Quality assurance in haematology

1. QUALITY ASSURANCE IN HAEMATOLOGY
Ishwar Bihana
Tutor Tech., Department of Haematology,
Postgraduate Institute of Medical Education & Research, Chandigarh.

2. Quality
Definition. A measure of excellence or,
• a state of being free from defects, deficiencies, and significant
variations,
• brought about by strict and consistent adherence to measurable
and verifiable standards
• to achieve uniformity of output
• that satisfies specific user requirements.

3. Introduction
All laboratory tests are susceptible to error.
It is virtually impossible ever to have a 0% error rate.
To have value for clinical decision making, an individual laboratory test
result must have a total error small enough to reflect the biological
condition being evaluated.
For e.g. the methodology used for estimating the hemoglobin of a patient
should yield consistent and accurate results, howsoever many times we
perform the test on the same sample.

4. Laboratory test results
(Good laboratory practice, GLP)
Clinical diagnosis
Patient management

5. QUALITY CONTROL
Describes the steps taken by a laboratory to ensure that the tests are
performed correctly. In simple words, this refers to ‘minimization of errors’.
Quality control activities span the testing process from the moment of
specimen collection until the time the physician receives the report.
The primary purpose of Quality Control is to statistically sample the
measurement process to verify that the method continues to perform within
the specifications consistent with acceptable systematic bias and imprecision.

6. Quality Assurance
Essentially similar to Quality Control
To ensure reliable test results with the necessary degree of precision and
accuracy.
However, it also includes steps taken by the lab to ensure that its Quality
Control is actually working.
This describes all the steps taken, both within and outside the laboratory to
achieve reliable results.

7. QUALITY ASSURANCE PROGRAMME INCLUDES :
Internal quality control
External quality assessment/ Proficiency testing
Standardisation.
Ensures adequate control of the pre-analytic and post-analytic stages from
specimen collection to the timely dispatch of an informative report.

8. Component 1
Internal Quality Control
◦ Measurements on specially prepared materials
◦ Repeated measurements on routine specimens
◦ Statistical analysis of the results obtained

9. Component 2
External Quality Assessment
 EQA is the analysis of performance by an outside agency using
specially supplied samples.
 Analysis is usually retrospective.
AIM: To achieve inter-laboratory comparability
 Schemes are usually organised nationally or regionally.
 National schemes are frequently known as NEQAS (National
External Quality Assessment Scheme) e.g. NABL in India.
 International EQA schemes, e.g. those organized by the WHO

10. Component 3
STANDARDIZATION
Material standard / reference preparation used to calibrate analytic
instruments and to assign a quantitative value to calibrators.
Prescribed by ‘WHO’
Reference method – is an exactly defined technique that is used in
association with the reference preparation
Provides sufficiently accurate and precise data for it to be used to assess the
validity of other methods.

11. Selected method:-
Directly comparable with an international reference
method.
Alternative to the reference method when an
international reference material is not available.
Should be used for evaluation and validation of a
proposed routine (working) method.
COMPONENT 3
STANDARDIZATION

12. Working (or Recommended) method :-
intended for use in routine practice, taking account of economy of labour
and materials and ease of performance.
Shown by a validation study with a reference method to be sufficiently
reliable for its intended purpose.
COMPONENT 3
STANDARDIZATION

13. Haemoglobin:
International reference preparation of haemiglobincyanide (HiCN)
has contributed to improved accuracy of haemoglobin
measurement used to assign a haemoglobin value to a lysate or a
whole blood preparation.
This is then used as the local secondary standard after
appropriate dilution
Examples of reference preparations

14. Controls
Substances used in routine practice for checking the
performance of an analytical process (or instrument).
They may or may not have a pre-assigned value.
Even though some control preparations have assigned
values, they should not be used as calibrators or
standards because:
◦ Assigned values are only approximations.
◦ Controls are stable for a limited time only.

15. Examples of in-house control preparations:
RBC parameters
Natural blood, collected in EDTA of no value as a reference
preparation because of its short life in the laboratory.
Blood in ACD or CPD at 4°C can be preserved for a few weeks.
 As MCV increases and some red cells lyse, so blood in ACD cannot be
regarded as a reference material .
 But it can be used as a control preparation to check the precision and
reliable functioning of a cell counting system over relatively short periods.

