Hematology blood analyzers are the workhorses of clinical laboratories. These high-performance instruments provide reliable RBC, platelet, and 5-component WBC counts that identify lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The number of nuclear erythrocytes and immature granulocytes are the 6th and 7th indicators. Although electrical impedance is still fundamental to the determination of total cell number and size, flow cytometry techniques have proven valuable in leukocyte differentiation and in the examination of blood on a hematological pathology analyzer.
Evolution of the analyzer
The first automated blood quantifiers introduced in the 1950s were based on Coulter's principle of electrical impedance, in whichcells, passing through a small hole, broke the electrical circuit. These were "prehistoric" analyzers that only counted and calculated the average volume of erythrocytes, average hemoglobin and its average density. Anyone who has ever counted cells knows that it is a very monotonous process and no two technicians will ever give the same result. Thus, the device eliminated this variability.
In the 1970s, automated analyzers entered the market, capable of determining 7 blood parameters and 3 components of the leukocyte formula (lymphocytes, monocytes and granulocytes). For the first time, manual leukogram counting was automated. In the 1980s, one tool could already calculate 10 parameters. The 1990s saw further improvements in leukocyte differentials using flow methods based on electrical impedance or light scattering properties.
Hematology analyzer manufacturers often seek to separate their instruments from competitors' products by focusing on a particular package of white blood cell differentiation or platelet counting technologies being used. However, experts in laboratory diagnostics argue that most models are difficult to distinguish, since they all use similar methods. They just add additional features to make them look different. For example, one automated hematology analyzer can determine leukocyte differentials by placing a fluorescent dye in the nucleus.cells and glow brightness measurements. The other can change the permeability and record the dye uptake rate. The third is able to measure the activity of the enzyme in a cell placed in a specific substrate. There is also a volumetric conduction and scattering method that analyzes blood in its "near natural" state.
New technologies are moving towards flow-through methods, where cells are examined in turn by an optical system that can measure many parameters never before measured. The problem is that each manufacturer wants to create their own method in order to maintain their identity. Therefore, they often excel in one area and lag behind in another.
Current State
According to experts, all hematology analyzers on the market are generally reliable. The differences between them are minor and relate to additional features that some may like, but some may not. However, the decision to buy an instrument usually depends on its price. While cost was not an issue in the past, today hematology is becoming a very competitive market and sometimes pricing (rather than best available technology) influences analyzer purchase.
The latest high performance models can be used as a standalone tool or as part of an automated multi-tool system. Fully automated laboratory includes hematology, chemistry and immunochemistry analyzers with automated inputs, outputs and refrigerationsettings.
Laboratory instruments depend on the blood being tested. Its different types require special modules. The hematological analyzer in veterinary medicine is configured to work with uniform elements of various animal species. For example, Idexx's ProCyte Dx can test blood samples from dogs, cats, horses, bulls, ferrets, rabbits, gerbils, pigs, guinea pigs, and minipigs.
Applying flow principles
The analyzers are comparable in certain areas, namely in determining the level of leukocytes and erythrocytes, hemoglobin and platelets. These are normal, typical indicators, largely the same. But are hematology analyzers exactly the same? Of course not. Some models are based on impedance principles, some use laser light scattering, and others use fluorescence flow cytometry. In the latter case, fluorescent dyes are used, which stain the unique characteristics of the cells so that they can be separated. Thus, it becomes possible to add additional parameters to leukocyte and erythrocyte formulas, including counting the number of nucleated erythrocytes and immature granulocytes. A new indicator is the level of hemoglobin in reticulocytes, which is used to monitor erythropoiesis and the immature fraction of platelets.
Progress in technology is starting to slow down as entire hematology platforms emerge. Still there are stillnumerous improvements. Almost standard now is a complete blood count with a count of nucleated erythrocytes. In addition, the accuracy of platelet counts has increased.
Another standard function of high-level analyzers is to determine the number of cells in biological fluids. Counting the number of leukocytes and erythrocytes is a laborious procedure. It is usually performed manually on a hemocytometer, is time consuming and requires skilled personnel.
