2. Quality Control Notes

Quality Assurance
Chemical Pathology
1

Objectives
Quality assurance (Q.A.):
A sound knowledge of principles and practice of
laboratory Quality Assurance and Good Laboratory
Practice (GLP).
Accreditation:
A sound knowledge of the Accreditation system
and process.
Quality Control (Q.C.):
Sound knowledge of all principles, procedures,
calculations and interpretation of all related Q.C.
data including CV/ SD/ LJ charts and % error.
Definitions: Trend, Shift, Systematic and random
error, accuracy, precision, specificity, sensitivity etc.
2

Quality Assurance
• Medical decisions are based, in part upon the
results of laboratory tests.
• The validity of the test results cannot be taken
for granted, but must be supported by
convincing evidence that the figures are
reliable.
• The assurance of accurate analytical work is
only one facet of the problem.
3

Quality Assurance
• Quality assurance involves every step of the
process, from
the initial ordering of a test and
collection of a patient sample,
to the analysis, and finally
to distribution of the test results to the proper
destination.
4

PRE ANALYTICAL (phlebotomy team)
• Correct patient
• Sample container
• Patient information
• Sample transport (time)
• Patient preparation(fasting, circadian rhythm,
drugs)
5

ANALYTICAL (laboratory)
• Select the most accurate and precise analytical
methods
• Select good instrument and institute a regular
maintenance program
• Institute a good quality control program
• Conduct continuing education sessions
• Adequately train and supervise
• Make available printed procedure for each
method
• Document the information for an appropriate
accrediting group
6

THE TECHNOLOGIST MUST:
• Follow essay direction explicitly
• Use proper control serum
• Always use sound analytical techniques
• Be conscientious in instrument maintenance
• Notify the superior immediately when
analytical problem develop, when the run is
out of control, and when results indicate the
presence of a life threatening situation
7

POST ANALYTICAL
[data processors
(LIS system)]
• This process is intricate, and constant vigilance
is required at all levels to ensure that accurate
results are delivered to physicians in timely
manner.
8

Variation
• Analytical variation refers to difference in the
analytical measurements of a specimen
systematic and random variation.
• A source of variation is systematic if it influences
all measurement in the same direction.
• Different laboratories, methods, instruments and
technicians are common source of systematic
analytical variation, also called analytical bias.
• Aging phenomena can also be sources of
systematic variation. Chemicals, reagents,
standards and instrument components may
deteriorate with time causing an increasing or
decreasing trend in laboratory results.
9

Random Variation
• Sources of random analytical variation influence
each measurement differently, in either positive
or negative direction and to a different extent in
magnitude.
• For example, multiple determinations on the
same specimen on the same system in the same
run vary in an unpredictable manner due to:
Random fluctuation in the electro optical
mechanism,
The fluid dispensing of the sample and reagent,
The temperature of the instruments, and
The evaporate of the sample reagent.
10

Accuracy
• Accuracy –is the deviation from the true result.
• i.e. the systematic error concept
• Constant systematic error is “an error that is
always in the same direction and of the same
magnitude even as the concentration of analyte
changes”.
• Proportional systematic error is” an error which is
always in one direction and whose magnitude is a
percentage of the concentration of analyte being
measured.
12

Accuracy-the total error concept
• Considers all types of error, both random and
systematic.
• The distribution of value around a central value
represents random error.
• The shift of the central value of the distribution
from the true value represents systematic error.
• The total error shows how large the error can be
when the random and systematic components
occur in the same direction
13

Precision
• Precision-reflects the reproducibility of the test
(the agreement of the results among themselves
when specimen is assayed many times).
• The less the variation, the greater the precision.
• Within- run precision is the variability found
when the same material is analyzed repeatedly in
one an analytical run
• Within- day precision is estimated when the same
material is analyzed repeatedly in several
different runs on the same working day.
• This variation is usually somewhat higher than
observed for within-run replicates.
14

