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Internal Quality Control in coagulation lab
Dr Ankit RaiyaniHaematology Department
Sahyadri Speciality Hospital
Presentation layout• Introduction to Quality Assurance and Quality Control• Definitions of common terms, Accuracy vs. Precision• Preparation/selection of reagents-
• PTT reagent PT reagent MNPT• QC for manual coagulation tests• Quality control procedures- schedule• Internal Quality Control• LJ charts• Westgard rules• Other measures for IQC
Introduction
In the haematology laboratory it is essential to ensure that the right test is carried out on the right specimen and that the correct results are delivered to the appropriate recipient without delay.
Quality assurance (QA) is defined as the overall programme for achieving these objectives.
Quality control (QC) is defined as measures that must be included during each assay run to verify that the test is working properly.
Quality Assurance vs. Quality Control
Quality Assurance Quality ControlAn overall
management plan to guarantee the
integrity of data(The “system”)
A series of analytical
measurements usedto assess thequality of the analytical data(The “tools”)
The Quality Assurance Cycle
• Data and Lab Management
• Safety• Customer
Service
Patient/Client PrepSample Collection
Sample Receipt and Accessioning
Sample TransportQuality Control
Record Keeping
ReportingPersonnel CompetencyTest Evaluations
Testing
Hurdles in Quality Assurance
6
Hurdles in Quality Assurance
7
Pre-analytical
Hurdles in Quality Assurance
8
Analytical
Hurdles in Quality Assurance
9
Post-analytical
Terms relating to quality control in clinical laboratory practicePrecision Can be controlled by replicate tests and by repeated tests on previously measured
specimens
Accuracy Can be checked only by the use of reference materials that have been assayed by reference methods
Controls Are preparations that are used for either internal quality control or external quality assessment. They should not be used as standards
Error The discrepancy between the result of a measurement and the true (or accepted true) value
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
Systemic error (bias)
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
Accuracy vs. Precision
Accuracy Precision
Meaning How well a measurement agrees with an accepted value
How well a series of measurements agree with each other
Achieved with…
Calibration Quality control
When low… Increases systemic errors
Increases random errors
Calibration
Calibration is a comparison of measuring equipment against a standard instrument of higher accuracy to detect, correlate, adjust, rectify and document the accuracy of the instrument being compared.
Preparation/selection of reagents
Reagent selection for PTT
LA-sensitive reagents- (low-phospholipid reagents)
• Siemens Actin FSL• IL HemosIL aPTT-SP • Stago PTT-LA
Intermediate responsiveness to LA
• Stago STAPTT-LA • IL HemosIl SynthASil
Low responsiveness to LA
• Siemens Actin FS • Stago C.K. Prest
Reagent selection for PTT
Selection of aPTT reagent depends on indication of aPTT--
• used for evaluating the factor VIII, IX, XI deficiency and effect of heparin• sensitive to deficiencies of factors VIII, IX and XI at concentrations of 0.35–0.4 iu/ml.• responsive to unfractionated heparin over the therapeutic range of approximately 0.3–0.7 iu/ml.• low responsiveness to LA
The routine laboratory aPTT
• High responsiveness to LA (silica+ low PL content)• Two phospholipid based clotting assays, one of which may be aPTT based, are required1
LA screening
• Used in labs with mixed sample load, when only one reagent is feasible/desired
Intermediate responsiveness to both factor levels and LA
1PENGO, V., TRIPODI, A., REBER, G., RAND, J. H., ORTEL, T. L., GALLI, M. and DE GROOT, P. G. (2009), Update of the ISTH guidelines for lupus anticoagulant detection. Journal of Thrombosis and Haemostasis, 7: 1737–1740.
Reagent selection for PT (Thromboplastin)Contains tissue factor and coagulant phospholipids
• Manufactured using recombinant human tissue factor produced in Escherichia coli and synthetic phospholipids, • Do not contain any other clotting factors such as prothrombin, factor VII and factor X. • Highly sensitive to factor deficiencies and oral anticoagulant-treated patient plasma samples
Should have ISI value as close to 1 as possible.
Source- human, bovine, rabbit brain/lung, human placenta, recombinant.
Animal/human source- potential hazard of viral, prion and other infections from handling
Recombinant thromboplastins-
What is ISI?
International Sensitivity Index
It is the quantitative measure, (in term of first international reference preparation of thromboplastin, human, combined, coded 67/40,) of the responsiveness of PT system to the defect induced by oral anticoagulants.
