Internal Quality Control

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Quality Control (QC)

• Lab QC refers to the measures that must be included

during each assay run to verify that the test is working
properly.

• Lab QC = Running controls and statistically analyzing the data

before releasing patient results
 Quality Control (QC)
• Quality Control is ensured by practicing correct procedures of Quality
assurance - Internal quality control and external quality assessment.

• Two procedures are complementary;

• Internal quality control primarily monitors day to day reproducibility, that is

precision, and detects frank errors (ACCURACY) in any one day’s procedures;

• External quality assessment primarily aims at detecting constant differences

(“BIAS”) between the laboratory results and those of others doing similar
work and also establishes under or over reporting of parameters.
 Internal Quality Control

• Clinicians deal with a wide-range of test results on a daily basis:

• Including routine POC testing, Clinical chemistry, Pathology, Microbiology, Histopathology,

Imaging/Radiology/Scans & other speciality testing etc:

Calibrator & Control.

Calibrator:

• May be same material as calibrators

• Specific to each kit/reagent lot
• May not identify shifts between reagent lots
• Can be used to calculate assay cut-off
• Levels typically well above or below assay cut-off
• Frequent lot changes don’t allow for long-term monitoring
• May not be similar to patient sample

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 Internal Quality Control

Controls
• Different from kit calibrators

• Independent of kit/reagent lot

• Identify shifts between reagent lots

• Designed to monitor the precision of the test system

• Levels are typically targeted to be reactive within a relevant range

• Same control lot can be used across multiple reagent lots allowing long-term monitoring
of assay performance

• Control may be treated same as a patient sample

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Internal Quality Control

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Comparing Results to the Appropriate Range

• Control results - compared to their own range of expected

results determined by the control manufacturer or
individual laboratory

• Patient values – compared to published reference values

or patient population reference ranges established within
the laboratory.
True Value, Precision and Accuracy

• True value – an ideal concept, which cannot be

achieved.

• Accepted True value – The value approximating the

‘True Value’; the difference between the two values is
negligible.

• Precision: Reproducibility

• Accuracy: Proximity to Accepted True Value

Precision and Accuracy

Low Accuracy,
High Precision

High Accuracy,
Low Precision

High Accuracy,
High Precision
 Error

• Error is the discrepancy between the result obtained in the

testing process and its ‘True Value’ / ‘Accepted True Value’

• Pre-Analytical Errors=40%
• Analytical Errors=20%
• Post-Analytical Errors=20%
 Pre-Analytical Errors
• Before the specimen is run.
• Examples: Clerical, patient ID, specimen selection and
contamination, improper storage of reagents and specimen,
improper transport, etc.

• Through Quality Assurance measures, the laboratory

tries to maintain control over these factors
• Well trained phlebotomy staff, nurses, and physicians
• Use of easy patient & specimen identification methods, such as
bar code identification.
 Analytical error

• Testing errors
• Random or indeterminate
• Hard or impossible to trace
• Examples: Electricity surge, One-time events, etc

• Systematic or determinant
• Identifiable cause
• Examples: Specimen carryover, contaminated reagents, instrument
component malfunction, dirty electrodes, etc.
• Through Quality Control measures, such as always running controls,
the laboratory limits these errors.
 Post-Analytical Errors
• After testing.
• Examples: Clerical, result reported on wrong patient, instrument to
host computer errors, etc.

• Quality Assurance measures such as comprehensive and easily read

Report sheets for manual tests must be implemented when
problems are identified.
 Qualitative QC vs. Quantitative QC
• Quantitative tests
• Measured quantity/concentration of analyte in the control
• Data must be graphed and evaluated by numerical statistics
• Examples: WBC count, Glucose, quantitative HCG

• Qualitative tests and Semi-Quantitative

• Qualitative=Pos/Neg or Present/Absent
• Semi-Quantitative=Small, Medium, Large
• Generally not statistically analyzed
• Examples: Clinitest, Acetest, qualitative HCG
 QC Data Analysis: Measures of Central tendency

• Measures of Central tendency

(how numerical values can be expressed as a central value )

• Mean - Average value
• Median - Middle observation
• Mode - Most frequent observation
 QC Data Analysis: Variance and Standard Deviation

• An important tool in the statistical analysis is

determining:

• Variance =

• Standard Deviation (SD) - a measure of the scatter around

the arithmetic average (mean) in a Gaussian distribution.