The next important step in hematology is the determination of the leukocyte formula. If earlier analyzers could only mark blast cells, immature granulocytes and atypical lymphocytes, now there is a need to count them. Many analysts mention them in the form of a research indicator. But most big companies are working on it.
Modern analyzers provide good quantitative but not qualitative information. They are good for counting particles and can categorize them as red blood cells, platelets, white blood cells. However, they are less reliable in qualitative estimates. For example, the analyzer may determine that it is a granulocyte, but it will not be as accurate in determining its stage of maturation. The next generation of lab instruments should be better able to measure this.
Today, all manufacturers have perfected the Coulter impedance principle technology and tuned their software to the point where they can extract as much data as possible. In the future, newtechnologies that use the functionality of the cell, as well as the synthesis of its surface protein, which indicates its functions and stage of development.
Cytometry border
Some analyzers use flow cytometric methods, in particular CD4 and CD8 antigen markers. Sysmex hematology analyzers come closest to this technology. Ultimately, there shouldn't be any difference between the two, but that requires someone to see the advantage.
A sign of possible integration is that what were considered standard tests, which have moved to flow cytometry, is making a comeback in hematology. For example, it would not be surprising if analyzers could perform fetal RBC counts, replacing the manual technique of the Kleinhauer-Bethke test. The test can be done by flow cytometry, but its return to the hematology laboratory will give it wider acceptance. It is likely that in the long run this terrible analysis in terms of accuracy will be more in line with what should be expected from diagnostics in the 21st century.
The line between hematology analyzers and flow cytometers is likely to shift for the foreseeable future as technology or methodologies advance. An example is the reticulocyte count. It was first performed by hand, then on a flow cytometer, after which it became a hematology tool when the technique was automated.
Prospects for Integration
According to experts, some simplecytometric tests can be adapted for the hematology analyzer. An obvious example is the detection of regular subsets of T cells, direct chronic or acute leukemia, where all cells are homogeneous with a very clear phenotypic profile. In blood analyzers, it is possible to accurately determine the scattering characteristics. Cases of mixed or truly small populations with unusual or more aberrant phenotypic profiles may be more complex.
However, some people doubt that hematology blood analyzers will become flow cytometers. The standard test costs much less and should remain simple. If, as a result of its conduct, a deviation from the norm is determined, then it is necessary to undergo other tests, but the clinic or doctor's office should not do this. If complex tests are run separately, they will not increase the cost of normal ones. Experts have doubts that screening for complex acute leukemia or the large panels used in flow cytometry will quickly return to the hematology lab.
Flow cytometry is expensive, but there are ways to reduce costs by combining reagents in different ways. Another factor that slows down the integration of the test into the hematology analyzer is the loss of revenue. People don't want to lose this business as their profits have already dwindled.
The reliability and reproducibility of flow analysis results is also important to consider. Methods based onimpedance, are workhorses in large laboratories. They must be reliable and fast. And you need to make sure that they are cost-effective. Their strength lies in the accuracy and reproducibility of the results. And as new applications in the field of cellular cytometry emerge, they still need to be proven and implemented. In-line technology requires good quality control and standardization of instruments and reagents. Without this, errors are possible. In addition, it is necessary to have trained personnel who know what they are doing and working with.
According to experts, there will be new indicators that will change laboratory hematology. Those instruments that can measure fluorescence are in a much better position because they have a higher degree of sensitivity and selectivity.
Software, rules and automation
While the visionaries are looking to the future, manufacturers today are forced to fight with competitors. In addition to highlighting differences in technology, companies differentiate their products with software that manages data and provides automatic validation of normal cells based on a set of rules set in the lab, greatly speeding up validation and giving staff more time to focus on abnormal cases..
At the analyzer level, it is difficult to distinguish the benefits of different products. To a certain extent, having software that plays a key role in obtaining the results of the analysis allows the product to stand out in the market. First of all, diagnostic companies go tomarket software to protect their business, but then they realize that information management systems are essential to their survival.
