Principle of automated hematology

1. Chapter 11 Automated
Hematology

2. Acknowledgements
 Addisa Ababa University
 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-
Ethiopia

3. Outline
 Hematology Whole Blood Cell Analyzers
 Instrumental Principles
 Initial Set-up
 Daily Start-up
 Calibration
 Quality Control
 Histograms
 Flagging
 Problem Solving – Troubleshooting
 Case Study

4. Objectives
At the end of this lesson, the students will be able to:
 Describe the general characteristics of automated
haematology analyzers
 State the different principles of automated analyzers
 Explain the process of calibration, parameters and
situations which necessitate calibration
 Describe the use of quality control

5. Cont’d
 Discuss detection of errors (flagging) and the remedial
actions with automated haematology analyzers
 Describe key aspects of automated haematology
analyzers, including histogram use and interpretation

6. What is Automated
Hematology?
• Is the performance of Hemotology Lab
investigations by using Automated Analyzer as
opposed to manual procedures.
• There are various Hematology analyzers
depending on operating principles and the
parameters they performanaly.

7. What do Automated Analyzers
Perform?
 Counting of WBCs, RBCs and Platelets
 Measurement of Hemoglobin
 Calculation of Hematological Indices (Absolute
Values)
 Some can perform Differential counts (3 part and
5 part)
 Some can also indicate abnormalities of RBCs,
Platelets and WBCs (Flags)

8. Advantages of Automated
Analysers
 They analyze and produce results within a very
short time.
 If properly used, they produce Precise and
Accurate results.
 They significantly increase the number of
patients to be served, making more efficient use
of laboratory resources.

9. CBC Performed on an Automated
Hematology Cell Analyzer
 Well mixed EDTA sample is used
 CBC is a group of tests (WBC, RBC, Hgb, Hct,
Red Cell Indices, Platelet Count, and automated
differential)
 Tests are performed simultaneously (usually in
less than a minute)
 When performance limits of automated
hematology analyzer is exceeded, manual
method of cell counting and blood smear review
must occur

10. Haematology Whole Blood
Analyzer

11. CELL-DYN 1800 Technical
Specifications
System
Features
 Utilizes impedence resistance to measure
human cells;18 parameters measured
 Open-sampling system with throughput of up
to 60 samples per hour; 30 µl aspiration
volume
 Accurate 3-part white blood cell differential
analysis
 Uses only 3 reagents, including cyanide free
lyse that are individually packaged and
monitored to eliminate waste and improve
operator safety
 Q.C. built in system- Levey-Jennings,
Westgard Rules and X B

12. Post-Draw
Stability
 48 hours (5 DIFF)
Throughput  60 samples / hour
Sample
Volume  30 µL (whole blood)
Sample
Identification  Numeric keyboard
Flags
 User definable high/low flagging of
patient results
Number of
Reagents  3 reagents only
CELL –DYN 1800
Technical Specifications

13. Precision
(Reproducibility)
Parameter CV% (95% confidence limit)
 WBC less than or equal to 2.5%
 RBC less than or equal to 1.7%
 HGB less than or equal to 1.2%
 MCV less than or equal to 1.5%
 PLT less than or equal to 6.0%
CELL-DYN 1800
Performance Specifications

14. Optical Detection Principle
 In the optical or hydrodynamic focusing method
of cell counting and cell sizing, laser light is used
 A diluted blood specimen passes in a steady
stream through which a beam of laser is focused
 As each cell passes through sensing zone of flow
cell, it scatters focused lights
 Scattered light is detected by a photodetector and
converted to an electrical pulse
 Number of pulses generated is directly
proportional to the number of cells passing
through the sensing zone in a specific time period

15. Electrical Impedance Principle
 Utilizes non-conductive properties of blood cells
 As blood cell passes through orifice of aperture it
displaces its own volume
 Increased resistance between electrodes results in an
electrical pulse
 RBCs and platelets counted together, separated by
pulse heights
 Hydrodynamic focusing forces cells to pass single file
through aperture

16. Haematology Automation
 Basic Technology: Automated CBC
 Cell counting and sizing (WBC,RBC,PLT)
 Electrical impedance method
 Optical detection principle
 Light scatter and/or absorption (with or without
cytochemistry)
 Haemoglobin: spectrophotometric method
 Cyanmethaemoglobin
 Cyanide free Haemoglobin

