Automated Cell-Counting Methods

• Routine automated instruments are common in labs due to their precision in counting larger cell numbers compared to manual methods
• Blood cells are counted, leukocytes differentiated, and other components calculated using automated cell-counting methods

Essential procedures before sample analysis

• Calibration ensures instrument accuracy using calibrator certified reference material (CRM)
• Quality control involves quality assurance measures to ensure the instrument functions correctly and reports valid results

Leukocyte Differentials by Analyzers

• Instruments are available offering 3, 5, or 7-part differentials
• Labs must have a policy for blood film preparation, visual examination, and cell counting when automated results are flagged as clinically significant
• Policy Needed:
  • Blood film preparation
  • Visual examination
  • Cell counting when automated results are flagged

Automated Cell-Counting Methods

• Erythrocytes, leukocytes, and platelets are counted by either electrical impedance or optical detection
• Hemoglobin (Hb) is measured using cyanmethemoglobin flow-cell method at 525 and 546 nm, or a cyanide-free compound

Electrical Impedance (Coulter) Principle

• Involves diluting the sample in an isotonic conductive solution to preserve cell shape and characteristics

• Dilutions depend on the instrument and methodology used

• Platelets are counted simultaneously with RBCs

• For WBC counting, the sample is mixed with a reagent to lyse RBCs

• A reagent lyses RBCs and converts hemoglobin to cyanmethemoglobin to measure hemoglobin and WBCs in one dilution

• Cells pass through an aperture with electrical current and change electrical resistance, which is counted as voltage pulses

• The number of generated pulses is proportional to the number of cells and the pulse amplitude is proportional to cell size

• Thresholds distinguish cell populations and subpopulations

• Hydrodynamic focusing reduces cell coincidence during counting

Electrical Impedance Errors

• Recirculation error: Cells re-circulate, producing an aberrant impulse
• Coincidence error: Cells pass simultaneously, counted as one large cell
• Non-central flow error: Cells pass off-center of aperture, appear larger than actual size
• Hydrodynamic focusing and laminar flow corrects these errors
• Combining flow cytometers with blood count analyzers addresses issues with white blood cell counting in the Coulter method
• Flow cytometers can determine size, shape, biochemistry, or antigenic components

Optical Detection Principle

• Blood cells flow in a single line via fluid dynamics

• Each cell passes through a flow cell with a focused laser, scattering light in various directions

• A detector captures forward scatter light (FSL) at 180° from the light source

• A second detector captures scattered (side scattered or SS) light at 90°

• Forward scatter correlates with cell volume/density

  • analogous to impedance counting.

• Right-angle deflection indicates the granularity of the cell cytoplasm

• Scattered light is converted into an electrical pulse via a photodetector and processed by a computer

• This allows differentiation between granulocytes, lymphocytes, and monocytes

• Forward Scatter: Light diffracted at narrow angles, proportional to cell size

• Side Scatter: Light deflected by obstacles (cytoplasmic granules, nucleus), proportional to cell granularity, detected by photodiodes at 90°

Flow Cytometer Components

• Fluidics system: Presents cells to lasers.

• Optical system: Collects light scatter information using photodiodes and photomultiplier tubes

• Efficient and better for weak signals

• Electronics system: Converts optical to digital signals for display and analysis

• Light signals are converted into voltage pulses proportional to the number of photons

• Output displayed as dot plots

• Graphic representation provides output details on erythrocyte, leukocyte, and platelet data

• Data display includes cell counts, RBC indices, and WBC differentials

• Characteristics differentiate WBC types and produce scatter plots for a five-part differential

• Side Scatter (SSC) and Forward Scatter (FSC) signals are interpreted to differentiate cells

Erythrocyte Histogram

• Reflects the native size of erythrocytes or other particles in their size range
• In a homogeneous cell population, the curve is symmetric (bell-shaped or gaussian)

Red Cell Distribution Width (RDW) Histogram

• Calculated from the histogram by dividing the SD by the mean of the red cell size distribution
• RDW-SD is not a statistical SD, but an arbitrary line at 20% height on the y-axis in femtoliters
• The RDW increases above normal in iron, B12, and folic acid deficiencies
• In hemoglobinopathies, RDW increases with the degree of anemia

Reticulocyte and Nucleated RBC Measurement

• Reticulocytes are measured with:
  • RNA fluorescent dyes with laser light to test reticulocyte and related parameters.
  • Supravital specific dyes to precipitate reticular material and be assessed by light absorbance and scatter.
• Nucleated RBCs are counted after stripping by combining light scatter and impedance for counting.
• Analyzed on the WBC site