Definitions, cont.
• Day-to –day precision is the variability found
when the same material is analysed repeatedly
on different days.
• Recovery-is “the ability of an analytical method to
correctly measure pure analyte when added to
the sample routinely analyzed“
• Analytical sensitivity is a measure of “the ability
of an analytical method to detect small quantities
of the measured component”.
• Detection limit is defined as the smallest single
results which, stated probability (commonly
95%), can be distinguished from suitable blank.
15

Definitions, cont.
• Blank readings are responses observed by the
measurement procedure due to reagent and the
sample constituent, exclusive of the desired
analyte.
• It may be useful to quantitative the blank
readings directly by making measurement of the
reagents solutions without sample
present(reagent blanks)and of sample dilutions at
least one reagent that initiates the reaction
(sample blanks).
• Analytical specificity - This is “the ability of an
analytical method to determine solely the
component(s) it purports to measure.”
16

Analytical range
• This is the “range of the concentration or the
other quantity in the specimen over which the
method is applicable without modification.”
• It is tested by a “linearity experiment “in which a
series of solution, usually standard representing a
wide concentration range, are analyzed by
analytical method.
• Ideally the standard curve (plot of response
versus analyte concentration) should be linear
and pass through line of origin.
• The analytical range should be wide enough to
include most (95%) of the expected clinical
specimens without pre-dilution.
17

Standard deviation
• The degree or precision of a measurement is
determined from statistical consideration of the
distribution of random error; it is best expressed
in terms of the standard deviation.
• A normal frequency curve (bell-shaped, Gaussian
curve) is obtained by plotting the values from
multiple analyses of a sample against the
frequency of occurrence, the standard
deviation(s) is derived from the following
formula;
18

Standard deviation
• SD= Σ (ẍ-x)2
N-1
• Where SD=1 standard deviation=sum of,
ẍ=mean (average value), x=any single value
observed, and N=total number observed
values.
• With a normal distribution, 68% of the values
are encompassed by ẍ ±1s, 95% by ẍ ± 2s and
99, 7% by ẍ ± 3s.
19

The procedure for calculating the
Standard deviation
• Calculate the mean of all values. ẍ = Σx/n
• Find the difference of each individual value
from mean (column 2).
• Square the difference (column 3).
• Add the entries in column 3 to obtain the sum
of the squares of the differences.
• Find the standard deviation(s) by using the
equation for SD.
• The standard deviation is greater when a
method is less precise. 21

Coefficient of variation
• The standard deviation is greater when a method
is less precise.
• The coefficient of variation (CV) expresses
deviation as a percentage of a mean value and is
more reliable means for comparing the precision
at different concentration levels:
• CV= (mean/SD)X100 and is expressed as %
• The precision of a method varies inversely with
the CV; the lower the CV the greater the
precision.
22

Coefficient of variation
• Although accuracy of the test is paramount in the
clinical laboratory precision is just as important.
• One way a laboratory can determine whether the
precision of a specific test is acceptable is to
compare it’s precision to that of another
laboratory performing the same test on the same
instrument using the same reagents (laboratory
peer group).
• An easy way to make thus comparison is to divide
the laboratory CV by laboratory peer group CV
obtained from an inter-laboratory comparison
report.
23

Preventive maintenance
• Another phase of quality control require regular
maintenance program for the various laboratory
instruments to ensure that they are in top working
condition such maintenance includes:
 regular calibration of spectrophotometer wavelengths,
 continuous recording of refrigerator and freezer
 temperature to ensure that requisite cold
temperatures are maintained,
 testing of water purity with a resistance meter,
 checking water bath temperature regularly, and
 calibration of micrometre.
 These seemingly small details may greatly affect
performance. 24

Preventive maintenance
• The maintenance of a good quality control
program is costly in both time and money.
• Control serum is not inexpensive, and much of
it is used in a year.
• The time invested by technologists in carrying
out tasks that bring in no revenue (analyzing
control serum, repeat testing of sample in a
run not in control, and calibrating glassware)
is considerable.
25

Characteristics to analytical methods
• Practicality characteristics. These are factors
(other than analytical performance) which
determine whether the method can be
implemented in the laboratory.
• They include the required equipment,
• work load, specimen handling, run size,
personnel skill, cost per test,
• methods of standardization and quality
control, space needs (including reagent
storage), and precaution and
• Procedures required for safety.
26

