Hematology analyzers are specialized, automated systems that count leucocytes, red cells and platelets in blood, and also determine hemoglobin and hematocrit levels. Hematology technology has come a long way in a relatively short space of time. In the 1950s, complete blood counts (CBC) were performed manually by a technician in front of a microscope. Hemoglobin was also measured manually using a cyanmethemoglobin method, which was slow and time consuming.
Modern day analyzers are capable of processing hundreds of samples an hour. Modular structures and advances in automation mean that systems can be built to accommodate numerous analyzers, slide maker/stainers and archiving facilities.
A CBC, also known as a full blood count (FBC), is usually the first test requested by physicians to assess general patient health. The CBC can be used to detect a wide range of pathological states including anemia, infection and hematological malignancy, as well as for the monitoring of cancer patients undergoing chemotherapy.
There is a wide variety of hematology analyzers on the market that are designed to meet the needs of every size laboratory, from the small analyzers to be used at point-of-care settings such as the physician’s office or ITU, to the high-throughput modular configurations designed to meet the demands of the large automated hematology laboratory.
Modern analyzers use electrical impedance methods, optical methods, or a mixture of both of these to count and classify white and red blood cells. Vacuum pump fluidic systems deliver perfectly precise volumes of diluents, samples and reagents to analysis chambers. Fully-automated analyzers have the advantage of being objective, high-throughput and cost-effective; they can flag up atypical results and have increased measurable parameters such as platelet distribution (PDW), red cell distribution width (RDW), nucleated red cells (NRBCs) and reticulocyte (RET) counts.
In 1953, WH Coulter obtained a patent for counting metal particles in suspension. He applied this technology to counting blood cells, and this led to the ability to automate hematology analysis. This technology transformed the clinical laboratory into what it is today, and it would be very difficult to imagine how the modern hematology department would cope without it.
Figure 1. The Coulter Principle (1)
Electrical impedance classifies red and white cells based on size. Electrically conductive diluent is passed through a narrow channel. As each cell passes through an aperture, it causes a momentary drop in the electrical current being passed through the channel (the cells are nonconductive). The number of particles in a given volume of fluid can be counted by measuring the number of pulses. The size of this pulse in electrical current is related to the size of the particle.
One of the problems associated with impedance counting is cell coincidence. This is when more than one cell passes through the impedance counting aperture at a time, and this phenomenon affects all impedance counting methods. The counting of two cells as one voltage pulse could result in falsely low counts, and so this is automatically corrected for using a built-in formula. In modern hematology analyzers, coincidence is usually minimized by the application of hydrodynamic focusing. This is when a thin, slow-moving stream of cell suspension is injected into a fast-moving stream of diluents, near to the aperture. The fast-moving diluent creates a sheath fluid, which surrounds the diluted sample and allows the cells to flow in single file through the sensing aperture.
Visible wavelength light is passed as a laser beam through the sample stream. As each cell passes through the sensing zone, the light is scattered and measured by a photo conductor, which converts it into an electrical impulse. The number of impulses generated is directly proportional to the number of cells. Light scatter is used to determine granularity, cell structure, size and shape. This enables a differential to be determined. Some analyzers have only one angle of light and are able to produce only one histogram. The more sophisticated analyzers use multi-angle optical scatter analysis (up to five different angles and pulse times), combined with flow cytometry to distinguish different cell populations and identify immature and atypical cells. These analyzers are capable of producing dozens of histograms, giving detailed information about the white cell population.
Traditional flow cytometry is considered to be the most effective method of differentiating cell types. This method of cell differentiation is, however, costly and time consuming. Some manufacturers have managed to adapt the basic techniques of flow cytometry to enable its incorporation into the automated hematology analyzer. Flow cytometry can provide information on forward light scatter, side scatter and side fluorescence to generate a WBC differential, NRBC, RETIC and optical platelets.
Figure 3. Sysmex, XT-1800 flow Cytometry technology.
Sysmex, Horiba, Beckman Coulter and Abbott Diagnostics have all incorporated flow cytometry technology into their analyzers. Flow cytometry methods enable analyzers to give more detailed information in the form of flags and messaging; for example, information on the presence of blasts, immature granulocytes, microcytosis, macrocytosis and anisocytosis.
