Automation in hematology - Part (1)
Automation is the process of using a machine to perform steps in laboratory testing.
The uses of automation:
- Cell counts
- diagnosis of Leukemias & Lymphomas
- diagnosis of Hemoglobinopathies
- Coagulation Abnormalities.
- Immunophenotyping.
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 overestimated; monocytes underestimated
- Slide cell distribution error: increased cell concentration along borders, also bigger cells found there i.e. monocytes, eosinophils, and neutrophils
- Statistical sampling error
Advantages of Automation
- Use a very small amount of sample and reagent.
- Increase the number of tests performed by one laboratorian in each period.
- Lessen the variation in results from one laboratorian to another
- The coefficient of variation is lowered.
- Reproducibility is increased.
- Accuracy is not dependent on the skills or workload of the operator on the day.
- Limitation of errors of manual analysis such as volumetric pipetting steps, calculation of results, and transcription of the result.
- Deviating results following on unusual characteristics of blood are “flagged” for subsequent review.
- No slide distribution error.
Disadvantages of automation
- Flagging
- RBC Morphology
- Erroneous results
- Expensive
Automated cell counters can provide
- CBC: WBC, RBC, RBC indices, Hgb, Hct, PLT
- WBC Differential: 5 “normal” white cell types
- RDW, PDW, MPV
- Reticulocyte count
- Nucleated red cell count
All automated cell counters are screening devices. Abnormalities must be confirmed by a blood film, staining, and scanning by an expert observer
Types of Automated Hematology Analyzers
1. Semi-automated analyzers:
- Measures only a few parameters
- Some steps like dilution of blood are carried out manually.
2. Fully automated analyzers
- They can count 8-20 variables including some new parameters which do not have any corresponding in manual methods.
- Requires only anticoagulated blood samples.
Principles of automated cell counters
- Impedance (conductivity) system (Coulter)
- Optical system (H*1)
- Both impedance and optical
- Selective lysis (e.g. lysis of red cells and counting of white cells).
- Special stains
Electrical impedance
The regular method for counting cells is electrical impedance, also known as the Coulter Principle. It is used in almost every hematology analyzer.
Whole blood is moved between two electrodes through an aperture so narrow that only one cell can pass through at a time. The impedance changes as a cell pass through. The change in impedance is equivalent to cell volume, resulting in a cell count and a measure of volume.
Impedance analysis produces CBCs and three-part WBC differentials but cannot able to differentiate between the nearly sized granular leukocytes: eosinophils, basophils, and neutrophils.
Counting rates of up to 10,000 cells per second can be obtained and a typical impedance analysis can be carried out in less than a minute.
Optical method
- Laser light is used
• A diluted blood specimen moves in a steady stream via which a beam of laser light is focused.
• As each cell moves through the sensing zone of the flow cell, it scatters the focused light
• Scattered light is discovered by a photodetector and converted to an electric impulse.
• The number of impulses generated is directly proportional to the number of cells moving through the sensing area in a specific period of time.
- The application of light scatters means that as a single cell passes across a laser light beam, the light will be reflected and scattered.
- The patterns of scattering are measured at various angles.
- Scattered light gives information about cell structure, shape, and reflectivity.
- These features can be used to differentiate the various types of blood cells and to produce scatter plots with a five-part differential.
Light scattering
- Cells counted as passed through a focused beam of light (LASER)
- Sum of diffraction (bending around corners), refraction (bending due to change in speed), and reflection (light rays turned back by obstruction).
- Multi-angle polarized scatter separation (M.A.P.S.S)
▪0°: an indicator of cell size.
▪10°: an indicator of cell structure and complexity.
▪90° polarized: indicates nuclear lobularity.
▪90° depolarized: differentiates eosinophils.
Types of Hematology Analyzers
- Smaller instruments: Measure WBCs, RBCs, Hgb, Hct, MCV, MCH, MCHC, and PLTs).
- Advanced cell counters: Add:
▪Red cell morphology information, RDW
▪Mean platelet volume
▪Leukocyte differential
- Today trends involve efforts to integrate as many analysis parameters as possible into one instrument platform, to decrease the need to run a single sample on multiple instruments (e.g. CD4 counts, smear preparation).
- like instruments are being incorporated into highly automated combined chemistry/hematology laboratories, where samples are automatically sorted, aliquoted, and brought to the appropriate instrument by a robotic track system.
