Hematology

Challenges in hematology diagnostics

Hematology is the medical specialty devoted to diseases of the blood. In contrast to clinical chemistry, hematology is more focused on counting cells and assessing morphology than measuring proteins. The primary hematology test is the complete blood count (CBC), used for patient screening. Unexpected CBC results typically trigger confirmatory or follow-up testing, For this article, hematology is separated into three eras: pre-automation, automation, and the future. 

The pre-automation era

The ancient Egyptians and Greeks had knowledge of blood, but it did not extend beyond knowing that blood was one of the “fluids of life.” Understanding advanced little until the middle of the seventeenth century, when the Dutch amateur scientist Antonie van Leeuwenhoek (1632-1723) invented a simple but effective microscope, and his countryman Jan Swammerdam (1637-1680) became the first to describe red blood cells (in 1658). Later, van Leeuwenhoek reported that blood also contained white cells. It was not until the mid-to-late 1800s, however, that the work of several scientists led to the recognition of platelets as cellular components of blood. 

In the last part of the nineteenth century and first part of the twentieth century, laboratory hematology rapidly developed thanks to the understanding that more and more diseases could be diagnosed using blood cell counts and morphologic features. It was the era of first descriptions of abnormalities in blood cells or blood composition associated with various diseases. Microscopy remained the principal driver of diagnosis for hematology laboratories until 1953, when the introduction of Wallace Coulter’s impedance method for counting particles in suspension was a major development. 

The automation era

Coulter’s method signaled the end of the manual testing era, in which technologists counted cells by microscope and hemocytometer. This was quickly followed by the world’s first automated five-part differential analyzer, the Technicon Hemalog-D, an instrument based on a combination of optical and cytochemical methods. In the 1980s, the first diagnostic imaging devices appeared. They were hampered, however, by the inadequate computing power at the time and did not achieve widespread use. However, this “dead end” of investigation continues to attract research and again is under active study. 

By the late 1990s, hematology analyzers had become increasingly sophisticated, offering as many as 36 measurands from a few microliters of blood. This sophistication included the incorporation of fluorescent stains to differentiate white cells and count immature red cells and platelets. 

Impedance technology

The principle of impedance technology is electrical resistance (or impedance) in which a known dilution of cells in suspension passes through a small orifice. The electrolyte-containing diluent serves as a conductor of a constant electrical current between two electrodes. As cells pass through the orifice, they impede the electrical current, which is detected as an increase in resistance. Each cell causes a resistance pulse, thus allowing for cell counting. In addition, the height of the resistance pulse is directly related to cell volume.

Because impedance measures only cell volume, it is not specific for white blood cells (WBC). They can only be counted after lysis of red blood cells (RBC), and the differentiation of WBC is limited to three basic types, based on their volume: lymphocytes (small WBC), neutrophils (large WBC), and so-called mid-cells. Abnormal WBC cannot be characterized by volume only; their presence can only be inferred if the three normal WBC types are not well separated, and most impedance analyzers raise an alarm flag in case of abnormal WBC distribution. Such a flag necessitates further investigation by microscopy or an alternative technology. WBC counting using impedance is prone to various interferences. 

Due to its technical simplicity, impedance technology is widely used in simple bedside/point-of-care instruments to generate a three-part WBC differential, along with RBC and platelet (PLT) counts. 

Some hematology testing manufacturers still rely on impedance technology for their high-end systems, though with additional technologies to improve the differential white cell count. These include radiofrequency, low angle(s) of light scatter, and fluorescence. Adding technologies for correlated measurements goes some way to mitigate the inherent limitations of impedance technology.

Optical technology

The principle of optical technology is light scattering by blood cells in suspension when they pass the optical flowcell illuminated by laser light. Using detectors that are placed under different angles relative to the incoming laser light, this technology allows measurement of different aspects of blood cells simultaneously. By correlating different optical signals generated by a single blood cell, detailed information is collected that allows for classifying the cell in a multi-dimensional space. The more information collected, the better a cell can be characterized. For example, forward scatter (very low angle) mainly depends on cell size; sideward scatter (90 degrees) predominantly reflects nuclear segmentation; and intermediate-angle scatter carries information on the presence and number of cytoplasmic granules.

Light scatter plus optical measurement of intracellular peroxidase activity through a cytochemical reaction was first used for classifying the usual WBC types. Because neutrophils and basophils cannot be separated using this approach, a second cytochemical channel was necessary for specifically measuring basophils. This optical technology with cytochemistry continues to be in use in some current hematology analyzers. 

Polarized light measurement also is an aid in differentiating neutrophils and eosinophils because the latter are normally the only blood cells that have the capacity of disturbing the polarization of the laser beam. Another technological advance was the use of fluorescence by a nuclear stain for identifying and enumerating nucleated RBC, apart from WBC.