Red blood cells (RBC) can be counted using impedance or optical methods. Even small hematology analyzers are capable of giving information on MCV, HCT, MCH and MCHC. Larger automated analyzers also give information on red cell morphology and the RDW. RBC histograms allow the user to look at red cell distribution. Red cells and platelets are usually counted together.
There are several situations that may lead to abnormal red cell counts; these include presence of cryoglobulins, cold agglutinins, and lipids. One abnormally measured parameter will lead to abnormalities in the calculated red cell indices; most significantly, the mean cell hemoglobin content (MCHC). The most sophisticated analyzers are able to flag the possible causes of spurious red cell counts and parameters. However, analyzers without the ability to produce white cell scattergrams will not be as sensitive to these situations.
Depending on the size and complexity of the analyzer, a 3-part, 5-part or 7-part differential might be reported.
Analyzers use a variety of technologies to determine this white cell differential. These can be broadly grouped into optical, impedance and flow cytometry methods, and often an analyzer might employ a combination of these methods to gather data.
In optical methods, the internal cell components might be stained with fluorescent dyes and then the emitted light captured. Alternatively, cytochemical stains might be employed to determine the internal composition of the cell (Horiba Pentra Series and Siemens ADVIA 2120).
Multi-angle light scatter technology can be utilized to give information on cell volume (forward scatter), internal cell structure (side scatter), and RNA/DNA content (side fluorescence). This is often referred to as VCS (Volume, Conductivity, and Light Scatter) technology.
The manufacturers of some of the most sophisticated analyzers have also managed to adapt flow cytometry principles to make it practical and cost effective in differential analysis. Flow combined with optical and/or impedance methods can be used to create detailed scatter plots, which determine the populations of each cell type.
Figure 6. Pentra DX Nexus, Horiba Medical
All large analyzers will give a 5-part differential, and in some cases, they might give a 7-part differential, which includes large immature cells (blasts and immature granulocytes) and atypical lymphocytes (including blasts). The increased amount of data being provided to the operator as a result of these sophisticated technologies reduces manual review rates and improves laboratory efficiency.
Figure 7. Abbotts M.A.P.S.S.™ (Multi-Angle Polarized Scatter Separation) technology applies a multi-step algorithm to the light scatter data to classify WBCs (3).
There are four common causes of cellular interferences in the white cell chamber. These are nucleated RBCs (nRBCs), giant platelets, intra-cellular parasites, and platelet clumps. These particles of interference lead to the same pattern on the WBC histogram, and hematology analyzers have traditionally been unable to distinguish between these.
The latest generation of analyzers now offers several additional histograms that are generated by two new angles of light. This additional information can be used to distinguish between the four types of cellular interference.
All automated hematology analyzers will provide a platelet count, either by impedance, optical methods or both of these methods combined. More sophisticated analyzers will also provide extra parameters such as the PDW. Platelets are often counted electrically using impedance. This count can then be optically confirmed in cases when an impedance count may be compromised, such as in the presence of giant platelets or platelet clumps. Some analyzers offer a combined electrical and optical platelet count as standard.
Fluorescence flow cytometry enables fluorescence platelet counts to be measured, as well as allowing the immature platelet fraction to be calculated, which can help clinicians to monitor thrombopoietic activity of the bone marrow. Platelet counts determined using flow cytometry can help to resolve impedance platelet flags. The Cell-Dyn Sapphire from Abbott Diagnostics offers a reportable CD61 immunoplatelet count in approximately five minutes.
Hemoglobin measurement is based on a modification of the traditional, manual hemoglobin-cyanide (HiCN) method. The sample is diluted with cyanmethemoglobin reagent. Potassium ferricyanide in the reagent converts the hemoglobin to methemoglobin. This combines with potassium cyanide to form stable cyanmethemoglobin, and the color intensity of the reaction is then read by a photodetector to give the concentration of hemoglobin. Some manufacturers use a non-toxic method such as sodium lauryl sulphate solution instead of HiCN.
