Quality in Transfusion Transmitted
Infections (TTI) Testing
Teaching Aim
To familiarize participants with essential quality elements
that govern TTI tests such as validation of test run, types of
control used and maintenance of equipments.
Why we require Quality in TTIs testing
 Blood is a source of TTIs (14 new viruses in
the past 28 years- one new virus every two
years!! )
 Quality is Essential to prevent TTIs
 Lack of quality can result either in TTI or
wastage of blood
 TTI testing is dependent on number of
variables which need to be controlled
Essential elements governing quality in
TTI
 Quality of the specimen used for testing
 Quality of kits used for testing
 Quality & Calibration of equipment used
 Use of SOPs for testing
 Type of Controls used while testing
 Interpretation of results
 Validation of results
 Record keeping
 Training of staff in SOPs
The quality of specimen used for testing
 Sample should be properly labeled
 Sample should be clear and sterile
 Lipemic, hemolysed and contaminated
specimens do not yield reliable results
 Bio-safety measures are very crucial to prevent
laboratory infections - handling and disposal.
 Avoid adding preservatives - some interfere
with test results
Quality of kits used for testing
 In general kits with highest sensitivity and
specificity should be used in a BTS for TTI
testing
 All kits and reagents should be used within the
expiration date
 kits which are used should have approval of
certifying authority (DCGI,NACO)
 Never interchange reagents from one kit to
another or one lot to another
Quality and calibration of equipments used
 Always use standard equipment in a BTS for TTI
testing - ELISA reader & washer, Micro-pipettes,
incubators, shakers etc.
 Periodic calibration of equipment is vital to
maintain quality  Periodic servicing of equipment is crucial for
optimal use of equipment- ELISA washers
 Proper documentation on equipment check and
its performance is essential- maintain records
Controls used in the assay for testing
 Internal kit controls : Include the positive
control, Negative control. At times may include a
calibrator provided by the manufacturer
 External controls: Include positive samples from
the laboratory either pooled or single, diluted or
undiluted. Essential to incorporate this to
monitor quality in testing procedures
 Intra-run and Inter-run reproducibility : three
slots/run and on three consecutive days
Validation
It is a way of assuring that a
system, process or
equipment is performing the
way it is supposed to do so.
Validation tools
• Include positive and negative controls in every
test run
• Include additional validation measures where
possible e.g.
- rapid test with internal control
- use of mechanical readers if available:
to reduce subjectivity
Interpretation of test results
 As rule all readings (both quantitative and qualitative)
and calculations should be checked by two individuals
 Validation of every run is essential for proper
interpretation of results
 Proper records - print outs of results, calculations of
cut off values, graphs, etc. should be maintained
 Any errors detected should be brought to the notice
of the concerned staff and corrective measures
instituted promptly
Controls
Internal controls
 Set of controls (Positive & Negative) provided
along with the kit
 To be used only in those batches of kit from which
they originate
 Do not detect minor deterioration of kits
Controls (contd..)
External controls
 Set of controls included from outside
 Positive (Borderline Reactive) & Negative
 Detect minor error in the assay performance
Sources of External Controls
- National
reference laboratories
- Commercial control panels
- In-house prepared external controls
- Pooled
test kit controls
- Samples collected from other laboratories
Preparation of In-house External Controls
NACO Guidelines
Select sero-positive serum/plasma
Retest the sample with another kit
Heat inactivate the sample @ 560C X 30 min
If plasma taken, re-calcify it to obtain serum
Make serial dilution of the sample with a sero-negative
serum
Making Suitable Dilutions
100 ul serum
in tube
Mix and Transfer
200 ul serum
in tube 1
100ul diluent in each
tube
Discard
Each tube is a 1:2 dilution
of the previous tube
Preparation of In-house External Controls
(contd…)
After test run, calculate ER for each dilution ELISA ratio =
sample OD / cut off OD
Select the dilution with ER b/w 1.5 to 2
Prepare external control aliquots of dilution selected above
Store at – 200C or below @ 1 year
Once thawed, control can be kept @ 2-80C, 1 wk
0.00
1:32768
1:16384
1:8192
1:4096
1:2048
2.00
1:1024
1:512
1.00
1:256
1:128
1:64
1:32
1:16
1:08
6.00
1:04
1:02
U.No.9607
E. RATIO
8.00
7.00
POS. CONTROL
o
5.00
4.00
3.00
E.P.C
.
CUT OFF
DILUTION
E.RATIO
Need for E ratio
Sample OD
Cut off OD
 E Ratio =
 Cut off values differ depending on the
principle of the test , manufacturer
guidelines and recommended protocol for
the calculation.
 Some degree of variation in internal controls
results in the variation in the cut off values
due to
◦ Variation in incubation condition
◦ Preparation of reagents
◦ Plate to plate and well to well variation (antigen
coating)
• OD of the controls would expectedly
influence the OD value of the test samples
in similar directions.
• However, the relative reactivity of the
given sample and the cut off would not
vary.
Preparation of QC charts/Levy Jennings charts
 Include at least 30 runs on the same graph
 Mean and ± 2SD is calculated plotted on
the graph.
 E ratios are plotted on the Y axis in chart
and consecutive dates of runs are plotted
on X axis.
