Example Of Comparison Of Impedance And Optical Based Counting Methods In Hematology Literature Review
Type of paper: Literature Review
Topic: Blood, Light, Development, Electricity, Size, Flow, Particle, Beam
There are many developments which have been seen counters. The current trends are mostly new developments which have come with eth enhancement of digital systems. There are many categories which have been developed with digital systems and processes. The choice and the method that is used for counting will depend on the theory used. This paper will focus on how impedance is used in counting technology. It will also look at the methods and procedures that have been followed while researching in impedance technologies.
Automated cell counters are classified asimpedance systems, optical systems and impedance and optical machines. They use a method referred as selective lysis for instance the lysis of red blood cells and counting of white cells.The electrical impedance method is known as the Coulter method (F, 2002). Cell counting and sizing is based on detection and measurement of electrical impedance changes produced by particles at ittraverses the small aperture. Blood particles are naturally unconductive but are suspended in an electrically conductive diluents. As the diluents is drawn through the aperture, the passage of eachindividual cell causes a momentarily increased impedance or resistance of the path of electric betweenthe two electrodes located at the opposite side of the aperture.
It has been a number of decades since the invention of the Coulter principle. The history of automatedhematology analysis started with Red Blood Cell counting. Segal notes that WBC counting andhemoglobin determination was facilitated by the development of lysing reagents. As technology advanced, a complete blood count was possible. Further developments lead to the development of theWBC differential and the common multi-parameter automated blood counting techniques of this time.The trend is moving towards development of next generation hematology analyzers. In this paper, literature review of impedance counting methods and optical methods is undertaken with focus on thedifferences and the similarities. Electric impedance method is the most common method used in hematology analyzers (Segal, 2005).Similarities
In electrical impedance method, biological cells are regarded as conductive resistivity cells. When RBC, WBC, or platelets pass through an aperture placed in an electrolytic solution, a change in impedance isthat is equivalent to the volume of the fluid is produced. Because cells are counted based on size alone, not all WBC can be separated from one another. Nucleated red blood cell and clumped platelets are relatively the same size as leukocytes and may be misclassified as WBCs and large platelets. Thiscreates and issue in the counting process as also small clump of platelets may be taken as erythrocytes
Rodak, (2002) explored the optical method. According to the author, laser light is used. A diluted bloodspecimen passes a steady stream through which a beam of laser light is illuminated. As each cell goesthrough the aperture, it scatters the focused light. Scattered light is detected by an photodetectorwhich converts it to an electric siganl. The amount of signals produced directly correlates with thenumber of cells traversing the light beam in the sensing zone in a particular time period.
The impedance based hematological method of counting was pioneered by Coulter Company andinvolves suspension of particles in an electrically conductive fluid such as saline water or emulsifier (Groner, 1995). Particles are forced to move through it. The merit with this method is that it issuitable for a broad range of particle sizes within 0.5 -300 micron and above with different orificeadjustments. It is also simple to calibrate with known size standards just as the optical counters. Bothmethods have quick analysis time and output repeatable results with many kinds of samples thatinclude non-spherical particles. The resolution is comparable to LALLs with adjacent narrow peaks thatdiffer by 15% and can be resolved.According to (Mueller, 2007), both methods have some common disadvantages. The dynamic size rangeis constraint to about 30 in a single run. This means that analysis is from 2% of the orifice to about 60% upon which the requirement for broader distribution requires pre-separation of samples as per theirsize. Samples are normally suspended in conductive fluids for the both methods but saline water may not be convenient for many types of samples. The underlying conductive medium behind these two methods is use of saline water with emulsifier. However, all particles undergoing the measurementsare not necessarily adapted to it.Hill, et al presumed that particles are normally electrical insulators so that each particle produces asharp spark in resistivity as it passes the orifice for the case of impedance counter. In the case ofoptical counting meter, the particles are deemed to be blocking agents of laser light so that a reductionin the light intensity reaching the sensor is equated to optical cross section of the particle. The lightintensity reaching the detector might be impacted by the properties of the sample (Hill, 2009).Another common disadvantage with the two methods is that it offers limited chances to measure sizesbelow 1-2 microns. Though the method is indicated to measure a minimum of 0.5 microns, experiencehas shown that measuring particles less than 2 micros is affected by stray oversize particles trapped inthe orifice. Also, particles less than 0.5 microns cannot be measured by the method under anycircumstance. Finally, both methods offer limited resolution in the lower limit of the instrument ascompared to the middle of the measurable range (Metzger).Optical counters are equivalent of the impedance method. However instead of the electrical signals, a laser beam is partially blocked as the particle passes by. The reduction in light intensity reaching adetector is a factor of the optical cross sectional are of the particle. This cross section is converted to a size distribution.