• SD= or Square Root of variance

 Determination of acceptable standard deviations limits:

•Performed by running a control at least 30 times on different days

and times.

•Calculate the mean and standard deviation (SD) by using standard

procedure.

•Acceptable limits are calculated by adding and subtracting the

value of 2SD to the observed mean.

•All control results are then plotted on Levy—Jennings chart.

 Criteria for acceptability of Results: :

In general the following are the rules observed for evaluating the control results:

(a) Both control (Normal and Abnormal levels) are within ± 2SD from the mean;

OR

(b) One control reads within ±2SD; the other between 2-3SD (once only).

Control runs are rejected if:

(a) One control is greater than ±3SD from the mean or,
(b) Both controls are greater than ±2SD from the mean, or
(c) One control is between 2—3SD on two successive runs.
Quality Control
• 95% confidence limit (± 2 SD) - 95% of all the results in a
Gaussian distribution
 How many points fall within 1SD?

• Another way of reviewing data

• Dispersal / or how the individual data points are distributed about
the central value
Levey-Jennings QC Graph (no points)
Levey-Jennings QC Chart for 1 month
Statistical concepts
• Shift – when there are 6 consecutive data results on the same
side of the mean
Statistical concepts
• Trend – when there is a consistent increase OR decrease in the
data points over a period of 6 days. (A line connecting the dots
will cross the mean.)
 Quality of Control Reference (guru)

• James O. Westgard, PhD

• University of Wisconsin
• Teaches in CLS program
• Director of Quality Management Services at the U of W
Hospital
• Westgard rules
• http://www.westgard.com/mltirule.htm
13s Westgard Rule
• 13s
• A single control measurement exceeds three standard
deviations from the target mean
• Action - Reject
12s Westgard Rule

• 12s
• A single control measurement exceeds two standard deviations
from the target mean
• Action – must consider other rule violations (trend, shift,
etc)
• This is a warning
22s Westgard Rule
• 22s
• Two consecutive control measurements exceed the same mean
plus 2S or the same mean minus 2S control limit.
• Action – Reject
R4s Westgard Rule
• R4s
• One control measurement in a group exceeds the mean plus 2S
and another exceeds the mean minus 2S.
• Action – Reject
41s Westgard Rule
• 41s
• Four consecutive control measurements exceed the same
mean plus 1S or the same mean minus 1S control limit.
• Action – Reject
 If QC FAILS?
One Possible Plan of Action: (your lab may advise another)
1. Look at vial of control. If using the last drops—reconstitute or open new
vial.
2. If plenty of QC left in vial, mix well, repour, rerun. Most issues resolve by
now. However…
3. If the QC fails again, check the volumes and expiration dates of the
reagents. Change out if necessary.
4. Calibrate the instrument, run cleaning sequence, or perform maintenance
as needed.
5. Rerun control.
6. If controls fail repeatedly after multiple efforts, call technical support.
Use discretion keeping in mind that controls, calibrators, and reagents are
expensive.
 References
https://www.who.int/medical_devices/diagnostics/WHO_EDL_2018.pdf

https://www.who.int/bulletin/volumes/95/9/16-187468/en/

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Thank You

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 External Quality Control:

It is a retrospective analysis of internal quality control results by an

external accredited agency.

Samples provided by the agency are analyzed and the results sent
back by each participant.

Results are evaluated by the agency and calculated, showing the

participant’s performance in comparison with the other laboratories
in the scheme.

We participate in NRL Australia External Quality Assessment Scheme.

