Calibration and Quality controls of automated hematology analyzer

Calibration and Quality controls of
Automated Hematology Analyzer
Dr. Pranav N Shirbhate..

CONTENTS
 History
 Automations in Hematology
 Need of automation
 Introduction to Quality assurance
 QC & calibration terminologies
 Requirements for QC and calibration
 Calibration of New/ Repaired automated Hematology Analyzer
 Quality control in Hematology – Internal and External
 Levey–Jennings (LJ) chart
 Application of Westgard’s Rules
 VIVA Ques at the End

CONTENTS
 History

HISTORY
Antonie van
Leeuwenhoek
Invented Microscope
Wallace Coulter :First
automated analyzer for
counting and sizing cells
and presented it in 1956
The world of cells was
colorless until Paul
Ehrlich stained blood cells

AUTOMATIONS IN HEMATOLOGY
Cell counts (Automated hematologyanalyzers)
Diagnosis of hemoglobinopathies (HPLC)
Immunophenotyping (Flow cytometry)
Coagulation (Coagulometers)

 Hemogram is a Backbone of any lab evaluation.
 The Core Laboratory report well over 140,000 CBCs and
48,000 peripheral smears per year at our Dept of Pathology, MGIMS Sevagram.
 It is a routine investigation for-
Anaemia,Polycythemia, Infection, Inflammation, allergy, malignancy,
bleeding tendency etc
All automated cell counters are screening devices. Abnormalities must be verified
by a blood film, staining and scanning by an expert observer.
But testing each and every sample out of theses 1.4 lac samples a year by manual
method is very laborious, cumbersome, so comes the need of automation.
NEED OF AUTOMATION

AUTOMATED TECHNIQUES OF BLOOD COUNTING
• Semi-automated instruments
- Require some steps, as dilution of blood samples .
- Often measure only a small number of variables
• Fully automated instruments
- Require only that an appropriate blood sample is presented to the instrument.
- They can measure 8-20 variables including some new parameters which do not
have any equivalent in manual methods.

INTRODUCTION
• Quality Assuarance (QA) is the sum of all those activities in which the laboratory is
engaged to ensure that information generated by laboratory is correct.
• QA is not restricted to the development and retention of quality control charts but
rather includes all aspects of laboratory activities that affects the results produced,
from the choice of methods, to the education of personnel, to the handling of
specimens and reporting results.
• The real purpose of QA activities is to determine how correct or incorrect the results
emanating from the lab are, and to allow those managing the lab to determine whether
or not the lab is fulfilling its functions satisfactorily.

INTRODUCTION
3 major activities of QA :
1 ) Preventive –
2 ) Assesment –
3 ) Corrective
1 ) Preventive –
those activities that are done prior to the examination of the specimen or sample and that
are intended to establish systems conducive to accuracy in analytical testing
 Preventive maintenance and calibration of instruments,
 Testing of media,
 Orientation and training of personnel

INTRODUCTION
3 major activities of QA :
2 ) Assessment –
those activities that are done during testing to determine whether the test systems are
performing correctly
 The use of standard and controls,
 Maintenance of control charts
3 ) Corrective – those activities that are done, when error is detected, to correct the
system
 equipment troubleshooting,
 recalibration of instruments

INTRODUCTION
QA in Hematology Laboratory
QA in Hematology lab is intended to ensure the reliability of the lab tests. The objective is
to achieve precision and accuracy;
So components of QA programme :
 Standardization/ Calibration
 Internal Quality Control ( IQC )
 External Quality Control ( EQC )/ Proficiency testing

DEFINITIONS IN TOTAL QUALITY MANAGEMENT

QC & CALIBRATION TERMINOLOGIES
Accuracy, I) A measure of agreement between the estimate of a value and a "true"
value; quantifiable in terms of departure from accuracy, expressed as
systematic error or bias;
II) Accuracy of an analytic process, n - expressed as the difference between
the average result obtainable by a method under specified conditions
and the result accepted as true or standard.
III) Accuracy of a result or control result, - Expressed as the difference
between a result and the "true" value.

