Automated Hematology

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Automated Hematology Dr.P.Dahim Health Reference
Laboratory
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• Principles of Automation
• Calibration
• Quality Control
• Flagging

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Hematology Automation

• Two General Principles
• Electronic resistance ( impedance)
• Light scattering

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Electronic Resistance (Impedance)

• 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 sensing zone

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Cell counting
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Light scattering

• Cells counted as passed through focused beam of
  light( LASER)
• Sum of diffraction(bending around corners),
  refraction (bending due to change in speed) and
  reflection (light rays turned back by
  obstruction).
• 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 differentiate eosinophils

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Principles of counting sizing

• Counting and sizing red cells ,WBCs , Plts by
  counting the number of pulses and height of
  pulses
• Measuring Hb by modified HiCN method
• Measuring MCV by computing the mean of height of
  pulses
• Hct (PCV ) derived from MCV RBC
• MCH derived from Hb RBC
• MCHC derived from Hb , RBC MCV

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• RDW SD or CV of individual measurement of
  red cell volume
• HDW The degree of variation in red cell
  hemoglobinization,CV of measurements of
  hemoglobin concentration of individual cells
• NRBC included in the WBC counting

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WBC Differential Count

• Impedance technology with current of various
  frequencies
• Light scattering light absorbance

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Reticulocyte counting

• Flourescence based methods

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Histograms

• RBC, PLT, and WBC plotted on histogram
• X-Axis
• Cell size in femtoliters (fL)
• Y-Axis
• of cells

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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

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• 1-????? ????
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  ?????
• 3- ????? ???? ????
• 3-??????? ? ???? ??????
• 4- ??? ?????? ????? ??????
• 5- ????? ?????? ????? ???? (?? ? ?????? ? ???????
  ??? )
•  
• ???? ????? ?? ???????? ? ????? ??? ??? ???? ??
  ?? ????? ?? ????? ????? ????? ?? ????????? ?????
  ???? .

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• 6- ??? ????? ???? ???????
• 7-??? ????? ?? ??????? ??????
• 8- ?????? ? ???? ????? ????? ?????? ???? ????
  ???????
• 9-????? ??? ??? ? ??? ??? ?????? ?? ????? ???

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???? ???????

• - ?????? ??????? ? ???? ?????
• - ????? ????? ????? ???? ?? ? ????? ????????
• - ????? ????? ???????????
• - ????? ????? ????? ?? ??? ???? ????? ???
• - ????? ????? ???? ? ??? ?? ??

16
???? ???

• ??? ?? ?????? ?? ????? ?? ????? ???? ???? ?????
  ??????? ?????? ?? ????? ????????? ???? ???????
  ???? ???? .

17
???? ???

• ?????? ????? ????? ?????? ? ?? ????? ?? ????? ??
  ?? ???? ?????? ??? ?? ?????? ? ???? ??????? ?
  ???? ????? ???? ??????? ???? ???? .

18
???? ???

• ???? ????? ????? ?? ??????? ?????? ? ?? ????
  ????? ????? ???????? ???? ? ????? ? ????? ???
  ????? ??????? ?? ????? ??? ???????? ????.

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???? ???

• ?? ??? ????? ????? ?? Back ground ??????
  ??????? ? ?? ???? ????? ??? ? ??????? ??? .????
  ????? ????? ????? ?? ?????? ?? ??????? ??????
  ???? ??? ??? ?????? ???? ???? ?????? ?????
  ?????? ?? ??? ??? ?? ?????
• RBC lt0/03 x 1012/l
• WBC lt0/04 x 109 / l
• Haemoglobin lt0/2g/dl
• Platelets lt5 x 109 /l

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Calibration

• Calibration provides the most accurate results
  possible.
• For best performance, calibrate all the CBC
  parameters.
• The WBC differential is calibrated at the
  factory. They do not require calibration in the
  laboratory.

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When to Calibrate

• 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

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Calibration

• Normal fresh blood samples
• Calibrators
  

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Accuracy

• 100 ??????? ??? ??????? - ??????? ??? ???? CF
  
• ??????? ??? ???????
  

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??????? ???????????