Reticulocytes are counted by staining the RNA in the red cells using supravital, nucleic acid or fluorescent dyes. The amount of fluorescence is directly proportional to the amount of RNA in the cell. Immature cells have larger amounts of RNA, while mature red cells do not contain RNA. The size and Hb content of reticulocytes is measured using light scatter and impedance. Fluorescent flow cytometry enables a reliable reticulocyte count at even extremely low concentrations, and in the presence of giant platelets. The immature reticulocyte fraction (IRF) provides an indication of early bone marrow regeneration. When bone marrow regeneration begins, the IRF will increase after only a few hours, whereas the circulating reticulocyte count may take two or three days to show an increase.
Some hematology analyzers for use at the point-of-care give only an Hb and HCT results. This can be useful for example on surgical and ITU wards.
Other point-of-care systems use dry hematology technology, which operate without bulky and costly reagent packs. Even small analyzers using this technology can give a white cell differential including granulocytes, lymphocytes and monocytes, as well as HCT, Hb, MCHC and platelet count.
Sysmex has a small hematology analyzer designed specifically for the point-of-care environment, which offers 19 reportable parameters including a 3-part differential.
Figure 11. pocHi 100 Hematology Analyzer, Sysmex
Horiba also offers a small, compact analyzer which can be used in hospital settings such as the ED and ITU, or in the laboratory, and gives a 3-part differential as well as RBC and PLT parameters which include a PDW, PCT and RDW.
Figure 12. ABX Micros ES 60, Horiba
There are numerous analyzers on the market that can automate slide preparation and staining, and several instruments that are capable of actually reviewing the slides themselves.
Slide maker strainers are usually sold as part of a hematology solution. Beckman Coulter, Sysmex, Abbott, Horiba and Siemens all offer these analyzers which can either be used alongside their hematology analyzers, or as stand-alone solutions.
Advances in digital imaging mean that it is now possible to automate the review of blood smears. These instruments review slides at low power, then classify the white blood cells. In some cases, they can also review red cell characteristics and platelet counts. Images of the cells are taken digitally and presented for review. These systems require a trained technician to review and approve the pre-classifications. The images are then stored digitally.
The advantages of systems such as these are that they reduce the manual review rate, they enable the user to remotely share images with other clinicians or laboratories; historical images from a single patient can be compared side by side; and the images can be stored with full traceability.
If a fully automated imaging system is beyond the scope of your laboratory, you could look at investing in a digital image capture system. With this option, the user is still able to share images remotely with other clinicians or laboratory staff, as well as storing images for future review.
The newest generations of hematology analyzers from Beckman Coulter incorporate Automated Intelligent Morphology (AIM) technology. This enhanced 3D high-definition cellular analysis solution utilizes flow cytometric technology to help lower manual review rates and increase laboratory efficiency.
As well as the core technology of the analyzers you are considering, there are additional factors to take into account, which can vary from instrument to instrument.
Increasingly, manufacturers are incorporating additional tests into the routine hematology analyzer, for example CD61 for immunoplatelet counts, and CD3/4/8 assays. These tests allow for increased analyzer utility and efficiency.
The advantages of digital morphology include lower manual review rates, increased efficiency and the ability to store digital data for traceability, sharing and future review. It is likely that we will increasingly see manufacturers partnering with digital imaging suppliers, and also developing this technology in-house. As this technology becomes more widely available, it will become increasingly accessible for hospital laboratories.
Latest generation analyzers are capable of giving detailed cellular information to the operator, such as blast cell differentials. Some are also able to give suggestions of pathological conditions (for example iron deficiency anemia). The additional information available to hematology laboratories through automated hematology technology increases the efficiency and productivity of the laboratory, and ultimately, when used correctly, improves patient care.
1. Figure 1: The Coulter Principle http://www.labplan.ie/3page.asp?Menu=191&Page=703&SubPage=28
2. Figure 7: Abbotts M.A.P.S.S.™ http://www.nearmedic.ru/upload/files/Doc_389_578.pdf
This guide is not inclusive of all hematology analyzers available on the market.
"This instrument is really user-friendly. I especially love that support staff can remotely diagnose and help resolve issues immediately. ”
Nery Berrios, MD Anderson Cancer Center