Bias/ systematic error –
• Systematic, signed deviation of the test result from the accepted reference value.
• (Defined as the difference between the expectation of the test results and an
accepted reference value.)
Calibration, n –
• The process of testing and adjustment of an instrument, kit, or test system, to
provide a known relationship between the measurement response and the value of
the substance being measured by the test procedure.

Calibrator, n - A (reference) material (e.g., solution, suspension) or
device of known quantitative/qualitative characteristics (e.g.,
concentration, activity, intensity, reactivity) used to calibrate,
graduate, or adjust a measurement procedure or to compare the
response obtained with the response of a test specimen/sample.
NOTE: The quantities of the analytes of interest in the calibrator are
known within limits ascertained during its preparation and are used
to establish the relationship of an analytical method's response to
the characteristic measured for all methods or restricted to some.
The calibrator must be traceable to a national or international
reference preparation or reference material when these are
available. (like eg NCCLS/CLSI)

ORGANIZATIONS INVOLVED IN STANDARDIZATION AND QUALITY
MANAGEMENT SYSTEMS

WHAT THESE ORGANIZATIONS ARE? FOR e.g. CLSI
CLSI/ NCCLS
The Clinical and Laboratory Standards Institute (CLSI) is a
volunteer-driven, membership-supported, not-for-
profit, standards development organization. CLSI promotes
the development and use of voluntary laboratory consensus
standards and guidelines within the health care community.
The organization’s mission is to develop clinical and
laboratory practices and promote their use worldwide.
The CLSI started as the National Committee on Clinical
Laboratory Standards (NCCLS) and changed to its current
name (CLSI) at the beginning of the year 2005.

NCCLS/ CLSI STANDARDS AND GUIDELINES
CLSI publishes voluntary consensus standards and guidelines, reports, and a number of companion
products.
Standards are documents developed through the consensus process that clearly identify specific,
essential requirements for materials, methods, or practices for use in an unmodified form. Standards
may, in addition, contain discretionary elements, which are clearly identified.
Guidelines are documents developed through the consensus process describing criteria for a general
operating practice, procedure, or material for voluntary use.
Guidelines may be used as written or modified by the user to fit specific needs.
Reports are documents that have not been subjected to consensus review and are released by the
appropriate consensus committee.
Companion products include any item provided or sold that is intended to be used in conjunction
with a CLSI standard or guideline. Examples include, but are not limited to, Quick Guides, Wall
Charts, software, and templates.

(Quality) control material, n - A device, solution, or lyophilized
preparation intended for use in the quality
control process;
NOTE: It should be similar to, and is analyzed along with, patient
specimens. If different, it should have a recognized, defined response
to analytic measurements.
Control materials
may or may not have known analyte concentrations (i.e., assigned
values) within specified limits (e.g. target value ± standard deviation).
Control materials are not used for calibration purposes.

Derived red cell indices, n - Quantities (see MCH, MCHC, MCV) that may be calculated from the
measurement of hemoglobin (Hb) concentration, packed (red) cell volume (PCV), and erythrocyte
(RBC) concentration.

Linearity, n - The ability (within a given range) to provide results that are directly proportional to
the concentration {amount} of the analyte in the test sample.

Precision, n - The closeness of agreement between
independent test results obtained under prescribed
{stipulated} conditions;
NOTE: Precision is not typically represented as a
numerical value but is expressed quantitatively in terms
of imprecision—the SD or the CV of the results in a set of
replicate Measurements.

Precision, n - The closeness of agreement between
independent test results obtained under prescribed
{stipulated} conditions;
NOTE: Precision is not typically represented as a
numerical value but is expressed quantitatively in terms
of imprecision—the SD or the CV of the results in a set of
replicate Measurements.
For practical purpose
• Accuracy: Refers to closeness to the true value
• Precision: Refers to reproducibility of test

Drift, -

Reference material, RM, n –
A material or substance, one or more of whose property values are sufficiently homogeneous
and well established to be used for the calibration of an apparatus, the assessment of a
measurement method, or for assigning values to materials;
Certified reference material, CRM, n - A reference material that has one or more values
certified by a technically valid procedure and is accompanied by, or is traceable to, a
certificate or other document that is issued by a certifying body;
NOTES: a) defines a CRM as a “reference material, accompanied by a certificate,
one or more of whose certified value is accompanied by an uncertainty at a stated level of
confidence”;
b) The term "Standard Reference Material (SRM)" is the name of a CRM, which is
the trademark name of a certified reference material that has been certified and is distributed by
the National Institute of Standards and Technology (NIST), a U.S. Government agency formerly
known as the National Bureau of Standards (NBS).