• Sd v ?(d2)- (?d)2 /n / n-1
• tn d vn 
• sd

_
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Precision check

• Purpose check reproducibility of instrument
• Precision quick method
• Run 5 replicates of one patients sample free of
  elevated bilirubin, lipemia, and hemoloysis
• Calculate CV or SD
• Refer to the methodology in procedure manual for
  expected CV or SD

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Quality Control

• Purpose of QC
• Assure proper functionality of instrumentation
• Monitoring the Integrity of the Calibration

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Quality Control Methods

• Assayed stabilized material (Commercial)
• Previously analyzed patient samples
• Easily obtained
• Cost effective
• Results and samples readily available

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Quality control

• Replicate tests (control samples)
• Control charts
• Duplicate Check tests
• Correlation system
• Delta check
• Daily means of MCV,MCH,MCHC
• Comparison the results with reference methods

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Replicate tests

• ????? ??? ??? ??????

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Control charts

• ??????? ?? ??? ?????
• ????? ?? ??????? ?? ?????? ?????? ????? ?????? ??
  ???????

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Quality Control

• OUT OF CONTROL!!!
• Repeat the assay ( One time occurrence )
• Check integrity of material
• Troubleshoot
• Verify instrumentation

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Duplicate test

• ??????? ???? 2-3 ????? ?? ???? ?????((Duplicate
  ?????? ?????? ?? ?? ????? ?????? ?????? ?? ??
  ???? ??????? ????? ?? ???? ?????? ?????? ????
  ????? . ?????? ?????? ??? ?? ?????? ??? ?? 2SD
  ?????? ??? ? ?????? ???? ???? ?????? ?? ???? ??
  ?????.
• SD v?d2
• 2n

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Check test

• ?? ?????? ??? ???? ?? ???3- 2????? ?????? ???
  ????? ????? ?? ?? ?????? ??? ???? ?? ??? ??
  ???????? ?????? ?? ????. ????? ???? ?? ??? ??
  ?????? ?? ??????? ?? ????? Duplicate test
  ???? ?????? ?? ????? . ?????? ????? ?? ??????
  2SD ???? ???? ?? ???? . ?? ???? ??????? ????? ??
  ?? ????? ????? ? ?????? ????? ?? ????? ???? ??
  ??? ?????? ????????? ????? ???????? ?????? ? ??
  ?????? ?? ????.
• ??? ?????? ???? ????? ????????? ? ???????? ????
  ????? ???? ? ?? ????? ???? ???? ????? ????????
  ???? ? ????? ?????? ???? ??? ???? ????????? ?????
  ??? ????? ????? ?????? ????? ?? ??? ?? 6 ????
  ???? ?????? ?????.

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Delta check

• Hb 2
  g/l
• PCV 0.05 L/L
  
• MCV gt6 fL
• MCH gt 5 pg
• WBC Normal to abnormal
• Platelets Reduced or increased
  by more than 50

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Daily means of MCV,MCH,MCHC

• ????? ?????????? ?? ????? ????? ???? (????? ????
  100 ?????
• ????? ?????? ??? ?? 3 ?? ???????? ???? ?????
  ???? ??? ? ???? ????? ??????????? ?????? ?? ????.

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Comparison the results with reference methods

• ????? ??????? ????? ???? ????? ?? ????? ?????
  ?????? ??? .

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ERRORS

• INACCURACY

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Reliability of Electronic Counters

• High Precision
• Accuracy?
• - Recirculation of cells
  
• - Red cell agglutination
  
• - Lipid droplets, Microorganisms,
• - Extraneous particles
  
• - Faulty maintenance
• - Incorrectly calibration
  

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Interfering substances

• Cold Agglutinins
• Cryoglobulins
• Lipemia
• Platelet Clumps
• RBC fragments
• NRBCs
• Clot

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WBC Count Interferences

• Unusual RBC abnormalities that resist lysing
• malarial parasites
• giant platelets, platelet clumps, NRBCs
• fragmented white cells, agglutinated white cells,
• lyse-resistant red cells,
• cryoglobulin, some extremely elevate proteins,
• unlysed particles greater than 35 fL in size

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RBC Count Interferences

• Very high WBC count,
• high concentration of very large platelets,
• Auto-agglutination

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Hgb

• Very high WBC count,
• Severe lipemia,
• certain unusual RBC abnormalities that resist
  lysing

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MCV

• Very high WBC count,
• high concentration of very large platelets,
• Auto-agglutination

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Flagging

• WBC Suspect flags
• Blasts
• Imm Grans
• Variant lymphs

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More Flagging

• RBC Suspect flags
• NRBCs
• Macrocytic RBCs
• Dimorphic RBC population
• Micro RBCs/RBC fragments
• RBC agglutination

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Troubleshooting Flagged Results

• Refer to the Instrument Manual to troubleshoot
  codes, flags, and messages displayed with patient
  results

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Thank you for your attention