Cell counter for hematology

1. 5 PART AUTOMATED CELL
COUNTER
PRESENTED BY,
DR. DIVYA DONEPUDI
MODERATOR,
DR. SHAILAJA PRABHALA

2. INTRODUCTION
Cell counting is a method used to determine the number of cells in a sample
17th Century
• Robert Hooke discovered cells in cork
19th century
• Visually counting cells using a microscope
• Hemocytometer was developed, which allowed for the accurate counting of blood cells
20th century
• Flow cytometry uses lasers and fluorescent dyes to count cells
Present
• Automated cell counting, including image analysis software and microfluidic devices

3. MANUAL COUNTING
Pros
•Low cost : Done with basic equipment
like a microscope and a
hemocytometer, making it a cost-
effective method.
•Flexibility : Counting cells from
various samples and use different
types of stains to visualize cells.
•Control : Control over the counting
process, making it easier to identify
specific cell types or distinguish
between living and dead cells.
Cons
•Time-consuming: Labor-intensive
process taking hours or even days
depending on the sample size.
•Subjectivity : Prone to human error,
and different researchers may count
cells differently, leading to variations
in results.
•Limited accuracy : Might be
inaccurate due to variations in the
size and shape of cells, as well as cell
clumping.

4. AUTOMATED COUNTING
Pros
•Speed: Much faster than manual counting,
with results obtained in a matter of
minutes.
•Accuracy: Less prone to human error,
resulting in more accurate and consistent
results across different experiments and
researchers.
•Multi-parameter analysis: Cell count,
viability, morphology, size, and
granularity, providing more detailed data
than manual counting.
Cons
•High cost: Requires specialized
equipment, which can be expensive and
may not be affordable for all labs.
•Sample limitations: Automated counters
may not be suitable for all types of
samples, as certain samples may require
manual processing or specialized
equipment.
•Complexity: Automated counting can be
complex, requiring training and expertise
to operate and interpret the results.

5. Features
Three-Part Automated Cell
Counter
Five-Part Automated Cell
Counter
Number of cell
types counted
Red blood cells, white blood cells
(Neutrophils, monocytes and
lymphocytes) and platelets
Red blood cells, Platelets White
blood cells (neutrophils,
lymphocytes, monocytes,
eosinophils, basophils)
Accuracy and
precision
Lower due to limited differentiation
capabilities
Higher due to wider range of cell
type differentiation
Cost
Less expensive due to simpler technology
and reagents
Expensive due to more complex
technology and reagents required
Sample volume
Requires a larger sample volume for
accurate results
Requires a smaller sample volume for
accurate results

6. COULTER & FLOW
CYTOMETRY

7. COULTER CELL COUNTER
In 1953, Wallace Coulter patented the Coulter principle in which
particles were counted in fluid which passed through an orifice.
Coulter Corporation introduced the first commercial cell counter, which
used the principle of impedance to count cells.
Following years : Various types of automated cell counters were
developed, including those that used light scattering, fluorescence, and
image analysis.

8. The Coulter Principle
• Works on Ohm’s law V = I *R
• As a cell passes through the
aperture, electrolytic solution is
displaced which is equal to size of
cell, increasing impedance of
electrolytic solution.
• Current is constant, therefore in the
formula the voltage V will increase.
• The bigger the cell, the more the
resistance, the greater the voltage.
• Each voltage spike is directly
proportional to the size of the cell.

10.  There are two chambers one measures Hb + WBCs and second chamber counts RBCs +
PLTs
 The aspirated whole blood specimen is divided into two aliquots and mixed with an
isotonic diluent
 The first dilution is delivered to the RBC aperture bath, and the second is delivered to the
WBC aperture bath.
 In the RBC chamber, both the RBCs and the platelets are counted and discriminated by
electrical impedance.
 Particles between 2 and 20 fL are counted as platelets (4.5 – 12.3 fL), and those greater than
36 fL are counted as RBCs (70-120 fL).
 A reagent to lyse RBCs and release hemoglobin is added to the WBC dilution before the
WBCs are counted by impedance in the WBC bath.
 The electrical pulses obtained in the counting cycles are sent to the analyzer for editing and
digital conversion.
WORKING OF CELL COUNTER

11. Raw data from Oscilloscope
X- Axis : Time
Y- Axis : cell size
Data from analyzer is
organized in an ascending
order
X-Axis : Occurrence
Y- Axis : Cell size

12. In the electrical impedance system, raw data is generated and the analyzer’s computer classifies the raw data
which is sorted and histograms are then smoothed (smooth curve) into distribution curve.
In the above graph,
X- Axis : Cell Size in fL
Y- Axis : Frequency of occurrence

13. FLOW CYTOMETRY
 Derived from the Greek words "cyto" (cell)
and "metry" (measurement) by using
“Flow”- movement of cells
 This technology enabled the use of
fluorescent markers to label specific
cellular components
 Allowing sorting and analysis of different
cell populations based on their fluorescence
properties.

