Automation In Haematology. Dept Of Lab Medicine Basavatarakam Indo American Hospital And Research Institute

1. Automation In Haematology
Dr. K.Siddartha
Mod: Dr. Faiq Ahmed
Dept Of Lab Medicine
Basavatarakam Indo American Hospital And Research
Institute

2. Automation in hematology includes
 Cell counts (Automated hematology analysers)
 Diagnosis of hemoglobinopathies by HPLC
 Flow cytometry
 Automated coagulometers
 Automated ESR

3. Automation in Cell Counters

4. Basic parameters – 3 part counter
• Haemoglobin
• RBC, PLT count
• Red cell indices
• RDW
• WBC with 3-part differential
NO. 4
DATUM: 9/10/95 15:11
MODE: VOLLBLUT
WBC 5,8 x 103/µl
RBC 4,84 x106/µl
HGB 13,7 g/dl
HCT 42,0 %
MCV 86,8 fl
MCH 28,3 pg
MCHC 32,6 g/dl
PLT 257 x103/µl
LYMPH% 31,2 %
MXD% 6,8 %
NEUT% 62,0 %
LYMPH# 1,8 x103/µl
MXD# 0,4 x103/µl
NEUT# 3,6 x103/µl
250
RBC
RDW-SD 40,0 fl
40
PLT
PDW 13,1 fl
MPV 10,4 fl
P-LCR 28,1 %
WBC
300

5. Hemoglobin estimation
V07063-part Diff technology
Haemoglobin molecule
RBC
Ammonium
salts
Fe2+
Fe2+
RBC
Fe2+
Fe2+
Lysis of RBC
Fe2+ Fe2+
Fe2+ Fe2+

6. V07063-part Diff technology
Haemoglobin molecule
Methemoglobin-complex
Stable coloumetric complex – directly proportional to Hb
Absorbance of solution is measured
Fe3+
Fe3+
Fe2+
Fe2+
O2
Oxidation
Fe2+ Fe2+
Fe2+ Fe2+

7. V07063-part Diff technology
As samples are passing through the
centre of the aperture with sheath flow
solution
DC for RBC & PLT
Hydrodynamic Focusing
 Recirculation and coincidence are
prevented
 Enhanced linearity & accuracy

8. V07063-part Diff technology
2-6 fl 12-30 fl
fixed at
12 fl
WL WU
100%
20%
 WBC detection: between 30 and 300 fL
 Leukocytes are separated in 3 parts:
lymphocytes, mixed cells (mono, eo, baso)
and neutrophils by discriminators: T1, T2
T1 T2
Leukocyte (WBC) Histogram

9. 5-part differential counters
Various technologies :-
Fluorescence Flowcytometry
Volume Conductivity Scatter
Peroxidase Staining

10. VOLUME MEASUREMENT
Volume of the cell is
done by using
Direct Current (DC)
across the two
electrode in a flow
cell.
Beckman Coulter VCS technology

11. CONDUCTIVITY MEASUREMENT
The
Radiofrequency
(RF) energy
penetrates into
cell and reveal
information about
its size and
internal structure.

12. SCATTER MEASUREMENT
As cells are passed in
single stream (flow
cell) they are struck
by laser which gets
scattered.
The light scatter at
angles between 10
and 70 deg is used by
VCS instruments.
The scattered light gives information about cell surface and
granularity

13. WBC and Differential
• Peroxidase Channel Stain Cells
With Peroxidase
:Eosinophils- Strong Staining
:Neutrophils- Medium Staining
:Monocytes- Weak Staining
:Lymphocytes and Basophils-
No Staining
:Large Unstained Cells (LUC) Eosinophils
Neutrophils
Monocytes
LUC
Lymphocytes
+
Basophils
Perox Activity
Volume
ADVIA TECHNOLOGY

14. The ADVIA WBC differential is calculated from a 3
step process.
Cells are stained by peroxidase reagent and analyzed for size
and peroxidase stain intensity.
Cell specific lysis reagents are used to separate basophils
from all other white cells.
Basos are subtracted from the lymph/baso cluster in the
perox channel to calculate the lymphocytes.
ADVIA TECHNOLOGY

15. Fluorescence Flow Cytometry
The intensity of scattered lights (FSC and SSC) reflects cell surface structure,
particle shape, nucleus form, refractive index and reflectivity of the cells.
The intensity of the side fluorescent light (SFL) mainly reflects the type and
amount of nucleic acids and cell organelles.