16. Examples of in-house control preparations:
RBC Parameters:
Suitably sized particles in stable suspension like fixed red cells and
spherical latex particles as substitutes for normal blood cells.
Permanently stabilized by glutaraldehyde fixation.
 Can be used to check the consistency of an instrument
 Causes red cells shrinkage
 The inflexible biconcave discs with flow properties different from those of
fresh RBCs cannot be used to calibrate an instrument

17. Control for TLC
Two types of material:
1. Leucocytes concentrated from human blood and fixed in glacial acetic
acid, sodium sulphate, sodium chloride and water.
2. Glutaraldehyde-fixed erythrocytes suspended in leucocyte-free
mammalian whole blood.
Turkey or chicken blood, in which the red cells are nucleated.

18. Calibration
Calibration is the determination of bias conversion
factor of an analytical process under specified
conditions, in order to obtain accurate measurement
results.
The accuracy over the operating range must be
established by appropriate use of reference methods,
reference materials and/or calibrators.

19. CALIBRATORS
Serve to check the accuracy of the instrument
Have a value assigned to them by a reliable reference centre
More expensive, shorter shelf life (24 hrs)

20. Good Laboratory Practice
Quality can be assured at
Pre-analytical stage
Analytical stage
Post-analytical stage

21. Quality management at the
Pre-analytical phase

22. Requisition form

23. Patient’ case no. / Name:______________________________________________
Date of Birth/Age:____________________________________________________
Sex: _______________________________________________________________
Referring Doctor’ name:_______________________________________________
Address
(OPD/WARD/Unit):___________________________________________________
__________________________________________________________________
Primary Sample Type: Blood/Fluid/Sputum/Stool/Microbiological Specimen/Slides/Tissue/_________________________________________________ (Any other specify)
Date and Time of Primary Sample Collection: ___________________________________________________________________________________________________
Date and Time of Receipt of Primary Sample by the Laboratory: ____________________________________________________________________________________
Clinical History of the patient:
________________________________________________________________
________________________________________________________________
Treatment History of Patient:
__________________________________________________________________
__________________________________________________________________
Examinations Requested Requisition number
1.
2.
3.
4.
5.
Requisition form Reference: Clause 5.4 (ISO 15189)
Signature ______________
Name of the organization
Complete address/telephone no./fax no./e mail/website

24. Example 1
sample collection
Stress and exercise
 Increases cell concentrations
 Increases coagulation factors (VIII) & also tissue
plasminogen activator (t-PA) with increased
fibrinolytic activity (2-4).
Reference:
(2) Standardization of blood specimen collection procedure for reference values. Clin Lab Haematol
4:83- 86, 1982.
(3) Van Assendelft OW, Simmons A. Specimen collection, handling, storage and variability, in Lewis
SM, Koepke JA (eds): Hematology Laboratory Management and Practice. Oxford, Butterworth
Heinemann, 1995, p 109-127.
(4) Dacie JV, Lewis SM. Practical Haematology, 8th ed.
Edinburgh, Churchill Livingstone, 1995, p 9-19.

25. Example 2
 Prolonged use of a tourniquet
 Haemoconcentration;
 The patient’s posture (standing, sitting or lying)
and even the position of the arm during
venous sampling will cause fluctuations of 5-
10% in the blood count(1).
Reference :
(1) International Committee for Standardization in Hematology.

26. Example 3
K3EDTA
Causes significant shrinking of the
red cells with a decrease of 1-2% in
the MCV.
.
K2EDTA
K2EDTA in a concentration of 1.5-2.2
mg/ml (4.55 ± 0.8 mmol/ml) as this
cause less cellular change(5)
.
Reference:
(5) Bachmann F. Molecular aspects of plasminogen, plasminogen activators and plasmin, in Bloom AL, Forbes CD, Thomas DP, Tuddenham
EGD (eds): Haemostasis and Thrombosis. Edinburgh, Churchill Livingstone, 1994, p 575-613

27. Example 4
Improper technique results in :
 Presence of microclots or platelet clumps
 Low platelet count reported by cell counter

28. Example 5
Shortening of APTT
Micro clots
Prolongation of PT & APTT
Excesssodiumcitrateconsumes
Ca+2 presentinreagents

29. Example 6
Prolongation of APTT
Contactwithheparin
Prolongation of PT & APTT
Delayedsample
leadsto
Factorsdeterioration

