Automation in finding hematologic counts...

2. Hematology Analyzer
Automated Cell Counting Instrumentation

3. Definition:
Hematology analyzers are computerized, highly specialized
and automated machines that count the number of
different kinds of white and red blood cells in a blood
sample.

4. History:
The original hematology
analyzers first appeared in
the 1950s, but the truly
functional and automated
versions of these machines
did not become available
for two more decades.
Before this time, cell
counts were performed
manually.

5. Disadvantages of manual cell
counting
Cell identification errors in manual counting:
Mostly associated with distinguishing lymphocytes from
monocytes
 Bands from segmented forms and abnormal cells
(variant lymphocytes from blasts)
 Lymphocytes overestimated
Monocytes underestimated
Slide cell distribution error
Increased cell concentration along edges

6. Advantages
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• They have a high level of precision for
cell counting and cell sizing greatly
superior to that of the manual
technology
• The results are generally accurate.
• No slide distribution error.
• Eliminate statistical variations
associated with manual count based on
high number of cells counted.
• Many parameters are available.
• All automated cell counters are
screening devices. (abnormalities)

7. Advantages..
•
•
•
•
•
•
Most current
hematology
analyzers provide
Red blood cell (RBC) counts
Hemoglobin in RBCs,
Hemocrit levels,
Platelet counts,
Corpuscular data,
Counts of five different types of white
blood cells.
• Some newer machines also measure
numbers of two specialized cell types
— immature white cells and
— nucleated red blood cells that are
necessary to confirm specific
diagnoses

8. Results:
The results they provide are collectively known as
complete blood counts (CBCs) .
complete complete blood with differentiation of cells
(CBCs with diff).
Some cell counters can process 120-150 samples per
hour .

9. Hematology analyzer
has revolutionised
Internal
medicine,
oncology
pediatrics

10. Types :
Automated techniques of blood counting:
I. Semi-automated instruments
Require some steps, as dilution of blood samples.
Often measure only a small number of variables.
II. Fully automated instruments
Require only that an appropriate blood sample is
presented to the instrument.
They can measure 8-20 variables including some new
parameters which do not have any equivalent in
manual methods.

11. Working
Hematology analyzers count cells by one of five general
cell techniques:
Flow cytometry
The use of fluorescent dyes
The electrical impedance method
The light scatter method
Radiofrequency

12. 1:Flow cytometry/Cell
sorting
o Flow cytometry is based on ejecting cells from a nozzle at
high speed in a fluid.
o Each cell passes through several laser beams so that
different optical properties can be measured.

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14. 2:Use of flourescent dyes
• Biochemical or antigenic properties are usually classified
using fluorescent dyes.

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Steps :
1.
2.
3.
4.
Labeling with flourescent dye.
A laser excites these fluorescent molecules.
They emit light at various wavelengths.
Amount of fluorescence can give an indication as to
what percentage of various cell types are present in the
sample.

17. 3:Electrical impedance
method
• Works on coulter principle:
A stream of cells in suspension passes through a small
aperture across which an electrical current is applied. Each cell
that passes alters the electrical impedance and can thus be
counted and sized.

18. Continue
• Particles such as blood cells are nonconductive
BUT
are suspended in an electrically conductive diluent.
•
As a dilute suspension of cells is drawn through the aperture,
the passage of each individual cell momentarily increases the
impedance (resistance) of the electrical path between two
electrodes that are located on each si.de of the aperture

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A blood cell's size ,
surface charge ,
concentration of the cells ,
shape of cells can be determined

20. 4:Optical scatter method
 The application of light scatter means that as a single cell
passes across a laser light beam
1. diffraction(bending around corners),
2. refraction (bending due to change in speed) and
3. reflection (light rays turned back by obstruction)
 Light scatter correlates to cell volume/size
Side angle/orthogonal light scatter correlates to degree of
internal complexity (granules and nucleus)

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The patterns of scatter are measured at various angles.
Multi angle polarized scatter separation (M.A.P.S.S)
•
0° :
indicator of cell size
• 10° :
indicator of cell structure and complexity
• 90° polarized:
indicates nuclear lobularity
• 90° depolarized:
differentiates eosinophils

23. Continue…
Scattered light provides information about
 cell structure,
 shape,
Reflectivity
These characteristics can be used to differentiate the
various types of blood cells and to produce scatter plots
with a five-part differential.

24. 5:Radiofrequency
Conductivity (RF) – proportional to cell
interior density (granules and nucleus)

25. Examples

26. o An automated retic count
 Fluoro-chromes combine with the RNA of the
reticulocytes.
Fluorescent cells can then be enumerated using a
flowcytometer.
 An automated retic counter also permits the
assessment of retic maturity since the more immature
reticulocytes have more RNA

27. o Coulter Instrumentation
Principles of Measurement
• Direct Measurement:
• RBC – Impedance
• WBC - Impedance
• Platelets – Impedance (2-20 fl)
• Hgb – mod. Cyanmethemoglobin (525 nm)
• MCV – mean RBC volume (histogram)
• Indirect Measurement:
• Hct, MCHC, and MCH (calculations)
• WBC differential
• VCS – volume, conductivity, scatter
• employs differential shrinkage
• Reticulocyte
• Supravital stain (new methylene blue)
• VCS

28. Coulter hematology analyzer

29. Sysmex Instrumentation
Principles of
Measurement
•Direct Measurement:
•RBC – DC detection (Impedance), hydrodynamic
focusing
•WBC – DC detection (Impedance),
hydrodynamic focusing
•Platelets – DC detection (Impedance),
hydrodynamic focusing
•(2-30 fl)
•Hgb – SLS-Hb (555 nm) (oxyhemoglobin +
sodium lauryl sulfate)
•HCT – cumulative pulse height detection (see
oscilloscope)
•Indirect Measurement:
•MCV, MCHC, and MCH (calculation)
•WBC differential
•DC/RF detection
•employs differential lysis
•Reticulocyte
•Fluorescent detection

30. Symax hematology analyzer

31. Automated and portable hematology
analyzers

32. Drawbacks
Some problems which could be faced:
 Two cells passing through the orifice at the same time,
counted as one cell.
 RBC agglutination(clump of cells) Counting bubbles
 or other particles as cells.

33. Questions????

34. Thank
you