16. WDF

18. WPC Scattergram
SSC
SFL
WBC
WBC
Blast
Abn Lymph
Blast
Abn Lymph
WPC channel
FSC

19. RET CHANNEL
Reticulocyte count
Reticulocyte fractions
(LFR, MRF, HRF)
Immature reticulocyte
fraction
Ret-He (reticulocyte Hb
content)
Platelet –O (fluorescent
platelets)
Fragmented red cells
(FRC)

21. SLS-Hemoglobin
Hemolytic reaction
between SLS and
RBC menbrane
1
2 3 4

22. • Photometrical analysis at 555 nm
• Lambert Beer Law: Absorbance α Hgb concentration
HGB Photometer
lens
Sample stream Cellpack
Flow cell photosensorLED

23. Hematocrit (Hct)
• Cumulative Pulse Height Detection
• Direct measurement, not calculated.
• Resistance change is detected as the height of the
pulse, proportional to size of RBC.
V
t
Total Volume
RBC Volume
RBC
RBC Volume
Total Volume
X 100 = Hct
Cumulative sum of all RBC pulses
RBC Volume

24. RBC indices
• Calculated from three basic parameter. RBC, HCT, Hb
Hct (%)
RBC (x 106/µl)
MCV (fl) =
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin(MCH)
Hb (g/dl)
RBC (x 106/µl)
MCH (pg) =
Hb (g/dl)
Hct (%)
MCHC (g/dl) =
Mean Cellular Hemoglobin Concentration (MCHC)

25. RBC- and PLT-Histograms
Platelet
MPV-8-12 fl
Red blood cells
80-100 fl

26. Normal RDW
Increased RDW
The RDW is determined by calculating the width of RBC histogram
in fl and is expressed as CV(12-14)or SD(40-46)

27.  The Mean of the platelet volume between the lower discriminator PL
and the upper discriminator PU
Pct (platelet crit) is equivalent to the sum of platelet impulses which are
individually detected by means of the impedance measurement
principle
Mean Platelet Volume (MPV)

28. From PLT histogram to PDW and P-LCR
Platelet Large Cell Ratio
(P-LCR)
 Percentage of large
platelets with a volume >12
fL
 (N= 15-35%)
Platelet Distribution Width
(PDW)
 The platelet distribution width
measured at 20% relative
height of the total height of the
curve (9-14fl)

29. Summary Principles
RBC/PLT HGB WNR WDF WPC
RET/
PLT-O
PLT-F
ChannelsTechnology
Sheath
Flow DC
Detection
Method
Histograms/
Scattergrams
SLS-
Hemoglobin
Method
Hemoglobin
Flow
Cytometry
method
Flow
Cytometry
method
Flow
Cytometry
method
Flow
Cytometry
method
Flow
Cytometry
method

31. Curve not ending at base line

37. Immature
granulocytes

38. http://www.pathpedia.com/education/eatlas/hist
opathology/blood_cells/rbc_agglutination.aspx

39. Plasmodium vivax
Hemazoin crystals
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013084/figure/F4/

40. Automation in Coagulation

41.  Screening test: PT, APTT, Fibrinogen and D- dimer.
Tests which look for abnormalities in coagulation &
gives direction for selecting the specific test and help
in the diagnosis and treatment
 Specific test: Platelet Aggregation studies, Specific
coagulation factor assay, Antithrombin ,Protein C
activity etc.,

42. Manual Methods
Reagents and samples are added manually.
Temperature is maintained by a water bath.
Values are measured manually by using
stopwatch.
 human error and are difficult to do when
there is large workload.

43. Semi Automated Analysers
 Has mechanism to automatically initiate timing device
upon addition of final reagent and internal mechanism
for detecting Clot formation.
 All reagents and samples are added manually by the
operator.
 Semi automated analyzer may or may not internally
maintain the temperature .
 E.g., Start®
(Stago Diagnostica)

44. Automated analyser
Human errors are eliminated(sample
identification, addition of sample and reagents
and end point detection)
Maintains and check the temperature.
Perform auto dilutions mainly for the single
factor assays.

