Automation in haematology

Dr.Titto rahim

Process of replacement of tasks performed by humans by computerized methods

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

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.

Automation in hematology

Speed with efficient handling of a large number of samples

Accuracy and precision in quantitative blood tests

Ability to perform multiple tests on a single platform

Significant reduction of labor requirements Invaluable for accurate determination of red

cell indices

Advantages

Flags : flagging of laboratory test result demands labor –intensive manual examination of a blood smear

Comments on red cell morphology cannot be generated . Abnormal red cell shapes (such as fragmented cells) cannot be recognized.

Erroneously increased or decreased results due to interfering factors

Expensive with high running costs

Disadvantage

Electrical impedance Light scatter Fluorescence Light absorption Electrical conductivity

Principle of working of Automation

The Coulter principle-electrical impedance

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.

Each cell flows in a single line through a flow cell . A laser device is focused on the flow cell as the laser light beam strikes a cell it is scattered in various directions.One detector captures the forward scatter light that is proportional to cell size and other captures the side scatter which is equal to cell granularity & complexity

Light scatter

Light absorption :Concentration of hemoglobin is measured by absorption spectrophotometry , after conversion of hemoglobin to cynmethmoglobin

Some analyzers use peroxidase cytochemistry to classify leukocytes

Electrical conductivity : Some analyzers use conductivity of high frequency current to determine physical and chemical composition of leucocytes for their classification .

Fluorescence: used to measure RNA , DNA and cell surface

Rbc count Hemoglobin Mean cell volume Mean cell hemoglobin Mean cell hemoglobin concentration WBC count WBC differential Platelet count Red cell distribution width Reticulocyte count

Parameters measured

Reticulocyte hemoglobin content Mean platelet volume Platelet distribution width Reticulated platelets

Contn……

◦ Hb is measured automatically by a modification of the manual (cyanide) method.

◦ To reduce toxicity of Cyanide some systems replace it by a non-toxic material Na- lauryl sulphate.

Hb Estimation

The RBCs are counted automatically by two methods

◦Aperture impedance: where cells are counted as they pass in a stream through an aperture.

◦Or by light scattering technologyThe precision of an electronic counting for

RBCs is much better than the manual count, and it is available in a fraction of time.

This made the use of RBC indices of more clinical relevance.

RBC Count

Red cell count and volume are directly measured by aperture impedance or light scatter analysis . In a red cell histogram , cell numbers are plotted on Y axis,white cell volume on X-axis

Analyzer counts those cells with volume of 36fl and 360 fl

MCV is used for anemia classification in microcytic, macrocytic and normocytic types

Red Cell Count and Volume

Mean cell hemoglobin =hemoglobin (g/l)/red cell count (10/ml)

Mean cell hemoglobin concentartion (g/dl)= hemoglobin(g/dl)/hemotocrit(%)Hemocrit (%) =mean cell volume(fl)/red cell

count (10 /ml)

Parametrs measured through calculation

RBC count Mean cell volume Red cell distribution width Hemoglobin Reticulocyte count WBC count Differential WBC count Platelet count Mean platelet volume

Parameters measured from histogram

Automated instruments produce volume distribution histograms which allow the presence of more than one population of cells to be appreciated.most instrument produce a quantitative measurement of variation in cell volume, an equivalent of the microscopic assessment of the degree of anisocytosis. This is known as the RDW.

Red cell Distribution Width (RDW)

The total WBC count is determined in whole blood in which red cells have been lysed.

Fully automated multichannel instruments perform WBC counting by either◦ Impedance◦ Or light scattering◦ Or both

Total WBC Count

Automated differential counters which are available now generally use flow cytometry incorporated into a full blood counter rather than being standard alone differential counters

Automated counters provide a three-part or five- to seven-part differential count.

Automated Differential Count

3-part differential usually cont ◦Granulocytes or large cells◦Lymphocytes or small cells◦Monocytes(mononuclear cells) or (middle cells)

5-part classify cells to◦Neutrophils◦Eosinophils◦Basophils◦Lymphocytes ◦Monocytes

Cont…

A sixth category designated “large unstained cells” include cells larger than normal and lack the peroxidase activity this include

◦Atypical lymphocytes◦Various other abnormal cells.

Other counters identifies 7 categories including

◦Large immature cells(composed of blasts and immature granulocytes)

◦Atypical lymphocytes(including blast cells).

Cont…

The accuracy of automated counters is less impressive than their precision.

In general automated differential counters are favorable to the manual in 2 conditions◦ Exam of normal blood samples◦ Flagging of abnormal samples

Cont….

Platelets can be counted in whole blood using the same tech. Of electrical or electro-optical detection as are employed for RBCs.

Other parameters include◦ MPV◦ PDW◦ Plateletcrit= MPV x platelet count.

Platelet Count

An automated retic count can be performed using the fact that various 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 RNAfluoresce more strongly than the mature retics found normally in PB.

Reticulocyte Count

They are precise but care should be taken so that they are also accurate.

Some 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

Reliability of Electronic Counters

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