30. Example 7
Withdraw the blood slowly using 22 or 21 gauze needles
Remove the needle from the syringe and deliver the blood gently into the
containers
Avoid vigorous mixing as it may cause foaming and hemolysis
Avoid hemolysis

31. Example 8
While drawing blood from indwelling lines or catheters
errors due to dilution and or contamination from
flushing solution should be avoided.
Reference : NABL 112, Issue 02, Page 18/39

32. Example 9
When an intravenous solution is being
administered in a patient's arm, blood should be
drawn from the opposite arm.
If an intravenous infusion is running in both arms,
samples may be drawn after the intravenous
infusion is turned off for at least two minutes
before venipuncture and applying the tourniquet
below the intravenous infusion site.
Reference : NABL 112, Issue 02, Page 19/39

33. Example 10
Avoid clotting of
blood collected
in anticoagulant
Mix well by
gently inverting
the tubes
8 to 10 times

34. Example 11
A serious, and potentially fatal, cause of
mishap is:
◦ collection from the wrong patient
◦ subsequent specimen mix-up
◦ transcription error
These can occur at any stage. It is essential to have
a cross-check procedure.

35. Sample receiving
◦Laboratory checks
quantity,
quality,
labeling,
request forms,
clot presence etc.

36. Example # 1
Quantity not
sufficient

37. Example # 2
Sample hemolysed / lipemic

38. Example # 3
Name on vacutainer and name on the requisition form do not match
X

39. Example # 4
Outside of container
contaminated by
specimen

40. Example # 5
Sample partially / fully clotted

41. Example # 6
Sample received without
requisition form
Or
Only Requisition form received
Mostly happen in OPD Collection
Centre
X

42. Sample rejection analysis
A feedback of this kind to the
concerned ward of the hospital
may enhance a positive attitude
towards quality improvement at
pre-analytical stage
Quality Indicator
Rejection analysis
helps in
‘Continual improvement’

43. Quality Management of the
Analytic Phase

44. PRECISION (REPRODUCIBILITY)
Definition
Precision refers to the reproducibility of a result.
Comparing QC terms to a target Figure illustrates
that the results are precise (close together) but not
accurate (they are not in the bull’s-eye).
Checking precision is required while
-calibration
-troubleshooting

45. ACCURACY
Definition
Closeness of a result to the true (accepted) value.
NOTE: Before determining accuracy, first determine
precision.
Comparing QC terms to a target Figure illustrates
that the results are accurate (in the bull’s-eye) and
precise (close together).
NOTE
You cannot have accuracy without precision.
However, you can have precision without accuracy.

46. NEITHER ACCURACY NOT PRECISION
This figure illustrates that the results are
neither accurate nor precise.
None of the results are close together,
and none of them are in the bull’s-eye.

47. Statistics involved…
Mean
Standard deviation (SD)
 ± 1SD
 ± 2SD
 ± 3SD
Coefficient of variation (%CV)

48. It is the average value of the various test results.
Mean = sum of observation / no of observation
Example…
PT in sec. of ten patients are : 13,13,16,14,13,12,14,14,13,16
Mean = (13+13+16+14+13+12+16+14+13+16)/10 = 140/10 = 14
MEAN

49. Theseareless frequently used...
MODE
Most important, frequently occurring
observation in a series.
Example…
PT in sec. of ten patients are :
12,13,14,13,18,13, 11,10,12,15
Mode = 13
MEDIAN
◦ It implies the mid value of the series.
◦ When all the observations of variable
are arranged in either ascending or
descending order, the middle
observation is known as median.
Example…
PT in sec. of the seven patients are
arranged in ascending order :
13,13,14,14,14,16,16
The fourth observation (14) is the
median in this series.

50. Standard Deviation
This is a statistical expression of the scatter or dispersion of value around the
central average value.
It indicates the variation in measurement obtained in lab test.
◦ X = single observed value
◦ 𝑿 = average
◦ n = total number of observations
◦ ∑ = sum of observations

51. Example…
= √[7/(10-1)]
= √7/9
= 0.8
Serial no. Observation of
PT in sec.
x
Deviation from
mean
X-x
Square of
deviation
(X-x)²
1 13 0 0
2 12 -1 1
3 14 +1 1
4 14 +1 1
5 14 +1 1
6 13 0 0
7 12 -1 1
8 13 0 0
9 12 -1 1
10 14 +1 1
TOTAL 130 1 7

52. Coefficient of Variation
Relates the SD to the actual measurement, so that measurements at different
levels can be compared.
 It is another way of expressing dispersion of result. It is a measure used to
compare relative variability…
CV = SD X 100%
mean
If CV,
<3% is ideal
<5% is acceptable.

