Automation

Replacement of manual methods by computerized methods is called as Automation.

Recent developments in electronic, robotics, computer technology and new analytical methods have been integrated to produce so called automated laboratory analyzers. This generation of equipment has greatly facilitated the work in busy clinical laboratories. Such equipment is usually expensive and requires expert engineers to maintain but has several advantages. Some of these are:

1. Manipulation of heavy workload with less manpower.

2. Reduction in time in completing the test.

3. Reduced consumption of reagents and microanalyses.

4. Precision and accuracy of results.

5. Integration of quality assurance into the test system.

6. Automatic printing of results thus eliminating clerical errors.

7. Distant communication of results.

8. Data storage and statistical analyses.

GUIDELINES FOR CHOOSING AN INSTRUMENT

The laboratory should define its budget and scope of daily work etc. It can choose instrument from amongst the market. Factors to be considered in making a choice include capital expenditure, running and maintenance costs, ready availability for reagents/accessories/spare parts, size of instrument, requirement of services (water, compressed air, drainage, electrical supply with a stable voltage), reagents availability, storage and back up services etc. A committee should consider whether to buy or lease the instrument. Alternatively, the machine may be used on a reagent rental basis. There is hardly a branch/department of Pathology where automation does not exist. Some examples of common automated equipment are as follows:

AUTOMATION IN HAEMATOLOGY

Several tests performed in hematology laboratory have been automated. Most important of these are blood counts, coagulation and blood grouping/cross matching.

AUTOMATION OF BLOOD COUNTS

Complete Blood Counts (CBC) form the main bulk of laboratory tests requested. By manual method it is difficult to do all of these with acceptable accuracy and precision. This was realized very early. In 1956 Wallace Coulter first described an electronic cell counter, which has revolutionized the hematology laboratory. Hematology analyzers are now available for the needs of laboratory of any size. The range varies from simple blood cell counts and red cell indices to partial or full differential count, histograms of cell sizes and reticulocyte count. It is important, particularly in our country, to ensure that proper after-sale services and spares are available with the supplier.

TYPES OF AUTOMATED CELL COUNTERS

Fully automated instruments

In these only an appropriate blood sample is presented to the instrument. Some are capable of aspirating the sample themselves from containers placed on a turntable or similar device.

Semi automated instruments

These require some steps, e.g., dilution, to be performed by the operator. They often measure a small number of components. These are mostly obsolete now.

Parameters included in Automated cell counters

WBC is white blood cells count

RBC is red blood cells count

HGB is hemoglobin

HCT is hematocrit

MCV is mean cell volume

MCH is mean cell hemoglobin

MCHC is mean cell hemoglobin concentration

PLT is platelets count

LYM %, MXD%, NEUT % are relative counts of differential leukocyte count

LYM #, MXD#, NEUT# are absolute counts of differential leukocyte count

RDW is red cells distribution width

PDW is platelets distribution width

MPV is mean platelet volume

PRINCIPLES OF AUTOMATED BLOOD COUNTING

1. Measurement of hemoglobin concentration

Most automated counters measure hemoglobin by a modification of the manual cyanomethaemoglobin method. Due to high throughput of the instruments, measurements of absorbance is made at a set time interval after mixing of the blood and the active reagents but before the reaction is completed. In order to achieve this standard HiCN technique is modified with respect to pH of reaction, temperature and concentration of the reagents. Usually a non-ionic detergent is used to ensure rapid cell lysis and to reduce turbidity. Alternatively, in some instruments sodium lauryl sulphate is used to measure hemoglobin. This is due to the fact that cyanide used in HiCN method is a highly toxic substance.

2. Particle (Cell) Counting

The two basic types of technologies used for blood cell counting are aperture (electrical) impedance counting and optical method (light scattering) counting. In these methods a large number of cells are counted rapidly. This leads to a high level of precision and reproducibility, which sharply contrasts with the results obtained for blood cell counting by manual techniques. These technologies have made RBC count, MCV and MCH of much greater clinical relevance.

a. Aperture impedance counting

Blood cells do not allow electrical current to pass through them, i.e. they impede (resist) the passage of electrical current. There are certain diluents, which allow electrical current to pass through them. This difference forms the basis of cell detection by this technology. The cells are highly diluted in a buffered electrolyte solution. This fluid passes through a small aperture. A constant current passes through two electrodes on either side of it. As a blood cell passes through it, electrical conductance in the aperture is decreased. This generates an electrical impulse. The magnitude of each pulse is proportional to the size of the particle. These impulses are sorted electronically and split to count WBC, RBC and platelets.

