I error
• The system rejects a valid result
• Hi h false
High f l rejection
j ti rate
t
133

• High false rejection rate (continue)

• LJ chart with inappropriately ‘SMALL’ SD /
CV
• Present with ‘BAD’ analytical performance
BUT actuallyy not
• Un-necessary troubleshooting
• Un-necessary re-run QC
• Pollute the planet with nonstop disposal of
plastic ware
• Inappropriate calibration while the analytical
performance has no problem
• Complicate the situation
• Do more harm than good
134
• High false rejection rate (continue)
• Refer out the service – send sample to other
laboratory
• Prolong turn around time
• Labile analyte might not be analyzed
• Significantly affect time dependant analyte
level determination – might be irreversibly
affected
• Diagnosis – e.g. cardiac troponin for MI
• Treatment monitoring – e.g. immuno-
suppressant drug for organ transplant
• Clinical management might be
significantly influenced
• Staff emotional stress 135

Third Party QCs

ABC C
Company A
Assayed
dCControll L
Levell 1 and
d2
Adopt package
insert QC range

???
Mean SD Appropriate
ABC
Co.
QC1

136
 ESTABLISH THIRD PARTY QC INTERVAL
Ensure [QUALITY] related activities are FIT for
[CLINICAL APPLICATION]

Analytical
A l ti l SD and
d CV are nott an
"isolated" figure but clinically related to
analyte
l t dependent
d d t within-subject
ithi bj t or
intra-individual biological variation

Analyte Specific Analytical Goal

Ensure [TEST RESULTS] are FIT for [CLINICAL

APPLICATION]
137

To manage
g imprecision,
p , the laboratory
y
can involve in two ways [PROACTIVELY] &
[CLINICALLY]

Assess CLINICAL appropriateness of

current running precision status

Establish risk management and control

measure to reduce the imprecision
reasonably and appropriately to FIT
CLINICAL need
138
Biological Variation
The natural variability in a laboratory parameter
due to physiological differences or
homeostatic changes within the same subject
and among different subjects over time.
Intra individual (CVi) – Differences in the same
Intra-individual
subject over time due to diurnal cycles,
physiological variability
variability, other rhythms
rhythms, biological
repair mechanism etc.
IInter-individual
t i di id l (CVg)
(CV ) – Differences
Diff among
subjects dues to differences in diet, genetics,
li i environment
living i t or iimmune status
t t etc.t
139

Imprecision

Analytical
y ((CVa)) Biological
Uncertainty of
Measurement Biological Variation
(Intra and Inter-individual)

Maximum Analytical
Imprecision
p Allowable
Analytical Goal /
Level of Clinical Fitness
(Analyte Specific) 140
Relationship between Biological
Variation (CVi) and Analytical Goal
Analytical Goal / Level Allowable
of Clinical Fitness Imprecision Criteria

Optimum ¼ of CVi
D i bl
Desirable ½ off CVi
Minimum ¾ of CVi

141

Desirable Specification for Total Error, Imprecision,

and Bias derived from intra
intra- and inter-individual
inter individual
biological variation

Sample Biological Variation

Analyte
Type CVi CVg
Plasma Glucose 4.5 5.8
Serum Sodium 0.6 0.7

Bilirubin – Conjugated
Serum 36.8 43.2
(Direct Bilirubin)

CVi
CVg 142
Clinically Applicable LJ Control Glucose
Chart
Intra-individual
Intra individual biological variation (CVi  4.5%)
4 5%)
Level of Clinical
Allowable
Fit
Fitness / Analytical
A l ti l
Imprecision Criteria
Goal
Optimum < 1.13%
1 13%
Desirable < 2.25%
Mi i
Minimum < 3.38%
3 38%

Three levels,
levels
choose
which one?
143

Total Error Allowable

[Allowable Limit of Performance]

144
Clinically Applicable LJ Control Glucose
Chart
Intra-individual
Intra individual biological variation (CVi  4.5%)
4 5%)
Level of Clinical
Allowable
Fit
Fitness / Analytical
A l ti l
Imprecision Criteria
Goal
Optimum < 1.13%
1 13%
Desirable < 2.25%
Mi i
Minimum < 3.38%
3 38%

Daily internal QC running CV

maximum allowable CANNOT
exceed 22.25%
25%
145

Clinically Applicable LJ Control Sodium

Chart
Intra-individual
Intra individual biological variation (CVi  0.6%)
Level of Clinical
Allowable
Fit
Fitness / Analytical
A l ti l
Imprecision Criteria
Goal
Optimum < 0.15%
0 15%
Desirable < 0.30%
Mi i
Minimum < 0.45%
0 45%

Daily internal QC running CV

maximum allowable CANNOT
exceed 00.45%
45%
146
Clinically Applicable LJ Control D-Bilirubin
Chart
Intra-individual
Intra individual biological variation (CVi  36.8%)
Level of Clinical
Allowable
Fit
Fitness / Analytical
A l ti l
Imprecision Criteria
Goal
Optimum < 9.2%
9 2%
Desirable < 18.4%
Mi i
Minimum < 27.6%
27 6%

