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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!!
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