3. I N D E X
PART 1
I. Necessity for Automation.
II. Advantages & Disadvantages of Automation.
III. Types of Automated Hematology Analyzers.
IV. Principles involved in Automation.
V. Pentra ES 60 Haematology Analyzer.
VI. Pentra DF Nexus Haematology Analyzer.
PART 2
I. Histograms.
II. Flags
III. Quality Control
4. Histograms
Graphical representation
of numerical data of
different cell populations
in a cell counter.
x-axis => cell size
y-axis => no: of cells
Gives info on
Average size
Distribution of size
5. Discrimination thresholds
Moving or fixed discriminator separates the
distribution curve for the volume
WBC Discriminator
The automated counter sets a LD flunctuating between 30-60 fl & an UD fixed at
300 fl.
WBC is calculated from particle counts > than this LD
RBC Discriminator
Has two flexible discriminators LD (25-75 fl) & UD (200-250 fl).
RBC is calculated from particle counts between this LD & UD.
Platelet Discriminator
Three discriminators – LD (2-6 fl), UD (12-30 fl) & a fixed discriminator (8-12 fl)
6. Estimation of RBC Count & MCV – RBC Histogram
Normal RBC distribution curve is Gaussian bell shaped curve.
Analyzer counts RBCs as those which ranges from 36-360 fl.
MCV is perpendicular line from peak of the curve to the base.
Peak of curve should fall within the normal MCV range of 80-100 fl.
There are 2 flexible discriminators – LD (25-75 fl) & UD (200-250 fl)
7. Abnormalities of RBC Histogram
Left shift of the curve in microcytosis.
Right shift of the curve in macrocytosis.
Bimodal peak of the curve in dimorphic population of cells.
9. Estimation of RDW
RDW is expressed as a coefficient of variation of RBC size distribution.
RDW- CV is a better indicator of anisocytosis than RDW-SD.
RBC distribution curve ll get wider as RBC vary more in size.
Normal Range – RDW-CV – 11.0-15.0% RDW-SD – 40.0 - 55.0 fL
11. Estimation of WBC Count
Hematology analyzers can generate a
3-part differential count – lymphocytes, monocytes & granulocytes
based on the principle of electrical impedence
Or
5-part differential count - lymphocytes, monocytes, neutrophils,
eosinophils & basophils
based on different principles –
light scatter
electrical impedence
electrical conductivity
peroxidase staining
12. Estimation of WBC Count – WBC Histogram
Cells > 35 fl are counted as WBCs in the WBC/Hb chamber.
Cells with volume 35-90 fl Lymphocytes.
Cells with volume 90-160 fl Mononuclear cells.
Cells with volume 160-450 fl Neutrophils.
13. Abnormalities of WBC Histogram
Peak to the left of lymphocyte peak – nucleated cells.
Peak between lymphocytes & monocytes – blast cells, eosinophilia, basophilia,
plasma cells & atypical lymphocytes.
Peak between monocytes & neutrophils – left shift
14. 2-6 fl 12-30 fl
fixed at
12 fl
Platelet Histogram
PL PU
PLT RBC
100%
20%
PLT size: 8-12 fL
PLT detection: between 2 and 30 fL
Fixed discriminator at 12 fL
15. Estimation of Platelet Count
Platelets are counted by the electrical impedence method in the RBC aperture.
Particles between 2 fl and less than 20 fl are classified as platelets by the analyzer.
MPV is a measurement of the average size of platelets found in blood Normal MPV is 7-10 fl.
↑ MPV ( > 10 fl) d/t destruction of platelets in circulation.
↓ MPV ( < 7 fl) d/t impaired production of platelets in circulation.
Plateletocrit (PCT) is volume of circulating platelets in a unit volume of blood. Normal PCT is 0.19-0.36%.
PCT ↑ in thrombocytosis and ↓ in thrombocytopenia.
PDW is a measure of variation in the size of platelets. Standard PDW ranges from 9 to 14 fL
↑ PDW is observed in megaloblastic anemia, CML & after chemotherapy.