Laboratory measurements Hb concentration
• Hb is estimated automatically by a modification of the manual (HiCN) method.
• To reduce the toxicity of HiCN some systems replace it by a nontoxic material Na- lauryl sulfate.
- The sample is diluted with Cyanmethemoglobin reagent
• Potassium ferricyanide in the reagent converts the hemoglobin iron from the ferrous state (Fe++) to the ferric state (Fe+++) to make methemoglobin, which then combines with potassium cyanide to form the stable cyanmethemoglobin.
- A photodetector detects color intensity at 546 nm
- The optical density of the solution is equivalent to the concentration of hemoglobin
RBC count
- The RBCs are estimated automatically by two methods:
•Aperture impedance: where cells are counted as they move in a stream through an aperture.
•Or by light scattering technology.
- The precision of an electronic counting for RBCs is better than the manual count, and it is available in a fraction of time.
- This made the use of RBC indices of more clinically applicable.
Reliability of electronic counters
- 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 an equivalent time counted together cell.
•RBC agglutination (clump of cells)
•Counting bubbles or other particles as cells.
PCV and red cell indices
- Pulse height analysis permits either the PCV or the MCV to be determined.
- MCV=PCV/RBC count
- MCH= Hb/RBC count
- MCHC= Hb/PCV
- MCH & MCHC are derived parameters.
Red cell distribution width (RDW)
- Automated instruments produce volume distribution histograms that allow the presence of more than one population of cells to be appreciated.
- Most instruments can give a quantitative measurement of variation in cell volume, an equivalent of the microscopic assessment of the degree of cell variations. This is known as the RDW.
Total WBC count
- The total WBC count is calculated in whole blood in which RBCs have been lysed.
- Fully automated multichannel instruments achieve WBC counting by:
•Impedance
•Light scattering
•Or both.
Automated differential count
- Automated differential counters which are accessible now generally use flow cytometry incorporated into a full blood counter rather than being standard alone differential counters
- Automated counters give a three-part or five- to seven-part differential count.
Differential cell counting
- 3-part differential usually count
•Granulocytes or large cells
•Lymphocytes or small cells
•Monocytes (mononuclear cells).
- 5-part classify cells to
•Neutrophils
•Eosinophils
•Basophils
•Lymphocytes
•Monocytes
- A sixth category nominated “large unstained cells” include cells larger than normal and without the peroxidase activity this include:
•Atypical lymphocytes
•Various other abnormal cells
- Other counters identify 7 categories including
•blasts and immature granulocytes
•Atypical lymphocytes (including blast cells)
- The analysis may be dependent on:
•Volume of the cell.
•Other physical characteristics of the cells.
•Occasionally the activity of cellular enzymes such as peroxidase.
- Technologies used
•Light scattering and absorbance
•Impedance measurement
- Automated differential counters apply flow cytometry to classify far more cells than is possible with a manual differential count.
Accuracy in blood cell counting
- The accuracy of automated counters is low impressive than their precision.
- In general, automated differential counters are favorable to the manual in 2 conditions
•Examination of normal blood samples.
•Flagging of abnormal samples.
Platelet count
- Platelets probably counted in whole blood by using the same technologies of electrical or electro-optical detection as are employed for RBCs.
- Other parameters include
•MPV
•PDW
•Plateletcrit = MPV x platelet count.
Reticulocyte count
- An automated reticulocyte counts probably achieved using the fact that various fluoro-chromes combine with the RNA of the reticulocytes. Fluorescent cells probably calculated using a flow cytometer.
- An automated reticulocyte counter also allows the assessment of reticulocyte maturation since the more immature reticulocytes have more RNA the take fluoresce more strongly than the mature ones found in peripheral blood.
Flow Cytometry
Laser flow cytometry is high-cost than impedance analysis, due to the need for expensive reagents, but returns detailed information about the morphology of blood cells.
It is a very good method for determining five-part WBC differentials.
A single-cell stream passes through a laser beam. The absorbance is calculated, and the scattered light is measured at multiple angles to identify the cell’s granularity, diameter, and inner complexity.
These are the same cell morphology features that can be discovered manually from a slide.
Fluorescent flow cytometry.
Adding fluorescent reagents increase the use of flow cytometry to measure specific cell populations.
Fluorescent dyes tell the nucleus-plasma ratio of each stained cell.
It is helpful for the analysis of platelets, nucleated RBCs, and reticulocytes.