17. Beckman-Coulter Haematology Whole
Blood Analyzers

18. Haematology Whole Blood
Analyser
ABX Pentra 60 C+

19. Data
Analysis
 26 Parameters: WBC, RBC, Hgb, Hct,
MCV, MCH, MCHC, RDW, PLT, MPV,
PCT, PDW
 Percentage and absolute counts of:
LYM, MON, NEUT, EOS, BASO, ALY
(Atypical Lymphocytes) and LIC (Large
Immature Cells)
 High-resolution leukocyte differential
matrix
 RBC, PLT, and Basophil histograms
 Quantitative and qualitative flags
ABX – Pentra 60 C+ Technical
Specifications

20. Post-Draw
Stability
 48 hours (5 DIFF)
Throughput  60 samples / hour
Sample
Volume
 55 µL (whole blood)
Sample
Identification
 Numeric keyboard
Flags  46 possible pathologic flags
Number of
Reagents
 5 reagents only
ABX – Pentra 60 C+ Technical
Specifications

21. Cytometry/Cytochemistry
DHSS
(Double Hydrodynamic Sequential
SystemTM )
Neutrophils
Eosinophils
Lymphocytes
Monocytes
Atypical
Lymphocytes
(ALY)
Large
Immature
Cells (LIC)
Volume
Absorbance

22. Sysmex Haematology Whole
Blood Analyzer

23. Initial Set-Up
“Out of the Box”
For the vendor’s service engineer to do:
 Check instrument for visual damage
 Check for any loose parts or connections
 Make sure all computer boards are properly
sealed
 Check the socket to verify proper voltage outlet

24. Delivery of Equipment

25. Cont’d
For the vendor’s service engineer
 Plug instrument power cord into (voltage
stabiliser) electrical supply
 Confirm the correct voltage on instrument
 Main power supply
 Photometric voltage
 Any other voltage supply that is pertinent to
instrument functions

26. Cont’d
For the vendor’s service engineer
 Permit instrument to stabilise/equilibrate
 Let all components reach proper temperature
 Set in any parameters that may be required
 Ranges
 Temperatures
 Adjustment for altitude if necessary

27. Ensure the Instrument is
Functioning Properly
Check the reagent containers for:
 Sufficient quantity
 Not beyond expiration date
 No precipitates, turbidity, particulate matter, or
unusual color
 Proper connections between the instrument and
the reagent containers
1

28. Cont’d
Check the waste container for:
 Sufficient capacity
 Proper connections
Perform daily startup per instrument manual
In addition to verifying daily startup results, verify
acceptable
 Reproducibility
 Carryover
 Control Results
3
2

29. Calibration
 Calibration fine tunes your Haematology
analyser and provides the most accurate results
possible
 In the normal process of tracking data for an
extended period of time, your laboratory can
make a specific decision to recalibrate a given
parameter.
 Never adjust to a specific value for an individual
sample

30. Cont’d
 For best performance, calibrate all the CBC
parameters
 The WBC differential is calibrated at the factory, it
does not require calibration in the laboratory

31. When to calibrate
You should calibrate your instrument
 At installation
 After the replacement of any component that
involves dilution characteristics or the
primary measurements (such as the apertures)
 When advised to do so by your service
representative

32. When to Check the Calibration
 As dictated by your laboratory procedures
 When controls begin to show evidence of
unusual trends
 When controls exceed the vendor’s defined
acceptable limits

33. Reproducibility Check
 Checks how close several results from the same
specimen are to each other (reproducibility)
 Reproducibility checks also known as precision checks
 Measurement is expressed as a percentage of deviation
from the mean which is called a Coefficient of Variation
or CV
 Reproducibility is not a measure of accuracy, but true
accuracy is not possible unless an instrument is precise
 Check instrument manual for procedure

34. Carryover Check
 Carryover is the transfer of the previous sample
to the current sample
 High to low carryover checks to verify the high
results of one sample do not affect the low
results of the next sample
 If carryover check does not pass, consult the
instrument manual for how to troubleshoot

35. Precision
Parameter Result Tolerance
 % LYM plus or minus 3.1%
 % MID plus or minus 1.6%
 % GRAN plus or minus 3.5%
CELL-DYN 1800
Performance Specifications

36. Daily Start-Up Procedures*
 Daily cleaning
 Background counts
 Electronic checks
 Check calibration
 Run controls
 Compare open and closed mode
sampling (use a normal patient
sample) * Check instrument
manual for procedures
Must be
within
specified
limits

37. Quality Control
 Purpose of QC
 Assures proper functionality of instrumentation
 Means of assuring accuracy of unknowns
 Monitors the integrity of the calibration
 When controls begin to show evidence of unusual
trends
 When controls exceed the vendor’s defined
acceptable limits