 

Sysmex KX-21 is an example of instrument working on this principle

b. Optical method (light scattering) counters

The blood cells scatter light to a variable extent and at various angles, depending upon their size, shape, nuclear lobes, presence of granules, etc. This forms the basis for blood cell detection and counting by electro-optical methods. The blood cells are suitably diluted. The diluted blood cell suspension is made to flow through an aperture in a way that the cells pass in a single file in front of a light source. The light is scattered by the cells. This scatter is measured by photomultiplier tube (PMT) or photodiode, which converts it into electrical impulse. These impulses are then sorted to count WBC, RBC, Platelets and differential count (Neutrophils, Lymphocytes Monocytes, Eosinophils and Basophils)

Forward Scattering:

Amount of light scattered in forward direction is detected by forward scatter channel. Intensity of forward scattering depend on size and shape of the particle

Reverse Scattering:

The amount of light scattered to the side is detected in side scatter channel. It describes inclusions within the cell and distinguish granulated from non granulated cells.

3. Automated WBC differentials

Automated blood counters have a WBC differential counting capability and provide three/five/seven part WBC differential counts. Abnormal cell populations may be flagged to be confirmed by microscopy. Three part differential counts are based on different volume of various cell types. In optical detection methodology this may be augmented using flowcytometry. In electrical impedance methodology, cells are further characterized with radio frequency current or low and high frequency electromagnetic current. Some counters use cytochemical stains to differentiate between various WBC.

4. Platelet counting

Platelets can be counted in whole blood using same techniques as employed for red blood cells. Usually platelets are counted in the same channel as used for red blood cell detection with a threshold set to separate red blood cells from platelets.

5. Reticulocyte counts

Reticulocytes contain RNA. There are fluorescent as well as traditional dyes, which combine with RNA, and reticulocytes can thus be counted.

Graphical representation of data

These instruments also produce a graphical representation of the data in the form of histograms or scatter plots. These may either be in colour or in black and white. These graphs provide further valuable information. These show patterns which correlate well with various abnormalities in the blood film. This alerts to the possibility of an abnormality, which can then be confirmed by examination of a blood film.

CALIBRATION OF HAEMATOLOGY AUTOANALYSERS

These machines are calibrated in the factory. However, calibrators are available which can be used to calibrate them when required. These calibrators are quite expensive. The manufacturer supplies details for calibration. The hemoglobin is calibrated using cyanmethemoglobin method, while the PCV is calibrated using the micro-hematocrit method.

EXAMPLES OF HAEMATOLOGY AUTOANALYSERS

The major manufacturers include

ü  Beckman Coulter,

ü  Sysmex

ü  Merck

ü  Technicon-Bayer

ü  Cell Dyn series of Abbott Diagnostics,

ü  Cobas of Roche Diagnostic systems

Various models are available by each manufacturer.

PRACTICAL IMPLICATIONS OF HAEMATOLOGY AUTOANALYSERS

These instruments, to be useful need proper maintenance and backup services. The laboratory should ensure proper internal quality control as well as external quality assessment of these machines. Various instruments use technologies like hydrodynamic focusing or sheath flow, electronic editing etc. These machines are usually closed system, with reagents, controls, calibrators all being supplied by the manufacturer himself.

AUTOMATION IN HAEMOSTASIS

Automated coagulation analyzers

A number of automated and semi-automated coagulation analyzers are available. The choice of an analyzer depends on the workload and cost implications. A thorough evaluation of the current range of analyzers is recommended before purchase.

Most equipment is based on clotting assays. Formation of fibrin clot results in change in optical density of the reaction mixture, Stoppage of moving bead by clot formation or by color production at the end. If coagulation analyzers are used it is important to ensure that the temperature control and the mechanism for detecting the end point are functioning properly. Although such instruments reduce observer error when a large number of samples are tested, it is important to apply proper quality control at all times to ensure accuracy and precision.

Types of Coagulation Automation

n  Electromechanical instrument (Stoppage of iron bead or current passage)

n  Optical density instrument (Increase in optical absorbance and decrease in optical transmittance)

n  Chromogenic clot detection instrument (Formation of color product)