inc Ch 13 AUTOMATED BLOOD CELL ANALYSIS

Electrical impedance/low-voltage direct current

Developed by Coulter in 1950s

● Principle: measurement of changes in electrical resistance produced by cells as they traverse a small aperture.

● Oscilloscope: display pulses that are generated by the cells as they interrupt the current between the external and internal electrode.

● Volume distribution histogram depicts the volume distribution of the cells counted.

● Lytic agents allow separation and quantitation of WBCs into three populations (Lymphocytes, Mononuclear cells, and Granulocytes).

Coulter Principle of Cell Counting

Cells suspended in an electrically conductive diluent are pulled through an aperture in an aperture tube

Low-voltage direct current

_ is applied between an external and internal electrode

Internal electrode

housed inside the aperture tube (+)

External electrode

suspended in the cell dilution in the aperture bath

(-)

Aperture current
Vacuum
Blood cell suspension
Aperture
Aperture bath
Aperture tube

Excluding internal and external electrode, Identify each pointed parts including arrows (in order

Oscilloscope

display pulses that are generated by the cells as they interrupt the current between the external and internal electrode

• shows voltage pulses of RBC

Isotonic saline

electrically conductive diluent like _ are pulled through an aperature tube

Volume distribution histogram

depicts the volume distribution of the cells counted or where the data are plotted on a frequency distribution graph

also used for evaluation of subgroups within a population: WBC, RBC, platelet population based on older Coulter analyzer

• X-axis is cell volume

• Y-axis is relative number of cells

Histogram

A graph shows the average volume of each population, size distribution graph. Can be helpful in diagnosis.
○ A ref that shows the size and relative number of the different types of blood cells.

• X-axis: size of cells.

• Y-axis: relative number of cells.

2-20 fL

Particles with volumes of _ were counted as platelets

> 36 fL

particles > _ were counted as RBCs

35-90 fL

particles with volumes between _ are lymphocytes

90-160 fL

particles with volumes between _ are mononuclears (monocytes, blasts, immature granulocytes, & reactive lymphocytes)

160-450 fL

particles with volumes between _ are granulocytes

Lytic agents

_ allow separation and quantitation of WBCs into three populations: Lymphocyte, Mononuclear cells, Granulocytes

Lymphocyte, Mononuclear cells, Granulocytes

Lytic agents allow separation and quantitation of WBCs into three populations: ENUMERATE

Coulter Model S

The first multiparameter automated blood cell analyzer that was released in the late 1960s

RF resistance/conductivity

modification by TOA Medical Electronics Company and is used with DC electrical impedance in some analyzers

Optical light scatter
Cytochemistry

introduced by Technicon Instruments Corporation in the 1970s, and ortho clinical diagnostics followed with laser-based optical analyzers.

Protein buildup & Carryover of cells

Sources of error: Coincident passage of more than one cell at a time through the aperture:

■ Causes artificially large pulses, falsely increased cell volumes, falsely decreased cell count

Protein buildup

Source of error: results in lower/falsely decreased cell counts, falsely elevated cell volumes

Burn circuits

Modern analyzers incorporate _ or other internal cleeaning systems to prevent slow protein build up

Carryover of cells

What source of error can be minimized by internal cleaning system from one specimen to the next?

isovolumetric sphering

This can prevent Bending, deforming, misaligning of RBCs without changing their volume

F, must be falsely elevated cell counts instead

T/F: Recirculation of cells back into the sensing zone

• Causes: erroneous pulses and falsely decreased cell counts

• Prevention: backwash or sweep-flow mechanism

Hydrodynamic focusing

this avoids many potential problems inherent in a rigid aperture system

• a process when laminar flow narrows specimen to align in a single file, passing thru aperture one at a time

Coincident passage loss

_ is statistically predictable (and mathematically correctable) because of its direct relationship to cell conc. and effective vol of the aperture

Coincidence correction

_ is completed by the cell analyzer computer before final printout of cell counts

Laminar flow

Minimizes pulse height irregularities with an outer sheath that surrounds & narrows specimen & directs cells to pass thru central axis of aperture.

• also narrows specimen stream to align cells in a single file calld hydrodynamic focusing

RADIOFREQUENCY CONDUCTIVITY

Low-voltage direct current (DC) impedance with radiofrequency (RF) conductivity to increase discrimination among cells

• Low-voltage DC impedance with radiofrequency (RF) Resistance

• The amplitude of the pulse depends on the cell density or internal complexity.

• Conductivity is reduced by high NC ratio, nuclear density and cytoplasmic granulation.

• When both DC and RF pulse signals are measured = total volume of the cell is proportional to the DC impedance

• Cell interior density is proportional to pulse height of change on RF signal.

• Conductivity is measured by a high frequency electromagnetic probe.

OPTICAL LIGHT SCATTER

• May be used as the primary methodology or in combination with other methods.

• Optical scatter systems (flow cytometers): a hydrodynamically focused sample stream is directed through a quartz flow cell past a focused light source

• Light source can either be tungsten-halogen lamp or helium-neon laser (light amplification by stimulated emission of radiation)

• ■ Laser light (monochromatic light): emitted at a single wavelength, and it differs from bright-field in its intensity, coherence and low divergence or spread.

• May be used to study WBC, RBC, and platelets

• Forward-angle light scatter (0°): ○ correlates with cell volume.

Optical scatter systems/flow cytometers

a hydrodynamically focused sample stream is directed through a quartz flow cell past a focused light source

Tungsten-halogen lamp
Helium-neon laser

Enumerate light sources (light amplification by stimulated emission of radiation under optical light scatter)

LASER LIGHT/MONOCHROMATIC LIGHT

emitted at a single wavelength, differs from bright-field in its intensity, its coherence and low divergence/spread

Forward-angle light scatter

• 0 degrees

• correlates with cell volume

Blocker bar

lenses are fitted with this to collect scattered light in the forward direction along the light source path

• prevents nonscattered source light from entering the detector

• series of filters & mirrors separate diff wavelengths & present them to photodetectors

Orthogonal light scatter/side scatter

• 90 degrees

• correlates with degree of internal complexity

• introduced to further characterize blood cells

• results from refraction & reflection of light from larger structures inside cell & correlates with degree of internal complexity

• allows differentiation of nuclear shape, density, and cytoplasmic granules

Forward low-angle scatter

• 2-3 degrees

• Mie theory: low-angle correlates with cell volume, while high-angle scatter correlates with refractive index of cell

forward-high angle scatter

• 5-15 degrees

Mie theory

this states that low-angle correlates with cell volume, while high-angle scatter correlates with refractive index of cell

Differential scatter

Combination of low-angle and high-angle forward light scatter

forward low-angle scatter
forward high-angle scatter

correlate with cell vol and refractive index with internal complexity