How to Manually Calculate Variance and Standard Deviation:
1.Subtract the mean from each score.
2.Square each Result
3.Sum all of the squares
4.Divide the sum of the squares by the number of data
points (N)
5.The result is the VARIANCE
6.Take the square root of the variance.
7.The result is the SD.
Assuring the Quality of Test and Calibration
Results - ISO/IEC 17025 – 5.9

• The laboratory shall have quality control

procedures for monitoring the validity of tests and
calibrations undertaken.
• The resulting data shall be recorded in such a way
that trends are detectable and, where practicable,
statistical techniques shall be applied to the
reviewing of the results.

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Assuring the Quality of Test and Calibration
Results - ISO/IEC 17025 – 5.9
• This monitoring shall be planned and reviewed and may
include, but not be limited to, the following:
• regular use of certified reference materials and/or internal
quality control using secondary reference materials;
• participation in interlaboratory comparison or proficiency-
testing programmes;
• replicate tests or calibrations using the same or different
methods;
• retesting or recalibration of retained items;
• correlation of results for different characteristics of an
item.

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Control Charts
• Powerful, easy-to-use technique for the control of
routine analyses
• ISO/IEC 17025 demands use wherever practicable
• It is hard to imagine quality management systems
in laboratories without control chart

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History
• Introduced by Shewhart in 1931
• Originally for industrial manufacturing processes
• For suddenly occurring changes and for slow but
constant worsening of the quality
• Immediate interventions reduce the risk of
production of rejects and complaints from the
clients

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Principle
• Take control samples during the process
• Measure a quality indicator
• Mark the measurement in a chart with warning and action
limits
concentration

upper action limit

upper warning limit

target value

lower warning limits

lower action limits

sample-# 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18

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Control Charts in Analytical
Science
• Assign a target value
• Certified value of a RM/CRM (if available)
• Mean of often repeated measurements of the control
sample (in most cases)

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Control Charts in Analytical
Science
• Warning / action limits
• If data are normally distributed
• 95.5% of the data are in µ ± 2σ
• 99.7% are in µ ± 3σ
• ± 2s is taken as warning limits
• x± 3s is taken as action limit

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Action Limits
• There is a probability of only (100-99.7) 0.3 % that
a (correct) measurement is outside the action limits
(3 out of 1000 measurements)
• Therefore the process should be stopped
immediately and searched for errors

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Warning Limits
• (100-95.5) 4.5% of the (correct) values are outside
the warning limits.
• This is not very unlikely.
• Therefore this is only for warning, no immediate
action required.

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Calculation of Standard Deviation
• Measurements marked in the control chart are
between-batch
• Standard deviation should also be between-batch
• Estimation from a pre-period of about 20 working
days
• Repeatability STD  too narrow limits
• Interlaboratory STD  too wide limits

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Limits  Fitness for Purpose
• Action and warning limits have to be compatible
with the fitness-for-purpose demands
• No blind use
• Limits should be adjusted to fit-for purpose
requirements

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Out-of-control Situation 1
• Suddenly deviating value, outside the action limits

concentration

upper action limit

upper warning limit

target value

lower warning limit

lower action limit

date

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Out-of-control Situation 2
• 2 of 3 successive values outside the warning limits

concentration

upper action limit

upper warning limit

target value

lower warning limit

lower action limit

date

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Out-of-control Situation 3
• 7 successive values on one side of the central line
Not so critical as 1 and 2
concentration

upper action limit

upper warning limit

target value

lower warning limit

lower action limit

date

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Out-of-control Situation 4
• 7 successive increasing or decreasing values
Not so critical as 1 and 2
concentration

upper action limit

upper warning limit

target value

lower warning limit

lower action limit

date

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Advantages of Graphical Display instead of
in a table
• Much faster

• More illustrative

• Clearer

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Different Control Charts
X-chart
• Synonyms are X-control chart, mean control chart or
average control chart
• Original Shewhart-chart with single values
• Mainly for precision check
• For trueness control synthetic samples with known
content or RM/CRM samples may be analysed
• It is also possible to use calibration parameters
(slope, intercept) to check the plausibility
(constancy) of the calibration