14.  Each cell will pass through a detection device called a flow cell.
 This flow cell will have a laser device focused on it and as the cell passes through the laser
light path, it will scatter light in several directions.
 The unit has one detector that captures the forward scatter light (FSL) and a second detector
that captures the light that is scattered (side scattered or SS) at 90°
Light scattering & Flow cytometry

16. Principle
Light Scattering Principle
Interaction of light with cells
Coulter Principle (Electrical Impedance)
Change in electrical impedance
Detection Photodetectors capture scattered light Electrodes measure impedance change
Advantages
- Information about cell size, shape,
granularity, and internal complexity
- Non-invasive
- Suitable for live cell analysis
- Highly accurate cell counts and size
measurements
- Less affected by debris or cell aggregates
- High resolution for small particles and cells
Limitations
- Affected by sample quality, debris, or
aggregates
- Limited resolution for very small cells or
particles
- Requires conductive liquid media and specific
calibration
- Limited information about cell shape, internal
complexity, and granularity
- Invasive method, may affect cell viability
Applications
- Cell counting
- Cell size and shape analysis
- Internal complexity and granularity analysis
- Cell counting
- Cell volume measurements
- Counting small particles or cells
COMPARISON BETWEEN LIGHT SCATTERING & COULTER

17. HORIBA YUMIZEN H550
Working
Principle
• Cytometry : DHSS (Double Hydrodynamic
Sequential System)
• Light Scattering
• Impedance
Sample
Management
• Automatic rack Mixing
• Only 3 reagents, Whitediff®, diluent,
cleaner

18. General out line of processing of one sample
Identify the
sample by
bar code
Mix the
sample
Pierce the cap of
sample tube and
aspirate the sample
Split the sample into
different channels
eg.RBC n Platelet counting
WBC –TC-DC counting
Retic counting
Haemoglobinometry
IN EACH CHANNEL:
make relevant dilution
Add reagent
Wait until the dilution
is ready for
measurement
Make
measuremen
t of each
channel
Flag if any
abnormal
reading
Display the
suitable result
to the
operator
Transfer the
data to lab
computer
system
They are equipped
with automatic
cleaning procedure

19. Patient
Preparation
Sample
Collection
Sample
Handling
Sample
Transport
Sample
receiving
Analysis
Results
Reports
Post
Analytic
Pre-
Analytic
Analytic
Factors affecting Quality

20. Interpretation

22. Normal RBC Histogram
RL – Lower Range wrt
detection of RBC
RU – Upper Range wrt.
detection of RBC
Mean Corpuscular Volume
(MCV) is calculated from the
area under the peak.
Red blood cell Distribution
Width (RDW) is also calculated
from the data used to calculate
the MCV
MCV =
𝐻𝐶𝑇 (𝐻𝑖𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡)
𝑅𝐵𝐶 (𝑅𝑒𝑑 𝐶𝑒𝑙𝑙 𝐶𝑜𝑢𝑛𝑡)
* 100
MCH=
𝐻𝑏 (𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛)
𝑅𝐵𝐶 (𝑅𝑒𝑑 𝐶𝑒𝑙𝑙 𝐶𝑜𝑢𝑛𝑡)
* 100
MCHC =
𝐻𝑏 (𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛)
𝐻𝐶𝑇 (𝐻𝑖𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡)
* 100

23. RDW-CV (%) = 100 x s/µ
RDW-CV: 11.5 – 14.5%
100 %
20 %
RDW-SD is not a statistical SD, but measured by
drawing an arbitrary line at a height of 20% on the
y-axis in femtoliters
RDW-SD: 35 - 45 fl
The RBC distribution width
gives a measure of
anisocytosis.
Red cell Distribution width (RDW)
100 %
s s
µ
Points of inflection
68,26 %
of all values
s - Standanrd
Deviation
µ - Mean

24. Abnormal RBC Histogram
Abnormal height at lower
discriminator RL (Lower
Range) flag
This flag is seen when the
LD exceeds the preset
height by greater than
10%

25. Abnormal RBC Histogram
Abnormal height at
upper discriminator
RU flag
This flag is seen
when the UD
exceeds the preset
height by greater
than 5%.

