Automated cell counters

1. BY: SIVARANJINI. N
GUIDE : DR. YOGITA.V. KARELIYA
AUTOMATED CELL COUNTERS:
PRINCIPLE AND TYPES

2. The complete blood count (CBC)
including DLC are the backbone of
any laboratory evaluation and
provide valuable information to
diagnose anemia, acute and chronic
illnesses, white cell disorders,
leukemias and platelet disorders.

3. Haematologic evaluation nearly always
requires-
⚫ Enumeration of the formed elements of
the
blood
⚫ Determination of the morphology of
each type.
Manual methods are
- time consuming,
- tedious
- imprecise.

4. Wallace H. Coulter an electrical engineer
and businessman while working with the
US Navy in the late 1940s developed and
first applied the Coulter Principle

5. TYPES OF CELL-COUNTERS
THREE PART FIVE PART SEVEN-PART
Differentiate
cells into three
categories
• Granulocytes
• Lymphocytes
• Monocytes/m
ixed cells
Differentiate
cells into the
five basic
Leukocyte types
1.Neutrophils
2.Eosinophils
3.Basophils
4.Lymphocytes
5.Monocytes
In addition are
able to
distinguish
1.Nucleated
RBCs, Abnormal
and atypical
cells and
immature cells

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. Principle
⚫Electrical impedance
⚫Optical light scatter
⚫Fluorescence
⚫Light absorption
⚫Electrical conductivity

9. ELECTRICAL IMPEDANCE

10. COULTER PRINCIPLE
⚫The poorly conductive blood cells are
suspended in a conductive diluent
(liquid).
⚫The diluent is passed through an electric
field created between two electrodes.
⚫The liquid passes through a small
aperture (hole).
⚫The passage of each particle through the
aperture momentarily increases the
impedance (resistance) of the electrical
path between the electrodes.
⚫The increase in impedance creates a
pulse that can be measured.

11. The number of pulses = blood cell count
The amplitude (height) of the pulse = Volume of cell
⚫Depending on this, frequency
distribution curves and size
distribution histograms are plotted.
⚫Threshold limit based on size of
RBCs, WBCs and platelets are
established to separate individual
cells.
⚫Applied in 3 part differential count

12. RBC SIDE
DILUTION
(1:50,000)
>30fl size
RBC
2-30fl size
PLATELETS

13. WBC SIDE
DILUTION
(1:500)
35-90 fl
lymphocytes 90-60fl
Monocytes,
eosinophils,
basophils
160-450fl
Neutrophils
LYSING
REAGENT

14. Discrimination threshold
⚫Moving or fixed discriminator
separates the distribution curves for the
volume. E.g.,
⚫Platelets: 8-12 fl, (range is 2-30 fl)
⚫RBCs: 80-100 fl (range is 40-250)
⚫WBC: 30-300 fl (after RBC lysis)
subdivided into,
⚫Lymphocytes: <50 fl
⚫Neutrophils: 100-250 fl
⚫Intermediate forms (eosinophils,
basophils, monocytes) : 50-100 fl
(T1-T2)

15. ⚫The Size
Distributio
n Curve
should
always start
on the base
line and
fall
between
the lower
and the
upper
discriminat
or

16. OPTICAL SCATTER

19. FORWARD ANGLE LIGHT
SCATTER
⚫Illuminating beam that has been bent to a
small angle from direction of the original
beam .It measures size or volume of cells

21. SIDE SCATTER
⚫The illuminating beam that is scattered by
particle to an angle of 90* from the
illuminating beam.
⚫This depends on cell's surface texture and
internal structure as well as to its size and
shape and granularity.
⚫It is sometimes referred to as a granularity
signal or an orthogonal light scatter signal

23. ⚫The patterns of scatter are measured at
various angle.
⚫Scattered light provides information
about cell
structure, shape, and reflectivity.
⚫These characteristics can be used to
differentiate the various types of blood
cells and to produce scatter
plots(scattergram) with a five-part
differential

24. OTHER METHODS
⚫Peroxidase based counter:
Used to count neutrophils. Lymphocytes
are not stained.
⚫Fluorescence based:
Reticulocyte and platelet count. Immature
platelet are detected best.
⚫Immunological based:
Accurate platelet count using CD41 and
CD61 antibodies.