16. I N D E X
PART 1
I. Necessity for Automation.
II. Advantages & Disadvantages of Automation.
III. Types of Automated Hematology Analyzers.
IV. Principles involved in Automation.
V. Pentra ES 60 Haematology Analyzer.
VI. Pentra DF Nexus Haematology Analyzer.
PART 2
I. Histograms.
II. Flags.
III. Quality Control.
17. Flagging
Flags are signals that occur when
an abnormal result is detected
by the automated blood
analyzer.
Flags are signaled by certain
‘asteriks’ on the report.
They reduce the False +ve &
False –ve results by mandating
the results of blood smear
examination.
18. RBC Flags
Seen when LD > preset
height by 10 %
Shown by
RBC Count
HCT, MCV,MCH,MCHC
Occurs when there is
platelet aggregation
RBC fragments
RL Flag
19. RBC Flags
Seen when UD > preset
height by 10%.
Shown by
RBC Count
HCT, MCV,MCH,MCHC
Occurs when there are
Cold agglutinins
RU Flag
20. RBC Flags
Shown by RDW-SD
Seen in
Post Blood tranfusion
Treated Fe deficiency anemia.
MP- Flag
21. WBC Flags
Generated when curve deviates
from the baseline on the LD.
Various causes for this are –
Platelet aggregates (clotted
sample, EDTA incompactibility)
Lyse-resistant RBCs.
Erythroblasts.
Cryoagglutinates.
Giant platelets.
WL Flag
22. WBC Flags
Generated when there is
deviation of the curve on the UD
or if it does not end at the
baseline.
Various causes for this are :-
Hyperleucocytosis.
WU Flag
23. WBC Flags - T1 & T2 Flags
Peak between T1-T2 : The middle cell count - Eosinophils, Monocytes, Blasts, promyelocytes,
myelocytes and metamyelocytes.
Peak between LD-T1 : Lymphocytes
Peak between T2-UD : Neutrophils.
T1 & T2 flags appears when discrimination between lymphocytes, middle cells & neutrophils is
not possible which happens in presence of abnormal/higher leucocyte counts as in Chronic
Myeloid Leukemia.
24. WBC Flags - F1, F2, F3 Flags
Sometimes, the cell Fractions
may be mixed.
F1 & F2 or F2 or F3 merge into
each other over large areas.
F1 (small cell inaccurate) flag :
Acute Lymphocytic Leukemia
F2 (middle cell inaccurate) flag :
eosinophil, Acute Myeloid
Leukemia, monocytosis, etc
F3 (large cell inaccurate) flag:
25. Platelet Flags
This occurs when the LD
> the preset height by
10%
Shown by
Platelet count
MPV
P-LCR
Occurs due to noise.
PL Flag
26. Platelet Flags
This occurs when the
UD> the preset height by
> 40%.
Occurs in
Hemolytic anemias with
fragmented cells.
Large platelets.
PU Flag
29. I N D E X
PART 1
I. Necessity for Automation.
II. Advantages & Disadvantages of Automation.
III. Types of Automated Hematology Analyzers.
IV. Principles involved in Automation.
V. Pentra ES 60 Haematology Analyzer.
VI. Pentra DF Nexus Haematology Analyzer.
PART 2
I. Histograms.
II. Flags
III. Quality Control
30. Quality Control / Quality Assurance/Quality Assessment
Quality Control => measures that must be included
during each assay run that the test is working properly.
Quality Assurance => overall program that ensures
that the final results reported by the lab are correct
Quality Assessment => means to determine the
quality of results generated by the lab
31. QUALITY CONTROL
Measure of precision – how well the measurement system reproduces the
same results over time and under varying operating conditions.
Designed to detect, reduce & correct deficiencies in a laboratory’s internal
analytical processes prior to release of patient’s reports.
Should approximate the same matrix as patient’s specimens, taking into
account properties such as viscosity, turbidity, composition and color.
Should be simple to use, with minimal vial to vial comparability.
Should be stable for long periods of time and available in large enough
quantities for a single batch to last at least 1 year.
32. Types of Quality Control
Internal
Continuous evaluation of reliability
of the daily works of the laboratory
with validation of tests.
Primary tool required is called a
control – a specimen with a
predetermined range of result values,
processed in the same manner as
patient sample.
If the result of a test on a control
sample is different from its known
value, this indicates a problem in the
equipment or the methods being
used.
External
Evaluation by an outside agency of
the comparability a laboratory's
testing to a source outside the
laboratory.
