automated cell counting instrumentation (1)

7/27/2019 Automated Cell Counting Instrumentation (1)

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1. Explain the different principles of automated cell counting.

2. Describe how the general principles are implemented on thedifferent instruments discussed.

3. Identify the hemogram parameters directly measured byanalyzers.

4. Explain the derivation of calculated or indirectly measured

hemogram parameters.5. Explain the derivation of the WBC differential count.

6. Interpret and compare patient data, including WBC and RBChistograms or cytograms or both, obtained from the major

hematology instruments.7. Explain the general principles of automated reticulocyte

counting.

8. Identify sources of error in automated cell counting anddetermine appropriate corrective action.


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AUTOMATION provides greater accuracy andprecision than manual methods.

Hematology analyzers provide the eight standardhematology parameters (CBC) plus a three-part orfive-part differential leukocyte count in less than 1minute on 200 uL of whole blood.

Automation allows more efficient workloadmanagement and more timely diagnosis andtreatment of disease.


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Low voltage direct current (DC) resistance

Developed by Coulter in the 1950s

PRINCIPLE : Cell counting is based on the detectionand measurement of changes in electrical resistanceproduced by cells as they traverse a small aperture.


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Aperture Diameter RBC/Platelet aperture is smaller than the WBC aperture

to increase platelet counting sensitivity

Protein build up : decreases the diameter of the orifice,

slowing the flow of cells

Burn Circuits or Internal Cleaning Systems Minimize protein build up and carry over of cells from

one sample to the next

Coincident Passage Causes count reduction


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Orientation of the cell in the center of the apertureand deformability of the RBC

Recirculation of cells back in the sensing zone createserroneous pulses and falsely elevated cell counts

Hydrodynamic focusing Avoids many potential problems


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Alternating current resistance

Low voltage DC maybe used in conjunction with RF

resistance, or resistance to a high voltageelectromagnetic current flowing between bothelectrodes simultaneously

The total volume of the cell is proportional to thechange in DC, the cell interior density is proportionalto pulse size or change in the RF signal.


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CONDUCTIVITY : attenuated by nucleus to cytoplasm ratio, nuclear density, and cytoplasmicgranulation


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Two dimensional distribution cytogram

Created by plotting impedance against conductivity

Displays cell populations as clusters, with the numberof dots in each cluster representing the concentrationof that cell type.


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Uses both laser and non-laser light

Latest technology


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Hydraulics System Includes aspirating unit, dispensers, diluters, mixing

chambers, aperture baths, or flow cells or both, and ahemoglobinometer

Pneumatics System Includes vacuums and pressures required for operating

the valves and moving the sample through thehydraulics system

Electrical Systems Controls operational sequences of the total system and

includes electronic analyzers and computing circuitryfor processing the data gathered


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Parameter Principle

WBC Impedance, hydrodynamic focusing

RBC Impedance

Hb Modified Cyanmethemoglobin at 525 nm

Hct (RBC x MCV)/10MCV Mean of RBC volume histogram

MCH (Hb/RBC) X 10

MCHC (Hb/Hct) X 100

Platelet Count Impedance (2-20 fL) : least squares fit of volume distributionhistogram (0-70 fL)

RDW CV (%) of RBC histogram : (SD/MCV) X 100

ReticulocyteCount

NMB-N, volume, conductivity, optical scatter


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Parameter Principle

WBC Hydrodynamic Focusing, DC Detection (impedance)

RBC Hydrodynamic Focusing, DC Detection (impedance)

Hb SLS Hb (555 nm)

Hct Cumulative Pulse Height DetectionMCV (Hct/RBC) X 10

MCH (Hb/RBC) X 10

MCHC (Hb/Hct) X 100

Platelet Count Hydrodynamic focusing, DC detection (impedance)approximately 2 30 fL

RDW RDW-SD (fL) or RDW-CV (%) available

ReticulocyteCount

Auramine O; fluorescence detection


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Parameter Principle

WBC Optical Scatter (primary count); Impedance (secondary count)

RBC Impedance

Hb Modified Cyanmethemoglobin (540 nm)

Hct (RBC X MCV)/ 10MCV Mean of RBC distribution histogram

MCH (Hb/RBC) X 10

MCHC (Hb/Hct) X 10

PlateletCount

Impedance (approximately 2 30 fL)

RDW Relative value, equivalent to CV

ReticulocyteCount

Propriety Stain (CD4K530), multiangular scatter, and fluorescencedetection


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Parameter Principles

WBC Hydrodynamic focusing, optical scatter and absorption

RBC Hydrodynamic focusing, laser low-angle (2 3 degree) and high-angle (5-15 degree) scatter

Hb Modified Cyanmethemoglobin (546 nm)

Hct (RBC X MCV)/10

MCV Mean of RBC volume histogram

MCH (Hb / RBC) X 10

MCHC (Hb/Hct) X 100

Platelet Count Hydrodynamic focusing, laser low-angle (2 3 degree) and high-angle (5-15 degree) scatter (1-60 fL)

RDW CV (%) of RBC histogram: (SD/MCV) X 100

ReticulocyteCount

Oxazine 750; low-angle (2-3 degree) and high-angle (5-15 degree)opticla scatter and absorbance


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Parameter Coulter Sysmex Abbott Siemens

Neutrophil VCS OS and FS MAPSS PS, OS, Abs

Lymphocyte VCS OS and FS MAPSS PS, OS, Abs

Monocytes VCS OS and FS MAPSS PS, OS, Abs

Eosinophils VCS OS and FS MAPSS PS, OS, Abs

Basophils VCS OS and FS MAPSS Differential lysis,laser low-angle(2-3 degree) andhigh angle (5-15degree) scatter


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ParametersAffected

RBC decreased, MCV, MCHC increased,grainy appearance

Rationale Agglutination of RBCs

InstrumentIndicators

Dual RBC population on RBC map, orright shift on RBC histogram

Corrective Action Warm sample to 37 degrees C and rerun


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ParametersAffected

Hb, MCH increased

Rationale Increased turbidity affects

spectrophotometric readings


Hb X 3 is not Hct+/-3, abnormalhistogram

Corrective Action Plasma replacement


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Parameters RBC, Hct decreased

Rationale RBCs lysed and can not be counted


Hb X 3 is not Hct+/-3, may show lipemiapattern on histogram

CorrectiveAction

Request new sample


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automated cell counting instrumentation (1)

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