Sensitivity – It is the smallest quantity of analyte that can be reproducibly distinguished from
background noise in a given assay system.
Specificity - For quantitative tests, the ability of an analytical method to determine only the
component it purports to measure or the extent to which the assay responds only to all
subsets of a specified analyte and not to other substances present in the sample.

Set point - The level (i.e., analyte concentration) at which an analyzer is calibrated.
Standard (measurement), n - An authoritative “document” setting forth criteria for
performance and characteristics.
• Standard, primary, n - A standard that is designated or widely acknowledged as having
the highest metro logical qualities and whose value is accepted without reference to other
standards of the same quantity.
• Standard, secondary, n - A standard whose value is assigned by comparison with a
primary standard of the same quantity.

GENERAL REQUIREMENTS FOR QC & CALIBRATION
1) Quality Control Manual
• All quality control procedures should be described in detail in a quality manual. Clinical
Laboratory Technical Procedure Manuals for more details.
• The quality manual should also include a general statement of the laboratory quality policy
and an acknowledgment that the policy is understood by all laboratory workers.
• The persons with authority to design and modify all laboratory procedures and the persons
with authority to approve such designs and changes should be listed.
2) Record Keeping
• Comparisons with previous performance should be provided before analyzing control
materials.
• Electronic verification and background measurement recorded.
• CBC/differential files should include control set-up, control run, and control review/report
files.

3) Retention of Records
There are three major files of information. Storage of this data base is usually made HIS.
• Laboratory order entry: The laboratory order entry data is maintained for a user-defined
number of days and is then purged.
• Workload data: Workload data is kept in active files for six months and is then archived.
These archives are updated every month and stored in a safe place with timely access to
permit review as the need arises.
• Laboratory data files: The duration of QC record storage depends on local hospital or clinic
policies or accreditation requirements, or should be a minimum of two years or more,
whichever is greater.
Proficiency testing results, noncommercial controls, or calibrators, if used,
must have complete documentation of standardization and performance characteristics.

4) Training and Qualifications
• Laboratory workers who are assigned to carry out QC procedures must have been trained
and records must indicate this.
• As per CSLS guidelines, personnel who are given responsibility for interpreting QC results
should not be the same personnel doing the routine work.
5) Environment
Maintain environment that reflects routine working conditions, ambient temperature.
A record of temperature be maintained.

6) Analyzer Identification labels

7) Preanalytic Variables
Uncontrolled preanalytic variables can exert a misleading effect , therefore a
checklist of possible error sources will help ensure that they will not be overlooked.
Analyte Instability
• Least likely to be seen in stabilized blood preparations and most likely to be seen in recently
collected blood specimens.
• Over-vigorous mixing may also play a role.
• Viral disease may cause reduced lymphocyte count due to cell fragility. Cold agglutination
may affect platelets as well as red cells.
(Detection of analyte instability depends on the demonstration of a difference between the mean
value of the first half of an iterative assay sequence and the mean value of the second half. To be
significant the difference between means should exceed standard error of the first mean at 95%
confidence.)

Analyte Nonuniformity
Caused by inadequate mixing. Fresh blood is less susceptible to this.
Mixing a specimen by rapid
shaking or vortexing or any method that causes frothing has be avoided.
Two components in nonuniformity.
1. nonuniform dispersion of the total cell mass throughout the plasma.
2. nonuniform distribution of the different cell types with respect to each other.
To achieve uniformity of a whole blood suspension, - 10 complete
inversions of the specimen container.
(A completely filled container gives poor mixing; an air bubble must be present. If the
specimen has been stored for 2 to 5 minutes, three additional inversions should precede the
assay.)