25. ERRORS
1. Recirculation error
Cells that re circulate through the edge of an
electrical field produce an aberrant impulse,
which is smaller than cell passing through the
aperture.
2. Coincidence error
Cells that pass through the aperture
simultaneously, or almost so, are counted and
sized as a single large cell called coincidence.
3. Non central flow error
Cell pass through the aperture off centre
produce aberrant impulses and appear larger
than their actual size

26. HYDRODYNAMIC FOCUSING AND
LAMINAR FLOW OVERCOMES
THESE ERRORS

27. ⚫ Principles of Measurement
Direct Measurement:
⚫ RBC –Impedance,
hydrodynamic focusing
⚫ WBC -Impedance,
hydrodynamic focusing
⚫ Platelets –Impedance (2-20 fl),
hydrodynamic focusing
⚫ Hb–mod.
Cyanmethemoglobin(525 nm)
⚫ MCV –mean RBC volume
(histogram)
Indirect Measurement:
⚫ Hct, MCHC, and MCH
(calculations)
⚫ RDW and MPV (CV of
respective histograms)
WBC differential
⚫ VCS –volume, conductivity,
scatter; employs differential
shrinkage
Reticulocyte
⚫ Supravital stain
⚫ Fluorescent detection

28. Parameters provided by automated
hematology analyzers
⦿Hemoglobin (Hb)
⦿Hematocrit (HCT)
⦿RBC count (RBC #)
⦿Platelet count (PLT #)
⦿WBC count (WBC #)
⦿WBC differential
(WBC %)
⚫ MCH (Hb/RBC
#)
⚫ MCV (HCT/RBC
#)
⚫ MCHC
(Hb/HCT)
⚫ RDW (RBC
volume)
⚫ MPV (Plt
TV/PLT)
⚫ Histograms

29. Hemoglobin estimation
Blood mixed with
RBC lysing agent
Cyan-meth
Hb method
Sulph-
meth Hb
method

30. Red Cell Indices

31. Hematocrit calculation
⚫ The volume of the red cells as compared
to the volume of the whole blood sample.
⚫The volume of each red cell is measured as
it is counted and a mean cell volume is
derived. i.e.
⚫(MCV) multiplied by the red cell count
(RBC(.
⚫Hematocrits are reported in L/L or the
traditional %.

32. ⦿Correlating Hemoglobin and Hematocrit
Values
◼The hemoglobin times three roughly equals the
hematocrit in most patients.
◼ Example: 14.8 x 3 = 44 (patient's hematocrit
result is 45 L/L)
◼11.0 x 3 = 33 (patient's hematocrit result is 32
L/L)
◼The exception to this rule is in patients with
hypochromic red cells. These patients will have
hematocrits that are more than three times the
hemoglobin

33. ⚫MCV The counter provides us with MCV
which is derived from the histogram (sum of
pulse height / sum of pulse)
⚫MCH is Mean Corpuscular Hemoglobin
weight in picograms. This is the average
weight of the hemoglobin in picograms in a red
cell.
MCH =hemoglobin in pg/L / red cell count in
millions/L
⚫MCHC is Mean Corpuscular Hemoglobin
Content. This indicates the average weight of
hemoglobin as compared to the cell size.
MCHC = (Hemoglobin in g/dL / HCT) x 100

34. X axis: Volume
of cells
Y axis: No. of
cells
LD: 25-75
fLUD: 200-250
fL
⚫ MEDIAN =
MCV

35. ⦿RDW: The RDW (red cell distribution
width) is a measurement of the width of
the bases of the RBC histogram.
⦿MPV: The MPV is a measure of the
average volume of platelets in a sample
and is analogous to the erythrocytic
MCV.
⦿Pct: (plateletcrit) analogues to HCT for
RBCs

38. Known interfering substances
⚫RBCs
1. High WBC count esp. with low RBC
count
2. Agglutinated RBCs
⚫ Hemoglobin
1. Increased turbidity of sample:
Leukocytosis, Lipemic sample, Fetal
blood.
⚫ MCV
1. RBC aggregation
2. Large platelets

39. ⚫Hematocrit : RBC agglutination
⚫RDW
1. Nutritional deficiencies
2. Blood transfusion
3. RBC agglutination

40. Interference with WBC counts
⚫High TC
1. Normoblasts
2. Lyse- resistant RBCs
⚫ In certain leukemias increased cell
fragility can give rise to low counts
⚫ Increased Lymphocytes
1. Nucleated RBCs
2. Lyse resistant RBCs

41. ⚫Increased monocytes
1. Increased large lymphocytes
2. Blast cells
⚫ Increased neutrophils
1. Blast cells
2. Eosinophilia

42. Interference with platelet
counts
⚫ High count
1. RBC fragments
2. Microcytic RBCs
3. WBC fragments
⚫Low count
1. Platelet
fragmentation
i.e.. in
chemotherapy
2. Platelet
agglutination

43. RBC histogram
Main RBC
population
RBC
doublets

44. ⚫Platelets have a size between 8 and 12 fl
and are counted between 2 and 30 fl.
⚫Erythrocytes have a size of 80-100 fl and
are counted between 25 and 250 fl.

