ABC

of automated

CBCDr.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 types1.Neutrophils2.Eosinophils3.Basophils4.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.

Optical Scatter

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 scattercorrelates to cell volume/size

Side scattercorrelates 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 GranulocytesMonocytesEosinophils NeutrophilsBasophils

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

Cyanmethhemoglobin method

Sodium Lauryl Sulphateadded

Sulphmeth homoglobinmethod

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

LDRBC

PLT

LDRBC

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.

UDRBC

The curve does not end at the base line.

UDRBC

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

• Microerythrocytes

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

• Microerythrocytes

• 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.

WBC Histogram

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

VCS Technology

COULTER TECHNOLOGY

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

3-D Cellular Analysis- VCS

Gate Software Technology

NORMAL DATAPLOT

DOT plot or Scattergram

Better Abnormal Cell Detection

LEUKOPENIA & THROMBOCYTOPENIA

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.

Newer Parameters(contd..)

• 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.

Newer Parameters(contd..)

• 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

The Retic Method

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 Control

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 MESSAGEAutomated 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