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HEMATOLOGY 1 22
AUTOMATED BLOOD CELL ANALYSIS

GENERAL PRINCIPLES OF AUTOMATED BLOOD CELL ANALYSIS

ELECTRONIC IMPEDANCE

 or low-voltage direct current (DC) resistance. Developed by Coulter in the 1950s. Most common methodology used.

RADIOFREQUENCY (RF)

 or al- ternating current resistance, is a modification sometimes used in conjunction with DC electronic impedance.

DARKFIELD OPTICAL SCANNING

 Introduced by Technicon Instruments Corporation in the 1960s.

OPTICAL SCATTER

 Ortho Clinical Diagnostics followed with a laser-based optical instrument in the 1970s.
 using both laser and non-laser light, is often employed in today’s hematology instrumentation.

PRINCIPAL INSTRUMENTS

COULTER PRINCIPLE

 Detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture.
 Blood cell suspension it is a solution that is composed of electrically conductive diluent
 2 chambers filled with a conductive buffered electrolyte solutions separated by glass tube having a small aperture
 Direct current is generated between the internal and external electrode
 Aperture for RBC/platelet is smaller than the WBC aperture
(1) The cells that are suspended in an electrically conductive diluent (saline) and are pulled through the aperture (orifice with a glass
tube)
(2) In the counting chamber or transducer assembly, the low frequency electrical current is applied
(3) Electrical resistance between two electrodes or impedance in the current occurs as the cell passes through the sensing aperture
(4) This causes voltage pulses that are measurable
OTHER DEVICES

Oscilloscope
 Screens or displays the pulses that are generated by the cell as they interrupt the current
 The number of pulses is proportional to the number of cells counted
 The height of the pulse is directly proportional to the volume of the cell
 allows the discrimination and counting of cells of specific volumes to the use of threshold circuits
Histogram
 Pulses are also collected and sorted according to their amplitude by pulse height analyzers
 Data are plotted on a frequency distribution graph or volume distribution histogram
o Y-axis - relative number (no. of the cells)
o X -axis – channel number equivalent to the specific volume or femtoliter (fL)
 It depicts the volume distribution of the cells counted
Pulse height is measured and categorized by pulse height analyzers;
 256 channels for WBC and RBC analysis.
 64 channels for platelet analysis

RADIOFREQUENCY

CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4

  Low-voltage DC impedance, may be used in conjunction with RF resistance, or resistance to a high voltage electromagnetic
current flowing between both electrodes simultaneously.
 Alternating current resistance - A modification used in conjunction with DC electronic impedance
 RFDC detection method
o Shows simultaneous use of direct current and radio frequency in one measurement system
o Conductivity – measured by high frequency is attenuated by nucleus or cytoplasm ratio, nuclear density, and
cytoplasmic granulation
 Two different cell properties – to create two- dimensional distribution cytogram or scatter plot
o Low voltage DC impedance
o RF resistance
 Cell distribution as clusters and concentration of cell type as number of dots can be seen
 Cell internal structure density:
o Nucleus: cytoplasm ratio
o Nuclear density
o Cytoplasmic granulation

REAGENTS AND SUPPLIES

1. Electrolyte
✓ Bufferred isotonic salt solution that may contain one or more preservatives
✓ Must be particle free
✓ Used to dilute cells and to flush the instrument
2. Lysing Reagent
✓ A detergent solution
✓ Used to lyse erythrocytes by dissolving their stroma when leukocyte counts are to be performed
3. Cleaning agent
✓ Used at regular intervals to remove protein build up from the aperture tube and electrodes
4. Sample Container
✓ May range from small glass beakers to plastic vials manufactured for this purpose.
✓ Must be particle free and their internal surfaces do not react or attract cells.

FLOW CYTOMETRY
 combines fluid dynamics, optics, laser science, highspeed computers, and fluorochrome-conjugated monoclonal antibodies
(MAbs) that rapidly classify groups of cells in heterogeneous mixtures\
 The principle of flow cytometry is based on cells being stained in suspension with an appropriate fluorochrome
 An immunologic reagent, a dye that stains a specific component, or some other marker with specific reactivity.
 Fluorescent dyes used in flow cytometry must bind or react specifically with the cellular component of interest (e.g.,
reticulocytes, peroxidase enzyme, DNA content).

PRINCIPLES OF FLOW CYTOMETRY

 A suspension of stained cells is pressurized using gas and transported through plastic tubing to a flow chamber within the
instrument
 In the flow chamber, the specimen is injected through a needle into a stream of physiologic saline called the sheath
 The sheath and specimen both exit the flow chamber through a 75-µm orifice.
 This laminar flow design confines the cells to the center of the saline sheath, with the cells moving in single file
 The stained cells then pass through the laser beam
 The laser activates the dye and the cell fluoresces
 Although the fluorescence is emitted throughout a 360-degree circle, it is usually collected by optical sensors located 90
degrees relative to the laser beam.
 The fluorescence information is then transmitted to a computer, which controls all decisions regarding data collection,
analysis, and cell sorting.