Autoagglutination
• Autoagglutination is a cause of unstable suspensions.
• If not visible to the eye, autoagglutination or rouleaux should be suspected if
1) reduced RBC together with elevated MCHC are encountered;
2) The MCV may be slightly to markedly increased;
3) The rule of thumb “3 x Hb (g/dL) = Hct (%) ±3" is not met.
• Its presence can usually be confirmed by microscopy, but be aware that a warm slide may
cause autoagglutinates to dissociate.
• Specimens with autoagglutination should not be used in quality control procedures.
• Refrigerated specimens should be brought to room temperature before analysis.

Anticoagulant Effect
• Insufficient blood collected into an evacuated container may cause shrinkage of all cell
types, but red cells usually become restored to the size dictated by their osmotic
equilibrium with the cell-counting diluent.
• Leukocytes, erythrocytes, and platelets (e.g., EDTA-induced aggregation) may be irreversibly
changed and cause errors in the differential count and platelet count and MCV.

CALIBRATION
• Calibration fine tunes your Hematology analyzer and provides the
most accurate results possible.
• In the normal process of tracking data for an extended period of time,
your laboratory can make a specific decision to recalibrate a given
parameter. Never adjust to a specific value for an individual sample.
• For best performance, calibrate all the CBC parameters.
The WBC differential is calibrated at the factory. They do not require
calibration in the laboratory.

CALIBRATION
We should calibrate our instrument:
*At installation
*After the replacement of any component that involves dilution
characteristics or the primary measurements (such as the apertures)
*When advised to do so by your service representative

BASIC OF CALIBRATION OF NEW/REPAIRED INSTRUMENT
Establish Baseline Parameters
• The analytical baseline data provide reference levels for judging the performance of the
analyzer.
• Baselines should be established or verified when a new analyzer is installed and when a
modification or repair is made that could significantly affect the analyzer performance.
• The product reference manual should provide instruction for frequency of recalibration
under normal operating conditions.
• Calibration or recalibration must be preceded by verification of the major analytical
variables, which are general conditions of imprecision and accuracy and the analyzer.
• Reportable range should be verified for a new installation and after major repair.
• To avoid the possibility of analyzer defect causing errors in the baseline procedures,
preventive maintenance should be performed shortly before calibration.

ESTABLISH BASELINE PARAMETERS
Imprecision Baseline
• Baseline imprecision is described by the standard deviation of replicate assays of the same
specimen.
Measure Baseline Imprecision
• Make n consecutive assays of the same well-mixed stabilized blood. The preferred number
of iterations is n=31.
• Fewer repeated assays (for example, n = 10) may be used, but if this is done, convert
standard deviation (SD) to confidence limits (CL) as Evaluate Baseline Imprecision.

• Record the results of each analyte
assay in a table.
• Note the need to record the
elapsed time of the measurement.

Evaluate Baseline Imprecision
• Imprecision may be stated as SD or CL . (95% confidence limit).
• If comparison is to be made 95% between standard deviations from different sources, e.g.,
between the laboratory estimate and a manufacturer's labeled value, be sure that each
measurement has been made with the same number of iterations (n).

Evaluate Baseline Imprecision
• If different values of n have been used, convert SD
to CL95% as follows:
• Multiply each SD by the value of t for (n-1) given in
this Appendix.
For e.g.,
• the laboratory used n = 31, but
the manufacturer's labeled SD is based on n = 16.
• Therefore, multiply the laboratory SD by 2.0423 and
the manufacturer's SD by 2.1315.
• This will convert both SD's to CL , making it possible
to compare them.

Accuracy Baseline
• The accuracy baseline is defined as the starting point against which future bias control
information will be compared.
• Verification of accuracy must be made immediately (less than 15 minutes) after calibration or
as directed by the manufacturer. If delayed, the data from the baseline verification process may
be contaminated by drift or other analyzer error.
• The term "calibration" means adjusting the analyzer to achieve zero bias, or verifying through a
similar process that zero bias already exists.
• The term “zero bias” means that any difference between the calibrator-assigned values and the
results of calibrator assay is due only to analytical imprecision.
• It is assumed that all automated hematology analyzers have a linear response over the
reportable range, that the limits of linearity are specified, and that zero analyte concentration
produces a zero analyzer response. These characteristics make it possible to calibrate analyzers
by a single-point method.