45. RBC Normal Histogram
RBC histogram starts at 50fl and ends at 150fl

46. Discriminant functions in
thalassemia

47. RU-Flag
⚫ Normoblasts
⚫ Cold
agglutinins:
disappears
on
incubating.

48. RL- Flag
When LD exceeds preset height by more than 10
%
Large platelets
Fragmented RBCs
Platelet aggregation

49. MP-Flag (multiple peaks)
⚫ Blood transfusion
⚫ Dimorphic anaemia
⚫ Treated IDA

50. Platelet histogram

51. ⚫The histogram
curve should lay
within the lower
and upper
platelet
discriminator (PL
& PU) and start
and end on the
base line.
⚫PLT counted
between 2 fl and
30 fl.
⚫1 flexible
Discriminator PL
2 to 6 fl.
⚫1 flexible

53. PL-Flag
⚫When LD exceeds
preset ht. by 10%.
⚫Cell Fragments
⚫Contamination
⚫Bacteria
⚫Noise

54. PU-Flag
⚫When UD exceeds
preset ht. by > 40%.
⚫Clotted Blood
⚫Fragmented RBCs
⚫Pseudo-
thrombocytosis
⚫Large platelets

56. WBC HISTOGRAM

60. WBC Normal Histogram
The WBC graph shows a normal distribution of Lymphocytes, Monocytes
and Granulocytes.
Lympho Mono Granulo

61. WL-Flag
⦿Curve does not start
at base line
⦿Causes:
⦿Platelet aggregation
⦿Lyse resistant RBCs
⦿cryoaggutinates
⦿nRBCs.
⦿Giant platelets

62. WU- Flag
⚫ Curve does not end at baseline
⚫ Immature WBCs
⚫ Hyperleucocytosis

63. T1 Flag
⚫When
discrimination
b/w lymphocytes
& middle cells is
not possible.----
T1 is not found
⚫Abnormal
leukocytes in
CML

64. T2 Flag
⚫ When
discrimination
b/w neutrophils
& middle cells is
not possible.----
T2 is not found.
⚫ Higher WBC
count in CML.

65. F 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,
monocytosis
⚫F3(Large cell data inaccurate):
Height of T2 exceeds limit of 50%

67. FIVE PART AND SEVEN PART
COUNTERS

69. ⚫VOLUME =SIZE
⚫CONDUCTIVITY = INTERNAL
COMPOSITION
⚫SCATTER= CELL SHAPE/ SURFACE

71. VOLUME
⚫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.

72. 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.

73. 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

80. BASOPHILIA

83. NEWER PARAMETERS
⚫ 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)
⚫ P-LCR(Platelet Large Cell Ratio):
% of platelets with a volume >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.

84. RETICULOCYTES

85. RETICULOCYTE INDICES
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
erythropoeisis.
⚫ 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.

86. Technical operations

87. ⚫Before starting sample run….
⚫Two essential procedures :
⚫Calibration
⚫Quality control.

88. ⚫ Accuracy:
Refers to closeness
to the true value. It
implies freedom
from error.
⚫ Precision:
Refers to
reproducibility of
test. It implies
freedom from
variation.

89. *Calibration*
(setting accuracy)
Calibration
-is done to standardize the instrument for
accuracy.
Calibrator
-Certified reference material (CRM) used to
calibrate a measurement on an analyzer.
Calibration factors
-If any deviation from calibration references is
observed necessary calibration correction factors
are applied to set the accuracy of the instrument

90. When to Calibrate
You should calibrate your instrument :
⮚ At installation.
⮚ When a different or new control material is used.
⮚ When results have changed.

