ABC of automated CBC

ABC
of automated
CBC
Dr.Parth S.Kaneria
Shanti Pathology Lab Junagadh

Pathology in old days
Antoni van Leeuwenhoek

Wallace H. Coulter an electrical engineer and businessman
while working with the US Navy in the late 1940s developed
and first applied the Coulter Principle.
Man who changed the world of counting...

Advantages
• Speed with efficient handling of large number of
samples.
• Accuracy and precision in
quantitative blood tests.
• Ability to perform multiple
tests on a single platform.
• Significant reduction of labor
requirements.
• Invaluable for accurate determination
of red cell indices.

Disadvantages
• Flagging of a laboratory test result demands labour
intensive manual examination of a blood smear
• Comments on red cell
morphology cannot be generated
• Platelet Clumps are counted as
single ,so low count.
• Erroneously increased or
decreased results due to
interfering factors
• Expensive with high running costs

TYPES OF AUTOMATED ANALYSER
Three part Five Part Seven Part
Differentiate cells
into three categories
1. Granulocytes
2. Lymphocytes
3. Monocytes/mixed
cells
Differentiate cells
into the five basic
leukocyte types
1.Neutrophils
2.Eosinophils
3.Basophils
4.Lymphocytes
5.Monocytes
In addition are able to
distinguish
1.Nucleated RBCs,
2Abnormal and
atypical cells and
immature cells

Counting Chambers
RBC/Platelet Chamber
WBC Chamber
Differential Chamber
Reticulocyte Channel

General Principles
• Introduction to Instrumentation
– Basic principles used :
• Electronic Impedance
• Radiofrequency
• Optical Scatter
a. Laser light scatter
b. Flow cytometry
c. Chemical dye method

The Coulter Principle
• The poorly conductive blood cells are suspended in a
conductive diluent (liquid).
• The diluent is passed through an electric field created
between two electrodes.
• The liquid passes through a small aperture (hole).
• The passage of each particle through the aperture
momentarily increases the impedance (resistance) of
the electrical path between the electrodes.
• The increase in impedance creates a pulse that can be
measured.
• The number of pulses = blood cell count
• The amplitude (height) of the pulse = Volume of cell

Electronic Impedance:
Disrupts electrical current by displacing fluid proportional
to the size of the cell.

Computing and generation of graph
• Each pulse is recorded as
an oscillation , the height
of which is proportional to
the volume and size of the
cell.
• These oscillations are
rearranged according to
volume interval to form a
histogram.

Low angle scatter 2o - 3o (Volume)
High angle scatter 5o - 15o
Forward-angle light scatter (FALS)
Illuminating beam that has been bent to a small angle from
direction of the original beam .It measures size or volume of cells

Side scatter (SSC)
• The illuminating beam that is scattered by particle
to an angle of 90* from the illuminating beam.
• This depends on cell's surface texture and internal
structure as well as to its size and shape and
granularity.
• It is sometimes referred to as a granularity signal or
an orthogonal light scatter signal.

Forward-angle light scatter
correlates to cell volume/size
Side scatter
correlates to degree of internal
complexity (granules and nucleus)

Radiofrequency
( impedance)
Impedance - size cells
Conductivity (RF) – proportional to cell
interior density (granules and nucleus)
Five-part WBC differential
•Scatter plot (RF X DC)
•Computer cluster analysis provides
absolute counts

ERRORS
1. Recirculation error
Cells that re circulate through the edge of an electrical
field produce an aberrant impulse, which is smaller than
cell passing through the aperture.
2. Coincidence error
Cells that pass through the aperture simultaneously, or
almost so, are counted and sized as a single large cell
called coincidence.
3. Non central flow error
Cell pass through the aperture off centre produce
aberrant impulses and appear larger than their actual size

1. Recirculation error
2. Coincidence error
3. Non central flow error
How to over come such errors ?

Hydrodynamic focusing and Laminar flow

COULTER VS FLOWCYTOMETER
• The Coulter-type volume signal is proportional
to the volume of a particle as well as to its
electrical characteristics.
• In flow cytometer signal is proportional to the
cross-sectional area of a particle as well as to
its refractive index.

