BAsic Trouble Shooting Guide

1. HAEMA
TOLOGY
CELL
COUNTERS

2. Wallace Coulter (1913-1998)
2
Wallace Coulter, an American electronics engineer,
developed the Coulter Principle of the counting and
sizing of microscopic particles (including blood cells)
during the late 1940’s into the early 1950’s. He
successfully secured a US patent for the first Coulter
counter in 1953
About the same time in Kobe, Japan, scientists were
developing using radio frequency to count cells.

3. Technologies Used in
Automated Cell Counters
3
Impedance Technology
Absorbance Spectrophotometry
Optical Technology
Fluorescent Technology
Conductivity (radio frequency)
Cytochemical staining
Monoclonal Antibodies are starting to be
utilised.

4. Impedance Technology
4
Earliest form of automated particle (cell) counting.
Based on cells being suspended in an electrically
conductive fluid.
Cells pass through an aperture of known size.
Hydrodynamic focussing is applied to keep diluted
specimen in the middle of the stream.
Current is applied across the aperture.

5. Impedance Technology cont’d
5
Cells impede current as they pass through the
aperture.
Each time a cell “impedes”, a pulse is created.
Number of pulses equal the number of cells
passing through the aperture.
Size of the pulse (resistance) is proportional to
the size of the cell.

6. Impedance Transducer
6

7. Pitfalls of Impedance Technology
7
Co-incidence Counting.
2 or more cells passing through the aperture at the same
time.
Non-axial Passage.
Cells “swirling” back into the sensing zone
Deformability of cells and different cell shapes.
Vacuum applied, pulling cells through the aperture distorts
the shape of the cell and hence the size of the cell.

8. Elliptocytes Passing through an
Impedance Transducer
Aperture
Elliptocytes
passing
through the
aperture
impedes
the current
differently
depending
on the
orientation
of the cell
as it
passes
through the
aperture.
Aperture
8

9. Aperture
However if we
make the cells
uniform, we see
that we remove
the issue of
shape and
orientation
having an effect
on the amount of
current impeded.
Aperture
9

10. Absorbance Spectrophotometry
10
 Used to measure the quantity of Haemoglobin
in blood.
Complete lysis of the red blood cells is required
to release Haemoglobin into solution.
Haemoglobin is measured at 540nm ( an LED
is the light source).
Haemoglobin is calculated by measuring the
absorbance readings at 540nm of both the lysed
specimen and the blank Haemoglobin reagent.
A Photodetector measures the amount of light
that passes through the lysed specimen and the
blank reagent.

11. Absorbance Spectrophotometry cont’d
11
The transmitted light signal is lower when the
Haemoglobin flow cell is filled with lysed
specimen than when filled with the blank
reagent. This difference is due to absorption by
the Haemoglobin complex and is used to
calculate the concentration of Haemoglobin in
the blood specimen.

12. Specimen- Related Interference
with Haemoglobin Calculations
12
High White Blood Cell counts (refer to the
manufacturer’s guidelines).
High level of plasma lipids.
High levels of bilirubin (obstructive jaundice).
Haemolysed specimens.

13. Optical Technology
13
Cells are counted and classified using flow
cytometry technology.
Lasers are used as the light beam source
and light scatter patterns at various angles
are measured and collated to classify cell
types.
Fluorescence technology also incorporated
to enumerate RNA (reticulocytes)and DNA
(nucleated red blood cells and white cell
abnormalities)

14. Optical Technology cont’d
14
Hydrodynamic focusing of cells is achieved by
sheath reagent being injected into the flow cell
at a greater rate than the diluted specimen. This
causes laminar flow which positions and spaces
the cells into the centre of the flow cell allowing
the cells to pass into the sensing zone.

15. Optical Technology Cont’d
15
The laser beam is perpendicular to the
path of the cells.
Light hits the cell causing light to scatter
at various angles. Hence the size of the
cell, it’s internal complexity and
granulation are measured.
If fluorescence technology is
incorporated, PMT’s will measure the
amount of fluorescence emitted.