Components of Flow Cytometry
1. Fluidics (The Flow System)
- The sample is introduced into a stream of sheath fluid within the flow chamber.
- They are pushed into the center of the stream forming a single file by the principle of HYDRODYNAMIC FOCUSING.
- The sample pressure is always greater than the sheath pressure guarantee a high flow rate, thus allowing more cells to enter the stream at a given moment.
- The high Flow rate is used for immunophenotyping analysis of cells.
- The low Flow rate used for DNA Analysis.
2. Optics
- Following cell carriage, a light source like the Argon- ion LASER is required to excite the cells.
- When light from a Laser Beam cross a cell at the ‘interrogation point’, 2 events occur -
•Light Scattering
•Fluorescence (Emission of Light)
- Light Scattered in the forward direction is detected in forwarding Scatter Channel ∝ to cell size and that scattered at 90° to the axis of Laser path is detected in Side Scatter Channel ∝ to the granularity of the cell.
- The cells labeled with fluorescence emit a momentary pulse of fluorescence.
- A system of optical mirrors and filters then directs the specified wavelengths of light to the designated photodetectors.
3. Electronics
- The photodetectors - photodiodes and
- photomultiplier tubes change the optical signals (photons) to corresponding electronic signals(electrons).
- The electronic signal build is proportional to the amount of light striking a cell.
- The electric current moves to the amplifier and is converted to a voltage pulse.
- The voltage pulse is allocated a digital value representing a channel by the Analog-to-Digital Converter (ADC).
- The channel no: is conveyed to the computer which displays it to the appropriate position on the data plot.
4. Data Analysis
- Data is collected and stored in the computer – can be displayed in various formats.
- Parameters – Forward Scatter, Side scatter, released fluorescence.
- Data plots – Single Parameter – Histogram Two Parameters – Dot Plot.
Gating
- A boundary that can be used to limit the analysis to a specific population within the sample.
- Could be:
•Inclusive – Choice of events that fall within the boundary.
•Exclusive - Choice of events that fall outside the boundary.
- Data selected by the gate is then presented in subsequent plots.
Sorting
Made up of collecting cells of interest (defined through criteria of size and fluorescence) for more analysis (microscopy /functional/ chemical analysis)
Applications of Flow Cytometry
1. Leukemias and lymphomas: Immunophenotyping (evaluation of cell surface markers), diagnosis, detection of minimal residual disease, and to identify prognostically important subgroups.
2. Paroxysmal nocturnal hemoglobinuria: Lack of CD 55 and CD 59.
3. Hematopoietic stem cell transplantation: Enumeration of CD34+ stem cells.
4. Feto-maternal hemorrhage: Detection and quantitation of fetal hemoglobin in the maternal blood sample.
5. Anemias: Reticulocyte count.
6. Human immunodeficiency virus infection: For enumeration of CD4+ lymphocytes
7. Histocompatibility cross-matching
Automated coagulation analyzer
Devices that diagnose the clotting mechanisms of hemostasis; used to detect clotting deficiencies linked to thromboembolic disease, thrombocytopenia, impaired liver function, hemophilia, von Willebrand disease, and other conditions. They are also used to observe the effect of certain drugs such as heparin, oral anticoagulants, and thrombolytic and antiplatelet agents on whole blood, as well as the effects of blood component therapy.
For quality control of the coagulometer, it is necessary to have a calibrated normal reference preparation tested alongside the patient’s sample.
Ensure that their temperature control and mechanism of detecting the endpoint are functions properly.
Endpoint detection mechanisms:
1. Electromechanical: (impedance steel ball)
In which the sample cuvette rotates, and a steel ball stay static in the magnetic field until the formation of fibrin strands around the ball produce movement, this detected by the change in the magnetic field and the coagulation time is record or
The steel ball is rotating under influence of the magnetic field until the formation of fibrin strands around the ball stop it is rotation, this is detected by a sensor, and coagulation time is recorded.
2. Photo-optical: (scattered light detecting for clotting assay)
The turbidity in the course of the formation of a fibrin clot is measured as scattered light intensity when exposed to light at a wavelength of 660nm.
3. Electrochemical: (INRatiometer- near point test device)
The device recognizes a change in electrical resistance when blood clots are formed.
Reported problems
Operators should consider the hazard of exposure to potentially infectious bloodborne pathogens during testing procedures and should use universal precautions, including wearing gloves, face shields or masks, and gowns.
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