38. Quality Control Methods
 Assayed or
unassayed stabilised
material (Commercial)
 Patient replicates
 Delta checks

39. Cont’d
 Assayed stabilised material
 Known values
 Can be purchased in volume
 Can be run over time
 Easily illustrates trends and shifts

40. QC method: Assayed or
Unassayed Stabilised Material
 Commercially available
 Known values (assayed only)
 Analyse low, normal and high control
 Results stored in the instrument computer
(Pentra only)
 Monitored with Levey-Jennings graphs
 Easily illustrates trends and shifts

41. Recording of Quality Control
Results on Levey-Jennings
Quality Control Charts

42. QC Method: Patient Replicates
 Previously analysed patient sample
 Easily obtained
 Cost effective
 Results and samples readily
available

43. QC Method: Delta Checks
 Compare a patient’s own leukocyte, haemoglobin, MCV,
and platelet values with previous results
 If difference between the two is greater than
laboratory-set limits, current result is flagged for
review

44. What should be done if QC
Results are Unacceptable?
 Verify instrument functioning
 Check for shifts and trends
 Check integrity of material
 Troubleshoot
 Repeat the assay

45. When a Control is Outside its
Expected Range
Ensure the control
 Material was mixed and warmed properly
 If not, mix it according to the package insert
 Identification information was entered correctly
 If using the numeric keypad, verify you
typed the correct information
 Setup information (assigned values and expected
ranges) matches the control package insert for
the current lot number being used
 If they do not match, change the control’s
information to match the package insert
1

46. Proper Mixing
Blood rotator: Proper mixing may be achieved by a rotator
or by hand.

47. When a Control is Outside its
Expected Range
If any of the problems existed, rerun the control;
otherwise, proceed to the next step
Rerun the control to ensure the problem was
not a statistical outlier
Ensure the control material was not
contaminated by running another vial or level of
control
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3
2

48. QC Corrective Action
Log Sheet
1. Write the date on the corrective action log
sheet
2. Write the condition, such as “WBC high on
normal control”
3. Write the initials of the person who noticed the
condition

49. Cont’d
4. Write the date the control was run
5. Write the action that you performed, such as
“repeated-back in range”
5. Write the initials of the person who performed
the corrective action

50. Histograms as seen on an
Automated Haematology
Analyser

51. General Histogram
Characteristics
 Graphic representations of cell frequencies (Y-
axis) versus cell sizes (X- axis) in femtoliters
(fL)
 Provide information about erythrocyte,
leukocyte, and platelet frequency and their
distributions about the mean, and also depict
presence of subpopulations

52. General Histogram
Characteristics
 Provide means of comparing sizes of patient’s
cells with normal populations
 Shifts in one direction or the other can be of
diagnostic importance

53. Histograms: Electrical
Impedance Method
 RBC,PLT, and WBC data are plotted in the form
of a histogram
 Cell number on Y-axis and cell size on X-axis
 MCV (mean cell size of all red cell pulses) are
derived from the RBC histogram
 HCT, MCH and MCHC are calculated by the
standard formulas

54. Histograms: Electrical
Impedance Method
 From WBC histogram, three cell populations
(lymphocytes, mononuclear cells and
granulocytes) are delineated on difference of
their cell size
 Lymphocytes – 35 to 90 fL
 Mononuclear cells – 90 to 160 fL
 Granulocytes – 160 to 450fL

55. Histogram
 WBC: Distribution with three individual peaks and
valleys at specific regions representing the
lymphocytes, monocytes, and granulocytes
 All curves normally start and end at baseline

56. Normal Histogram
 WBC: Trimodal distribution with individual
peaks and valleys at specific regions
LYMPHS = 35-90 fL MONOS = 90-160 fL GRANS = 160-450 fL
 RBC: Unimodal, > 36 fL
 PLT: Unimodal, 2-20 fL (fitted 0-70 fL)
 All curves normally start and end at baseline
 All curves normally represent Gaussian
distributions

57. Histograms
 RBC, PLT, and WBC
plotted on histogram
 X-Axis
 Cell size in femtoliters
(fL)
 Y-Axis
 # of cells

58. WBC/Coulter Histogram as a
Quality Control Tool
Abnormality / Indicator Probable Cause Comment
WBC histogram (lymph peak) does
not start at baseline
Giant platelets, nRBC, Plt
clumping
Review smear,
correct for nRBC
Elevation of left portion of
granulocyte peak
Left Shift Review smear
Elevation of right portion of
granulocyte peak
Neutrophilia Review smear
Trail extending downward at
extreme left, or lymph peak not
starting at baseline
nRBC, Plt clumping,
unlysed RBC, cryoproteins,
parasites
Review smear
and for nRBC
Peak to left of lymph peak or lymph
peak widening towards left
nRBC
Review smear &
correct nRBC
Widening of lymph peak to right
Atypical lymphs, blasts,
plasma cells, hairy cells,
eosinophilia, basophilia
Review smear
Wider mono peak
Monocytosis, plasma cells,
eosinophilia, basophilia,
blasts
Review smear

