1. Automated cell counters- THE BASICs
Pankhi Dutta MD DM (haematopath)
Consultant haematopathologist
SevenHills Hospital, Mumbai.

2. 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

3. Basic parameters – 3 part counter
NO. 4
DATUM: 9/10/95 15:11
MODE: VOLLBLUT
WBC 5,8 x 103/µl
RBC 4,84 x106/µl
HGB 13,7 g/dl
HCT 42,0 %
MCV 86,8 fl
MCH 28,3 pg
MCHC 32,6 g/dl
PLT 257 x103/µl
LYMPH% 31,2 %
MXD% 6,8 %
NEUT% 62,0 %
LYMPH# 1,8 x103/µl
MXD# 0,4 x103/µl
NEUT# 3,6 x103/µl
250
RBC
RDW-SD 40,0 fl 40
PLT
PDW 13,1 fl
MPV 10,4 fl
P-LCR 28,1 %
WBC
300
Haemoglobin
RBC, WBC, PLT count
Red cell indices
RDW
3-part differential
Histograms

4. 3-part differential analyser
Two chambers
Hb + WBCs
RBCs + PLTs
Vacuum
Blood cell
DC supply
Registor (constant current)
Internal electrode
External electrode
Aperture
Transducer chamber
Blood cell suspension

5. Haemoglobin estimation
1. Lysis of RBC
Haemoglobin molecule
Fe2+  
Fe2+
Fe2+  
Fe2+
Ammonium salts    
Fe2+
Fe2+
Fe2+
Fe2+
RBC
RBC
3-part Diff technology
V0706

6. 2. Change of conformity Haemoglobin molecule RBC RBC       Fe2+
3-part Diff technology
V0706

7. 3. Oxidation Haemoglobin molecule O2       Methemoglobin-complex
Fe3+
Fe2+  
Fe2+
Fe2+  
Fe2+
Fe2+
Fe2+
Methemoglobin-complex
Stable coloumetric complex – directly proportional to Hb
Absorbance of solution is measured against standard
3-part Diff technology
V0706

8. DC detection method Particle counting
DC - direct current
- impedance principle
- volumetric measurement
WBC count and 3-part differential
RBC count
PLT count
3-part Diff technology
V0706

9. DC Detection Method aperture vacuum internal external electrode
U = R x I
Impulse
3-part Diff technology
V0706

10. Impedance Principle External Electrode Internal Electrode Aperture
V = Voltage
C = Current
R = Resistance
V = R x C
XT-Series Product file
Chapter V. Principles and Technologies
Date: , TTO – page 10

11. Impedance Principle Aperture External Electrode Internal Electrode
V = Voltage
C = Current
R = Resistance
V = R x C
XT-Series Product file
Chapter V. Principles and Technologies
Date: , TTO – page 11

12. Problems- recirculation and coincidence
pulse A pulse B pulse C
aperture
cells

13. Hydrodynamic Focusing
for RBC & PLT
Samples are passing through the centre of the aperture with sheath flow solution
 Recirculation and coincidence are prevented
 Enhanced linearity & accuracy
3-part Diff technology
V0706

14. DC Detection Method From pulse to histogram: pulse diagram time 1 2 34 5 6 7 8 9 10 11 12 13 14
pulse height
3-part Diff technology
V0706

15. DC Detection Method 30 20 10 Cumulative Distribution Curve Histogram 14 5 6 7 8 9 10 11 12 13 14 30
cells 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10
Cumulative
Distribution Curve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 4 1 1 2 3 4 5 4 3 2 1
Histogram
3-part Diff technology
V0706

16. NO. 4
DATUM: 9/10/95 15:11
MODE: VOLLBLUT
WBC 5,8 x 103/µl
RBC 4,84 x106/µl
HGB 13,7 g/dl
HCT 42,0 %
MCV 86,8 fl
MCH 28,3 pg
MCHC 32,6 g/dl
PLT 257 x103/µl
LYMPH% 31,2 %
MXD% 6,8 %
NEUT% 62,0 %
LYMPH# 1,8 x103/µl
MXD# 0,4 x103/µl
NEUT# 3,6 x103/µl
250
RBC
RDW-SD 40,0 fl 40
PLT
PDW 13,1 fl
MPV 10,4 fl
P-LCR 28,1 %
WBC
300