This comparison can be made to
the performance of a peer group of
laboratories or to the performance
of a reference laboratory.
The analysis of performance is
retrospective.
33. Accuracy and Precision
Accuracy is a measure of rightness.
It refers to closeness to the true value.
Precision is a measure of exactness.
It refers to reproducibility of the test.
34. Control
A solution that contains the same constituents as those being
analyzed in the patient sample.
Commercially produced pooled RBCs/sera or stabilized
anticoagulated whole blood.
Should have same test properties as that of a blood sample.
At least 1 control specimen should be used for every batch.
If large specimens, use 1 control for every 20 specimens.
For most tests, a “normal” control and an “abnormal” control are
analyzed with each patient test or batch of tests.
The results are compared with the manufacturer’s range of values
and plotted on a Levey-Jennings Chart.
35. Levey Jennings Graphs
A graphical method for displaying control
results and evaluating whether a procedure is
in-control or out-of-control
Control values are plotted versus time
Lines are drawn from point to point to accent
any trends, shifts, or random excursions
Consecutive values of control are recorded and
the standard deviation is calculated.
The mean and ± 2 SD are plotted on a Levey-
Jennings chart.
As long as the control value are between the ± 2
SD lines on the L-J chart, the control values are
“in control”
If they are outside the ± 2 SD lines, they are 'out
of control'
36. Westgard Control Rules
Proposed by Dr. James Westgard
on lab quality control.
Set the basis for evaluating
analytical run for medical
laboratories.
There are 6 basic rules.
37. 12s Rule . Warning rule to trigger careful inspection of the control data
A single control measurement exceeding 2 standard deviations of control limits either
above or below the mean.
No cause for rejecting a run
38. 13s rule
A single control measurement exceeds the mean plus 3s or the mean minus 3s
control limit.
The run must be rejected.
39. 22s rule
2 consecutive control measurements exceed exceeds the +2SD or -2SD
control limit .
The run must be rejected.
40. R4s rule
1 control measurement exceed the +2SD and the other exceeds the -2SD
control limit .
The run must be rejected.
41. 41s rule
4 consecutive control measurements exceed exceeds the +1SD or -1SD
control limit .
The run must be rejected.
42. 10x rule
10 consecutive quality control results for one level of control are on one
side of the mean
The run must be rejected.
43. ‘Out of Control’
Stop testing
Identify and correct the issue.
Repeat testing on patient samples and controls.
Don’t report patient results until the issue is sorted out
and the controls indicate proper performance
44. Errors
Systematic error is evidenced by a change in the mean of
control values.
The change in mean maybe
• Gradual - demonstrated as a trend in control values.
• Abrupt – demonstrated as a shift in control values.
Random error is any deviation away from the expected
result.
45. Errors
Trend Shift
Gradual loss of reliability in test
system.
Causes
Deterioration of instrument light
source.
Gradual accumulation of debris in
sample/reagent tubing and
electrode surfaces.
Aging of reagents.
Gradual deterioration of –
Control materials.
Incubation chamber
temperature.
Light filter integrity.
Calibration.
Sudden/dramatic +ve or –ve change in
test system
Causes
Sudden failure or change in the light
source.
Change in reagent formulation.
Major instrument maintenance.
Sudden change in incubation
temperature.
Change in room temperature or
humidity.
Failure in sampling system.
Failure in reagent dispense system.
Inaccurate calibration/recalibration.
46. Calibrators
Determines the accuracy and precision of the
analyzer using a specifically formulated product
in order to recover each parameter within close
tolerances of known target values and limits.
Finetunes the hematology analyzer to provide the
most accurate results possible.
Coefficients of variation and percent difference
recovery must be within their specified limits.
47. Why to Calibrate?
Calibration is necessary
To ensure readings from an instrument
are consistent with other measurements.
To determine the accuracy of the
instrument readings.
To establish the reliability of the
instrument i.e. that it can be trusted.
48. When to Calibrate?
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
Editor's Notes
Hemoglobin (gm/dl) × 3 = PCV
Red cell count (million/cmm) × 9 = PCV
REFERENCE RANGES
• Adult males: 40-50%
• Adult females (nonpregnant): 38-45%
• Adult females (pregnant): 36-42%
• Children 6 to 12 years: 37-46%
• Children 6 months to 6 years: 36-42%
• Infants 2 to 6 months: 32-42%
• Newborns: 44-60%
CRITICAL VALUES
• Packed cell volume: < 20% or > 60%
In a RBC histogram,
cell numbers are plotted on Y-axis
cell volume are on X-axis.