53. Techniques for internal quality control
1. Use of controls and control charts
2. Duplicate test on patient specimens
3. Delta check
4. Use of normal hematological data
5. Use of patient data
6. Correlation check

54. In 1931,
Dr. Walter Shewhart, a
scientist at the Bell Telephone
Laboratories, proposed
applying statistical based
control charts to interpret
industrial manufacturing
processes.
Use of control and control charts
In 1950,
S. Levey &
E.R. Jennings
suggested the use in
the clinical laboratory.

55. Once the QC results are entered into the QC log, they
should be plotted on the Levey-Jennings chart. When
the results are plotted, an assessment can be made
about the quality of the run. The technologist
performing the test should look for systematic error
and random error.

56. 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 in control values or it may
be abrupt and demonstrated as a
shift in control values.

59. Two applications to this rule: within-run , across runs
Violation of the
within-run application
indicates that
systematic error is
present and that it
affects potentially the
entire analytical
curve.
Violation of the
across run application
indicates that only a
single portion of the
analytical curve is
affected by the error

60. If there is at least a 4s difference between control values within a single run,
the rule is violated for random error

63. 2. QC by Duplicate Tests on Patients' Specimens
 Another way of checking the precision of routine work
 Test 5–10 consecutive specimens in duplicate
 The differences in the paired results are calculated and the mean and SD is
derived
 The duplicate tests should not differ from each other by >2 SD (<5 % should
fall outside 2 SD)
 If the test is always badly done or has an inherent fault, the SD is wide.
 Detects random errors
Disadvantage
 It does not detect incorrect calibration
 Less sensitive to gradual drift

64. 3 Delta Check Reference:Lewis:DacieandLewisPracticalHaematology,10thed.,Pg665
A formal way of testing for aberrant results is known as `delta check`.
The blood count parameters should not differ from recent tests in the
previous 2-3 weeks by more than a certain amount.
For Hb and RBC For WBC For Platelet count
10 % 20-25 % 50 %
Assuming that the patient’s clinical condition has not altered significantly

65. Example of delta check
54 yr old male, on follow up for AML in complete remission for the
last 4 years.
His platelet count on the automated analyzer was 83,000/μl. The
previous platelet count 2 weeks ago was 233,000/μl.
Should this report be released?
FAILED delta check.
Examine smear (correlation check).

66. 4. Use of normal hematological data
In healthy individuals, blood counts remain constant day by day
Blood counts from selected healthy individuals are monitored, their means
and SD over time are calculated
The means should not vary by more than 2 SD
Significant differences in mean signify a consistent error e.g. incorrect
calibration
Random error- results in increase in SD , mean remains unaffected

67. 5. Use of Patient Data for Quality Control
Principle - In labs where at least 100 patients are investigated daily, the
daily means should not differ significantly if the population remains
stable.
Assuming that the sample population is stable any significant change in
the means of the red cell indices will indicate a change in instrument
calibration or a drift owing to a fault in its function.

68. 6. QC by Correlation Checks
 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.
 Example
 Decreasing blast counts on successive blood films may be due to
hemodilution by hydration, institution of chemotherapy etc (clinical
correlation check)
 A low MCHC should be confirmed by demonstrating hypochromic red cells
on a Romanowsky-stained blood (morphological correlation check)
 Hemoglobin is usually 1/3rd of hematocrit in health (biological correlation
check)

69. EXTERNAL QUALITY ASSESSMENT (EQA)

70. EQA
EQA is an important complement to internal control.
Even after adequate internal control, errors arise which
are only detectable by objective external assessment

71. EQA
PRINCIPLE :-
Same material is sent from a national or regional centre to
numerous participating laboratories (at least 20).
Results that are returned to the EQAS centre are analysed, a
target value and an acceptable range around the target are
established, and the performance of the individual participants is
judged.