Daily internal QC running CV

maximum allowable CANNOT
exceed 9
9.2%
2%
147

Glucose D-Bilirubin

148
Clinically Applicable LJ Control
Chart

There is NO SINGLE daily

i t
internall QC running
i CV
maximum allowable fit for
all analytes

Clinically Applicable
SD / CV  Internal
I t l QC
Rangeg
149

Assess / Evaluate / Quantify

QUALITY
150
 (TEa – Bias) / CV
Allowable Limit of
Performance

151

Allowable Limit of Performance &

Intra-individual Biological Variation

Analyte Intra-individual Biological Variation

Na 0.6%
DBIL 36.8%

Analyte Allowable Limit of Performance

Na +/- 3 up to 150 mmol/L; +/- 2% > 150 mmol/L
DBIL +/- 3 up
p to 15 umol/L;; +/- 20% > 15 umol/L
152
 (TEa – Bias) / CV
Clinically
Cli i ll Applicable
A li bl LJ
Control Chart

153

Identify BAD Performing Analytes

Clinically
Performance Review
Was the laboratory’s
Analyte CVa(%)
analytical performance
of Na and DBIL
EQUALLY well ?
Na 2

DBIL 2

154
 P f
Performance R
Review
i

TEa
Analyte CVa(%) Grade
(ALP)

Na 2 2 1 Bad

DBIL 2 20 10 Good

Remark: Sigma
S >=6  Good,
G <3  Bad
155

P f
Performance R
Review
i

<3σ

156
Relate Assay Required Performance
Clinically

Performance Review Has Lab A

performed better
CVa than Lab B?
DBIL Daily
y QC
(%)
Thrice & 3
Lab A levels@
1
Once and 2
Lab B levels
3

157

P f
Performance R
Review
i

DBIL CVa(%) TEa Grade

(ALP)

Lab A 1 20 20 Good

Lab B 3 20 7 Good

Remark: Sigma
S >=6  Good,
G <3  Bad
158
 P f
Performance R
Review
i

Total No.
CVa of Daily
DBIL Grade Daily QC Resource
(%) QC
Analysis

Thrice & 3
Lab A 1 20 Good
levels@ 9

Once and 2
Lab B 3 7 Good
l
levels
l 2

Remark: Sigma >=6  Good, <3  Bad

159

Quality
Q y Achievements

160
161

162
Set-up Clear Achievable and Glucose
Realistic Target
Level of Clinical Allowable
Fitness / Imprecision
y
Analytical Goal Criteria
TEa (ALP)
Optimum < 1.13%
Desirable < 2.25% 8%
Minimum < 3.38%

Increase level of clinical fitness from

[Desirable] to [Optimum] 163

Set-up Clear Achievable and Sodium

Realistic Target
Level of Clinical Allowable
Fitness / Imprecision
y
Analytical Goal Criteria
TEa (ALP)
Optimum < 0.15%
Desirable < 0.30% 2%
Minimum < 0.45%

TEa / CV
Is it
realistic?
164
Analyte(s) Need Attention

165

Review Peer’s
Peer s Performance

CV (%)
Bench
Mark
166
 Review Peer’s
Peer s Performance

Our Laboratory
Sigma Value: 3
Peers Sigma =
ALP / CV
C
= 2% / 1% = 2
167

Allocate Resources Wisely

y & Reasonably
y

Un-necessaryy
troubleshooting

Un-necessary re-run
QC
168
 Regular
electrode
l t d
replacement
169

170
 P f
Professional
i l Recognitions
R iti

After a year of implementation due to

attainment of HIGH SCORING in the
key performance indicator, the
l b
laboratory
t was categorized
t i d by
b the
th
Royal College Pathologists of
Australasian Quality Assurance
Program (RCPAQAP) as GOOD
PERFORMING laboratory.
171

P f
Professional
i l Recognitions
R iti

In subsequent year because of past

record and well documented evidence
of GOOD ANALYTICAL
PERFORMANCE in i the
th RCPAQAP,
RCPAQAP th the
laboratoryy was selected to be one of
the REFERENCE INSTITUTES
worldwide for TARGET VALUE set-up
set up
of FIVE ANALYTES.
172
 P f
Professional
i l Recognitions
R iti

Phosphate
P t i
Protein
Lactate
Urea
Glucose
LOCAL RECORD
173

Clinically Applicable LJ Control Glucose

Chart
Level of Clinical Allowable
Fitness / Imprecision
y
Analytical Goal Criteria
TEa (ALP)
Optimum < 1.13%
Desirable < 2.25% 8%
Minimum < 3.38%

Increase level of clinical fitness from

[Desirable] to [Optimum] 174
 2019 May - July

175

Clinically Applicable LJ Control Glucose

Chart
Point
o t of
o Care
Ca e
Testing (PoCT)
Service

176
 Sigma
g Performance of > 50
Glucometers
(Low level internal quality control
materials
t i l off glucose
l llevell ~ 3.5
35
mmol/L) (n > 80,000)
Glucometers

Sigma value mean 5.9

(95% CI: 5.0 – 9.0)
No. of G

Error rate: 0.00036 – 0.00050%

177
Sigma Value

Sigma
g Performance of > 50
Glucometers
(High level internal quality control
materials
t i l off glucose
l llevell ~ 16.0
16 0
mmol/L) (n: > 80,000)
Glucometers

Sigma value mean 7.9

(95% CI: 6.0 – 12.0)
No. of G

Error rate: < 0.00036%

178
Sigma Value
Cli i ll Applicable
Clinically A li bl LJ Control
C t l Chart
Ch t

Sigma Metric Incorporated Internal

Q lit Control
Quality C t l Plan
Pl

High Quality Medical Testing

Laboratory AND Point of Care Glucose
Testing Service
179