AUTOMATED CELL COUNTERS- THE BASICS

Pankhi Dutta MD DM (haematopath)Consultant haematopathologistSevenHills Hospital, Mumbai.

Introduction

Automated cell counter-backbone of the haemat lab

Wallace Coulter in 1956 – impedance method

Various technologies today More accurate, more precise reports

at a faster rate Basic CBC + newer parameters Inherent technological limitations

Basic parameters – 3 part counter

Haemoglobin RBC, WBC, PLT count Red cell indices RDW 3-part differential Histograms

NO. 4DATUM: 9/10/95 15:11MODE: VOLLBLUTWBC 5,8 x 103/µlRBC 4,84 x106/µlHGB 13,7 g/dlHCT 42,0 %MCV 86,8 flMCH 28,3 pgMCHC 32,6 g/dlPLT 257 x103/µl

LYMPH% 31,2 %MXD% 6,8 %NEUT% 62,0 %LYMPH# 1,8 x103/µlMXD# 0,4 x103/µlNEUT# 3,6 x103/µl

250

RBC

RDW-SD 40,0 fl

40

PLT

PDW 13,1 flMPV 10,4 flP-LCR 28,1 %

WBC

300

3-part differential analyser Two chambers Hb + WBCs RBCs + PLTs

Vacuum

Blood cell

DC supply

Registor(constant current)

Internal electrodeExternal electrode

Aperture

Transducer chamber

Blood cell suspension

V07063-part Diff technology

Haemoglobin molecule

RBC

Ammonium salts

Fe2+Fe2+

RBC

Fe2+ Fe2+

1. Lysis of RBC

Fe2+ Fe2+ Fe2+ Fe2+

Haemoglobin estimation

V07063-part Diff technology

Haemoglobin molecule

RBC

Fe2+Fe2+

RBC

Fe2+ Fe2+

Fe2+ Fe2+ Fe2+ Fe2+

2. Change of conformity

V07063-part Diff technology

Haemoglobin molecule

Methemoglobin-complex

Stable coloumetric complex – directly proportional to Hb

Absorbance of solution is measured against standard

Fe3+ Fe3+

Fe2+Fe2+

O2

3. Oxidation

Fe2+ Fe2+ Fe2+ Fe2+

DC detection method

Particle counting DC - direct current - impedance principle

- volumetric measurement

WBC count and 3-part differential

RBC count PLT count V07063-part Diff technology

DC Detection Method

V07063-part Diff technology

external electrode

internal electrode

aperture

vacuum

U = R x I

Impulse

Impedance Principle

External Electrode

Internal Electrode

Aperture

V = R x C V = VoltageC = CurrentR = Resistance

Impedance Principle

V = R x C V = VoltageC = CurrentR = Resistance

External Electrode

Internal Electrode

Aperture

Problems- recirculation and coincidence

ABC

pulse A pulse B pulse C

aperture

cells

V07063-part Diff technology

Samples are passing through the centre of the aperture with sheath flow solution

for RBC & PLT

Hydrodynamic Focusing

Recirculation and coincidence are prevented Enhanced linearity & accuracy

V07063-part Diff technology

1 2 3 4 5 6 7 8 9 10 11 12 13 14tim

e

pulse height

From pulse to histogram: pulse diagram

DC Detection Method

V07063-part Diff technology

1 2 3 4 5 6 7 8 9 1011121314

Histogram

10

20

30

1 2 3 4 5 6 7 8 9 1011121314

Cumulative Distribution Curve

4 1 0 0 0 1 2 3 4 5 3 2 14

cells

1 2 3 4 5 6 7 8 9 1011121314

DC Detection Method

NO. 4DATUM: 9/10/95 15:11MODE: VOLLBLUTWBC 5,8 x 103/µlRBC 4,84 x106/µlHGB 13,7 g/dlHCT 42,0 %MCV 86,8 flMCH 28,3 pgMCHC 32,6 g/dlPLT 257 x103/µl

LYMPH% 31,2 %MXD% 6,8 %NEUT% 62,0 %LYMPH# 1,8 x103/µlMXD# 0,4 x103/µlNEUT# 3,6 x103/µl

250

RBC

RDW-SD 40,0 fl

40

PLT

PDW 13,1 flMPV 10,4 flP-LCR 28,1 %

WBC

300

V07063-part Diff technology

25-75 fl 200-250 fl

Erythrocyte (RBC) Histogram

RBC detection: between 25 and 250 fL Distribution curves are separated by flexible

discriminators: RL & RU

RL RU

RBCPLT

V07063-part Diff technology

25-75 fl 200-250 fl

The histogram curve should start and end at the base line within the discriminators