PRINCIPLES
• Principles of Measurement
– Direct Measurement:
• RBC – Impedance, hydrodynamic
focusing
• WBC - Impedance, hydrodynamic
focusing
• Platelets – Impedance (2-20 fl),
hydrodynamic focusing
• Hgb – mod. Cyanmethemoglobin (525
nm)
• MCV – mean RBC volume (histogram)
– Indirect Measurement:
• Hct, MCHC, and MCH (calculations)
– RDW and MPV (CV of respective histograms)
– WBC differential
• VCS – volume, conductivity, scatter
• employs differential shrinkage
– Reticulocyte
• Supravital stain
• Fluorescent detection
• Flags

TWO SIDES OF INSTRUMENTS
(THREE PART INSTRUMENTS)
RBC SIDE WBC SIDE
(DILu-1:50,000) (DILu-1:500)
>30 fl size 2-30 fl size lysing reagent
RBC PLETELET
35-90 fl 90-160 fl 160-450 fl
Lymphocytes Mononuclear cells Granulocytes
Monocytes
Eosinophils Neutrophils
Basophils

Erythroid Parameters
• Measured parameters:
Hemoglobin
RBC count
MCV
RDW
• Calculated parameters
• These are calculated values and depend on the values that go into their
calculation.
• HCT(%) = MCV x RBC concentration
• MCH(pg) = Hb concentration / RBC concentration
• MCHC(g/dl) = Hb concentration / HCT

Hemoglobin Estimation
• Estimated from a part of the blood mixed with lysing
agent to lyse the RBCs.(the same can be used for
WBC counts)
Lyse treated blood
Cyanide Solution
added
Cyanmeth
hemoglobin method
Sodium Lauryl Sulphate
added
Sulphmeth homoglobin
method

HGBColorimetry for HB estimation

X axis: Volume of cells
Y axis: No. of cells
LD: 25-75 fL UD: 200-250 fL
RBC Histogram
Median =MCV
+1SD

RBC- and PLT-Histograms
1. Platelets have a size between 8 and 12 fl and are counted
between 2 and 30 fl.
2. Erythrocytes have a size of 80-100 fl and are counted
between 25 and 250 fl.

The Size Distribution Curve should always start on
the base line and fall between the lower and the
upper discriminator.

Histograms FLAGS Interpretation

Erythrocyte histogram Flagging
Possible causes:
• Giant platelets
• Microerythrocytes
• Fragmentocytes or dysplastic RBC
• Platelet clumps
Mark “ RL “, abnormal height at lower discriminator
LD
RBC
PLT
LD
RBC
The curve does not start at the basis line.

Erythrocyte histogram Flagging
Note:
All results marked with “ RL “ should be controlled.
Explanation:
RBC agglutination might cause a low incorrect RBC count and effect also the
parameter Hb, MCV, MCH and MCHC. In case of cold agglutinates warm the
sample up to 37°C.
(MCHC should drop back to normal value if the problem is solved)
Mark “ RU “, abnormal height at upper discriminator.
UD
RBC
The curve does not end at the base line.
UD
RBC
Possible causes:
• Cold agglutination
• RBC agglutination
• Rouleux formation

Possible causes:
• Iron deficiency in recovery (therapy)
• Dimorphic picture
• Multiple RBC transfusions
• Extreme leukocytosis (> 600 x 10³/µl)
Mark “ MP “: multiple peak of RBC histogram
RBC
Note:
Parameter: RBC, MCV, RDW-SD & RDW-CV are flagged.
Explanation:
Often: Extreme anisocytosis is found. In case of anisocytosis the RBC result is not
affected.
Seldom: extreme high numbers of leukocytes may cause high incorrect RBC results.
Therefore all RBC parameters should be controlled.