Calculation of ISI
Calibration of a test thromboplastin must be against a reference thromboplastin of the same species e.g. human against human, rabbit against rabbit
Tests are performed By using reference thromboplastin and test thromboplastinon 20 normal donors not on anticoagulants and 60 patients who have been on oral anticoagulant treatment for at least 6 weeks and has stable INR.
Prothrombin Times are performed in duplicate for each sample and the mean for each pair of tests derived.
If there is more than a 10% difference in the clotting times between duplicate samples, the tests on that plasma sample should be repeated
Mean of each pair of PT results are plotted on double-log paper with the reference sample on the Y axis and the test plasma on the X-axis.
• A best fit line is drawn with points above the highest recorded PT and the lowest PT (see figure above). The slope of the line is calculated
• Slope= AC/BC
• ISItest = ISIStandard X Slope
• INR= (PT/MNPT)ISI
Preparation of pooled normal plasma and derivation of MNPTCitrated blood samples from at least 20 healthy volunteers (with PT, aPTT in normal range) should be collected. Equal numbers of male and female (not pregnant, not on OC pills) volunteers.
Platelet poor plasma should be prepared. After pooling it should be immediately frozen at -80⁰ C in aliquots.
The mean of PT of individual samples is calculated and taken as mean normal prothrombin time (MNPT).
SD is calculated and reference range is taken as -2 SD to +2 SD.
This pooled plasma is used as control with each batch of samples.
MNPT calculation- 18/6/15
No PT PTT1 11.5 28.62 12.4 27.63 10.5 29.24 10.6 26.25 10.6 32.16 11.7 29.87 11.1 30.78 12.2 31.69 11.9 30.9
10 11.3 31.511 10.6 29.112 12.3 29.713 10 26.314 11.9 28.715 11.6 33.316 11.2 31.417 10.5 28.218 12.5 28.719 10.6 27.820 11.1 27.721 10.9 29.522 11.3 31.923 11.3 29.924 12.5 32
total 272.1 712.4Arithmetic Mean 11.3 29.6SD 0.724 1.899
Range 9.8 to 12.7 25.8 to 33.4
QC for manual coagulation tests
All tests and controls should be performed in duplicates.
• Serviced and Calibrated every 6 months or after break down• Put on sturdy platform to reduce vibrations.• Sample load should be well balanced.
Operator skill
• Temperature- 37⁰ C (checked in all four corners)• Transparent walls for transillumination• Filled with distilled water till indicated level.
Required instruments should be properly calibrated.
• Calibrated. • Disposable tips.
Centrifuge
Waterbaths
Pipette
Calibration of centrifuge
Tachometer is used for calibration of RPM.
Actual RPM is measured with tachometer at uniform intervals (minimum 5) for 5 times.
Difference between actual and set values is calculated.
If difference is more than +/- 5%, then repeat the test. If repeat reading again shows significant difference corrective measures are required.
Timer and Temperature, when applicable, should also be calibrated.
Calibration of pipettes
Analytical balance is used to weigh pipetted volume in controlled environment. Distilled water is used as substrate.
The pipette is checked 5 times with the maximum volume (nominal volume), the minimum volume or 10% of the maximum volume, whichever is higher.
With multichannel pipettes, both volumes are tested with the two edge channels
Conversion of mass to volume V = (w + e) x Z
• V = Volume (µl), w = Weight (mg), e = Evaporation loss (mg), • Z = Conversion factor for mg/µl conversion (temperature, pressure)
Acceptable accuracy and precision range varies with pipette capacity.
Quality control procedures
Quality control procedures
The procedures that should be included in a quality assurance programme vary with the tests undertaken, the instruments used and (especially if these include a fully automatic system) the size of the laboratory and the numbers of specimens handled.
Some control procedures should be performed daily and other performance checks should be done at appropriate intervals.
The latter is particularly important when there is a change in staff and after maintenance service or repair has been carried out on equipment.
Schedule for quality control procedures
1. Calibration with reference standards
Instruments, pipettes6-month intervals or more frequently if control chart or EQA indicates bias or fluctuation in results and after any repair/service
Others: thermometers, scales, etc Annually
2. Control chart with control materialDaily or more frequently with each batch of specimensDuplicate tests on two or three patients’ samples: if control chart or delta check shows discrepancies
3. Analysis of patients’ resultsCumulative results: following previous tests and if changes in clinical state
4. EQAS performanceAssessment monthly
Components of Quality control
Internal quality control (IQC) • Monitoring the haematology test procedures to ensure continual
evaluation of the reliability of the daily work of the laboratory with validation of tests before reports are released
External quality assessment (EQA) • Evaluation by an outside agency of the between-laboratory and
between-method comparability. It can be organized nationally, regionally or internationally
Internal Quality Control
IQC is based on monitoring the haematology test procedures that are performed in the laboratory and includes measurements on specially prepared materials and repeated measurements on routine specimens, together with daily statistical analysis of the data.