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Different Control Charts
Blank Value Chart
• Analysis of a sample, which can be assumed to not
contain the analyte (blank)
• Special form of the X-chart
• Information about
• The contamination of reagents
• The state of the analytical system
• Contamination from environment (molecular biology
laboratories)
• Enter direct measurements of signals, not
calculated values

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Different Control Charts
Recovery Rate Chart - I
• Reflects influence of the sample matrix
• Principle:
• Analyse actual sample (unspiked)
• Spike this sample with a known amount of analyte (ΔX)
• Analyse again
• Recovery rate:

 xspiked  xunspiked 
RR     100%
  x 
 expected 
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Different Control Charts
Recovery Rate Chart - II
• Detects only proportional systematic errors
• Constant systematic errors remain undetected
• Spiked analyte might be bound differently to the
sample matrix  better recovery rate for the spike
• Target value: around 100%

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Different Control Charts
Range Chart
• Synonyms are R-chart or Precision chart.
• Absolute difference between the highest and lowest
value of multiple analyses
• Repeatability Precision check
• Control chart has only upper limits
concentration

upper action limit

upper warning limit

target value

sample-# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Different Control Charts
Difference Chart - I
• Uses difference with its sign
• Analyse actual sample at the beginning of a series
• Analyse same sample at the end of the series
• Calculate difference (2nd value – 1st value)
• Mark in control chart with the sign

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Different Control Charts
Difference Chart - II
• Target value: around 0
• Otherwise: drift in the analyses during the series
• Appropriate for repeatability precision and drift
check

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Different Control Charts
Cusum Chart - I
• Highly sophisticated control chart
• Cusum = cumulative sum = sum of all differences
from one target value
• Target value is subtracted from every control
analyses and difference added to the sum of all
previous differences

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Different Control Charts - Cusum Chart - II
T = 80 s = 2.5 90

Nr. x x-T Cusum

1 82 +2 +2
85

2 79 -1 +1
3 80 0 +1
80

4 78 -2 -1 75
5 82 +2 +1
6 79 -1 0 70

7 80 0 0 0 2 4 6 8 10 12 14 16

8 79 -1 -1 30
9 78 -2 -3
10 80 0 -3 20

11 76 -4 -7 10

12 77 -3 -10
13 76 -4 -14
0
0 2 4 6 8 10 12 14 16

14 76 -4 -18 -10

15 75 -5 -23 -20

-30

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Different Control Charts - Cusum Chart - III
• V-mask as indicator for out-of-control situation

30 30

20
in control 20
out of control
10 10

0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
-10 -10

-20 -20

-30 -30

 Choose d and  so that

  Very few false alarms occur when the process is
d
under control but
 An important change in the process mean is
quickly detected

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Different Control Charts
Cusum Chart - IV
• Advantages
• It indicates at what point the process went out of control
• The average run length is shorter
• Number of points that have to be plotted before a change in
the process mean is detected
• The size of a change in the process mean can be
estimated from the average slope

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 Different Control Charts
Target Control Charts - I
• In the contrary to classical control charts of the
Shewhart-type the target control charts operates with
fixed quality criterions and without statistically
evaluated values
• The limits for this type of control charts are given by
external prescribed and independent quality criterions
(fitness for purpose)

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 Different Control Charts
Target Control Charts - II
• All types of classical control chart (X-chart, blank value,
recovery, R-, R%-chart etc.) can be used as a target
control chart
• A target control chart is appropriate if:
• There is no normal distribution of the values from the control sample due
to persisting out of control situations (e.g. blank values)
• There are not enough data available for the statistical calculation of the
limits (rarely analysed parameters)
• There are external prescribed limits which have to be applied to ensure
the quality of analytical values

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 Different Control Charts
Target Control Charts - III
• The control samples for the target control charts are
the same as for the classical control charts
• The limits might be given by
• Requirements from legislation
• Standards of analytical methods and requirements for internal quality
control
• The (minimum) laboratory-specific precision and trueness of the
analytical value, which have to be ensured
• The evaluation of laboratory-internal known data of the same sample
type