RL RU

RBCPLT

Erythrocyte (RBC) Histogram

V07063-part Diff technology

25-75 fl 200-250 fl

In case of abnormal histogram curves the flag messages: RL; RU or MP are generated and results must be checked

RL : Abnormal height at lower discriminator RU : Abnormal height at upper discriminator MP : (Multi Peak) RBC Anisocytosis

RL RU

RBCPLT

Example:RL flag message

100%

20%

Abnormal Erythrocyte (RBC) Histogram

V07063-part Diff technology

2-6 fl 12-30 flfixed at

12 fl

PL PU

PLT RBC

100%

20%

PLT detection: between 2 and 30 fL Fixed discriminator at 12 fL

Platelet (Plt) Histogram

V07063-part Diff technology

2-6 fl 12-30 fl

PL PU

PLT RBC

100%

20%

In case of abnormal histogram curves the flag messages: PL; PU or MP are generated and results must be checked

PL : Abnormal height at Lower discriminator PU : Abnormal height at Upper discriminator MP : (Multi Peak) Platelet Anisocytosis

Example:abnormal PLT curvePU message

Abnormal Platelet (Plt) Histogram

Lysing reaction to the WBCs

Structure of WBS

Mitochondria

Nucleus

Nucleolus

Cell membrane

Ribosome

Cytoplasm

Lysing reaction on the WBC

Leukocyte (WBC) Histogram

Before lysing reaction

0 2 4 6 8 10 12 14 16 18 20 22

NeutrophileBasophileEosinophileMonocyteLymphocyte

Cell size in µm10 - 15 9 - 1411 - 1612 - 20 7 - 12

Lysing reaction and WBC

After lysing reaction

0 50 100 150 200 250 300

Lymphocyte

Monocyten Basophile Eosinophile

Neutrophile 30 - 80 60 - 120 70 - 130 80 - 140120 - 250

Cell volume in fl

LymphocyteMonocyteBasophile Eosinophile Neutrophile

Leukocyte (WBC) Histogram

V07063-part Diff technology

2-6 fl 12-30 flfixed at

12 fl

WL WU

100%

20%

WBC detection: between 30 and 300 fL Leukocytes are separated in 3 parts:

lymphocytes, mixed cells (mono, eo, baso)and neutrophils by discriminators: T1, T2

T1 T2

Leukocyte (WBC) Histogram

V07063-part Diff technology

~30 fl -300 fl

WL WU

100%

20%

The histogram curve should start within the lower and upper discriminator at the base line

Abnormal curves are flagged with WL, WU, T1, T2, F1, F2 results must be checked

T1 T2Example:abnormal WBC curveWL message in case of Lyse resistant RBC

Abnormal Leukocyte (WBC) Histogram

ABOUT 3-PART DIFFERENTIAL COUNTERS

QUESTIONS?

5-part differential countersVarious technologies :-

Fluorescence flowcytometryVolume Conductivity ScatterPeroxidase staining

VOLUME MEASUREMENT

VCS utilises the Coulter Principle of counting and sizing to measure the volume of the cell by using Direct Current (DC) across the two electrode in a flow cell.

Beckman Coulter

CONDUCTIVITY MEASUREMENT

Cell exposed to RF, the RF energy penetrates into cell and reveal information about its size and internal structure.

SCATTER MEASUREMENT

As cells are pass in single stream (flow cell) they are struck by laser strike which gets scattered.

The light scatter at angles between 10 and 70 deg is used by VCS instruments.

The scattered light gives information about cell surface and granularity

3D Data Analysis

Lymphs

Monos

Basos

NRBCs

Eos

Neuts

ADVIA TECHNOLOGY

WBC and Differential Peroxidase Channel Stain Cells

With Peroxidase

:Eosinophils- Strong Staining

:Neutrophils- Medium Staining

:Monocytes- Weak Staining

:Lymphocytes and Basophils-

No Staining

:Large Unstained Cells (LUC)

No staining Also Measure Cell Size Using Low

Angle ScatterPlot 2D Scattergram To Give 4 Part

Differential

Eosinophils

Neutrophils

Monocytes

LUC

Lymphocytes + Basophils

Perox Activity

Volu

me

The ADVIA WBC differential is calculated from a 3 step process.

• Cells are stained by peroxidase reagent and analyzed for size and peroxidase stain intensity.

• Cell specific lysis reagents are used to separate basophils from all other white cells.

• Basos are subtracted from the lymph/baso cluster in the perox channel to calculate the lymphs.

ADVIA TECHNOLOGY

Sysmex X-class analyzers-Fluorescence flow cytometry

Fluorescence flow cytometry- (light scatter and fluorescent dyes)

49

Differential-

FSc vs SSc (baso channel)

SFL vs SSc (diff channel)

ACAS / Centroids

SSC

SFL

GhostNeut + Ba

MonoLymph

Eo

1. The first centroids are provided:

The starting position of centroids has been determined from thousands of samples. These values are stored in the instrument and are used as the starting position for cluster analysis.