45. ¶ Storage of large no. of patient data and control
result
¶ Generates flagging for sample abnormality and
instrumental malfunctioning.
¶ Batch processing of multiple samples for single
test or multiple tests on a single sample can be
performed

46. Clotting assay
Functional assay
 In this sample and reagent are mixed together to form a
clot.
 End point of the reaction i.e., clot is detected by
Mechanical
Optical detection
Electrochemical

47. Mechanical method
Magnetic steel ball method
 The sample is introduced to a cuvette that has a small
steel ball inside
 The cuvette continuously moves when reagent and
sample is added
 The fibrin strands begin to form and attach to the moving
ball
 There is a break in contact with the magnetic sensors
when the steel ball becomes incorporated into a fibrin
clot as the cuvette
 Clotting time is recorded.

49. Optical detection method
• As the plasma sample clots, it becomes more
optically dense and the amount of light falling on a
photosensitive detector decreases.
• The drop or change in light is determined as the
endpoint
= turbidometric method

50. Nephelometric method
The nephelometer uses a light -emitting diode at a
high wavelength (>600 nm) to detect
variations in light scatter as fibrin clot is formed.
When the light rays encounter insoluble complexes
such as fibrin strands, they are scattered at 900 angle.

51. Chromogenic Method
• Chromogenic or amidolytic methodology is based on the
use of specific color producing substance = chromophore.
• Normally used chromophore is para-nitroaniline (405nm)

52. Immunologic Method
• Immunologic assays are based on Ag-Ab reactions.
• Latex Micro particles are coated with a specific Ab
directed against Ag.
• A beam of monochromatic light is then passed through
the suspension of micro latex particles.
• Formation of agglutination lead to increase in turbidity of
the test solution and increase in Absorbance which, in
turn, is proportional to the antigen level present in the
sample, which is read from a standard curve.

53. Electro chemical
• The INRatio single-use test strip has a
sample well where blood is applied,
three channels through which the blood
sample flows to reach the testing areas.
• Reagents start the coagulation process.
• The device detects a change in
electrical resistance when blood clots.
INRatio meter

54.  Uses stimulators of platelet
adhesion and aggregation in an
environment that stimulates an
injured blood vessel wall.
 More sensitive screening test
than the bleeding time method
 Nonspecific test- not diagnostic
for any single disorder
Platelet Function Analyser

55.  The instrument adds citrated blood to a
reservoir with collagen/epinephrine on
a bioactive membrane
 A pressure sensor detects the
formation of a platelet plug on the
membrane
 The time it takes to close the aperture
in the membrane with the platelet plug
is recorded.
 The result is a function of platelet
count, platelet activity, VWF activity.
Platelet Function Analyser

56. Platelet Aggregometry
• Platelet aggregometry involves a
series of tests performed on whole
blood or platelet-rich plasma, using
several agonists (platelet activators).
• The agonist is added to the
suspension and a dynamic measure
of platelet clumping is recorded.
• Simultaneously to platelet
aggregation, luminometry test can
be performed. In that case, ATP
release is assayed using a
luminescent marker.

57. Thrombelastography (TEG)
•Sample of citrated whole blood is
placed in a cup which has a pin
carefully connected to a torsion
wire.
•As the cup rotates in a back and
forth movement, the aggregates
formed within the cup cause the wire
to become more rigidly placed and
reflects the strength of the
aggregates formed within the cup.
•The movement or lack of movement
is reflected via either an optical or
magnetic detector
•A graphic presentation is produced

58. TEG Graphic Result
R=Time of latency
from start of test to
initial fibrin
formation
K=Time taken to
achieve certain
level of clot
strength
Alpha angle
=measures the
speed at which
fibrin build up and
cross linking takes
placeMA=maximum amplitude

59. Typical TEG Graph Patterns

60. Illustrates function and dysfunction in the
Hemostatic system
Allows physicians to give appropriate amounts of
FFP, Cryo, and platelets to control hemorrhage
Reduces unnecessary use of blood products
Allows effective management of hypercoaguability
Differentiates surgical from pathological bleeding
Uses of TEG

61. Flowcytometry

62. Flow cytometry
• Immunophenotyping of cells is one of the most important
clinical application of Flow cytometery.
• Flow cytometery is the methodology used to detect cell
surface antigens using monoclonal antibodies conjugated
with different fluorochromes.

63. Uses of flowcytometry
• Diagnosis.
• Assess response to therapy.
• Prognosis and detection of minimal residual
disease.