Characteristics to analytical methods
• Reliability characteristics. These properties
relate to the method, including the precision,
accuracy analytical sensitivity,
• Analytical specificity,
• Recovery,
• Interference,
• Blank readings,
• Linear range,
• Sample interaction, and
• Reagent stability. 27

Calculate %Error
• % Error= (ẍ - x) x100
ẍ
ẍ= mean value x= Value
• E.g.: ẍ=100 x=95
• % Error= 100-95 x 100
100
=5%
28

Control of analytical quality using
control materials
• The performance of analytical methods can be
monitored by analysing specimens whose
concentrations are known and then comparing
the observed values with the known value.
• The known values are usually represented by a
range of acceptable values, or upper and lower
limits for a control specimen (control limits).
• When the observed values fall within the control
limits, this should assure the analyst that should
the analytical method is working properly.
• When the observed value fall outside the control
limits the analyst should be alerted to the
possibility of problems in the analytical
determination. 29

Control materials
• The known specimens that are analysed for
quality control purpose are called “control
materials” they need to be stable material,
available in aliquots or vials that can be analysed
periodically over a long period of time.
• There should be a little vial-to-vial variation so
that differences between repeated
measurements can be attributed to analytical
method alone.
• A control material should preferably have the
same matrix as the test specimen of interest; for
example, a protein matrix may be best when
serum is the material to the analysed by the
analytical method.
30

Control materials cont.
• The concentration of analyte in difference control
material should be normal and abnormal normal
ranges, corresponding to the concentrations that
are critical in the medical interpretation of the
test results (medical decision levels).
• Control material can be prepared in the
laboratory from unused sera, but for safety,
stability and economy, most laboratories choose
to the purchase control sera or “control product”.
• Commercial product are generally supplied as
lyophilised materials that are reconstituted by
adding water or a specific dilute solution .
• Material are also available that have matrices
representing urine, spinal fluid and whole blood.
31

• In the selection of commercial control material,
there are several considerations besides the
matrix of the material.
• Stability is critical because it is often desirable to
purchase a year`s supply of one manufacturing
batch or lot.
• Different batches (or lot numbers) of the same
material will have different concentrations, which
require new estimate of the mean and standard
deviation for each of the analytes of interest.
• The size of the aliquots or the vials must be
adequate for the analytical methods to be
monitored. 32

• Control products may be purchased as assayed or
unassayed materials.
• Assayed materials are accompanied by a list of
values (mean and standard deviation) for the
concentrations that are expected for that
material.
• Values may be specified for several of the
common analytical methods and preferably for a
reference method for each analyte.
• Assayed materials are more expensive because of
the work required to establish the values.
• Even when assayed materials are selected, it is
advisable to determine the mean and standard
deviation in the user `s own laboratory.
33

Procedure to be followed when
reconstituting a lyophilized calibrator
1. Open the vial carefully; avoiding any loss of
the material, (vial is under vacuum).
2. Reconstitute with an accurately measured
volume of distilled water or supplied dilute
using a volumetric glass pipette (not an
automatic pipette). The water or dilute must
be at room temperature (20-25°C).
3. Replace rubber stopper and leave to stand
for 30 min out of sun light.
34

Procedure to be followed when
reconstituting a lyophilized calibrator
4. Swirl gently several times during this
reconstitution period to ensure that the content
is completely dissolved. Do not shake the vial.
5. Prior to use, mix the content by inverting the vial,
avoiding the formation of form ensure that no
lyophilized material remains un-reconstituted.
6. Label with date of reconstitution and the name
of the person.
35

External quality assurance (inter-
laboratory comparison program)
• Inter-laboratory in an inter-laboratory quality
control comparison program is highly
recommended and requirement for laboratory
accreditation.
• Without such programs the laboratory
becomes a statistical island and has no means
to regularly verify the accuracy of its work.
• The laboratory needs to regularly asses in
accuracy and imprecision.
36