91. ⮚ Calibration should be done at least once
per year as per NABL guidelines
⮚ Automated hematology analyzers should
be calibrated using calibrators provided
by manufacturers
⮚ Controls lack accuracy so not
recommended as calibrators
⮚ If calibrators are not available then
controls must be assigned values reliably
by reference method and can be used as
calibrators

92. Pre calibration check
⚫ Total maintenance of the instrument
⚫ Reagents(replenish or replace)
⚫ Calibrator(check for expiry)
Calibration procedure
Done by company personal.
⚫ Precision check
⚫ Carryover check
⚫ Calibration in both (open and closed modes)
Post calibration validation
⚫ Run calibrator as samples
⚫ Run 3 level controls

93. PRECISION CHECK
(N=10)
Sample Requirements - For reproducibility studies, ensure
the patient for the sample that is being tested:
⚫ Is receiving no medication
⚫ Has normal hematologic parameters, with a WBC count
of 5.0 ± 1.0.
⚫ Has normal erythrocyte, leukocyte, and platelet
morphology and, if you want to check the Diff
parameters, with Diff values
⮚ Neutrophils 40 to 72%
⮚ Lymphocytes 17 to 45%
⮚ Monocytes 4 to 12%
⮚ Eosinophils 0 to 10%
⮚ Basophils 0 to 1%
Ensure you have enough normal whole blood from a single
donor for 11 cycles.

94. Quality Control

95. QUALITY CONTROL
⚫ QA is the sum of all those activities in which the
laboratory is engaged to ensure that information
generated by laboratory is correct.
⚫ QA is not restricted to the development and
retention of quality control charts but rather
includes all aspects of laboratory activities that
affects the results produced, from the choice of
methods, to the education of personnel, to the
handling of specimens and reporting results.

96. PURPOSE OF QC
⚫Assure proper functionality of
instrumentation
⚫Means of assuring accuracy of unknowns
⚫Monitoring the integrity of the calibration
- When controls begin to show evidence of
unusual trends
- When controls exceed the manufacturer’s
defined acceptable limits

97. ⚫ QA in hematology lab is intended to ensure
the reliability of the lab tests.
⚫ The objective is to achieve precision
and accuracy

98. ⚫3 components of QA programme :
1 ) Internal Quality Control ( IQC )
2 ) External Quality Control ( EQC )
3 ) Standardization

99. CONTROLS
Prepared in house or obtained commercially.
10 consecutive values of control recorded and Mean &
SD calculated.
3 concentration (high, normal, low) of control to be
analyzed.
The results are plotted on a graph to create Levey-
Jennings Chart.

100. Retained sample testing
⚫ Retained sample is yesterdays patient sample
having normal value for the measurable
parameters and preserved at 4’c
⚫ The same preserved sample is run on next day
after the commercial control and values are
compared with the yesterdays value. The values
should fall within the acceptable limit for the
observed parameters. i.e. for WBC+/- 0.75
,Hb+/-0.4 and platelet +/-30.
⚫ Then the same sample can be used through out
the day as a control for monitoring the
performance of the analyser.

101. ⚫Continuous detection and rectification
of the Analytical Process.
⚫Reagent-Equipment-Personnel-
Specimen
⚫Ensure the degree of both precision &
accuracy of your results
⚫Assure the quality and clinical
applicability of your laboratory reports
⚫Generate objective evidence of your
analytical performance.
Impact of Internal Quality
Control

102. 2 ) External Quality Control ( EQC)
{Proficiency Testing}
⚫ Is the objective evaluation by an outside
agency of the performance by a number of
laboratories on material which is supplied
specially for the purpose
⚫ is usually organized on a national or
regional basis
⚫ By comparing results from different
laboratories it is possible to establish
between-laboratory and between-method
performances.
⚫ Use:
⚫ Quality assessment of laboratory.
⚫ Identifying and recommending best

103. INTER-LAB QUALITY
CONTROL
⚫Regional level external quality control.
⚫We send same sample after running it in
our lab for testing into another lab.
⚫Results are compared.
⚫If they are in acceptable limit, then it
confirms accuracy and precision of lab.
⚫If results are out of acceptable range, then
we must take corrective actions like
calibration.

104. EQAS
⚫External quality assurance services
⚫National level external quality control
⚫Reference sample are sent by AIMS to
numerous laboratories of the country.
⚫Mean and SD is calculated from the results.
⚫Proficiency of the labs is evaluated from
whether their results are within the range
of mean +/- 2SD or not.

105. 3 ) Standardization
⚫ Refers to both materials and methods
⚫ A material standard or reference
preparation is used to calibrate analytic
instruments and to assign a quantitative
value to calibrators
⚫ A reference method is an exactly defined
technique which provides sufficiently
accurate and precise data for it to be used
to assess the validity of other methods
⚫ International reference preparations are
not freely available for routine use but are
intended to act as standards for assigning
values to commercial ( or lab produced )

106. Thank you