3. History
Antonie van Leeuwenhoek
Invented Microscope
The world of cells was colorless
until Paul Ehrlich stained blood cells
Wallace Coulter :First automated analyzer for
counting and sizing cells and presented it in 1956
4. Automation In Hematology
Cell counts(Automated hematology analyzers)
Diagnosis of hemoglobinopathies(HPLC)
Immunophenotyping(Flow cytometry)
Coagulation(Coagulometers)
5. Automated Hematology
Analyzers
Hemogram-Backbone of any lab evaluation
As a routine investigation
Including anaemia, polycythemia,
infection, inflammation,
allergy, drug toxicity, malignancy,
bleeding tendency etc
Aim-to study RBC, WBC series and platelets
6. Advantages
Speed with efficient handling of large number of samples
Accuracy and precision in
quantitative blood tests
Ability to perform multiple
tests on a single platform
Significant reduction of labor
requirements
Invaluable for accurate determination
of red cell indices
7. Disadvantages
Flagging of a laboratory test result demands labour intensive
manual examination of a blood smear
Comments on red cell morphology
cannot be generated
Platelet Clumps are counted as single.
so low count.
Erroneously increased or decreased
results due to interfering factors
Expensive with high running costs
8. Types of counters
Semi automated :
Some steps carried out manually like dilution of blood
Measures only a few parameters
Fully automated:
Require only anticoagulated blood samples.
Measures multiple parameters
9.
10. Components of a cell counter
3 Basic components
Hydraulics:
Includes aspirating unit, dispencers,diluters,mixing
chambers, aperture baths &
hemoglobinometer
Pneumatics :
Vacuums & pressure for
operating valves
Electronics :
Analyzer &computing circuit
11. Principles of Working
Electrical Impedance
Optical Light Scatter
Fluorescence
Light absorption
Electrical conductivity
?!!@#@#
12. Electrical Impedance
First introduced by Wallace
Coulter
Blood cells are poor conductor of
electricity
2 chambers filled with a
conductive
buffered electrolyte solution
Separated by a small aperture
DC current between two
electrodes
13. Electrical Impedance
Diluant displacement causes
potential
difference
Voltage pulse displayed on
an osciloscope
No. of impulse = No. of cells
Height = vol. of cells
Freq dist curve & size dist
histograms
Requisite – High dilution
14. Optical Light Scatter
Each cell flows in a single line
through a flow cell
A laser device focussed
On striking on cells scattering
in different directions
Sensor capture & multiplies
Forward angle light scatter
(FALS)-Cell Size
Side scatter(SS)-Granularity
15. Other Methods
Peroxide based counters:
MPO is used to count neutrophils.Lymphocytes not stained
Fluroscence based:
Retic and platelet count.Immature pltlets detected best
Immunological based:
Accurate platelet count using CD41/CD61 antibodies
20. IP Messages
Interpretive messages
Assist the laboratory in
screening for abnormal
samples that may need
verification
Seen at the bottom end of
hemogram
21. Indicators that may appear after the
data
@ : Data is outside the linearity limit
* : Data is doubtful
+ or – :Data is outside the reference limits.
---- : Data doesn’t appear due to analysis error or
abnormal sample
++++ : Data exceeds display limit.
22. Discrimination Thresholds
WBC Discriminator
WBC LOWER discriminator-the optimum position in 30 - 60 fL .WBC
is calculated from the particle counts more than this LOWER
discriminator.
RBC Discriminator
RBC LOWER discriminator- optimum position in 25 -75 fL and UPPER
discriminator, 200 - 250 fL,.
RBC is calculated from the particle counts between this LOWER
discriminator and UPPER discriminator.
PLATELET Discriminator
PLT LOWER discriminator, the optimum position in 2 – 6 fL and and
UPPER discriminator- 12 - 30 fL,
23. Histograms
These are the graphical presentation
of numerical datas of different cell
populations in a cell counter.On the
X-axis is the cell size and on the Y-
axis is the number of cells.
Used to determine:
The average size
Distribution of size
Detect subpopulation
24. Flagging System
Whenever any significant
abnormalities of any cell
present , signalled by
certain ‘asteriks’ on the
report.
Every instrument has its
own flagging system.