External quality assurance (inter-
laboratory comparison program)
• One of the easiest methods to asses
inaccuracy and imprecision is to compare
within laboratory methods means and
standard deviation to other laboratories using
the same instrument and method (peer
group).
• External quality assurance is important for
maintaining the long term accuracy of the
analytical methods.
37

Method groups and modes
• Statistical analysis
• The aim of the analysis is to compare like with
like most practical statistically meaningful
level.
• Method
• The method, where possible encompasses all
results within a particular methodology (essay
Technology) when using the same reagent or
instrument. These are your exact peer
comparisons
38

Method groups and modes
• Method group
• Different instruments utilizing the same,
specific measurement technology (for
example, enzymatic methods for creatinine)
are assigned to the method group.
• Mode
• Within an analyte, method groups are
separated, where appropriate, into modes to
provide a separate analysis for distinct,
different chemistry principles.
39

Reports
• Every one or two weeks you will receive your
report giving methods comparisons and
statistics.
• Your deviation is also plotted on a Levey-
Jennings chart.
• At the end of the cycle, when all the samples
have been assayed as assessment of your
laboratory performance during the cycle is
given in terms of imprecision and bias.
40

Participation in the program
• Advantages
• Comparison of your results (method means and
standard deviations) to other laboratories using
the same instrument and method (peer group).
• You can evaluate the performance of all
instruments and methods for a specific analyte.
• Receive weekly or two weekly reports and an end
of cycle survey of your performances.
• Give you confidence in your results.
41

Participation in the program
• Disadvantages
• Cost factors.
• The serum pack consists of the freeze dried
material and must be reconstituted, which
may results in errors.
• Report feedback is usually two weeks after
sample date.
42

Definitions
• Calibrator-has an assigned value that is established by
the manufacturer or the user by reference methods. A
calibrator is used to standardise or calibrate the
method or instrument. It is often used to adjust an
instrument to certain values (calibrate) prior running a
sample
• Controls-are samples that are closely resemble the
real. Span the clinically important range of the analyte
concentration.
• For the assessment and precision of analytical method.
Goes through all stages.
43

Internal quality assurance programme
(Inter-laboratory comparison)
• Internal quality assurance focuses on
monitoring your (single) laboratory
performance and is necessary for the daily
monitoring of the precision of the analytical
method.
• Internal quality control-a program that varies
the validity of laboratory results (observation).
• It is planned and carried out as part of the
daily regular routine within the laboratory.
44

External quality assurance programs
(Inter-laboratory comparison)
• External quality assurance is important for
maintaining the long-term accuracy of the
analytical method means and standard deviation
to other laboratories preferably using the same
instruments and method. (Peer group
comparison).
• External quality control- a program in which an
external agency provides unknown sample for
analysis.
• The results are submitted to an independent
evaluation of “acceptance “ or “not acceptable”
performance.
45

Internal Quality Assurance
• In contrast to external quality assurance, internal
quality assurance focus on monitoring a single
laboratory (intra-laboratory programs).
• Internal quality assurance is necessary for the
daily monitoring of the precision and(accuracy)of
the analytical method.
• Limitations of internal quality assurance is that
the problems detected are only the changes in
performance between the present operation and
the "stable" operation that was characterized
during the baseline period when the analytical
method was thought to be working properly.
47

Quality Control Chart
• A quality control chart is established for each
constituent in the control serum.
• In the most commonly used form, the Levey-
Jennings chart, the concentration is plotted on
the ordinate, with a black line drawn across
the chart at the mean value, blue lines at± 1s,
orange lines at ± 2s, and red lines at ± 3s.
• The days of the month are plotted on the
abscissa (x-axis).
48

Quality Control Chart
• The chart is hung in a convenient location or
kept in a notebook at the workbench, and
each value obtained on the control serum is
recorded on the chart every time an analysis is
made.
• Sometimes when control values are plotted
regularly, one can see that a method is getting
out of control even while the values are still
within 2s of the mean.
49

Systematic Error
• Systematic error is evidenced by a change in
the mean of the control values.
• The change in the mean may be gradual and
demonstrated as a trend or it may be abrupt
and demonstrated as a shift.
50