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 Different Control Charts
Target Control Charts - IV
• Constructed with an upper and lower limit
• Pre-period is not necessary
• Out-of-control only, if the analytical value is higher or
lower than the respective limit
• Nevertheless trends in the analytical quality should be
identified and steps should be taken against them

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 Different Control Charts
Target Control Charts - V (example)
only two limits and one out-of-control situation
Control period
Ammonia RM (µmol/l) Date Value Comment / Out-of-control situation / Action
17 16.05.2003 12,61 Check WB/O v. 14.5./KB v. 15.5.
Values mean upper CL upper WL lower WL lower CL mean+1s mean-1s
20.05.2003 12,96 Check DB 1,2,6
16 21.05.2003 12,36 Check DB 10, 16, 19
22.05.2003 12,66 Check SH v. 21.5.03
27.05.2003 12,58 Check RB
15 11.06.2003 11,45 Check UW/O/KB v. 10.6
13.06.2003 12,28 Check UW/O/KB Wdh.

14 13.06.2003 12,28 Check O / SH v. 11.6.

16.06.2003 12,05 Check WB v. 12.6.03
17.06.2003 12,93 Check RB/DB
13 18.06.2003 13,13 Check O/KB
19.06.2003 12,79 Check DB
GB/S/P
12 24.06.2003 12,47 Check
25.06.2003 12,07 Check OB/O/GB
26.06.2003 12,6 Check RB
11 02.07.2003 12,37 Check O/UW/KB v. 1.7.03
08.07.2003 13,06 Check O/KB/RB QCl neu
09.07.2003 13,29 OB/O/GB
10 Check
10.07.2003 13,75 Check KH P 9.7. / SH 3.7.03
11.07.2003 13,88 Check GB/S/P
9 15.07.2003 15,62 Check Out of Control A DB
15.07.2003 14,3 Check DB Wdhl QCl neu
16.07.2003 13,01 O v. 2.7.
8 Check
16.05.2003

20.05.2003

21.05.2003

22.05.2003

27.05.2003

11.06.2003

13.06.2003

13.06.2003

16.06.2003

17.06.2003

18.06.2003

19.06.2003

24.06.2003

25.06.2003

26.06.2003

02.07.2003

08.07.2003

09.07.2003

10.07.2003

11.07.2003

15.07.2003

15.07.2003

16.07.2003

18.07.2003

18.07.2003 14,09 Check WB v. 2.7.03

Check
Check
Check

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EXCEL-Tool for Control Charts
ExcelKontrol 2.1
• X-/mean-charts
 Blank value chart
 Range chart with absolute ranges
 Range chart with relative ranges
 Recovery rate chart
 Differences chart

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Control Samples
• No control chart without control samples
• Requirements:
• Must be suitable for monitoring over a longer time period
• Should be representative for matrix and analyte conc.
• Concentration should be in the region of analytically
important values (limits!), if possible
• Amount must be sufficient for a longer time period
• Must be stable for several months
• No losses due to the container
• No changes due to taking subsamples

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Control Samples
Standard Solutions
• To verify the calibration
• Control sample must be completely independent
from calibration solutions
• Influence of sample matrix cannot be detected
• Limited control for precision (no matrix effect)
• Very limited control for trueness

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Control Samples
Blank Samples
• Samples which probably do not contain the analyte
• To detect errors due to
• Changes in reagents
• New batches of reagents
• Carryover errors
• Drift of apparatus parameters
• Blank value at the start and at the end allow
identification of some systematic trends

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Control Samples
Real Samples
• Multiple analyses for range and differences charts
• If necessary separate charts for different matrices
• Rapid precision control
• No trueness check

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Control Samples
Real Samples Spiked with Analyte
• For recovery rate control chart
• Detection of matrix influence
• If necessary separate charts for different matrices
• Substance for spiking must be representative for
the analyte in the sample (binding form!)
• Limited check for trueness

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Control Samples
Synthetic Samples
• Synthetically mixed samples
• In very rare cases representative for real samples
• If this is possible  precision and trueness check

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Control Samples
Reference Materials
• CRM are ideal control samples, but
• Often too expensive or
• Not available
• In-house reference materials are a good alternative
• Can be checked regularly against a CRM
• If the value is well known  good possibility for
trueness check
• Retained sample material from interlaboratory
tests

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Which One?
• There are a lot of possibilities
• Which one is appropriate?
• How many are necessary?