72. EQA
It is important that surveys should be performed at regular
intervals, although their frequency may vary & depends on
 Diagnostic importance of the particular test,
 How frequently they are requested
 Their technical reliability
Main function :-
To ensure reliable performance by individual laboratories and to
achieve harmonisation or concordance between laboratories.

73. EQA Schemes Available
UK NEQAS – Specimens are distributed at 4 weekly intervals for
blood counts and every 3 months for most other tests
In India:
◦ CBC & PS: Indian Society of Hematology and Transfusion Medicine & AIIMS
◦ Coagulation: CMC Vellore conduct EQAS under National Accreditation
Board for Testing and Calibration Laboratories (NABL)
◦ Mumbai Haematology Group Inter-lab Comparison Programme for Flow
Cytometry

74. EXTERNAL QUALITY ASSESSMENT (EQA)
Other complementary functions :-
Collecting information on the reliability of particular methods, materials,
and equipment
Providing information on performance required for the purpose of licensing
or accreditation
Identifying laboratories whose performance provides a benchmarking
standard.
Recommending state-of-the-art procedures for various analytic tests,
organizing workshops for education and training of laboratory staff, and
advising on best-practice guidelines

75. ANALYSIS OF EQA DATA

76. Deviation Index
Adjusted mean: Exclude results that are outside 3SD
Trimmed SD: Use SD of best performance, reference lab, or of a
select group of labs (e.g. first 30 labs )
< 0.5 - Excellent performance
0.5 – 1.0 - Satisfactory
1.0- 2.0 - Acceptable
> 2.0 - Suggests that the analyser should be checked
> 3.0 - Serious defect

77. Out-with-consensus Method
 The median is calculated and all the participant results are then ranked in
five grades:
 Group A: 25% of all results that are immediately adjacent to and above the
median and 25% that are immediately adjacent to and below the median
 Group B: The next 10% on each side of A
 Group C: The next 5% on each side of B
 Group D: The next 5% on each side of C
 Group E: The final 5% on each side of D (& non-participation)
 Unsatisfactory performance is designated when the combination in 2
consecutive exercises is DD, EC, ED or EE.

78. Youden (xy) Plot
 Useful for relating measurements on two
samples in a survey to provide a graphic
display
 Distinguishes between a consistent bias
and random error.
 The range of SDs calculated from the
overall results with sample A and sample B,
respectively, are drawn on the x axis and
the y axis.
 Results in the central square are
satisfactory; those in B demonstrate a
consistent bias with measurements that
are too low (B1) or too high (B2), whereas
results in other areas indicate random
errors in the 2 sample
Sample A

79. Clinical Significance
In assessing performance of a participant in an EQA
cycle, using limits based on the SD in some cases may
be too rigid and in other cases it may be too lenient.
To ensure that results are clinically reliable, they should
be within a certain percentage of the assigned value.
This must take account of unavoidable imprecision of
the method and normal diurnal variations.

80. Clinical Significance
The following limits are adequate to meet these requirements
in practice:
Hb and RBC (by cell counter) 3-4%
PCV, MCV, MCH, MCHC 4–5%
Leucocyte count 8–10%
Platelet count 10–15%
Vitamin B12, folate, iron, ferritin 20%
HbA2 and HbF quantitation 5%

81. Lab accreditation
Formal recognition, authorization and registration of a laboratory
that has demonstrated its capability, competence and credibility
to carry out the tasks it is claiming to be able to do.
Provides feedback to laboratories as to whether they are
performing their work in accordance with international criteria for
technical competence.
External accreditation is mandatory by law for laboratories in
many advanced countries.
In India, external accreditation is voluntary.

82. Aims Of Lab Accreditation
To provide third-party certification
To improve customer (clinicians and patients) confidence in the
test reports.
The National Accreditation Board for Testing & Calibration
Laboratories (NABL) is an autonomous body under the aegis of
the Dept. of Science & Technology, Govt. of India and is
registered under the Societies Act.
Provides accreditation to testing & calibration laboratories.

83. Sample (post analysis)

84. Sample retention
◦Repeat examination
◦Additional or further examinations

85. Record (retention)

86. Record retention
Reason for retention
◦ prompt retrieval of the information
Retention time
◦ the length of time that reported data are retained
may vary as long as medically relevant.

87. THANK YOU