26. Abnormal RBC Histogram
Abnormal distribution
width (DW)
Abnormal histogram
distribution RDW – SD
or RDW – CV is flagged.

27. RBC anisocytosis-
Multiple peaks
(MP)
Abnormal RBC Histogram

28. • Lower discriminator
(LD) fluctuating
between 30 and 60 fL
• Upper discriminator
(UD) fixed at 300 fL.
• The number of cells
between the UD and the
LD is the WBC count.
• WBC histograms
consist of two troughs,
T1 between 78 and 114
fL and T2 < 150 fL.
 The peak between the LD and T1 represents small cells, i.e lymphocytes. The volume of the cells range from
35 to 90 fL.
 The peak that lies between T1 and T2 represents the mid cell count which includes the eosinophils, basophils,
monocytes, blasts and promyelocytes. Volume of the cell ranges from 90 to 160 fL.
 The peak after T2 represents neutrophils. Volume ranging between 160 to 300 fL.
Normal WBC Histogram

29. Abnormal WBC Histogram
Abnormal
Curve in Front
of the Lower
Discriminator

30. Abnormal WBC Histogram
Abnormal Curve at the Lower Discriminator–Wl Flag
This appears when height of LD is greater than the preset 20% of Y-axis. As a result of this the WBC count,
W-SCR (small cell region), W-MCR (mid cell region) and W-LCR (large cell region) will show a Wl flag

31. Abnormal WBC Histogram
Abnormal Curve at the Upper Discriminator–WU Flag
This appears when the height of UD is greater than the preset 10% on the Y-axis. As a result the WBC count
will show a WU flag. It occurs when there is insufficient WBC lysing.

32. Abnormal WBC Histogram
Abnormal Curve at T1 Level—T1 Flag
 The T1 and T2 discriminators are flexible and will be set automatically according to the sample.
 In extreme pathological condition discrimination between 3 population is not possible.
 Flag T1 occurs when discrimination between lymphocytes and mid cell population is not done as in
abnormal leukocytosis like chronic myeloid leukemia.

33. Abnormal WBC Histogram
Abnormal Curve and Flagings—F1, F2 and F3 Flag
F1 flag appears when the relative height of T1 exceeds preset limit of 40%. F1 flag means that the small
cell and middle cell data may be inaccurate. It may occur in acute lymphoblastic leukemia.
F2 flag appears when the
relative heights exceed
the preset of T1 (40%) or
T2 (50%). F2 flag means
that the middle cell data
is inaccurate. This often
occurs in eosinophilia,
acute myeloid leukemia,
monocytosis, etc.
F3 flag appears when the
T2 exceeds the preset
limit of 50%. F3 Flag
means that the large cell
data is inaccurate.

34. Abnormal WBC Histogram
Abnormal Curve at T2—T2 Flag
Flag T2 appear when discrimination between mixed cell and neutrophil could not be done, as in
chronic lymphocytic leukemia.

35. Normal Platelet Histogram
Mean platelet volume (MPV)
This is a mathematical calculation to
determine the average size of the
platelets. The average MPV range = 7.4
to 10.4 fL.
MPV = PCtx1000/Plt fl
Platelet distribution width (PDW)
The platelet distribution width (PDW) is
the width of the curve of distribution of
platelets related to the different sizes
produced by these cells

36. Abnormal Platelet Histogram
PL Flag
This occurs when the lower discriminator exceeds the preset height by 10% the platelet count, MPV
and P-LCR will show the PL flag.

37. Abnormal Platelet Histogram
PU Flag
This occurs when the upper discriminator exceeds the preset height by more than 40%.

38. Abnormal Platelet Histogram
MP Flag (Multipeaks in PLT Histogram)

39. WBC Differential Scattergram

41. Reference
• The ABC of CBC Interpretation of Complete Blood Count and Histograms, DP Lokwani.
• Flow Cytometry Animation GIF | Gfycat
• Flow Cytometry Animation GIF | Gfycat
• https://www.horiba.com/gbr/medical/products/detail/action/show/Product/yumizen-h550-1851/
• https://www.semanticscholar.org/paper/Overview-of-Automated-Hematology-Analyzer-XE-2100-
Inoue/bcda80fdb303611a9d1db457346128a252f447d5/figure/6