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Explain the different principles of automated cell counting. Describe how the general principles are implemented on the different instruments discussed. Identify the hemogram parameters directly measured by analyzers. Explain the derivation of calculated or indirectly measured hemogram parameters. Explain the derivation of the WBC differential count. Interpret and compare patient data, including WBC and RBC histograms or cytograms or both, obtained from the major hematology instruments. Explain the general principles of automated reticulocyte counting. Identify sources of error in automated cell counting and determine appropriate corrective action.

 AUTOMATION provides greater accuracy and

precision than manual methods.

Hematology analyzers provide the eight standard

hematology parameters (CBC) plus a three-part or five-part differential leukocyte count in less than 1 minute on 200 uL of whole blood.
Automation allows more efficient workload

management and more timely diagnosis and treatment of disease.

 Low voltage direct current (DC) resistance Developed by Coulter in the 1950s PRINCIPLE : Cell counting is based on the detection

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

 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

 Orientation of the cell in the center of the aperture

and deformability of the RBC

Recirculation of cells back in the sensing zone creates

erroneous pulses and falsely elevated cell counts

Hydrodynamic focusing
Avoids many potential problems

 Alternating current resistance Low voltage DC maybe used in conjunction with RF

resistance, or resistance to a high voltage electromagnetic current flowing between both electrodes simultaneously
The total volume of the cell is proportional to the

change in DC, the cell interior density is proportional to pulse size or change in the RF signal.

 CONDUCTIVITY : attenuated by nucleus to

cytoplasm ratio, nuclear density, and cytoplasmic granulation

 Two dimensional distribution cytogram Created by plotting impedance against conductivity Displays cell populations as clusters, with the number

of dots in each cluster representing the concentration of that cell type.

 Uses both laser and non-laser light Latest technology

 Hydraulics System
Includes aspirating unit, dispensers, diluters, mixing

chambers, aperture baths, or flow cells or both, and a hemoglobinometer

Pneumatics System
Includes vacuums and pressures required for operating

the valves and moving the sample through the hydraulics system
Electrical Systems
Controls operational sequences of the total system and

includes electronic analyzers and computing circuitry for processing the data gathered

Parameter

Principle

WBC
RBC Hb Hct MCV

Impedance, hydrodynamic focusing

Impedance Modified Cyanmethemoglobin at 525 nm (RBC x MCV)/10 Mean of RBC volume histogram

MCH
MCHC

(Hb/RBC) X 10
(Hb/Hct) X 100

Platelet Count Impedance (2-20 fL) : least squares fit of volume distribution histogram (0-70 fL)
RDW CV (%) of RBC histogram : (SD/MCV) X 100

Reticulocyte Count

NMB-N, volume, conductivity, optical scatter

Parameter

Principle

WBC
RBC Hb Hct MCV

Hydrodynamic Focusing, DC Detection (impedance)

Hydrodynamic Focusing, DC Detection (impedance) SLS Hb (555 nm) Cumulative Pulse Height Detection (Hct/RBC) X 10

MCH
MCHC

(Hb/RBC) X 10
(Hb/Hct) X 100

Platelet Count
RDW

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

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

Reticulocyte Count

Auramine O; fluorescence detection

Parameter

Principle

WBC
RBC Hb Hct MCV

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

Impedance Modified Cyanmethemoglobin (540 nm) (RBC X MCV)/ 10 Mean of RBC distribution histogram

MCH
MCHC

(Hb/RBC) X 10
(Hb/Hct) X 10

Platelet Count
RDW

Impedance (approximately 2 30 fL)

Relative value, equivalent to CV

Reticulocyte Count

Propriety Stain (CD4K530), multiangular scatter, and fluorescence detection

Parameter

Principles

WBC
RBC

Hydrodynamic focusing, optical scatter and absorption

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

Hb
Hct

Modified Cyanmethemoglobin (546 nm)

(RBC X MCV)/10

MCV
MCH MCHC Platelet Count

Mean of RBC volume histogram

(Hb / RBC) X 10 (Hb/Hct) X 100 Hydrodynamic focusing, laser low-angle (2 3 degree) and highangle (5-15 degree) scatter (1-60 fL)

RDW
Reticulocyte Count

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

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

Parameter Neutrophil

Coulter VCS

Sysmex OS and FS

Abbott MAPSS

Siemens PS, OS, Abs

Lymphocyte Monocytes
Eosinophils Basophils

VCS VCS
VCS VCS

OS and FS OS and FS
OS and FS OS and FS

MAPSS MAPSS
MAPSS MAPSS

PS, OS, Abs PS, OS, Abs

PS, OS, Abs Differential lysis, laser low-angle (2-3 degree) and high angle (5-15 degree) scatter

Parameters Affected

RBC decreased, MCV, MCHC increased, grainy appearance

Rationale Instrument Indicators

Corrective Action

Agglutination of RBCs Dual RBC population on RBC map, or right shift on RBC histogram
Warm sample to 37 degrees C and rerun

Parameters Affected

Hb, MCH increased

Rationale

Increased turbidity affects spectrophotometric readings

Hb X 3 is not Hct+/-3, abnormal histogram

Instrument Indicators

Corrective Action

Plasma replacement

Parameters

RBC, Hct decreased

Rationale
Instrument Indicators

RBCs lysed and can not be counted

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

Corrective Action

Request new sample