Reference-Assayed Whole Blood Calibration
Rarely used today in medical institutions. Not recommended.
Standardized Stabilized Blood Calibration Method
Frequency of calibration: - As per product reference manual.
- Usually calibration is recommended after a major repair or after a
significant change of base line accuracy (drift).
- In the latter case, the cause of excessive drift should be corrected
before proceeding with calibration.
Iteration of calibrator assays: The number of assays will influence the precision of the calibration
factor.
Greater assay iteration will result in a lower SEM.

• More replication will yield a more precise calibration set point and more accurate subsequent
patient assays but, because of the square root function, the rewards of increasing replication will
reduced.
• Furthermore, excessive mixing associated with increased replication may damage
cells and introduce other errors.
• It is suggested that 10 consecutive assays of the calibrator will give a satisfactory result.
For example,
the SD for WBC was found to be 0.2 x 109/L. The SEM of ten
calibrator assays will then be 0.2 ÷ /10 or 0.2 ÷ 3.16 = ±0.063 x 109/L.
This error, in combination with the analyzer SD will be propagated (included in) to patient assays. To
obtain the 95% assay bias range, use the root sum square variance method as..

Reportable Range
• Reportable range is the range of assay values over which accuracy is ensured; hence its
reportable to the patient’s record. (accuracy of values beyond reportable range is not ensured).
• It describes only the effect of analyte concentration and does not include possible error due to
interferences, such as agglutination or hemolysis.
• Upper and lower limits of reportable range for a newly installed analyzer can be verified by
recovery of the assigned values of abnormal high and abnormal low stabilized blood controls.
• Assays of sequential dilutions of blood between these limits do not contribute information that
is helpful in establishing extension of this reportable range.
• Reassurance of midrange linearity is given by the recovery of the assigned value of the normal
stabilized blood control and by the verification of the calibration set point.
For e.g., this happens a lot of time in leukemia cases where WBC values go beyond reportable
range. Analyzer counts the cells but certainty about the accuracy can be questioned.

CALIBRATION COMPLETION
Technician from authorized / standardized calibration party Runs the blacks cycle to test values
within range.

• He feeds all these (SD, CV, SEM and Base
line parameters) values to analyzer
software, and helps its synchronization
with HIS/ Hospital software.
• Calibration of the analyzer completes only
when we receive calibration certificate
clearly mentioning the CRM body like
CSLS/NCCLS.

Now our machine/ analyzer is ready to be used for routine runs.

DAILY MAINTENANCE
• Daily cleaning
• Electronic checks
• Compare open and closed mode sampling (using a normal patient sample)
• Run controls

ENSURE THE INSTRUMENT IS FUNCTIONING PROPERLY
• By Checking the reagent containers for:
o Sufficient quantity
o Not beyond expiration date
o No precipitates, turbidity, particulate matter, or unusual colour
o Proper connections between the instrument and the reagent containers
• Check the waste container for: *Sufficient capacity and * Proper connections
• Perform daily startup and Background counts
• In addition to verifying daily startup results, verify acceptable:
o Reproducibility
o Carryover
o Control Results

QUALITY CONTROL
Internal Quality Control –
The set of procedures undertaken in a laboratory for the continuous
assessment of work carried out in the laboratory and evaluation of tests to decide whether these
are reliable enough for their results to be released to the requesting physician.
NOTE: The main object is to ensure day-to-day consistency of measurements or observations, if
possible in agreement with an agreed-on indicator of truth, such as a control material with assigned
values.

QUALITY CONTROL
Internal Quality Control
• Once a performance baseline has been established, its control is based on methods that will
detect loss of accuracy or loss of precision, or both.
• Control methods must be sensitive enough to reveal loss of performance that could compromise
patient assay values, but they cannot be oversensitive to the point of signaling error when none
exists.
• As a minimum, a performance control system must provide the following information:
 Analytical imprecision has not deteriorated with time or use.
 Bias has remained within the limits established at the time of calibration.