ACAS / Mahalanobis Distance2. Cluster analysis of scattergram

If a cell is detected, distances between this signal and the given centroids are calculated (Mahalanobis distance). This distance reveals to which given cell population the signal belongs.

SSC

SFL

Ghost

Neut + Ba

MonoLymph

Eo

1. calculated centroids Mahalanobis-Distance

Differential fluorescent staining- Immature granulocytes(Sysmex)

Diff scattergram: IG positive vs IG negative

IG MASTER

Reticulocyte parameters (RET channel)

Separate channel

Polymethine dye stains N.A. in WBCs, nRBCs , retics & platelets.

Size vs fluorescence

Retic count

Retic channel

Reticulocyte count Reticulocyte fractions (LFR, MRF, HRF) Immature reticulocyte fraction Ret-He (reticulocyte Hb content) Platelet –O (fluorescent platelets) Fragmented red cells (FRC)

An example of efficient multitasking!!

New haematological parameter can predict iron deficiency where classical serum tests fail

Reticulocyte HaemoglobinEquivalent--- Ret-HeCHr- Siemens

(FDA approved)

Ret-He

Ret-He - Hb content equivalent of reticulocytes

Units of “pg”(normal range- 28 – 35 pg) Monitors state of iron supply during the

course of erythropoiesis, provides information on availability of functional iron.

Can classify hypochromic anemia, classical vs functional ID, helps to select optimal therapy & to monitor response to EPO and iron treatment.

Spurious platelet counts-Siegenthaler & Spertini, NEJM May 2006

48 yr old M with severe burns

Automated CBC- Hct-37%, MCV-91fl, RDW-15.8%, WBC-7,400/ul, PLT count- 274000/ul

PS – microspherocytes and spherocytes

Manual platelet count- 85,000/ul

Overcoming the problems with impedance counting

Manual method- haemocytometer with phase contrast

(Time consuming, laborious, operator dependency is more)

Flowcytometric method using RBC/PLT ratio (anti CD41, anti CD61) Am J Clin Path 2001

(Expensive, requires a flowcytometer and experience with FCM)

Optical (fluorescent) platelets (good correlation with reference methods)

Sysmex fluorescent PLT-O results are unmatched by ordinary platelet technologies of other analyzers

Fluorescent PLT (platelet-O)

Microcytic RBCGiant PLT

PLT abn. Distribution giant thrombocytes

IPF – Immature Platelet Fraction

Immature PLT are identified by its increase in fluorescence (more RNA), FSC is also higher.

IPF

Peripheral smear examination still required!!

Thrombocytopenia

Platelet clump(EDTA induced)

Platelet count after collection in citrate !!

Criteria for smear review

Smear review- increases manual work, reduces TAT & productivity

Need to reduce smear review rate without risk of missing anything significant

Different labs – different criteria

Criteria depend upon patient population, type of analyser in use, etc.

Consensus rules for smear review

International consensus group for hematology review – ISLH, 2002 ( Dr. Berend Houwen)

Laid down rules for action following automated CBC including smear review

Rules tested in 15 labs (13,298 samples)

Data analysed, rules refined, 43 rules laid down

Guidelines for individual laboratories

Review : Criteria for automated CBC & WBC diff analysis

Examples of some consensus rules (Lab Haematol, 2005)

Rule no Parameter Primary And/or Action

1 neonate 1st sample Slide review

10 MCV <75fl or >105fl

Specimen <24hrs old

Slide review

15 RDW >22 1st time Slide review

16 No WBC diff/incomplete

Manual diff & slide review

7 Platelet <100 or >1000

1st time Slide review

flagged normalAnalyser

Routine technician

? Abnormal RBCs? Atypical mononuclear WBCs

•Common RBC abnormality•Granulocyte left shift•Atyipcal (variant) lymphocytes•normoblasts

Sr. technician

? Blasts? Organisms

•Myelocytes•Plasma cells•Dohle bodies•Targets•Auer rods

Physician Diagnostic cells

Report

Report

Report

Report

Examiner Differentiation

Hierarchial blood film evaluation

QUIZ-

Fragments ?

Eosinophilia

Iron Deficiency Anemia

Photo of slide

Lymphocytosis

Summary Automated cell counters-backbone of the

diagnostic laboratory Fast, accurate, precise Impedance and various other

technologies All have various limitations Accurate information on the technologies

helpful to recognize problematic areas Maintenance, calibration, QC procedures Slides still need to be reviewed (criteria) Finally………

IT IS THE MAN BEHIND THE MACHINE WHO MATTERS MOST!!

THANK YOU!!