64. Indications
• Acute leukemias
• Chronic lymphoproliferative disorders
• Myelodysplastic syndrome
• Plasma cell dyscrasias
• Red cell disorders
• Platelet disorders
PNH
Fetal cell estimation
Hereditary spherocytosis
Reticulocyte estimation

65. BD FACSCalibur

66. The common elements in all Flowcytometers
• A light source
• Fluid lines and controls to direct a liquid stream
containing particles through the focused light beam
• An electronic network for detecting the light signals
coming from the particles as they pass through the
light beam and then converting the signals to
numbers that are proportional to light intensity
• A computer for recording the numbers derived from
the electronic detectors and then analyzing them

67.  The specimen must be in a
monodisperse suspension.
 In this, isotonic fluid is forced
under pressure where a fluid
column with laminar flow and a
high flow rate is generated (so-
called sheath fluid).
 The sample is introduced into
the flow cell in the center of the
sheath fluid, creating a coaxial
stream so that they are
presented to the light beam one
at a time.

68. • The emitted light is focused by a lens
onto fiber optic cables and transmitted to
octagonal detectors.
• The sensors convert the photons to
electrical impulses that are proportional
to the number of photons received and to
the number of fluorochrome molecules
bound to the cell.

69. Sample preparation and processing
 Types of samples 
 Anticoagulants  EDTA (stable upto 24 hrs)
Acid citrate dextrose (upto 72 hrs)
 Sample storage  ideally within 48 hrs
<40C and >300C should be avoided
Peripheral blood
BMA
Body fluids
Lymphnode
aspirates

70. 100 l of sample

Add 8l of antibody
(30 min)
2ml of lysing sol
(15 min)
Centrifuge 1500 rpm for 5min

Supernatant fluid discard

2ml of sheath fluid

Centrifuge 1500 rpm for 5min

Supernatant fluid discard

Add 0.5 ml of sheath fluid

Acquire into flow machine
Sample preparation and processing

71. For cytoplasmic antibodies
100 l of sample

Add 8l of antibody
(30 min)
2ml of lysing sol
(15 min)
Centrifuge 1500 rpm for 5min

Supernatant fluid discard

Permealisation sol 0.5ml
(15 min)
1ml of sheath fluid

Centrifuge 1500 rpm for 5
min

Supernatant fluid discard

10l of antibody
(15 min)
1ml sheath fluid

Centrifuge 1500 rpm for
5min

Supernatant fluid discard

0.5ml of sheath fluid

Acquire

72. • A pair of light
scatter channels
provides an
approx. measure
of cell size
{Forward
scatter(FS)} and
granularity {Side
scatter(SS)}.

73. Automation in ESR

75. Fully automated analyzer
No reagents required – no waste.
Direct use of primary EDTA tubes – no contact with blood.
Therefore the EDTA tube can be used unchanged for
further analyses after erythrocyte sedimentation.
Compatible with all the commonly used EDTA tubes in
the market.
Photometric infrared (950nm) reading prevents
interference caused by lipids or bilirubin in the sample.

76. High Performance Liquid Chromatography

77. • HPLC is a chromatographic technique that can
separate a mixture of compounds.
• Type of liquid chromatography where the sample is
forced through a column that is packed with a
stationary phase composed of irregularly or
spherically shaped particles, a porous monolithic
layer.

78. Principle:-
Depends on interchange of charged groups
on the ion exchange material with charged
groups on Hb molecule.

79. Carboxyl groups attached to a resin base
Direction of flow Detector
Cation Exchange Cartridge

80. Whole blood + hemolysate
Introduction
Positively charged hemoglobin fragments in
the hemolysate attach to the carboxyl
groups at varying strengths.

81. Starting Gradient:
Low Ionic Strength Buffer
 The gradient starts with a low % of Buffer
 At this gradient, hemoglobin fragments with
an ionic strength lower than the buffer
gradient, such as HbF, are displaced from
the cartridge and pass into the detector

82. Ending Gradient:
High Ionic Strength Buffer
• As the % of the Ionic Strength of Buffer
increases, the more hemoglobin fragments
will be displaced
• Once the gradient is 100% all remaining
hemoglobin fragments, including any variant
hemoglobins such as S, D and C, will be
removed

83. • Separated components pass through dual
wavelength detectors.
• Absorbance is measured at 415 nm and
the data is displayed as chromatogram.
• Finally it is converted into peaks as per the
retention time
• Separated Hb’s with % is displayed.
Retention time: Time in min
from the sample injection to the
max point of elution peak of Hb

84. Retention time for Hb
HPLC of abnormal Hb

85. Conclusion
• The automation in hematology is efficient, lack inter
observer variability and size distribution errors.
• They give an estimate of many variables which are
manually not possible and produce data with
increased reliability, precision and accuracy.
• Data can be stored in automated analysers.