RDW-CV (%) = 100 x
s/µ
µ = L2 + L1 / 2
s = L2 – L1 / 2
100 %
L1 µ
Turning points
Normal value: 11 - 16 %
100 %
20 %
RDW-SD is calculated in
20 % of the total height of
the distribution curve.
Normal value: 37 - 46 fl
Clinical relevant > 60 fl
Erythrocyte histogram Distribution width (RDW)
RDW-CV
RDW-SD
L2
Note:
RDW-CV can be used as a marker for anisocytosis
Note:
RDW-SD can be used as a marker for anisocytosis

Mark “DW “: abnormal histogram distribution (distribution width)
(only RDW-SD or RDW-CV is flagged)
RBCRBC
Explanation:
The flag “DW” is shown in case of abnormal histogram curve.
The overall height of the curve represents 100%. The width is
calculated on the 20% line of the curve. If the histogram curve does
not match the 20% line either on the lower (RL) or upper (RU)
discriminator. The flag “DW” is generated for RDW-SD or RDW-
CV and these results can not be calculated.
Possible causes:
• same as RL or RU
Histogram curve does not match
the 20% line twice

100 fl
100 fl
Left shift in Microcytic Anaemia
Right shift in Macrocytic Anaemia

Cold agglutinins
Because in this case erythrocytes have passed
through the detector as clusters of several cells, the
RBC, HCT,MCH, MCV, MCHC and RDW values
are abnormal. The RBC histogram shows a second
peak.

Calculating reported parameters
• MPV Mean of 2D-PLT Vol histogram
(Mean Platelet Volume)
• LargeP- LCR Platelets with volumes greater than 20 fL
(Large Platelets)
The Platelet Method

• The histogram curve should lay within the lower and upper
platelet discriminator (PL & PU) and start and end on the
base line.
• PLT counted between 2 fl and 30 fl.
1 flexible Discriminator PL 2 to 6 fl.
1 flexible Discriminator PU 12-30 fl.
1 fixed Discriminator at 12 fl
Thrombocyte histogram

Parameter of the thrombocyte
histogram
MPV = mean PLT volume
reference range: 8 - 12 fl
P-LCR = ratio of large platelets
Reference range 15 - 35 %
 Increase could be a sign
for:
• PLT Clumps
• Giant PLT
12 fl
LD UD
PLT P-LCR
100 %
20 %
Pct (%)
PLT (x 103/µl)
MPV (fl) =
PDW = platelet distribution width
calculated at 20 % of peak height
Reference range: 9 - 14 fl
Increase could be a sign for:
• PLT Clumps
• Fragments

Note :
Check blank (background check)!
Initiate auto rinse or check sample.
Explanation:
In case of high background numbers (blank), check reagent for
contamination (bacteria). Check expiry date.
In order the background check is within range, the patient
sample should be checked – platelet results might be incorrect
high due to cell fragments or bacteria's. In some cases also
platelet aggregates might cause the problem. In this case the
histogram curve would also show an abnormal distribution at
the upper discriminator. Platelet aggregation might cause low
incorrect platelet results.
Flag display: mark „PL“ is shown with higher priority than
„PU“.
Mark “ PL “, abnormal height at lower discriminator
Possible cause:
• High blank value
• Cell fragments
• High numbers of bacteria
• Contaminated reagent
• Platelet aggregation
PLT
The curve does not start at the basis line.
Thrombocyte histogram Flagging

Mark “ PU “: abnormal height at upper discriminator
Explanation:
In case of platelet aggregation, the PLT count is false low. Check EDTA incombatibility – e.g. re-
collect the sample and use citrate as anticoagulant to avoid clogging caused by EDTA.
In case of giant platelets, the PLT count might be incorrect low. PLT results should be confirmed
with alternative methods: e.g. chamber counting.
Possible Cause :
• PLT clumps
EDTA-incombatibility
Clotted sample
• Giant Platelets (False low)
• Microerythrocytes (False High)
• Fragmentocytes or dysplastic RBC

Possible Cause:
• Platelet anisocytosis
• Recovery after
chemotherapy
• Platelet aggregation
• Platelet transfusion
Mark “ MP “: multi peaks in PLT histogram
Note:
Parameter: PLT, MPV, PDW and P-LCR is flagged.
Explanation:
In case of platelet anisocytosis the PLT result is not affected.
Seldom: Multiple peaks can be seen in some cases of platelet aggregation (jagged
curve). In case of PLT aggregation the PLT result might be incorrect low. Therefore
recollect the sample, In case of EDTA incompatibility sodium citrate as anticoagulant
can prevent platelet clumping.