With each generation of analyzers, the software improves significantly. New computing power provides much better selectivity in the manual calculation of the leukocyte formula. The possibility of reducing the amount of work with a microscope is very important. If there is an accurate instrument, then it is enough just to examine pathological cells on a hematological analyzer, which increases the efficiency of the work of specialists. And modern devices allow you to achieve this. This is exactly what the lab needs: ease of use, efficiency and reduced microscope work.
It is of concern that some clinical laboratory physicians are focusing their efforts on improving technology rather than optimizing it to make sound medical decisions. You can buy the most bizarre lab instrument in the world, but if you constantly double-check the results, then this eliminates the possibilities of the technologist. Abnormalities are not errors, and labs that automatically validate only the “No abnormal cells found” result from the hematology analyzer are acting illogically.
Each laboratory should define criteria for which tests should be reviewed and which should be manually processed. Thus, the total amount of non-automated labor is reduced. There is a time to work with abnormalleukograms.
The software allows laboratories to set rules for auto-validation and identification of suspicious samples based on the location of the sample or study group. For example, if the lab processes a large number of cancer samples, the system can be configured to automatically analyze blood on a hematology pathology analyzer.
It is important not only to automatically confirm normal results, but also to reduce the number of false positives. Manual analysis is the most technically difficult. This is the most labor intensive process. It is necessary to reduce the time that the laboratory assistant spends with the microscope, limiting it to only abnormal cases.
Equipment manufacturers offer high-performance automation systems for large laboratories to help cope with staffing shortages. In this case, the laboratory assistant places the samples in an automatic line. The system then sends the tubes to the analyzer and onward for further testing or to a temperature-controlled “warehouse” where samples can be quickly taken for additional testing. Automated smear application and staining modules also reduce staff time. For example, the Mindray CAL 8000 hematology analyzer uses the SC-120 swab processing module, which can handle 40 µl samples with a load of 180 slides. All glasses are heated before and after staining. This optimizes quality and reduces the risk of personnel infection.
Degree of automation inhematology laboratories will increase, and the number of staff will decrease. There is a need for complex systems in which one can put samples, switch jobs, and only come back to review truly anomalous samples.
Most automation systems are customizable to each lab, with standardized configurations available in some cases. Some laboratories use their own software with their own information system and anomalous sampling algorithms. But you should avoid automation for the sake of automation. Large investments in the robotic project of a modern expensive high-tech automatic laboratory are in vain due to the elementary mistake of repeating the blood test of each sample with an abnormal result.
Automated counting
Most automatic hematology analyzers measure or calculate the following parameters: hemoglobin, hematocrit, red blood cell count and average volume, average hemoglobin, average cell hemoglobin concentration, platelet count and average volume, and leukocyte count.
Hemoglobin is measured directly from a whole blood sample using a hemoglobin cyanometer method.
When examining a hematology analyzer, the count of red blood cells, white blood cells and platelets can be done in several ways. Many meters use the electrical impedance method. Heis based on the change in conductivity when cells pass through small holes. The sizes of the latter differ for erythrocytes, leukocytes and platelets. The change in conductivity results in an electrical impulse that can be detected and recorded. This method also allows you to measure the volume of the cell. Determination of the leukocyte formula requires lysis of erythrocytes. The different leukocyte populations are then identified by flow cytometry.
The Mindray VS-6800 hematological analyzer, for example, after exposure to the samples with reagents, examines them based on laser light scattering and fluorescence data. To better identify and differentiate blood cell populations, especially to detect abnormalities not detected by other methods, a 3D diagram is built. The BC-6800 Hematology Analyzer provides data on immature granulocytes (including promyelocytes, myelocytes, and metamyelocytes), fluorescent cell populations (such as blasts and atypical lymphocytes), immature reticulocytes, and infected erythrocytes in addition to standard tests.
In Nihon Kohden's MEK-9100K hematology analyzer, blood cells are perfectly aligned by a hydrodynamically focused flow before passing through the high-precision impedance counting port. In addition, this method completely eliminates the risk of recounting cells, which greatly improves the accuracy of studies.