• The laboratory manager has to decide!

• But there can be assistance

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Choice of Control Charts - I
• The more frequent a specific analysis is done the
more sense a control chart makes
• If the analyses are always done with the same
sample matrix, the sample preparation should be
included. If the sample matrix varies, the control
chart can be limited to the measurement only

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Choice of Control Charts - II
• Some standards or decrees (authority decisions)
include obligatory measurement of control samples
or multiple measurements. Then it is only a
minimal additional effort to document these
measurements in control charts
• In some cases the daily calibration gives values
(slope and/or intercept) that can be integrated into
a control chart with little effort

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Benefits of Using Control Charts
• A very powerful tool for internal quality control
• Changes in the quality of analyses can be detected
very rapidly
• Good possibility to demonstrate ones quality and
proficiency to clients and auditors

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Definitions (1)

• Quality Control - QC refers to the measures that must be included

during each assay run to verify that the test is working properly.
• Quality Assurance - QA is defined as the overall program that ensures
that the final results reported by the laboratory are correct.
• “The aim of quality control is simply to ensure that the results
generated by the test are correct. However, quality assurance is
concerned with much more: that the right test is carried out on the right
specimen, and that the right result and right interpretation is delivered to
the right person at the right time”
Definitions (2)

• Quality Assessment - quality assessment (also known as

proficiency testing) is a means to determine the quality of
the results generated by the laboratory. Quality assessment
is a challenge to the effectiveness of the QA and QC
programs.

• Quality Assessment may be external or internal, examples

of external programs include NEQAS, HKMTA, and Q-
probes.
Variables that affect the quality of
results
• The educational background and training of the
laboratory personnel
• The condition of the specimens
• The controls used in the test runs
• Reagents
• Equipment
• The interpretation of the results
• The transcription of results
• The reporting of results
Errors in measurement

• True value - this is an ideal concept which cannot be

achieved.

• Accepted true value - the value approximating the true

value, the difference between the two values is
negligible.

• Error - the discrepancy between the result of a

measurement and the true (or accepted true value).
 Sources of error

• Input data required - such as standards used, calibration values, and

values of physical constants.
• Inherent characteristics of the quantity being measured - e.g. CFT
and HAI titre.
• Instruments used - accuracy, repeatability.
• Observer fallibility - reading errors, blunders, equipment selection,
analysis and computation errors.
• Environment - any external influences affecting the measurement.
• Theory assumed - validity of mathematical methods and
approximations.
Random Error

• An error which varies in an unpredictable manner, in magnitude

and sign, when a large number of measurements of the same
quantity are made under effectively identical conditions.
• Random errors create a characteristic spread of results for any test
method and cannot be accounted for by applying corrections.
Random errors are difficult to eliminate but repetition reduces the
influences of random errors.
• Examples of random errors include errors in pipetting and changes
in incubation period. Random errors can be minimized by training,
supervision and adherence to standard operating procedures.
Random Errors

x x
x x
True x x x x
Value x x x
x x x
x
x

x
Systematic Error

• An error which, in the course of a number of measurements of

the same value of a given quantity, remains constant when
measurements are made under the same conditions, or varies
according to a definite law when conditions change.
• Systematic errors create a characteristic bias in the test results
and can be accounted for by applying a correction.
• Systematic errors may be induced by factors such as variations in
incubation temperature, blockage of plate washer, change in the
reagent batch or modifications in testing method.
Systematic Errors

x
x x x x x x x
True x
Value
Internal Quality Control Program for
Serological Testing

An internal quality control program depend on the use of

internal quality control (IQC) specimens, Shewhart Control
Charts, and the use of statistical methods for interpretation.