The distribution curve should be within the discriminators. The curve
should start and end at the basis line.
• The LD is flexible, but can not be lower than 30 fl.
• The WBC-channel shows Leukocytes and Thrombocytes
( Erythrocytes are lysed).
• The volume of the Thrombocyts is usually between 8 - 12 fl, therefore
the LD at the WBC-Histogramm seperates the Leukocytes from the
Thrombocytes. (Thrombocytes were not counted).

Flag “ WL “, Curve does not begin at the basis line
Possible causes :
• PLT Clumps EDTA-Incombatibility coagulated Sample
• High osmotic resistant (Erythrocytes not lysed)
• Erythroblasts
• Cold agglutinate

Flag “ WU “, Curve does not end at the base line.
High leukocyte count

T1 and T2 are valley discriminators defined by the plateau.
This discriminators separates the Leukocytes populations.
• The discriminators are flexible and will be set automatically
according the sample.
• In special cases is a separation from the valley
discriminators not possible.

T1 could not be detected
High no. of large or atypical
lymphocytes
T1 was detected but not T2
Neutrophilic hypolobation
eg. Pelger Huet and
pseudo Pelger Huet anomaly.

This is a case of WBC agglutination, which occurs rather
rarely. The histogram does not show a clear tri-modal pattern,
with particles present in the region above 250 fl . The count of
leukocytes is likely to be falsely low. Depending on the nature
of leucocytes antibodies, agglutination may be dissolvable and
measurement may become possible upon incubation the at
37 o C or upon washing the samples with isotonic saline.

The valley between the erythrocytes ghost area and the small
leucocytes area exceeds the limit, and WL flags are given.
NRBC are likely to contribute significantly to the population
on the WBC histogram, therefore most of them are counted as
leukocytes. corrected by the following equation:
corrected WBC Count = measured WBC Count x 100
(100 + NRBC count )
NRBC Count: The number of NRBC per 100 leukocytes.

ABN / INDICATOR PROBABLE
CAUSE
COMMENT
WBC histogram
(lymph peak) does
not start at baseline
Giant platelets,
NRBC, Plt clumping
Review smear,
correct WBC for
NRBC
Elevation of left
portion of
granulocyte
Left Shift Review smear
Elevation of right
portion of
granulocyte peak
Neutrophilia Review smear
WBC Histogram

Five groups of leukocytes (stained)
What’s 5AND 7-part diff?
Neutrophil Eosinophil Basophil
Lymphocyte Monocyte

VOLUME
As opposed to using 0ø light loss to estimate cell
size,VCS utilizes the Coulter Principle of (DC)
Impedance to physically measure the volume that the
entire cell displaces in an isotonic diluent.
This method accurately sizes all cell types regardless
of their orientation in the light path.

CONDUCTIVITY
Alternating current in the radio frequency (RF) range
short circuits the bipolar lipid layer of a cell's
membrane allowing the energy to penetrate the cell.
This powerful probe is used to collect information
about cell size and internal structure, including
chemical composition and nuclear volume.

LASER LIGHT SCATTER
When a cell is struck by the coherent light of a
LASER beam, the scattered light spreads out in all
directions.
Using a proprietary new detector, median angle light
scatter (MALS) signals, are collected to obtain
information about cellular granularity, nuclear
lobularity and cell surface structure

Better Abnormal Cell Detection

NEW PARAMETERS
• Nucleated RBCs
• Immature granulocyte
• Haematopoitic progenitor cells
• Immature reticulocyte fraction
• RBC fragments (schistoytes)
• Reticulated platelets
• Reticulocyte indics
• Malarial parasites

Newer Parameters(contd..)
• Cellular Hb Concentration Mean(CHCM):
Uses Light scatter technology.
True estimate of hypochromia in IDA.
• Hb Distribution Width:
Degree of variation in red cell hemoglobinization.
Range-1.82 to 2.64.
• Nucleated Red Cells:
nRBCs identified,separated & corrected count obtained.
WBCs have high fluorescence & forward scatter.