Primarily a demonstration of Precision.
It ensures continual checks that the established reliability of the laboratory’s work does not fluctuate and that reports are validated before they are released
IQC includes
Control charts with tests on control materials
Duplicate tests on a proportion of the specimens
Consistency of mean values of patient data
Correlation check
Control charts
These were first applied in clinical chemistry by Levey and Jennings.
They are now widely used in haematology for both automated and manual procedures.
Samples of the control specimen are included in every batch of patients’ specimens and the results are checked on a control chart.
To check precision, it is not necessary to know the exact value of the control specimen.
ControlsLevel 1 controls- normal controls
Level 2 controls- low abnormal controls
Level 3 controls- high abnormal controls (not used routinely)
Minimum of one level QC at least once a day.
Patient samples >25 per day Employ 2 levels of QC at least once a day.
Patient samples >75 per day Employ 2 levels of QC at least twice a day at appropriate intervals
Controls are intended to simulate random sampling, they must be treated exactly like the patients’ specimens.
Preparation of LJ charts
Mean value and standard deviation (SD) of the control specimen should first be established in the laboratory where the tests are performed.
Using arithmetic graph paper, a horizontal line is drawn to represent the mean (as a base), and on an appropriate scale of quantity and unit, lines representing +2SD and −2SD are drawn above and below the mean.
The results of successive control sample measurements are plotted. If the test is satisfactory, sequential results oscillate about the mean value and <5% of the results fall outside 2SD.
LJ chart PT / low abnormal May 2015
LJ chart PT / low abnormal June 2015
LJ chart PT / low abnormal June 2015
Reason ??
LJ chart aPTT / NCPNP
LJ chart F VIIIc / NCPNP
Westgard rules
“Multirule Quality Control” developed by Dr. James O. Westgard based on statistical concepts
Uses a combination of decision criteria or control rules
Allows determination of whether an analytical run is “in-control” or “out-of-control”
Westgard multirule system
12S rule
13S rule
22S rule
R4S rule
41S rule
10X rule
Westgard 12S rule
“warning rule”
One of two control results falls outside ±2SD
Alerts tech to possible problems
Not cause for rejecting a run
Must then evaluate the 13S rule
12S Rule = A warning to trigger careful inspection of the control data
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
12S rule violation
Westgard – 13S Rule
If either of the two control results falls outside of ±3SD, rule is violated
Run must be rejected
If 13S not violated, check 22S
13S Rule = Reject the run when a single control measurement exceeds the +3SD or -3SD control limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
13S rule violation
Westgard – 22S Rule
2 consecutive control values for the same level fall outside of ±2SD in the same direction, or
Both controls in the same run exceed ±2SD
Patient results cannot be reported
Requires corrective action
22S Rule = Reject the run when 2 consecutive control measurements exceed the same +2SD or -2SD control limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
22S rule violation
Westgard – R4S Rule
One control exceeds the mean by –2SD, and the other control exceeds the mean by +2SD
The range between the two results will therefore exceed 4 SD
Random error has occurred, test run must be rejected
R4S Rule = Reject the run when 1 control measurement exceed the +2SD and the other exceeds the -2SD control limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
R4S rule violation
Westgard – 41S Rule
Requires control data from previous runs
Four consecutive QC results for one level of control are outside ±1SD, or
Both levels of control have consecutive results that are outside ±1SD
Westgard – 10X Rule
Requires control data from previous runs
Ten consecutive QC results for one level of control are on one side of the mean, or
Both levels of control have five consecutive results that are on the same side of the mean
10x Rule = Reject the run when 10 consecutive control measurements fall on one side of the mean
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
10x rule violation
Alternate ways of checking the precision of routine workDuplicate tests on patients’ samples
• Test of precision can be performed by repeat analysis on samples from two or three patients
Correlation Check
• any unexpected result of a test must be checked to see whether it can be explained on clinical grounds or whether it correlates with other tests
“Delta” check
• Blood count from the same patient checked repeatedly over 2-3 weeks• Values should not change significantly providing the patients condition has not altered significantly
Patient data
• Need enough through put (i.e. >100 samples /day)• MCV, MCHC, MCH should not vary, providing the patient population is stable and samples from different sets of patients are not batched together.• Mean of 20 successive patients can be plotted and any drift in the calibration of the analyzer will be recognized
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