Internal Quality Control Specimens

IQC specimens comprises either (1) in-house patient sera

(single or pooled clinical samples), or (2) international serum
standards with values within each clinically significant ranges.
Shewhart Control Charts
A Shewhart Control Chart depend on the use of IQC specimens and is
developed in the following manner:-

• Put up the IQC specimen for at least 20 or more assay runs and record
down the O.D./cut-off value or antibody titre (whichever is applicable).
• Calculate the mean and standard deviations (s.d.)
• Make a plot with the assay run on the x-axis, and O.D./cut-off or
antibody titre on the y axis.
• Draw the following lines across the y-axis: mean, -3, -2, -2, 1, 2, and 3
s.d.
• Plot the O.D./cut-off obtained for the IQC specimen for subsequent assay
runs
• Major events such as changes in the batch no. of the kit and instruments
used should be recorded on the chart.
Westgard rules

• The formulation of Westgard rules were based on statistical methods.

Westgard rules are commonly used to analyse data in Shewhart
control charts.
• Westgard rules are used to define specific performance limits for a
particular assay and can be use to detect both random and systematic
errors.
• There are six commonly used Westgard rules of which three are
warning rules and the other three mandatory rules.
• The violation of warning rules should trigger a review of test
procedures, reagent performance and equipment calibration.
• The violation of mandatory rules should result in the rejection of the
results obtained with patients’ serum samples in that assay.
 Shewhart Chart
100

80

60
Antibody Units

+3 sd
40 +2 sd
+1 sd
20 Target value
-1 sd
0
-2 sd
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
-3 sd

Assay Run
VZV IgG ELISA: Target Value = 49 U/ml
Warning rules

• Warning 12SD : It is violated if the IQC value exceeds the

mean by 2SD. It is an event likely to occur normally in less
than 5% of cases.
• Warning 22SD : It detects systematic errors and is violated
when two consecutive IQC values exceed the mean on the
same side of the mean by 2SD.
• Warning 41SD : It is violated if four consecutive IQC values
exceed the same limit (mean  1SD) and this may indicate the
need to perform instrument maintenance or reagent calibration.
Mandatory rules

• Mandatory 13SD : It is violated when the IQC value exceeds

the mean by 3SD. The assay run is regarded as out of control.
• Mandatory R4SD : It is only applied when the IQC is tested in
duplicate. This rule is violated when the difference in SD
between the duplicates exceeds 4SD.
• Mandatory 10x : This rule is violated when the last 10
consecutive IQC values are on the same side of the mean or
target value.
 Westgard Rules: 1 3SD
100

80

60
Antibody Units

+3 sd
40 +2 sd
+1 sd
20 Target value
-1 sd
0
-2 sd
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
-3 sd

Assay Run
VZV IgG ELISA: Target Value = 49 U/ml
 Westgard Rules: 10X
100

80

60
Antibody Units

+3 sd
40 +2 sd
+1 sd
20 Target value
-1 sd
0
-2 sd
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
-3 sd

Assay Run
VZV IgG ELISA: Target Value = 49 U/ml
 Follow-up action in the event of a violation

There are three options as to the action to be taken in the event of a

violation of a Westgard rule:

• Accept the test run in its entirety - this usually applies when
only a warning rule is violated.
• Reject the whole test run - this applies only when a
mandatory rule is violated.
• Enlarge the greyzone and thus re-test range for that particular
assay run - this option can be considered in the event of a
violation of either a warning or mandatory rule.
Accreditation of Pathology
Laboratories
• There is a movement towards the accreditation of medical testing
laboratories worldwide.
• Accreditation is an external audit of an applicant department’s
organization and quality assurance program.
• Examples of accreditation authorities include the CPA (College of
American Pathologists) in the U.S., CPA (Clinical Pathology
Accreditation) in the UK, NATA in Australia.
• ISO 15189 is used as the standard for all accreditation bodies in
order to provide uniformity of standards and cross-recognition.