A brief discussion about the instrument. The purpose of it. the way of handling..

1. HAEMATOLOGY ANALYZER
Automated Cell Counting Instrumentation
Presented By:
Sukanya Halder
M.Tech, Biotechnology
KIIT School Of Biotechnology

2. 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.

3. 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.

4. 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

5. Continue…
• 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)

6. 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

7.  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 .

8. Internal
medicine,
pediatricsoncology

9. 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.

10. 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

11. 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.

13.  Biochemical or antigenic properties are usually
classified using fluorescent dyes.

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

16.  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.

17. 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

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

19. 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)

21. 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

22. 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.

23. Conductivity (RF) – proportional to cell
interior density (granules and nucleus)

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

26. 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

27. Coulter hematology analyzer

28. 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

29. Symax hematology analyzer

31. 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.