Other methods have been combined with light scatter. Initially, they used multiple channels, each with its own reagents in order to achieve the desired specificity of the WBC differential. In the latest generations of some analyzers, nuclear fluorescence is combined with light scatter and radiofrequency for constructing a five-part WBC differential. 

Platelet analysis using optical technology is less prone to interference than impedance technology. Yet separating large or giant platelets from microcytic RBC or RBC fragments remains a challenge that requires additional measurements for obtaining adequate specificity. Multi-angle light scatter and fluorescent dyes have been introduced for this goal with reasonable success. Alternatively, using CD61 monoclonal antibodies conjugated with a fluorescent label provides absolute specificity to ensure reliable results at very low counts.

Digital imaging

Digital imaging of traditionally stained blood cells on a glass slide is gaining more widespread use now that digital cameras and powerful computers have become more affordable. Apart from these methods, two interesting alternatives are currently under development and will soon be offered to the market. One solution is based upon ink-jet volumetric printing of blood cells on a slide and then staining them and subsequently analyzing the pictures using digitalized pattern recognition. Another solution for point-of-care hematology testing uses a compact device that images blood cells suspended in a counting chamber after staining with fluorescent dye(s).  

Other future developments

In the near term, information technology will play a growing role as an adjunct to technological developments in hematology analyzers. There is a tendency for new hematology analyzers to employ increasingly more detectors than the older models, for improved specificity in cell classification and for more accurate flagging of abnormal blood cells. In other words, blood-cell analysis will become more multi-dimensional, and this multitude of cellular information will rapidly expand. Moreover, a more detailed look into the characteristics of a certain cell can possibly yield more information than just WBC, such as data associated with cellular activation pathways and apoptosis or other cellular processes. It is even conceivable that information that is connected to certain disease states will be available. Thus, advanced information technology could be useful for unveiling information that is “hidden” in signals that register, but currently are used only for cell counting and classification.

Is newer better for the CBC?

As automated hematology systems became more sophisticated, laboratory managers coping with increasing workloads for CBC tests placed greater emphasis on systems that offered superior throughput and low testing cost. Automation is still a must, but how much more innovation is needed for CBC testing, and how much more is a CBC test worth in today’s healthcare environment?

The aging of the global population is fueling rapid growth in healthcare services, and CBC testing is expected to increase rapidly as a result. Innovations for CBC, therefore, must allow laboratories to process high volumes of CBC tests, reduce retesting rates, and provide nearly 100 percent first-time reliable results at lower cost.

Clearly, the priorities have shifted. In the 1970s, laboratories demanded analytics, throughput, and reliability; today the emphasis is on reliability, price, and throughput. The overwhelming majority of hematology tests today are CBCs ordered by primary care physicians. Labs need fast and reliable CBC results to serve burgeoning primary-care demand. However, this basic testing must be low-cost, both in the purchase price of the test and in cost per test result. Laboratories want CBC tests that offer excellent first-time reliability with minimal indeterminate results that require repeat testing. 

The post-automation era

With increasing emphasis on reliability and price, what does the post-automation hematology era look like? We can expect developments in two major areas: improvement in conventional hematology analyzers to boost efficiency and the emergence of imaging devices that can deliver a CBC as well as a differential white cell count from a smear. The focus will be to increase first-pass efficiency (the percentage of typical samples that can be automatically reported without further operator intervention) to beyond the current 80 percent rate. 

We can anticipate that vendors will introduce improved algorithms, detection systems, and reagents to improve blast, immature granulocyte, and NRBC detection, which will further improve FPE. Another major theme for development will be automation: 

1. the use of robots and tracks to move samples around the laboratory; and 
2. simplification of interfaces on instruments to harmonize training needs. 

For example, common functions, such as replacing reagents or checking QC files, should have similar procedures on chemistry and hematology platforms, which will reduce the training burden for lab staff. 

Another area for improvement is “error trapping.” We can expect instruments to need 100uL or less for a CBC, which will reduce short-sample rejections. Clot and short sample detection mechanisms will also improve to reduce the risk of generating or releasing spurious results. 

Imaging for hematology is still in its infancy. The accuracy and precision of pre-classifier devices will be improved through innovations in algorithm development, but deriving cell counts and hemoglobin measurement from a smear remain significant technical challenges that can be expected to take some time to solve. Increasing throughput on imaging devices is another challenge that will need to be solved before such instruments can replace conventional analyzers.

Analytic performance for reportable parameters is generally good on all major instruments today. Though instruments today can generate > 30 measurands, the emphasis in the future will be on correlating measurands to clinical or economic outcomes.