37. F1,F2,F3 Flags
No other flags are present
But valleys are far from base line
F1(small cell inaccurate):Height of
T1 exceeds limit of 40%
ALL
F2(medium cell
inaccurate):Heights of T1 & T2
exceeds limit of 40% & 50%
Respectively AML,Eosinophilia
F3(Large cell data inaccuraate)
Height of T2 exceeds limit of 50%
F1 FF2 F3
46. Volume
As opposed to using 0ø light loss to estimate cell
size,VCS utilizes 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.
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 cell
size and internal structure, including chemical
composition and nuclear volume.
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 (MALS) signals, are
collected to obtain information about cellular
granularity, nuclear lobularity and cell surface
structure
48. Newer Parameters(contd..)
Cellular Hb Concentration Mean(CHCM):
Uses Light scatter technology.
True estimate of hypochromia in IDA.
Hb Distribution Width:
Degree of variation in red cell hemoglobinization.
Range-1.82 to 2.64.
Nucleated Red Cells:
nRBCs identified,separated & corrected count obtained.
WBCs have high fluorescence & forward scatter.
49. Newer Parameters(contd..)
Reticuloctes:
Various dyes & flurochromes bind with RNA
RNA content- 3 Maturation stages; LFR,MFR & HFR
Immature reticulocyte Fraction(IRF):
Sum of MFR & HFR.
Early and sensitive index for erythropoisis.
Reticulocyte Hb Equivalent(RET-He):
Hb content of freshly prepared RBCs.
Real time information on Fe supply to erythropoiesis.
Early detection of Fe deficiency.
Differentiate IDA & ACD.
Monitoring of erythropoietin & Fe therapy.
50. Newer Parameters(contd..)
P-LCR(Platelet Large Cell Ratio):
% of platelets with a vol >12fl.
Due to platelet aggregates,microerythrocytes,giant platelets.
Reticulated Platelets /Immature Platelet Fraction(IPF):
Newly produced platelets that have remains of RNA in their
cytoplasm.
Reflects rate of thrombopoiesis.
51. WBC Research Population Data Case Study – Malaria Parasites(Normal plot and
Research Population Data compared to a patient infected with malaria type
Plasmodium falciparum. Note the increased size and variation of the lymph's and
Monocyte's.)
NORMAL
Normal
Normal MO
Normal LY
Macrophage
Parasitized
RBC
MALARIA
MP Positive
Reactive LY
53. Terminologies
Quality Assessment:
Adequate control of the pre & post analytical from
sample collection to report dispatch.
Quality Control:
Measures that must be included during each assay
run to verify the test working properly.
Proficiency Testing:
Determines the quality of results generated by lab.
54. Internal Quality control:
Continuous evaluation of the
reliability of the daily works of
the lab with validation of tests.
External Quality Control:
Evaluation by an outside agency
of between-laboratory &
between-method comparability.
55. Accuracy & Precision
Accuracy:
Refers to closeness to the true
value
Precision:
Refers to reproducibility of
test
1 2
3 4
56. Controls & Calibrators
Controls:
Substances used to check the precision .
Analyzed either daily or along each batch.
Should have same test properties as blood
samples.
Stabilized anticoagulated whole blood or
pooled red cells.
3 conc.-high,normal ,low
Calibrators:
Check the accuracy.
Value assigned to them by a reliable ref.
center.
57. precision
Use of controls:
Most convenient & accurate procedure.
Prepared in house or obtained commercially.
10 consecutive values of control recorded and Mean & SD
calculated.
3 conc of control to be analyzed.
Plotted in Levey-Jennings Chart.
59. Control Values and Decision
Consider using Westgard Control Rules
Use premise that 95.5% of control values should fall
within ±2SD
Commonly applied when two levels of control are used
Use in a sequential fashion
61. 12S Rule = A warning to trigger careful inspection
of the control data
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
12S rule
violation
62. 13S Rule = Reject the run when a single control
measurement exceeds the +3SD or -3SD control limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
13S rule
violation
63. 22S Rule = Reject the run when 2 consecutive control
measurements exceed the same +2SD or -2SD control
limit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
22S rule
violation
65. 10x Rule = Reject the run when 10 consecutive
control measurements fall on one side of the mean
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Mean
Day
+1SD
+2SD
+3SD
-1SD
-2SD
-3SD
10x rule
violation
66. What to do when Control Value is
out of limit?
“out of control”
Stop testing
Identify and correct problem
Repeat testing on patient samples and
controls
Do not report patient results until
problem is solved and controls indicate
proper performance
67. Accreditation
Certification by a duly recognized authority of
facilities, capability, objectivity, competence and
integrity of an agency.