The analyzer counts cells between 36 fl and 360 fl as red cells. Although leukocytes are present and counted along with red cells in the diluting
fluid, their number is not statistically significant. Only if leukocyte count is markedly elevated (>50,000/μl), histogram and the red cell count will be affected.
Area of the peak between 60 fl and 125 fl is used for calculation of mean cell volume and red cell distribution width.
Abnormalities in red cell histogram include: (1) Left shift of the curve in microcytosis, (2) Right shift of the curve in macrocytosis, and (3) Bimodal peak of the curve in double (dimorphic) population of red cells.
REFERENCE RANGES
• Mean cell volume: 80-100 fl
• Mean cell hemoglobin: 27-32 pg
• Mean cell hemoglobin concentration: 30-35 g/dl
• Red cell distribution width: 9.0-14.5
Red Blood Cell Distribution Width (RDW): Definition and Calculation
The red cell distribution width (RDW) is a measurement derived from the red blood cell distribution curves generated on automated hematology analyzers and is an indicator of variation in red blood cell (RBC) size within a blood sample. The RDW is used along with the indices (MCV, MCH, MCHC) to describe a population of RBCs. The RDW measures the deviation of the RBC width, not the actual width or size of individual cells.
The two RDW measurements currently in use are the red cell distribution width - coefficient of variation (RDW-CV) and the red cell distribution width - standard deviation (RDW-SD).
The RDW-CV is a calculation based on both the width of the distribution curve and the mean cell size. It is calculated by dividing the standard deviation of the mean cell size by the MCV of the red cells and multiplying by 100 to convert to a percentage. A normal range for the RDW-CV is approximately 11.0 - 15.0%. Because it is a calculation, the RDW-CV is dependent not only on the width of the distribution curve but also the MCV of the red cell population and may not always reflect the actual variation in red cell size.
Be aware that:
A homogenous population of red cells with a narrow distribution curve and low MCV may have an elevated RDW-CV
A heterogeneous population of red cells with a broad distribution curve and a high MCV may have a normal RDW-CV.
The RDW-SD is an actual measurement of the width of the red cell distribution curve in femtoliters (fL). The width of the distribution curve is measured at the point that is 20% above the baseline. Since the RDW-SD is an actual measurement, it is not influenced by the MCV and more accurately reflects the red cell size variance. The normal RDW-SD range for adults is 40.0 - 55.0 fL.
MAHA – MicroAngiopathic Hemolytic Anemia
ImmNE1 – Band forms
ImmNE2 – Immature neutrophils
Platelets are difficult to count because of it small size, marked variation in size, tendency to aggregation and overlapping of size with microcytic red cells, cellular fragments and other debris.
A low MPV indicates average size of platelets is small; older platelets are generally smaller than younger ones and generally a low MPV may mean that a condition is affecting the production of platelets by the bone marrow.
A high MPV indicates a high no: of larger, younger platelets in the blood: this may be d/t the bone marrow producing and releasing platelets rapidly into circulation.
MP – Multiple peaks
The aim of quality control is simply to ensure that the results generated by the test are correct.
However, quality assurance is concerned with much more: that the right test is carried out on the right specimen, and that the right result and right interpretation is delivered to the right person at the right time”
Quality assessment (also known as proficiency testing) is a challenge to the effectiveness of the QA and QC programs
Accuracy is a measure of rightness. Precision is a measure of exactness. Precision vs. Accuracy
The manufacturer has analyzed each lot of serum for a variety of test components and the expected range of assay values for each component is provided to the laboratory when shipped.
Whenever a patient’s test or a batch of tests are performed and the control(s) is “in control”, the values obtained for the patient test(s) are determined to be “acceptable” and can be released to the doctor as accurate.
Whenever a patient’s test or a batch of tests are performed and the control(s) is “out of control”, the values obtained for the patient test(s) are determined to be “not acceptable” and CANNNOT be released to the doctor as accurate until the problem is identified and resolved.