 Change of operator and batch of assay
should be recorded
Levy-Jennings Control Chart
1
h
tc
a
B
3
h
tc
a
B
2
h
tc
a
B
+
2SD
Outlier
Mean
Mean
+2SD
Mean
2SD
Mean
-2SD
Outlier
E Ratio of External control sample
obtained on each day of testing
Outlier
(New operator)
Calculation of Cut off
The cut off value for ELISA is calculated according
to the formula given in kit insert of test.
For eg:
(The mean of negative control (OD) reading at 405 nm) + 0.2
= 0.005 + 0.2
= 0.205
• Less than 0.205 is Negative
• More than 0.205 is Positive
Calculation of Mean: ELISA Tests
• Collect optical density (OD) values for Controls
for each assay run.
• Collect cutoff (CO) value for each run.
• Calculate ratio of OD to CO (OD/CO) for each
• Use these ratio values to calculate the Mean, SD
and CV%
Mean is calculated as
X - ∑X
n
=
Sum total of E ratio
Number of reactivity
•Standard deviation = Each of t he individual values (E
ratio) are compared with the mean (X) to find out the
deviations from the mean.
•If the E ration is x1 then the deviation will be X1 ~ X
which is expressed as ‘d’. The deviations are then
squared.
These squared deviations are added and expressed as
∑d2 or ∑(Xn - X)2 or The result is then divided by the
number of readings.
•The square root of the above value is taken to find out
SD =
∑ (Xn – x )2
n
Coefficient of Variation –
- it is expressed as the percentage and the following
formula is used.
SD
x 100
CV (%) =
Mean (X)
- CV less than 10% is considered as an indication of
little variation
L J chart –Scope and application
Detection of the following
•Systematic variation
•Random variation
•Lot to lot variation
•Day to day variation
Applications of control charts
 Highlight the outliers (values outside +/- 2 SD)
 Reveal batch to batch variation
 Reveal operator to operator variation
 Changes in assay performance even when
test runs are valid
Systematic Variation
 Trend-Results change gradually in either
direction indicating slowly changing
parameters-deteriorating reagents,
equipment failing
 Shift-Results fall sharply on one side of
the mean indicating a major change has
occurred
Chart showing shift and trend
Interpretation of aberrant results
 Control values of six consecutive runs fall on
one side of mean(SHIFT)
◦
◦
◦
◦
Switching to new lot of kits
New reagents
Changes in incubation temperature
New technical hand
 Six consecutive points distributed in on
general direction (TREND)
◦ Deterioration of reagents
◦ Slowly faltering equipment.
Random Variation
 Observance of one result significantly different
from other results without any pattern
 Causes
o Transcription errors
o Sample mix-up
o Poor pipette precision
o Poor mixing of samples
o Reader not calibrated
o Washing inconsistent
Common source of errors in
TTI testing
 Transcription errors- mislabeling, data entry
 Errors in addition of samples to the plate interchanging specimens
 Lack of equipment maintenance & calibration Fungus on filters, volumes
 Poor technique
 Sudden change of staff / kit used in the BTS
 Lack of periodic training and updating of SOPs/
manuals
Error management in TTI testing
 Transcription errors- mislabeling, data entry Vigilance
 Errors in addition of samples to the plate interchanging specimens - Vacant slot in rack
 Lack of equipment maintenance & calibration Fungus on filters, volumes - Fixing responsibility &
supervision
 Poor technique - Training of staff, Supervision
 Sudden change of staff / kit used in the BTS - Training
 Lack of periodic training and updating of SOPs/
manuals - Vigilance and Supervision
Quality Control of Equipment
ELISA Reader
ELISA Reader….contd.
Photometric instrument
filter should be protected from moisture and
fungal growth
keep silica gel packs in the filter box
Calibration is done every six months (supplier)
OD of special plates & standard colour solution
are recorded
Results of OD should be within 10% of expected
Daily check – negative & positive controls added
to each run
ELISA Washer
After UseFill the rinse bottle with about 500 ml of
distilled water.
Dispose off the unused wash bufffer. Rinse
with distilled water, a couple of times and
leave about 500 ml in the wash bottle.
Fix the cap tightly.
Water baths & Incubators
Daily recording of temp. using a calibrated
thermometer
Acceptable results are the expected temp.
± a narrow range (± 0.5°C) predetermined
by the laboratory
Pipette
Maintenance of Pipette
 Do not dispense volatile / corrosive materials as
it will disturb the vaccum
 Volume should be increased or decreased
gradually
 Pipette must always be returned to zero position
after use
 Always store pipettes in vertical or erect position
in a holder
 Pipette snout must cleaned regularly with moist
filter paper after use
Calibration of Pipette
All items at ambient room temp.
Record the weight of empty beaker
Record the temp. of tube with distilled water
Pipette a known volume of distilled water
(expected volume)
Record:
[wt of beaker + D.W.] – wt of empty beaker
= weight of D.W.
Calibration ….contd.
weight of water
 Delivered vol. =
temp. factor x sp. gravity of water
 Repeat this 10 times, changing the tip
 Calculate mean, SD and CV
expected vol. – delivered vol. x 100
 % Deviation =
expected vol.
% deviation
CV
< 1.5%
< 1%
Learning outcome
• To understand the essential quality elements
in TTI testing
• Interpretation of test results
• Importance of validation
• Maintenance of equipments in TTI testing lab