59. RBC/Coulter Histogram as a
Quality Control Tool
Abnormality / Indicator Probable Cause Comment
Left of curve does not touch
baseline
Schistocytes and extremely
small red cells
Review smear
FBC and Platelet
histogram
Bimodal peak
Transfused cells,
therapeutic response
Review Smear
Right portion of curve extended Red cell autoagglutination
Review FBC &
Smear
Left shift of curve Microcytes
Review smear &
FBC
Right shift of curve Macrocytes
Review smear &
FBC

60. Platelet/Coulter Histogram as a
Quality Control Tool
Abnormality / Indicator Probable Cause Comment
Peak or spike at left end of
histogram (2-8 Fl)
Cytoplasmic
fragments
Review smear
Spike towards right end of
histogram
Schistocytes,
microcytes, giant
platelets
Review smear + FBC
( MCV &  RDW)
( MPV &  PDW)
Bimodal peak
Cytoplasmic
fragments
Review smear

61. Flagged/Abnormal Results
 Questionable or abnormal results are flagged for
verification by additional means for WBCs,
RBCs, and/or platelets
 Different types of flags exist for different
instruments - consult instrument manual
 Smear should be reviewed when flags exist
according to standard operating procedures

62. Review of Standard Formulas
for RBC Parameter
HCT (l/l) = RBC(x10¹²/L) x MCV (fL)
MCH (pg/RBC) =
HGB (g/dL) x10
RBC (x 10¹²/L)
MCHC(g/dL) =
HGB (g/dL) x 10
HCT(l/l)
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2
1

63. Preventive Maintenance

64. Automated Haematology-
Training Checklist
 Locate important procedures in the appropriate manual
including:
 Startup and shutdown procedures
 QC and calibration procedures
 Sample analysis procedures
 Reagent replacement procedures
 Printer paper and ribbon replacement procedures
 Troubleshooting procedures

65. Cont’d
 Identify basic instrument modules/components
and describe their functions
 Correctly perform startup, clean cycle, and
shutdown procedures
 Explain the importance of the startup, clean
cycle, and shutdown procedures and the
frequency with which they should be performed
 Perform appropriate Quality Control checks

66. Cont’d
 Review, delete and set up control files
 Perform all preliminary procedures required for
calibration
 Perform the CBC calibration and document the
results according to your laboratory’s protocol
 Perform sample analysis in the primary,
secondary, predilute modes

67. Cont’d
 Recognise the basic principles of parameter
derivation and state if a parameter is directly
measured, derived from a histogram, or
computed
 Review data and recognise if a scatterplot is
normal or abnormal
 Explain what a particular flag indicates
 Recognise an instrument problem based on
abnormal sample results, abnormal startup or
system test results, abnormal control results,
and/or error messages

68. Vendors for Haematology
Whole Blood Analysers
 Abbott (http://www.abbott.com)
 Cell-Dyn 1200, Cell-Dyn 1700, Cell-Dyn 3200, Cell-
Dyn 4000
 ABX Diagnostics (http://www.abx.com)
 ABX Micros 60 and ABX Pentra 60 C+ and ABX
Pentra 80 ( Tanzania standard for ART laboratories)
 Beckman-Coulter (http://www.beckmancoulter.com)
 Ac.T series, STKS, Gen-S,MAXM,HmX
 Sysmex (http://www.Sysmex.com)
 SE- series; K-series; T-series

69. Knowledge Check
WBC Clumping
1. What results are affected?
2. What resolutions may be attempted to obtain
reliable results?

70. Knowledge Check Answer
WBC Clumping
1. What results are affected?
Answer: 1. False decrease in WBC

71. Knowledge Check Answer
2. What resolutions may be attempted to obtain reliable
results?
Answers:
 Redraw another EDTA specimen and review results
 Collect citrated blood for CBC or CBC/PLT (multiply
WBC and PLT by 1.11 to correct for anticoagulant
dilution)
 Incubate blood 37 ºC for 10-30 minutes and reanalyze
 Estimate WBC from smear and perform manual
differential
 Perform a manual count to verify
 Append appropriate comment to the result, “WBC
Clumps”

72. Summary
 Haematology whole blood cell analysers
 Instrumental principles
 Initial set-up
 Daily start-up
 Calibration
 Quality control
 Histograms