17. Erythrocyte (RBC) HistogramRL RU
PLT
RBC
25-75 fl fl
RBC detection: between 25 and 250 fL
Distribution curves are separated by flexible discriminators: RL & RU
3-part Diff technology
V0706

18. Erythrocyte (RBC) HistogramRL RU
PLT
RBC
25-75 fl fl
The histogram curve should start and end at the base line within the discriminators
3-part Diff technology
V0706

19. Abnormal Erythrocyte (RBC) HistogramRL RU
100%
Example:
RL flag message
PLT
RBC
20%
25-75 fl 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
3-part Diff technology
V0706

20. Platelet (Plt) HistogramPU
100%
PLT
RBC
20%
fixed at
12 fl
2-6 fl
12-30 fl
PLT detection: between 2 and 30 fL
Fixed discriminator at 12 fL
3-part Diff technology
V0706

21. Abnormal Platelet (Plt) HistogramPU
Example: abnormal PLT curve PU message
100%
PLT
RBC
20%
2-6 fl
12-30 fl
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
3-part Diff technology
V0706

29. Leukocyte (WBC) Histogram
Lysing reaction to the WBCs
Structure of WBS
Lysing reaction on the WBC
Mitochondria
Nucleus
Nucleolus
Cell membrane
Ribosome
Cytoplasm

30. Leukocyte (WBC) Histogram
Lysing reaction and WBC
Cell size in µm
Before lysing reaction
Neutrophile
Basophile
Eosinophile
Monocyte
Lymphocyte
9 - 14
7 - 12
After lysing reaction
Lymphocyte
Monocyten
Basophile
Eosinophile
Neutrophile
Cell volume in fl
Lymphocyte
Monocyte
Basophile
Eosinophile
Neutrophile

31. Leukocyte (WBC) HistogramWL WU T1 T2
100%
20%
fixed at
12 fl
2-6 fl
12-30 fl
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
3-part Diff technology
V0706

32. Abnormal Leukocyte (WBC) HistogramWL WU T1 T2
Example: abnormal WBC curve WL message in case of Lyse resistant RBC
100%
20%
~30 fl
-300 fl
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
3-part Diff technology
V0706

39. about 3-part differential counters
QUESTIONS?
about 3-part differential counters

40. 5-part differential counters
Various technologies :-
Fluorescence flowcytometry
Volume Conductivity Scatter
Peroxidase staining

41. Beckman Coulter 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.
VOLUME:
As opposed to using 0 light loss to estimate cell size, VCS utilises 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.

42. CONDUCTIVITY MEASUREMENT
Cell exposed to RF, the RF energy penetrates into cell and reveal information about its size and internal structure.
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 the internal structure of the cell, including chemical composition and nuclear volume.
OPACITY:
By correcting the Conductivity signal so that it is no longer influenced by cell size, we obtain a measurement that is related only to the internal structure of the cell. This allows VCS Technology to separate cells of similar size, but different internal composition. It also allows the instrument to calculate the Nuclear/Cytoplasmic ratio - a feature useful in distinguishing variant Lymphocytes from normal Lymphs.
Opacity is a transformation of data derived from the ratio of RF/DC components obtained during data acquisition. It is calculated for every cell measurement and has the effect of removing the size component yielding a measurement which is more related to the internal conductivity of the cell.This parameter is plotted on the X axis of DF2 plot.
Opacity  RF/DC

43. 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.
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 signals are collected to obtain information about cellular granularity, nuclear lobularity and cell surface structure.
ROTATED LIGHT SCATTER (RLS):
Using a method similar to that used in Opacity, Coulter eliminates the size component of the light scatter signals. In so doing, we were able to determine the optimum angle of scatter for each cell type and design a scatter detector that covered this range (10 to 70). This allows VCS Technology to accurately separate what would normally be mixed cell types (such as Neutrophils and Eosinophils) into distinct clusters without mathematical manipulation. It also enhances the separation between the non granular cell types.
Rotated Light scatter  Log10 (LS) / DC
The scattered light gives information about cell surface and granularity