• Reticuloctes:
Various dyes & flurochromes bind with RNA
RNA content- 3 Maturation stages; LFR,MFR & HFR
 Immature reticulocyte Fraction(IRF):
Sum of MFR & HFR.
Early and sensitive index for erythropoisis.
 Reticulocyte Hb Equivalent(RET-He):
Hb content of freshly prepared RBCs.
Real time information on Fe supply to erythropoiesis.
Early detection of Fe deficiency.
Differentiate IDA & ACD.
Monitoring of erythropoietin & Fe therapy.

• P-LCR(Platelet Large Cell Ratio):
% of platelets with a vol >12fl.
Due to platelet aggregates,microerythrocytes,giant platelets.
• Reticulated Platelets /Immature Platelet Fraction(IPF):
Newly produced platelets that have remains of RNA in their
cytoplasm.
Reflects rate of thrombopoiesis.

WBC Research Population Data Case Study – Malaria Parasites(Normal plot and Research
Population Data compared to a patient infected with malaria type Plasmodium
falciparum. Note the increased size and variation of the lymph's and Monocyte's.)
NORMAL
Normal
Normal MO
Noral LY
Macrophage
Parasitized RBC
MALARIA
MP Positive
Reactive LY

Peroxide based counters:
MPO is used to count neutrophils.Lymphocytes
not stained
Fluroscence based:
Retic and platelet count.Immature platlets
detected best
Immunological based:
Accurate platelet count using CD41/CD61
antibodies

Must to do before running the sample
• CBC specimens must be checked for clots (visually, by
applicator sticks, or by automated analyzer histogram
inspection or flags), significant in-vitro haemolysis and
interfering lipaemia before reporting results.
• CBC processing, either automated or manual, should
be done within 8 hours but in no case later than 24
hours of sample collection, as storage beyond 24 hours
results in erroneous data on automated / semi-
automated Haematology analyzers
• Blood samples must be adequately mixed before
analysis.

Quality Assessment:
Adequate control of the pre & post analytical from
sample collection to report dispatch.
Quality Control:
Measures that must be included during each assay
run to verify the test working properly.
Proficiency Testing:
Determines the quality of results generated by lab.
Terminologies

Accuracy & Precision
• Accuracy:
Refers to closeness to the
true value
• Precision:
Refers to reproducibility
of test
1 2
3 4

Internal Quality control:
Continuous evaluation of the reliability of the daily
works of the lab with validation of tests.
External Quality Control:
Evaluation by an outside agency of between-
laboratory & between-method comparability.
TYPES

CBC Quality Control
• Commercial Controls:
• 3 levels (low, normal, high)
• Values stored in instrument computer
• Levey-Jennings graph generated and stored for each
parameter
• Delta Checks
• When the Laboratory Information System (LIS) and the
instrument are interfaced (connected) delta checks are
conducted by the LIS on selected parameters.
–Current values compared to most previous result
–Differences greater than the limits set within the LIS
are flagged

Controls & Calibrators
• Controls:
Substances used to check the precision .
Analyzed either daily or along each
batch.
Should have same test properties as
blood samples.
Stabilized anticoagulated whole blood or
pooled red cells.
3 conc.-high,normal ,low
• Levey-Jennings graph generated and
stored for each parameter
• Calibrators:
Check the accuracy.
Value assigned to them by a reliable ref.
center.