INTERNAL QUALITY CONTROL
• The term “internal" indicates quality control procedures that take place within the laboratory
immediately before, during, or immediately after an analysis run.
• The procedures must be carried out as an integral part of patient assays.
• Control assays performed only once daily may not provide enough information to ensure
accuracy and consistency of results.
• When the analyzer is restarted after complete shut-down, precision and set point should be
verified to ensure that satisfactory analytical capability has not been lost.
• Internal quality control is distinguished from external quality control. The latter is administered
by a third party, such as an authorized proficiency testing body, based on blind assay of a
stabilized control material, or by a manufacturer offering statistical peer comparison of routine
stabilized blood control results.
• Analyzers of differing designs and operating principles will have differing propensities for failure
or for performance deterioration.

Quality Control Using Stabilized Blood
• Stabilized blood controls should be similar to calibrators,
except that assigned values, if provided, may be conferred by
calibrated analyzers using stabilized blood calibrators instead
of reference-assayed whole blood.
• Manufacturers' assigned values pose a temptation to use
controls as calibrators and to "unblind" some types of
internal and external performance reporting.
• Controls should have 2-3 levels of analyte concentration.
These levels should reflect performance at normal analyte
levels and at, or
close to, the low limits of the reportable range.

Quality Control Using Stabilized Blood
• Stabilized blood controls must be labeled for use
with specified analyzers.
• The laboratory should document that the product
it uses is so labeled.
• We are using Bio-Rad 3 level of control LOW/ NORMAL/
HIGH.

Frequency of Use
• We have to decide how many assays should separate controls.
This decision should be made on the basis of work load and
experience with the drift characteristics of the analyzer(s).
• If the results of daily control assays are within laboratory-
defined limits, such as ±2 SD of the assigned or conferred
value, it can be assumed that the results of patient assays
performed within a control-bracketed work segment are valid
and reportable.
• The fewer the control assays relative to patient specimens, the
greater will be the number of assumed invalid results in the
event of an out-of-control condition.

Frequency of Use
• As a minimum, assays of normal and abnormal levels of
control
material should be made at the beginning of each run and one
assay of normal level control made at the end of the run to
ensure that patient results have been satisfactory.
We at our institution running these three control samples
at the start of the new day of analyzer.

Charts are prepared with the values shown by analyzer for that day.

Test Replication / Precision on patients sample
• The mean of replicate assays of control material is more
informative than a single measurement.
• Paired assays are recommended. Not only does the mean of
the pair give a more exact value, but the difference between
members of a pair also gives an estimate of imprecision.

Test Replication / Precision on patients sample
Overlap is a special case of test replication. When a new lot of control
material is received, it should be tested in parallel with the currently used
lot for 4 days.
This will establish the ratio of the assigned values of the incoming
lot to the assigned values of the currently used material.
Differences in assigned values of the two lots should not be
taken as an indication to recalibrate.
We are doing precision bi-weekly ie on Tuesday and Friday at start of new
day for the analyzer with random fresh EDTA sample. (Overlap - not done)

Correlation Check
Correlation check implies that any unexpected result of a test must be checked
to see whether it can be explained on clinical ground or whether it correlates
with other tests.
E.g. 1) Unexpectedly higher Hb needs explanation of blood transfusion or by
hemorrhage respectively.
2) Low MCHC confirmed by demonstrating hypochromic red cells on
Romanowsky stained blood film
3) High MCV must correlate with macrocytosis.
4)Marked leukocytosis, thrombocytopenia and thrombocytosis, needs
blood film examination
We do correlation check for each and every sample we receive in our laboratory.

Checking of aberrant result
Recording blood count data on cumulative report for or charts is a good clinical
practice and provides an inbuilt quality control system by making it easy to
detect any aberrant result when compared with previously determined base
line.
E.g. Useful in detecting the occasional wild error caused by
1) incorrect labelling of specimen,
2) Inadequate suspension of blood before sampling,
3) Partial clotting of a blood sample,
4) Deterioration on storage.
We have HIS (Hospital Information System), which is helpful in providing previous
(cumulative) as well as other investigation done by patients which are very handy,
while maintaining internal quality of reporting.

Quality Control Methodology
• The basis of the method is continued conformance of
control assay values to the assigned values of the stabilized
blood product. These values may be manufacturer-assigned
or assigned by the laboratory, using an analyzer that has
satisfied the requirements of base line accuracy.
• Recovery of assigned values within the confidence limits of
the analytical process, combined with the confidence limits
of the assigned values, will provide assurance that analyzer
accuracy and precision are acceptable.