CELLULAR FEATURES MEASURED BY FLOW

CYTOMETRY
Cell size or volume
DNA content

CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4

 Cytoplasmic Granularity
Cell-surface antigens
Intracellular enzymes
RNA content

Emerging Clinical applications of Flow Cytometry

Detection of small populations of cells
Determination of cell surface phenomena
Evaluation of leukocyte function
Evaluation of intracellular metabolism
Cytogenetics
Semen analysis
Detection of autoantibodies
Measurement of cytotoxicity
Analysis of platelet function

ERRORS IN AUTOMATION

INSTRUMENTAL ERRORS:
 Negative Errors - Excessive Lysis of RBC’s
 Positive Errors
▪ Bubbles caused by shaking

CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4

 HISTOGRAM
 Graphical representation of numerical data of different cell populations in a cell counter
 Gives information on:
CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4
 o Average size of cell
o Distribution of size
 X axis = volume of cell
 Y axis = number of cells

 Discriminators - separates the distribution curve for the volume

 WBC Discriminator
Lower Discriminator = 30-60 fL
Upper Discriminator = fixed at 300 fL
 RBC Discriminator
Lower Discriminator = 25-75 fL
Upper Discriminator = 200-250 fL
 Platelet Discriminator
Lower Discriminator = 2-6 fL
Upper Discriminator 12-30 fL
Fixed discriminator 12 fL

RBC HISTOGRAM

 Platelets have volume between 8-12 fL and counted between 2-25 fL

 The value here in the figure is within the lower and upper discriminator
 RBCs have volume 80-100 fL and are counted between 25- 250 fL
 The value here in the figure is within the lower and upper discriminator
 A platelet size distribution plot is produced using a threshold.
 One fixed at 12 fL and the other two are allowed to hunt the upper and lower ends of the platelet population between certain
limits.
 The lower platelet size threshold may move between 2-6 fL, the higher between 12 - 30 fL.
 The importance of this different threshold is to distinguish the platelets from the small red blood cells that is located in the
upper end of the platelet population, and the debris at the lower end testing.
 To distinguish their particular boundaries.

 Microcytosis
 Shift to the Left
 Presence of microcytosis - smaller RBCs
 Macrocytosis
 Shift to the right

CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4

  More than the normal distribution
 Macrocytosis

 Dimorphic Population
 There is a bimodal peak of the curve.
 Two population of sizes of red blood cells.

WBC HISTOGRAM

 Cells >35 fL - WBC in the WBC/Hb chamber

 Lymphocytes = 35-90 Fl
 MID cells = 90-160 fL
 Refers to monocytes, basophil, and eosinophils
 Neutrophils = 160-450 fL

Abnormal WBC Distribution Curve

 When a particular parameter peaks

 This diagram shows the different types of leucocytosis.

 Increased neutrophil = neutrophilia
 Increased lymphocyte = lymphocytosis

 MID cells is difficult to detect or distinguish when there is a presence of leukocytosis

 WBC distribution of that particular curve are all distributed in the same region.
 It cannot be distinguished whether there is an increase in monocyte, basophil or eosinophil.
 all three cells could be the reason of the increase of that particular parameter.

CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4

WBC Scatterplot

 X axis = side scatter and will detect the granularity of the cell
 The more it is going to the right, the more granular
 Y axis = forward scatter that will reflect the size of the cell.
 Going upward, the larger the size
Interpretation:
 Lymphocytes (red) – smaller and less granular
 smaller in have few internal components or granules than the monocyte (yellow) and granulocyte (purple).
 Granulocyte (purple)
 most granular population and have the most side scatter, and appear farthest to the right of the scatter plot.

CALIBRATION
 Calibrate every morning before running tests
 To ensure readings from an instrument are consistent with other measurements
 To determine accuracy of the Instrument readings
 To establish reliability of the instrument
 Determines the accuracy and precision of the analyzers
 “Tuning” of the instrument

Basophil-Lobularity (BASO) Channel

 cells are treated with a reagent containing a nonionic surfactant in an acidic solution.
 Basophils are particularly resistant to lysis in this temperature-controlled reaction, whereas RBCs and platelets lyse and other
leukocytes (nonbasophils) are stripped of their cytoplasm.
 Laser optics, using the same two-angle (2 to 3 degrees and 5 to 15 degrees) forward scattering system of the RBC/platelet
channel, is used to analyze the treated cells.
 High-angle scatter (proportional to nuclear complexity) is plotted on the x-axis, and low-angle scatter (proportional to cell
volume) is plotted on the y-axis.
 Basophils fall above a horizontal threshold on the cytogram. The stripped nuclei fall below the basophils, with segmented cells to
the right and mononuclear cells to the left along the x-axis. Blast cells uniquely cluster below the mononuclear cells.
 The nucleated RBC method is based on the physical charac- teristics of volume and density of the nucleated RBC nuclei.
 These characteristics allow counting in both WBC channels on the ADVIA 2120, and algorithms are applied to determine the
absolute number and percentage of nucleated RBCs.
 Information from the PEROX and BASO channels is used to generate differential morphology flags indicating the possible
presence of reactive lymphocytes, blasts, left shift, immature granulo- cytes, nucleated RBCs, or large platelets or platelet
clumps.
SPECIMEN LIMITATION
CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4
 Limitations resulting from inherent specimen problems include those related to the presence of cold agglutinins,
icterus, and lipemia.
 Cold agglutinins manifest as a classic pattern of increased MCV (often greater than 130 fL), markedly decreased RBC
count, and increased MCHC (often greater than 40 g/dL).
 Careful examination of the histograms or cytograms from the instruments may yield clues to this abnormality.
 Icterus and lipemia directly affect hemoglobin measurements and related indices.

CHRISTIANA YSABEL RODRIGUEZ / 3Y1 – 4