16. Benefits of Optical Technology
16
Laser light can be focused on individual cells.
More than 2 measurements can be made.
-more information can be gathered giving better separation of
cell populations.
Cells are passed in single file through the flow cell.
- no issue with backflow into sensing zone
More realistic results. E.g. Mean Cell Volume.

17. Optical Technology – Flow Cytometry
Laser Beam
Focussed on
individual
cells.
Scattered laser
light, which is
picked up by a
series of
detectors,
allowing us to
classify cells on
their scatter
characteristics.
Sheath Fluid Surrounding sample stream
Cells pass through flow
cell in single file due to
HYDRODYNAMIC
FOCUSSING
17

18. Fluorescent Technology
18
Fluorescence is obtained when light is emitted
at a wavelength that is higher than that of the
light source
RNA fluorescence measured Reticulocytes
DNA fluorescence measures white cell viability,
nucleated red blood cells and fragile white
blood cells (eg. Smudge cells)

19. Analyser Running to Manufacturer’s
Specifications
19
Analyser background limits essentially zero.
No carryover following high cell counts.
Good precision.
Commercial Quality Controls running within
limits.
Patient Moving Averages within acceptable
limits

20. Cell Dyn Sapphire
20
 Processes 106 specimens per hour in CBC mode, and
only requires 117ul of blood (both open and closed
modes). If there are a lot of cytopenic patients
processed, there will be fewer processed per hour
 Processes 76 specimens per hour in CBC/Reticulocyte
mode, and still only requires 117ul of blood (for both
modes).
 WBC linearity is up to 250 x 109/L.
 Platelet linearity is up to 2,000 x 109/L.
 Utilises impedance, absorbance spectrophotometry,
optical and fluorescence technologies (3 colour
fluorescence).
 Nucleated red cells counted as part of an FBC analysis (no
reflex testing required) utilising DNA fluorescence

21. Cell Dyn Sapphire Cont’d
21
 Has an Argon ion solid phase (blue) laser with a
wavelength of 488nm.
 Ability to run specimens in an extended lyse mode if
lyse resistant red cells present.
 Red cells are “sphered” to enable consistent MCV
measurement.
 Fully automated and intergrated Monoclonal Antibody
testing for CD61 platelet counts and CD3,4 and 8 for
T-cell lymphocyte typing. Kits are available for CD64
Neutrophil sepsis marker and Hb F for foetal cell
enumeration. CD34 stem cell counting is at the
developmental stage.
 White cell viability measured using DNA fluorescence.

22. Multi Angle Polarised
Scatter Separation
Hydrodynamic focusing
22

23. Red Blood Cell and Platelet Counting
23
Red cell count and platelet counts are obtained
by both impedance and optical methods.
Optical platelets are enumerated using 2
dimensional light scatter at 7°and 90º using the
complexity of the platelets to effectively separate
the platelets from small or fragmented red blood
cells

24. Benefits Of Reporting the Optical
Platelet Count
24
Interfering substances NOT included in the optical
platelet count. These include: circulating blast
fragments, lipids, some protein complexes and
cryoglobulins.
Already stated improved separation between
platelets and fragmented and/or microcytic red
cells.
The lower the platelet count, the longer the count
period to give a more accurate count.

25. Impedance Red Cell and Platelet plots
vs. Optical Platelet plots
25

26. New Software Upgrades
26
Reticulated platelet counts processed when a
specimen is processed in CBC/Reticulocyte
mode
Reportable extended White cell Differential
Extended red cell parameters including red cell
cytograms
Extended Reticulocyte parameters

27. Normal Patient Reports from the
Cell Dyn Sapphire
27

28. Sysmex XE-5000
28
 Haemoglobin is measured by absorbance
spectrophotometry (cyanide free)using the SLS
method (reagent is sodium-lauryl-sulphate).
 Red cells are measured by impedance technology
 Platelets are measured by impedance and optical
light scatter technology.
 White cell count and differential using 3
technologies.
 Nucleated red blood cells and Reticulocytes are
measured using Fluorescence technology.
 The laser is a semiconductor type.