Quality Control Methodology
• Any disagreement that exceeds these expected range, ie
2SD, limits calls for further investigation to determine
whether it is a random event, loss of precision, loss of
accuracy, or deterioration of the control material.
• It is recommended that result recovery ranges be
expressed as ±2
SD, or preferably 95% of CL of the mean value.

Interpretation of Results
An interpretive method for out-of-limits control result should be able to distinguish among the
following:
 Random event,
 Loss of precision, and
 Loss of accuracy.

Random event versus imprecision:
• With control boundaries defined as ±2 SD, one control value
in exceed these limits as a random event.
• Exclude random event by two further assays of the same
type of control (normal, high, or low) that gave the out-of-
limit result.
• If the difference between these assay results is less than the
established ±2 SD limits, the out-of-limit control result was
probably a random event and requires no further action.

Random event versus imprecision:
• If the difference is greater than ±2 SD, assume precision loss
and confirm this by two further assays.
• At least two of the total of five results will exceed ±2 SD limits
if precision has deteriorated.
• In this case, patients’ results that preceded the out-of-limit
condition should be reviewed in order to determine
reportability.

Loss of accuracy:
• An out-of-limits control result due to loss of accuracy (bias change) can
be verified by the repeated assay method given above. If inaccuracy has
developed, the mean of the replicate results will differ by at least 1 SD
from the mean of previous control results.
• If the bias change affects only one of the three control levels, suspect a
deterioration of the control material.
• If the bias change affects the red cell indices, inspect the weighted
moving average values for supporting evidence of change in RBC, Hb, or
MCV.
• Several consecutive similar results on one side of the mean = calibration
fault causing consistent bias.

EXTERNAL QUALITY CONTROL (EQA) / PROFICIENCY TESTING

EXTERNAL QUALITY CONTROL (EQA) / PROFICIENCY TESTING
• EQA complements internal quality control and is a basic requirement
for clinical laboratory certification and accreditation.
• Certification is a procedure by which a third party gives written
assurance that a product, process or service conforms to specific
requirements.
• Accreditation is a procedure by which an authoritative body (NABL)
gives formal recognition that a body or person is competent to carry
out specific tasks.

EXTERNAL QUALITY CONTROL (EQA)

EXTERNAL QUALITY CONTROL (EQA)
• EQA has following important principles and criteria.

SHEWHART OR LEVEY-JENNINGS CHART ie Internal Quality Control
• Dates of analysis plotted along
the X-axis and control values
plotted on Y- axis.
• Mean and one, two and three
standard deviation limits are
also marked on Y-axis.
• Distance from the mean is
measured in standard
deviations.

WESTGARD RULES TO BE FOLLOWED
13s rule
13s refers to a control rule that is commonly
used with a Levey-Jennings chart when the
control limits are set as the mean plus 3s and
the mean minus 3s.
A run is rejected when a single control
measurement exceeds the mean plus 3s or
the mean minus 3s control limit.

12s rule
12s refers to the control rule that is commonly used
with a LJ chart when the control limits are set as the
mean plus/minus 2s.
In the original Westgard multirule QC procedure,
this rule is used as a warning rule to trigger careful
inspection of the control data by the following
rejection rules.

22s rule
22s - reject when 2 consecutive control
measurements exceed the same mean plus 2s or
the same mean minus 2s control limit.
Warning

R4s rule
R4s - reject when 1 control measurement in a group
exceeds the mean plus 2s and another exceeds the
mean minus 2s.
This rule should only be interpreted within-run, not
between-run. The graphic should really imply that
points 5 and 6 are within the same run.

41s rule
41s - reject when 4 consecutive control
measurements exceed the same mean plus 1s
or the same mean minus 1s control limit.