Laser flow cytometry has been termed by Rodak (2002) as the most accurate and recent automated cellanalyzer technology. The method is utilized in many laboratories to determine the leukocyte differentialcount. As cells traverse a laser beam, a pattern of light scattered by the individual cells is counted(Segal, 2005). Such information such as the cell size, cytoplasmic content, nuclear characteristicand others are recorded to provide a true 5-part optical differential of the leukocyte. Metzger explored the difference between analyzers using dog and cat reticulocyte, a gold method for classifying anemia. The authorindicated that the technology is slightly expensive than other available methods and are limited toreference labs alone.According to Metzger, the main features in in-house hematology analyzers vary greatly. Thus, practitioners should weight the advantages and disadvantages before deciding on an appropriate instrument. The author posits that regardless of instrumentation, veterans should interpret analyzer results based on a peripheral blood film evaluation which give invaluable information on cellular morphological changes (Ghanayem, 1990).The difference between the two counting method can be illustrated by the functioning of the SYSMEX XE-2100 counter. The machine uses both impedance and flow cytometry. Its output is 150 samples per hour. The counter uses optical fluorescent platelets when impedance count is not reliable. It has unique features that can enumerate and not just flag for immature granulocytes. Flow cytometry is preferredfor WBC differential, reticulocytes and optical platelets. The number of RBC and platelets are determined by electric impedance method. In samples with large platelets and RBC or RBC fragments, platelet counts by light scattering are preferred. So the inference is, optical counting method is morepreferred to flag off irregular blood components.Electrical impedance method has leads to the production of M-shaped signals as a result of non-axialcell flow in the sensing zone. This can lead to misleading measurements. Electrical impedance baseddevices such as Sysmex products KX-21 and K-4500 lack hydrodynamic focusing but uses an editedcircuitry to detect and eliminate M-shaped signals. Coincidence loss is also possible and is eliminatedautomatically (Johnson, 2002). Since the frequency and shapes of M-shaped signals is dependent on thedeformability of the cells, the individual characteristic of a cell has distinct features with varyingdegrees of deformability.In the high end electrical impedance analyzers such as XE-2100, SE Series, NE series, the requirementfor editing circuitry has been removed by hydrodynamic focusing methods. The method minimizescoincidence and occurrence M-shaped signals, hence outputting reliable distribution curves and countresults. Also, accurate sensing on the sheath flow in the sensing zone necessitates precision components and sophisticated fluid control. The occurrence of small air babbles produced near the sensing zonehas the capacity to disturb accurate focusing and cause electric noise. Thus, a special surfactant isadded to the sheath reagent in electrical impedance method to produce a small number of bubbleswhich are rapidly removed from the sensing unit (Metzger).ConclusionHematology analyzers have traditionally used electric impedance method for quite a while. Theapplication stated from experimental analyzers and advanced to clinical laboratory analyzers withsubstantial benefits to modern health. Recent application s in medical use include electric impedance hematology analyzers for blood stem cell identification, RBC deformability, blast-formation andlymphocytes and assessment of platelet membrane functions among others. Additionally, particle sizedistribution analyzers exist for use in industrial applications such as quality control maintenance offood, drugs, cosmetics, oil and fats and many other products.While there exist small differences between the two methods, the two types of analyzers have many commonsimilarities. The similarities noted in the two methods include simplicity in using concept andcalibration, gives persistent results, and its suitability for a wide range of sizes. Both methods seem tohave some limitations in their assumption of conductive fluid. The contents of the cell are determined by studying the angle of scattering. Information on cell size produced by low angle light scattering and information on internalcomposition of the cell produced by high angle scattered light. It is possible to develop a scattergramwhich differentiates the various blood components and count red blood cells, platelets andreticulocytes. The light scattering method in optical counting is depending on two theories relative to the wavelength of incident and the diameter of objective: Raleigh and Mie theories. Their applicationis well established in the development of counters in the industry. Developments and innovation in theindustry has seen the development of hybrid hematology counters which employ impedance and lightscattering method. An example of such as counting device is the XE-2100 from Sysmex.
Bourner, G. D. (2005). Performance evaluation of the latest fully automated hematology analyzers in a large, commercial laboratory setting: a 4-way, side-by-side study. . Laboratory Hematology , 11(4), 285-297.
F, R. B. (2002). Diagnostic Hematoology. Saunders.
Fujimoto, K. (2009). Principles of measurement in hematology analyzers manufactured by Sysmex Corporation. . Sysmex Journal International, , 9(1; SEAS SUM) 31-44.
Ghanayem, B. I. (1990). Comparison of the hematologic effects of 2-butoxyethanol using two types of hematology analyzers. Toxicology and applied pharmacology , 106(2), 341-345.
Groner, W. &. ( 1995). Practical guide to modern hematology analyzers. John Wiley & Sons.
Hill, V. L. (2009). Evaluation of the performance of the Sysmex XT-2000i hematology analyzer with whole blood specimens stored at room temperature. . Lab Medicine , 40(12).
Johnson, M. S. (2002). Three-way evaluation of high-throughput hematology analyzers—Beckman Coulter LH 750, Abbott Cell-Dyn 4000, and Sysmex XE-2100. . Laboratory Hematology , 8(4), 230-238.
Metzger, F. Diagnostic Insight: Choosing an in-house hematology analyzer. PA: State College.
Mueller, T. D. (2007). Utility of whole blood impedance aggregometry for the assessment of clopidogrel action using the novel Multiplate® analyzer—comparison with two flow cytometric methods. . Thrombosis research , 121(2), 249-258.
Segal, H. C. (2005). Accuracy of platelet counting haematology analysers in severe thrombocytopenia and potential impact on platelet transfusion. British journal of haematology Journal .