29. White Cell Count and Differential on
the Sysmex XE-5000
29
Uses Impedance Technology, direct current
(DC).
 Radio frequency technology (RF).
Optical and fluorescent light scatter.
Neutrophils, Lymphocytes and Monocytes
initially sorted by size and light scatter patterns
following lysis of red cells and platelets.
Eosinophils are classified by a different light
scatter due to their granulation. Basophils are
counted using low angle forward scatter and
side scatter at 90º after further lysis of all other
white cells except Basophils.

30. Sysmex XE-5000
30
Capable of processing 150 specimens per hour
on CBC, CBC+NRBC, CBC+
NRBC+WBC5DIFF modes.
113 specimens per hour in
CBC+NRBC+WBC5DIFF+RET and
CBC+RET modes.
Approximately 38 body fluid analysis per hour.
Requires 200uL of blood in the closed mode.
Requires 130uL of blood in the open mode

31. Radio Frequency and Direct Current
Methods
31

32. White Cell Count and Differential -
Optical
 Values for white cell
differential derived
from the scatterplots.
 Scatterplots are a
graphical
representation of
electronic data
generated by the laser
/cell interaction.
 Each coloured area
represents a
reportable parameter
(the dotted regions
indicate flagging
areas).
32

33. Special Features of the XE- 5000
33
Ability to quantitate the RNA content of platelets
when processing specimens in the reticulocyte
mode.
Measurement of stem cells (Human Progenitor
Cells) utilising the DC/RF white cell counts and
differentials.
Body Fluid analysis
High fluorescent Lymphocytes – reactive B
Lymphocytes
Additional red cell parameters ie % microcytes
and % macrocytes

34. Technicon Advia 2120i
34
CBC
CBC/Diff
CBC/Diff/Retic
CBC/Retic
Retic
120 Samples/hr
120 Samples/hr
120 Samples/hr
74 Samples/hr
74 Samples/hr
Reagents used specific to request

35. Advia 2120i - Improved Efficiency
35
Enhanced Linearity
WBC
RBC
Hb
Plt
Retic
0.02 - 400 x 10^9/L
0-7.5 x 10^12/L
0 - 225 g/L
5 - 3500 x 10^9/L
0 - 24.5%
175uL of blood required for processing in both the
open and closed modes.

36. White Blood Cell Technology
Dual WBC counts and Differentials.
Optical Light Scatter.
Peroxidase Staining.
Ce
ll
Size
Peroxidase Activity
Nuclear Complexity
36
Ce
ll
Size

37. Red Blood Cell Technology
Cellular Haemoglobin and Volume of rbc’s.
Dual Haemoglobin.
Accurate Morphology - rbc differential.
Macro
120fL
60fL
28g/dL 41g/dL
Micro
HgbHyCpooncentration(
g
H
/
d
y
p
L
)
e
r
37
RBC
Volume
(fL)

38. 38
Red Blood Cell Cytogram:
Visual Analysis
Normal -Thalassemia Trait
Iron Deficiency Anemia Sickle Cell Anemia

39. 2 Dimensional Platelet Analysis
Linearity - 5 to 3500 x109 PLT/L
Size range - 1 to 60 fL
Refractive Index - 1.35 to 1.40
Large PLTs included
RBC fragments excluded
39

40. Reticulocytes and Nucleated Red
Blood Cells
40
Nucleated red blood cells are automatically
counted when specimens are run in the
CBC/DIFF mode (based on size and intensity of
the nucleus).
Reticulocytes are measured in a specific
reticulocyte channel, and additional reticulocyte
parameters are derived such as the cellular
haemoglobin reticulocyte.

41. Special Features of the Advia 2120i
41
CSF Assay - FDA approved cell count and
differential
Cellular Haemoglobin cross checked with the
colorimetric Haemoglobin method.
Red cell differential - %hypo, %hyper,
% micro and % macro.
Cross checked WCC - Peroxidase and Baso
channel.