10x rule
10x - reject when 10 consecutive control
measurements fall on one side of the mean.
Note – There are plenty more Westgard’s rules. Can be
accessed at https://www.westgard.com/mltirule.htm

CONTENTS
 History
 NABL

NABL
National Accreditation Board for Testing & Calibration
Laboratories (NABL) is an autonomous society providing Accreditation
(Recognition) of Technical competence of a testing, calibration, medical
laboratory & Proficiency testing provider (PTP) & Reference Material
Producer (RMP) for a specific scope following
IEC/ISO17025:2005,
ISO 15189:2012,
IEC/ISO 17043:2010 &
IEC/ISO 17034:2016 Standards
It has Mutual Recognition Arrangements with
Asia Pacific Laboratory Accreditation Cooperation (APLAC),
Mutual Recognition Arrangement (MRA),
International Laboratory Accreditation Cooperation (ILAC).

NABL
• NABL provides accreditation in all major fields of Science and
Engineering such as Biological, Chemical, Electrical, Electronics,
Mechanical, Fluid-Flow and many more.
• NABL also provides accreditation for medical testing laboratories.
• In addition, NABL also offers accreditation for Proficiency testing
providers & Reference Material producers and is now signatory to
APLAC MRA for both.

NABL
Benefits of accreditation
• NABL is a Full member (ILAC MRA signatory) to ILAC as well as APLAC
Mutual Recognition Arrangements (MRA) ,based on mutual evaluation
and acceptance of other MRA Partner laboratory accreditation systems.
• This facilitate acceptance of test / calibration results between countries.

NABL
Benefits of accreditation
• This developing system of international mutual recognition agreements
between accreditation bodies has enabled accredited laboratories to
achieve a form of international recognition.
• In medical field NABL accredation works as VISA for the any report.
• Any report report reported in NABL accredited lab, makes it valid and
gives confidence to overseas treating physician/surgeon about reports
being standardized.

SO, ………………………. TO SUM-UP

WITHOUT UNDERSTANDING CALIBRATION AND QUALITY CONTROL

TAKE HOME MESSAGE
Automation in Hematology is like Vitamin
tablets.
It is a supplement and not
the substitute to Manual methods.

REFERENCES
1. Standard P. Calibration and Quality Control of Automated Hematology Analyzers; Proposed Standard NCCLS.
Vol. 19. 2008.
2. Saiket M. World of Automation. In: Automated cell counter and quality control. 2014. p. 1–24.
3. Novak E, Guenther B. Automation in. In: Automation in Hematology. 2014. p. 369–94.
4. Anon, (2018). WestgardQC. [online] Available at: https://www.westgard.com/mltirule.htm [Accessed 2 Mar.
2018].
5. En.wikipedia.org. (2018). National Accreditation Board for Testing and Calibration Laboratories. [online]
Available at:
https://en.wikipedia.org/wiki/National_Accreditation_Board_for_Testing_and_Calibration_Laboratories
[Accessed 2 Mar. 2018].
6. Dacie, Lewis. Practical Haematology. Edition.12. 2012

VIVA
Controls Calibrators
Values ranges upto 2SD Values are fixed
Prepaired by various
companies, 3rd party
controls are also available
Calibrators are prepaired by analyser
manufacturer and is approved by CLSI
like authorities
After opening can be used
for 35 times within 25 days
After opening, can be used for 20times
within 1-2hr
Cant be used for calibrating
the machine Used to calibrate the machine
Used for Quality control
No expertise needed Needs handling by expertise

VIVA
What if the control values are continuously coming on one side (eg between 1sd and
2sd or >2SD) of mean???
Check for Sample error or Systemic Error
For sample error: 1) Check for the control sample ,whether it is viable/out of expiry date or not?
2) Can use another level of control for checking whether this problem is with
same level or with all levels?
If, values between 1sd and 2sd are acceptable, just has to calculate New Lab mean which will be
shifted a slight towards 1SD or the multiplying factor is derived to keep mean same.
For Systemic error: Values continuously >2SD are not acceptable, after checking all the sources of
error machine has to be recalibrated by service engineer.

VIVA
What is Within run and Between run?
Within run is
Values of the sample/ mean of the sample values is derived after running the sample
continuously (without a gap).
Eg If we running control sample at the start of day then run 5 times continuously without gap.
Between run is
Values of the sample/ mean of the sample values is derived after running the sample, for
specified times, not continuously, but with a gaps.
Eg If we have to run control sample for 5 times a day then, we run it 1 time after each hour gap or
between running other routine samples. (like stratified random sampling )

Calibration and Quality controls of automated hematology analyzer

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