Leukocyte Histogram

• RBC lysis and partial WBC lysis is used
• Size-referenced histograms classify leukocytes by size after RBC lysis, not displaying the native cell size
• The lytic reagent causes a cytochemical reaction
• Cytoplasm collapse leads to differential shrinkage, sorting cells by relative nuclear size
• The histogram differentiates lymphocytes, mononuclear cells, and granulocytes and monocytes
• Mononuclear cells include blasts and immature cells

Platelet Histograms

• Platelet counting and sizing methods reflect the native cell size using electrical impedance and optical systems
  • Electrical impedance: Count/size done in the RBC aperture
  • Optical system:
    • Determines platelet count and frequency distribution by the forward light scatter pattern in the flow cell

RBC and PLT Histograms

• Plt & RBC distribution curves separated with a moving auto discriminator at the plateau

• RBCs Histogram:

  • Erythrocytes have a size of (80-100 fl) and are counted between (25-250 fl)
  • Distribution curves are separated by flexible discriminators:
    • RL and RU (RL: 25-75 fL & RU: 200-250 fL)

• Platelet Histogram:

  • Platelets have a size (8-12 fl) and are counted between (2 -30 fl)
  • Three discriminators are used LD , UD and a fixed discriminator at 12fl
    • LD: 2–6 fL
    • UD: 12-30 fL

• Size distribution curve should start/end on the basis line, located within lower/upper discriminator

Parameters from Thrombocyte Histogram

• MPV (Mean Platelet Volume):
  • Reference range: 8 - 12 fl
  • P-LCR = ratio of large platelets
    • Reference range 15 - 35%
  • Increase indicates:
    • PLT Clumps
    • Giant PLT
    • Microerythrocytes
    • Immature platelets
• PDW (Platelet Distribution Width):
  • Calculated at 20% of peak height
  • Reference range: 9 - 14 fl
  • Increase indicates:
    • PLT Clumps
    • Microerythrocytes
    • Fragments

Flags in automated cell counters:

PL Flag

• Lower discriminator exceeds preset height by 10%.
  • Platelet count, MPV, P-LCR show PL flag.
  • High background numbers (check for reagent contamination/expiry). If background check in range, check patient sample - High platelet results means possible cell fragments/bacteria
  • Platelet aggregates might lower discriminator, incorrect PLT results.
  • Treat sample to prevent clotting if caused by EDTA incompatibility.
  • "PL" shown with priority over "PU".

PU Flag

• Upper discriminator > 40% over preset height.
• Aggregation lowers PLT count.
• Check EDTA compatibility (re-collect with Citrate).
• Giant PLTs lower count.
• Extreme Microcytosis/fragmented RBCs raise count.

MP Flag

• Multi peaks in PLT histogram means -
  • Platelet anisocytosis.
  • Recovery from chemo.
  • Platelet aggregation

Flags for abnormal height

• Lower discriminator (RL)
  • Means >10% higher than preset height.
  • May indicate platelet clumps, RBC fragments, giant platelets or microerythrocytes.
• The Curve doesn't start to the baseline because the lower discriminator Is triggered incorrectly.
• Higher readings may mean platelet result is falsely high.
  • Perform alt PLT test
• Upper discriminator (RU) Flag - Means >5% higher than preset height.

RU may mean;

-  RBC agglutination 
 - Check RBC count, MCV/HCH which will all be incorrect

Flags for Abnormal Distribution

• Flag "DW" means curve doesn't match set lines, so couldn't calculate Causes for this:

• In recover from anemia

• Anisocytosis can mean incorrect RBC result

Flags when multiple Peaks are recognized (MP) are;

 -  Post transfusion
  -Anisocytosis
     - Extreme leukocytosis can cause high WBC

Discriminator Peaks

• The distributor histogram should start close to 0, and spread without two limits, with two peaks. - F1 is for lymphocytes. - F2 Is for for monocytes. - F3 Is for granulocytes. Curve may not match standards because;

WL - Extreme Leukocytosis flag

• This is because WBC are aggregating
  • A Pre-dilution may help assess the sample
• In the absence of these Flags all cell readings are not affected*

T1 flag means;

• Couldn't distinguish Lymphocytes from Mixed cells

T2 flag means;

• Couldn't distinguish mixed cells from neutrophils

• May need to use Microscope film for additional information

• All WBC readings may be incorrect because readings weren't differentiated correctly.