44. 3D Data Analysis
Lymphs
Monos
Basos
NRBCs
Eos
Neuts

45. 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
Volume
Monocytes
Neutrophils
LUC
Lymphocytes +
Basophils
Eosinophils
Perox Activity 45

46. ADVIA TECHNOLOGY
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.

47. Sysmex X-class analyzers-Fluorescence flow cytometry
Jaldeep Bhansali 47

48. Fluorescence flow cytometry- (light scatter and fluorescent dyes)

49. Differential- FSc vs SSc (baso channel) Differential scattergrams
SFL vs SSc (diff channel)
FSc vs SSc (baso channel)
Differential scattergrams
Two scattergrams are used for the WBC differential
1. Diff Channel for the measurement of lymphocytes, monocytes, neutrophils, and eosinophils. The cell populations are separated by the side scatter based upon the internal structure of the cells and side fluorescence based upon the RNA and DNA content of the cells.
The neutrophile population contains also the basophile which are counted in the WBC/BASO channel
2. WBC/BASO channel for the measurement of the total white blood cells and the basophils. The cells are separated by internal structure (side scatter) and cell size (forward scatter)

50. ACAS / Centroids
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.
SSC
SFL
Mono
Lymph
Ghost
Neut + Ba Eo
XT-Series Product file
Chapter V. Principles and Technologies
Date: , TTO – page 50

51. ACAS / Mahalanobis Distance
2. 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
1. calculated centroids
Mahalanobis-
Distance
Mono
Lymph
Ghost
Neut + Ba Eo
XT-Series Product file
Chapter V. Principles and Technologies
Date: , TTO – page 51

52. Differential fluorescent staining- Immature granulocytes(Sysmex)
Jaldeep Bhansali 52

53. IG MASTER Diff scattergram: IG positive vs IG negative

54. Reticulocyte parameters (RET channel)
Separate channel
Polymethine dye stains N.A. in WBCs, nRBCs , retics & platelets.
Size vs fluorescence
Retic count

55. 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!!

56. New haematological parameter can predict iron deficiency where classical serum tests fail
Reticulocyte Haemoglobin
Equivalent--- Ret-He
CHr- Siemens
(FDA approved)

57. 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.

58. 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 /ul
PS – microspherocytes and spherocytes
Manual platelet count- 85,000/ul

59. 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)

60. Optical (fluorescent) platelets (good correlation with reference methods)
Sysmex fluorescent PLT-O results are unmatched by ordinary platelet technologies of other analyzers
Jaldeep Bhansali 60

61. Fluorescent PLT (platelet-O)
Giant PLT
Microcytic RBC

62. PLT abn. Distribution giant thrombocytes

63. IPF – Immature Platelet Fraction
Immature PLT are identified by its increase in fluorescence (more RNA), FSC is also higher.

67. Peripheral smear examination still required!!

68. Thrombocytopenia

69. Platelet clump(EDTA induced)

70. Platelet count after collection in citrate !!

71. 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.

72. 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

73. Review : Criteria for automated CBC & WBC diff analysis

74. 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 15
RDW
>22
1st time 16
No WBC diff/incomplete
Manual diff & slide review 7
Platelet
<100 or >1000

75. Hierarchial blood film evaluation
Examiner
Differentiation
Analyser
flagged
normal
? Abnormal RBCs
? Atypical mononuclear WBCs
Common RBC abnormality
Granulocyte left shift
Atyipcal (variant) lymphocytes
normoblasts
Routine technician
Sr. technician
? Blasts
? Organisms
Myelocytes
Plasma cells
Dohle bodies
Targets
Auer rods
Physician
Diagnostic cells
Report
Report
Report
Report

76. QUIZ-

81. Fragments ?

83. Eosinophilia

85. Iron Deficiency Anemia

86. Photo of slide

87. Lymphocytosis

88. 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………

89. IT IS THE MAN BEHIND THE MACHINE WHO MATTERS MOST!!
THANK YOU!!