Hematology Analyzer Automated Cell Counting Instrumentation

Presented to: Mam Raheela

Presented by: NAYAB ARSHAD 2011-Dvme-001`

Definition:Hematology analyzers are computerized, highly specialized and automated machines that count the number of different kinds of white and red blood cells in a blood sample.

History:

The original hematology analyzers first appeared in

the 1950s, but the truly functional and automated versions of these machines did not become available for two more decades.

Before this time, cell counts were performed

manually.

Disadvantages of manual cell counting Cell identification errors in manual counting:Mostly associated with distinguishing lymphocytes from

monocytes Bands from segmented forms and abnormal cells

(variant lymphocytes from blasts) Lymphocytes overestimatedMonocytes underestimatedSlide cell distribution errorIncreased cell concentration along edges

Advantages

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…

• They have a high level of precision for cell counting and cell sizing greatly superior to that of the manual technology

• The results are generally accurate.• No slide distribution error.• Eliminate statistical variations

associated with manual count based on high number of cells counted.

• Many parameters are available.• All automated cell counters are

screening devices. (abnormalities)

Advantages..

Most current hemato

logy analyze

rs provide

• Red blood cell (RBC) counts• Hemoglobin in RBCs, • Hemocrit levels, • Platelet counts,• Corpuscular data,• Counts of five different types of white

blood cells. • Some newer machines also measure

numbers of two specialized cell types — immature white cells and— nucleated red blood cells that are necessary to confirm specific diagnoses

Results:The results they provide are collectively known as

complete blood counts (CBCs) .complete complete blood with differentiation of cells

(CBCs with diff). Some cell counters can process 120-150 samples per

hour .

Hematology analyzer has revolutionised

Internal medicine,

pediatrics oncology

Types :Automated techniques of blood counting:

I. Semi-automated instruments Require some steps, as dilution of blood samples. Often measure only a small number of variables.

II. Fully automated instruments Require only that an appropriate blood sample is presented to the instrument. They can measure 8-20 variables including some new parameters which do not have any equivalent in manual methods.

WorkingHematology analyzers count cells by one of five general cell techniques:Flow cytometryThe use of fluorescent dyes The electrical impedance methodThe light scatter methodRadiofrequency

1:Flow cytometry/Cell sorting

o Flow cytometry is based on ejecting cells from a nozzle at high speed in a fluid.

o Each cell passes through several laser beams so that different optical properties can be measured.

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2:Use of flourescent dyes

• Biochemical or antigenic properties are usually classified using fluorescent dyes.

Continue….Steps :1. Labeling with flourescent dye.2. A laser excites these fluorescent molecules.3. They emit light at various wavelengths.4. Amount of fluorescence can give an indication as to

what percentage of various cell types are present in the sample.

3:Electrical impedance method• Works on coulter principle:

A stream of cells in suspension passes through a small aperture across which an electrical current is applied. Each cell that passes alters the electrical impedance and can thus be counted and sized.

Continue• Particles such as blood cells are nonconductive

BUT are suspended in an electrically conductive diluent.

• As a dilute suspension of cells is drawn through the aperture, the passage of each individual cell momentarily increases the impedance (resistance) of the electrical path between two electrodes that are located on each si.de of the aperture

Continue…A blood cell's size , surface charge , concentration of the cells , shape of cells can be determined

4:Optical scatter method The application of light scatter means that as a single cell

passes across a laser light beam1. diffraction(bending around corners),2. refraction (bending due to change in speed) and3. reflection (light rays turned back by obstruction) Light scatter correlates to cell volume/size

Side angle/orthogonal light scatter correlates to degree of internal complexity (granules and nucleus)

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Continue …The patterns of scatter are measured at various angles. Multi angle polarized scatter separation (M.A.P.S.S)

• 0° :indicator of cell size • 10° :

indicator of cell structure and complexity • 90° polarized:

indicates nuclear lobularity • 90° depolarized:

differentiates eosinophils

Continue…Scattered light provides information about

cell structure, shape, Reflectivity

These characteristics can be used to differentiate the various types of blood cells and to produce scatter plots with a five-part differential.

5:Radiofrequency

Conductivity (RF) – proportional to cell interior density (granules and nucleus)

Examples

o An automated retic count Fluoro-chromes combine with the RNA of the

reticulocytes. Fluorescent cells can then be enumerated using a

flowcytometer. An automated retic counter also permits the

assessment of retic maturity since the more immature reticulocytes have more RNA

o Coulter Instrumentation

Principles of Measurement

•Direct Measurement:•RBC – Impedance•WBC - Impedance•Platelets – Impedance (2-20 fl)•Hgb – mod. Cyanmethemoglobin (525 nm)•MCV – mean RBC volume (histogram)•Indirect Measurement:•Hct, MCHC, and MCH (calculations)•WBC differential •VCS – volume, conductivity, scatter•employs differential shrinkage•Reticulocyte•Supravital stain (new methylene blue)•VCS

Coulter hematology analyzer

Sysmex Instrumentation

Principles of Measurement

• Direct Measurement:• RBC – DC detection (Impedance), hydrodynamic

focusing• WBC – DC detection (Impedance), hydrodynamic

focusing• Platelets – DC detection (Impedance),

hydrodynamic focusing• (2-30 fl)• Hgb – SLS-Hb (555 nm) (oxyhemoglobin +

sodium lauryl sulfate)• HCT – cumulative pulse height detection (see

oscilloscope)• Indirect Measurement:• MCV, MCHC, and MCH (calculation)

• WBC differential • DC/RF detection• employs differential lysis

• Reticulocyte• Fluorescent detection

Symax hematology analyzer

Automated and portable hematology analyzers

DrawbacksSome problems which could be faced: Two cells passing through the orifice at the same time,

counted as one cell. RBC agglutination(clump of cells) Counting bubbles or other particles as cells.

Questions????

Thank you