Trend
• Trend: gradual often subtle, increase or
decrease in control values and possibly patient
values.
• A trend indicates a gradual loss of reliability in
the test system. Trends are usually subtle.
51

Trend
• Causes of trending may include:
Deterioration of the instrument light source
Gradual accumulation of debris in
sample/reagent tubing
Gradual accumulation of debris on electrode
surfaces
Aging of reagents
Gradual deterioration of control materials
Gradual deterioration of incubation chamber
temperature (enzymes only)
Gradual deterioration of light filter integrity
52

Shift
• Shift: a sudden and eventually stable change in
control values and possibly patient values
• Abrupt changes in the control mean are defined
as shifts. Shifts in QC data represent a sudden
and dramatic positive or negative change in test
system performance. Shifts may be caused by:
53

Shift
• Shifts may be caused by:
Sudden failure or change in the light source
Change in reagent formulation
Change of reagent lot
Major instrument maintenance
Sudden change in incubation temperature
(enzymes only)
Change in room temperature or humidity
Failure in the sampling system
Failure in reagent dispense system
Inaccurate calibration/recalibration
54

Westgard Rules
• Westgard devised a shorthand notation for
expressing quality control rules.
• Most of the quality control rules can be
expressed as NL where N represents the
number of control observations to be
evaluated and L represents the statistical limit
for evaluating the control observations.
• Thus 13s represents a control rule which is
violated when one control observation
exceeds the ±3s control limits,
55

12s Rule
• 12s : The only warning rule which is violated when
one control observation is outside the ±2s limits.
• Remember that in the absence of added
analytical error, about 4.5% of all quality control,
results will fall between the 2s and 3s limits.
• This rule merely warns that random error or
systematic error may be present in the test
system.
57

12s Rule
• The relationship between this value and other
control results within the current and previous
analytical runs must be examined.
• If no relationship can be found and no source
of error can be identified, it must be assumed
that a single control value outside the ±2s
limits is an acceptable random error.
58

13s Rule
• 13s : This rule identifies unacceptable random
error or possibly the beginning of a large
systematic error.
• Any QC result outside ±3s violates this rule.
60

• Violation of any of the following rules may be
cause to reject the entire run and re-test
patient and QC samples.
61

22s Rule
• 22s This rule identifies systematic error only.
The criteria for violation of this rule are:
• Two consecutive QC results, Greater than 2s,
On the same side of the mean.
• There are two applications to this rule: within
run and across runs.
63

R4s Rule
• This rule identifies random error only, and is
applied only within the current run.
• If there is at least a 4s difference between
control values within a single run, the rule is
violated for random error.
65

R4s Rule
• For example, assume both Level I and Level II
have been assayed within the current run.
• Level I is +2.8s above the mean and Level II is -
1.3s below the mean.
• The total difference between the two control
levels is greater than 4s; i.e., [+2.8s - (-1.3s)] =
4.1 s.
66

• Violation of any of the following rules does
not necessarily require rejection of the
analytical run.
• These violations typically identify smaller
systematic error or analytical bias which is
not often clinically significant or relevant.
• Analytical bias may be eliminated by
performing calibration or instrument
maintenance.
67

41s Rule
• The criteria which must be met to violate this
rule are:
• Three consecutive results, Greater than 1 s,
On the same side of the mean
• 41s The criteria which must be met to violate
this rule are:
• Four consecutive results, Greater than 1s, On
the same side of the mean.
69

10ẍ Rule
• 7ẍ, 8ẍ, 9ẍ, 10ẍ, and 12ẍ.
• These rules are violated when there are:
• 7 or 8, or 9, or 10, or 12 control results, on the
same side of the mean regardless of the
specific standard deviation in which they are
located.
71

10ẍ Rule
• The 7ẍ control rule is far more sensitive to
analytical bias than the 12ẍ and the chances
of finding seven consecutive control
observations on one side of the mean are
much higher than finding twelve.
• It is extremely important that each individual
laboratory be aware of highly sensitive rules
like 7ẍ, 8ẍ, and 9ẍ and apply them sparingly, if
at all.
72

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