Levey-Jennings Chart
Mean
1 SD
2 SD
3 SD
1 SD
2 SD
3 SD

Control Values and Decision
 Consider using Westgard Control Rules
 Use premise that 95.5% of control values
should fall within ±2SD
 Commonly applied when two levels of control
are used
 Use in a sequential fashion

12S Rule = A warning to trigger careful inspection of
the control data
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
12S rule
violation

13S Rule = Reject the run when a single control
measurement exceeds the +3SD or -3SD control limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
13S rule
violation

22S Rule = Reject the run when 2 consecutive control
measurements exceed the same +2SD or -2SD control limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
22S rule
violation

10x Rule = Reject the run when 10 consecutive control
measurements fall on one side of the mean
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
10x rule
violation

What to do when Control Value is out of limit?
• In these situations, precision of routine work can be
monitored by performing duplicate tests on patient
samples.
• SD of differences between results on 20 duplicate
samples is determined and +2SD limits specified.
Subsequent duplicate values should be within these
defined limits.
• Patient data can also be used to monitor precision in a
laboratory performing >100 samples a day. Day-to-Day
variation in MCV, MCH and MCHC should be analyzed
using Bull's algorithm. This facility is available in the
software of many auto analyzers.
• The use of stable controls, however, is the method of
choice.

• OUT OF CONTROL!!!
–Repeat the assay ( One time occurrence )
–Check for trends (Delta check)
(from Levy Jennings chart)
–Check integrity of material
–Troubleshoot
–Verify instrumentation

Specimen-Related Problems
• An instrument problem is differentiated from a specimen-
related problem by running a control.
• If the control results are acceptable, the problem is
probably specimen-related.
• Check for:
– clots
– hemolysis
– lipemia

Instrument Problems
• If the control shows similar problems, it indicates an
instrument problem.
– Electronic?
– Pneumatic / Hydraulic?
– Reagent?
• Because it is easiest to detect a problem in the electronic
subsystem and hardest to detect a problem in the reagent
subsystem, the subsystems are usually checked in the
following order: electronic, pneumatic / hydraulic, reagent.

Reagent Troubleshooting
• A reagent problem can be as obvious as
precipitate in the reagent tubing.
• In the less obvious cases, the most effective
way of detecting a problem is by keeping a log
of the lot numbers with the opening and
expiration dates of the reagents in use, and
knowing how each reagent affects the data.
• Refer to the labeling information with your
reagents for details.

Calibration
1.It is done to compensate for any inaccuracies of the
pneumatic hydraulic and electric systems
2. Calibration fine tunes your hematology analyzer
and provides the most accurate results possible.
3. Automated Haematology analyzers should be
calibrated using calibrators„ that have traceability to
standard reference material or methods.
4.Controls are not used for calibration

Calibration
• 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.

When to Calibrate
You should calibrate your 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.

Blood Sample for Calibration
• 4 ml specimen are obtained from three
hematological normal volunteer
in k2 edta

Hb Estimation
• By Cyan meth hemoglobin method
mean value is taken

ICSH Reference methods for PCV
The reference PCV is
Standard whole blood haemoglobin concentration
-----------------------------------------------------------------
Packed red cell haemoglobin concentration
following centrifugation in a microhaematocrit centrifuge
1.The measurement on packed red cells is performed on
cells obtained from the middle of the column of red
cells
2. where there is little trapping of plasma or white cell
contamination.
3.It therefore produces a measurement that does not
include trapped plasma.

RBC AND WBC
TOTAL COUNT
• The reference method for the RBC and
WBC employs a semi-automated
single-channel aperture-impedance
method with accurate coincidence
correction being achieved by
extrapolation from counts on serial
dilutions.

PLATELET
1.The platelet count can be determined by flow
cytometry using a fluorochrome – labelled
monoclonal antibody, mixture of CD41, CD42a or
CD61,
2.That binds specifically to platelets and dilution errors
do not affect the count
4.when there is an inherited platelet membrane defect
with absence of one of the platelet glycoproteins,
5.The relevant monoclonal antibody will not bind to
platelets, hence the use of two antibodies

CARRY HOME MESSAGE
Automated analyzers provide rapid and useful information of the cell
count, morphology and cell function.
Automation is a supplement and not a
substitute to manual methods

ABC of automated CBC

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