Celltac G DynaScatter laser optical technology allows you to get a leukocyte formula in an almost natural state. ATThe MEK-9100K hematology analyzer uses a 3-angle scattering detector. From one angle, you can determine the number of leukocytes, from another you can get information about the structure of the cell and the complexity of nucleochromatin particles, and from the side - data on internal granularity and globularity. 3D graphical information is calculated by Nihon Kohden's exclusive algorithm.
Flow Cytometry
Carried out for blood samples, any biological fluid, dispersed bone marrow aspirate, destroyed tissue. Flow cytometry is a method that characterizes cells by size, shape, biochemical or antigenic composition.
The principle of this study is as follows. The cells move in turn through the cuvette where they are exposed to a beam of intense light. The blood cells scatter light in all directions. Forward scattering resulting from diffraction correlates with cell volume. Lateral scattering (at right angles) is the result of refraction and approximately characterizes its internal granularity. Forward and side scatter data can identify, for example, populations of neutrophils and lymphocytes that differ in size and granularity.
Fluorescence is also used to detect different populations in flow cytometry. Monoclonal antibodies used to identify cytoplasmic and cell surface antigens are most often labeled with fluorescent compounds. For example, fluoresceinor R-phycoerythrin have different emission spectra, allowing to identify the formed elements by the color of the glow. The cell suspension is incubated with two monoclonal antibodies, each labeled with a different fluorochrome. As blood cells with bound antibodies pass through the cuvette, the 488 nm laser excites the fluorescent compounds, causing them to glow at specific wavelengths. The lens and filter system detects light and converts it into an electrical signal that can be analyzed by a computer. Different elements of the blood are characterized by different side and forward scattering and the intensity of the emitted light at certain wavelengths. Data composed of thousands of events is collected, analyzed and summarized in a histogram. Flow cytometry is used in the diagnosis of leukemias and lymphomas. The use of various antibody markers allows for precise cell identification.
The Sysmex hematology analyzer uses sodium lauryl sulfate to test hemoglobin. It is a non-cyanide method with a very short reaction time. Hemoglobin is determined in a separate channel, which minimizes interference from high concentrations of leukocytes.
Reagents
When choosing a blood test instrument, consider how many reagents are required for a hematology analyzer, as well as their cost and safety requirements. Can they be purchased from any supplier or only from the manufacturer? For example, Erba ELite 3 measures 20 parameters with just three environmentally friendly and freecyanide reagents. The Beckman Coulter DxH 800 and DxH 600 models use only 5 reagents for all applications, including nucleated erythrocytes and reticulocyte counts. ABX Pentra 60 is a hematology analyzer with 4 reagents and 1 diluent.
The frequency of reagent replacement is also important. For example, the Siemens ADVIA 120 has a stockpile of analytical and wash chemicals for 1,850 tests.
Automated analyzer optimization
In the opinion of experts, too much attention is paid to the improvement of laboratory instruments and not enough - to optimize the use of automated and manual technologies. Part of the problem is that hematology labs are trained in anatomical pathology rather than laboratory medicine.
Many specialists perform the functions of verification, not interpretation. The laboratory should have 2 functions: to be responsible for the results of the analysis and to interpret them. The next step will be the practice of evidence-based medicine. If, after running 10,000 tests, there is no evidence that they could not be automatically verified with exactly the same results, then this should not be done. At the same time, if 10,000 analyzes provided new medical information, then they should be revised in the light of new knowledge. So far, evidence-based practice is at the initial level.
Staff training
Another problem is to help laboratory assistants not only study the instructions for the hematology analyzer,but also to understand the information received with its help. Most specialists do not have such knowledge of technology. In addition, the understanding of the graphical representation of data is limited. Its correlation with morphological findings needs to be emphasized so that more information can be extracted. Even a complete blood count becomes too complex, generating a huge amount of data. All this information must be integrated. The benefits of more data must be weighed against the added complexity it brings. This does not mean that laboratories should not accept high-tech advances. It is necessary to combine them with the improvement of medical practice.