42. Beckman Coulter UniCel DxH 800
42
Utilises Impedance technology for White Cell
Counts, platelet counts and red cell counts.
Utilises Volume, Conductivity and Scatter (VCS)
principles for white cell differential and NRBC(5
angles of scatter)
Random access reticulocyte testing using new
methylene blue staining of reticulocytes and
analysis using VCS technology
Monoclonal capabilities

43. UniCel DxH 800
43
Processes 100 specimens per hour in CBC and
CBC/Diff modes (can take longer if there are a
lot of cytopenic patients).
Processes 45 specimens per hour in
CBC/Reticulocyte mode.
White cells linear from 0 – 400 x109/L
Platelet counts linear from 0-3,000x109/L
Sample path the same in open and closed
modes and requires 165uL of blood
Pre-dilute mode available - 50uL of blood to
200uL of diluent

44. VCS Technology - Volume
V is for Volume and is obtained when the
impedance white cell count is performed
and the cells are grouped by size.
44

45. VCS Technology - Conductivity
Conductivity is a measurement of cellular
internal content using a high-frequency
electromagnetic probe. The information gained
by this method looks at the cell size, the nucleus
to cytoplasm ratio as well as cell granularity
and chemical composition.
45

46. VCS Technology - Scatter
Scatter is the measurement of the light
scattering characteristics of a cell , which
gives valuable information on the
granularity and the nuclear structure of a
cell. The laser is a Helium-Neon Laser.
46

47. 47
DxH 800 – Multi-transducer module –
29 data measurements for each cell
Direct Current
Radio Frequency :
Opacity
Stretched Opacity
5 angles of Light Scatter :
Axial Light loss
Low angle light scatter
Median angle light
scatter
Lower Median angle
light scatter
Upper Median angle
light scatter

48. The VCS Cube
48

49. VCS Technology - cont’d
49
Eosinophils separated from other
populations.
Monocytes identified as a separate
population and then classified.
Neutrophils are separated from
Lymphocytes and Basophils.
Finally Lymphocytes and Basophils are
classified.
2-Dimensional separation completed.
Actual 3-Dimensional display.

50. Example of a Normal
Scatterplot
50

51. UniCel DxH 800
51
Body Fluid analysis for CSF, serous and
synovial fluids.
White cell differentials by monoclonal flow
cytometry technology (for white cells flagged as
being abnormal).
Red cell counts automatically corrected in the
presence of high White cell counts and other
interfering substances.

52. Normal Neonate on the CD Sapphire
Neonatal
specimens
are run in an
extended
lyse mode.
Note high
MCV and
Hb and low
number of
NRBC’s
52

53. Patient with Red Cell Agglutination - RT
RCC 2.16x1012/L
Hb 111g/L
MCV 81.0 Fl
MCH 51.4pg
MCHC 634g/L
RDW 16.3%CV
WCC 8.9x109/L
Platelets 392x109/L
53

54. Patient with Red Cell agglutination –
warmed to 37°C
RCC 4.21x1012/L
Hb 107g/L
MCV 82.9fL
MCH 25.5pg
MCHC 308g/L
RDW 15.0%CV
WCC 10.5x109/L
Platelets 361x109/L
54

55. Patient with Platelet Clumping
Patient with
known
Platelet clumping
with EDTA
collection
Platelet count of
57 x 109/L
55

56. Patient with lyse resistant red cells –
in CBC mode
WBC 31.4 x 109/L
Absolute Lymphocyte
22.0 x 109/L
Analyser gave a
“resistant red cell” flag
56

57. Patient with lyse resistant Red Cells – ran in
extended lyse mode
WBC is now 15.2 x 109/L
Lymphocyte Absolute
count is 6.0 x 109/l
Resistant red cell flag has
now disappeared
57

58. Acquired Education
58
Spend as much time in the laboratory as
possible.
Familiarise yourself completely with analyser
capabilities (and limitations) and information
given by the analyser.
Analyser flagging needs to be as accurate as
possible – incorrect flagging (either false
positive or negative flags) have a drastic effect
on blood film review rates.
Talk to the staff responsible for running and
maintaining the analysers.

59. Where to Next?
59
Integration of multiple analysers including automated
slide makers and stainers with a “tracking” system
especially in institutions with an extremley heavy
workload. Already in use in the largest Private
Laboratories
Further development of use of monoclonal antibodies –
reducing the workload for Flow Cytometry Depts.
?????????? And that’s an exciting aspect.

60. The true stars of this
presentation
60