Consolidation will continue, resulting in a polarized testing environment. On the one hand there will be fewer large centralized testing facilities, with satellite labs offering a limited or stat menu in the ER, ICUs, and specialized clinics. The needs of consolidated and satellite labs are different and will require customized approaches by manufacturers to meet them. For example, instrument operators in satellite labs will typically be nurses, EMTs, or clinicians who will need simple instruments that make generation of spurious results highly unlikely.

Larger labs will need high throughput instruments and automated “rules-based” reflex testing.

In the foreseeable future, hematology testing may not be characterized as much by technology innovation but in finding ways to perform higher volumes of highly accurate routine and follow-up tests in both traditional laboratories and alternative care sites.

Johannes Hoffmann and Nigel Llewellyn-Smith are scientific affairs managers for Abbott.

The digital era of hematology is coming of age

By Jurgen Riedl, PhD

Morphological examination of a peripheral blood smear is still an essential component in the detection of hematological diseases.^1,2 In essence, three morphological entities should be appreciated morphologically in a peripheral blood smear: leukocytes (e.g., blast cell detection in the case of myelodysplastic syndromes [MDS] or acute leukemias); red blood cells (RBC; e.g., sickle cells with regard to sickle cell anemia); and thrombocytes/platelets (e.g., in the case of MDS or myeloproliferative neoplasmata [MPN]). 

Traditionally, morphological examination of the peripheral blood smear has been performed by manual use of a microscope. Although this approach is common, it has certain drawbacks/disadvantages. For one, it takes time to acquire expertise in hematological morphology, approximately six to seven years for peripheral blood smears and bone marrow aspirates. Also, manual microscopical examination is labor-intensive and displays a very high inter-observer variability. Furthermore, microscopy carries with it an unavoidable lack of standardization. However, to date the gold standard for morphology is still manual microscopy by an experienced professional.

The advent of digital morphology

For more than a decade, digital morphology/imaging has been a viable option.^3,4 There are various systems to perform automated morphological analysis of the peripheral blood smear, but their overall approach is similar. The systems consist of a microscope of some kind with various lenses, combined with a digital camera system and attached computer software. First, the system looks for a good monolayer in a peripheral blood smear sample. Next, individual leukocytes are photographed by the digital camera. Finally, the computer software automatically classifies cells on the basis of a certain well-defined set of parameters. The systems use that approach to morphologically analyze the red blood cells, and hopefully future applications will address thrombocyte/platelet morphology. 

After pre-classification by the system (i.e., without intervention of a morphology expert), an expert performs post-classification to check the pre-classification by the system. During post-classification, it is, for instance, possible to reclassify cells in the case of a misclassification by the system. After the post-classification process, the slide is signed and the results are reported digitally to the laboratory information system (LIS). The ultimate goal will be a completely automated digital differential, including leukocyte, red blood cell, and thrombocyte morphological analysis and subsequent classification.

The use of digital microscopy systems introduces, next to the cell counter, an additional excellent screening tool for pathology—that is, a pathology filter which filters disease from non-disease, and thus patient from non-patient.

Advantages of digital morphology

In our laboratory we have effectively replaced one full-time employment (FTE) morphological expert technician with a digital morphology (DM) system over a four-year time period. Several advantages have become apparent after the introduction of the DM system. First, a preliminary differential can be generated by the system 24/7 in a fully automated manner. The system provides a pre-classification of the differential in a standardized fashion, thereby facilitating a quicker review for the observer. The use of a computer screen enables discussion with colleagues on an individual cell level on the lab floor or even between laboratories via the Internet. It is even possible to post-classify a differential generated on a DM system in a remote laboratory, thereby rendering it useful for a multi-location laboratory setting. This remote access is becoming increasingly common. 

Moreover, digital imaging leads to a more standardized way of performing a differential, and quality surveys can be performed in a digital manner, thereby automatically reducing costs. Digital images can easily be stored and used for educational purposes or even be put in a patient report/dossier digitally. To sum up, digital imaging brings standardization, speed, and cost reduction to the morphological analysis of the peripheral blood smear.

The next exciting challenge will be the integration of cell counter results with digital imaging results, ultimately leading to a faster detection of hematological malignancies and a higher analytical sensitivity and specificity.

Digital technology in action

Thrombotic thrombocytopenic purpura (TTP) is a rare disease, characterized by a microangiopathic hemolytic anemia with a thrombocytopenia in combination with neurological symptoms and/or kidney failure. TTP is an emergency that, without proper intervention, will ultimately lead to the patient’s death. 

The correct and fast detection and subsequent diagnosis of TTP is essential. Moreover, the correct and fast detection of schistocytes/fragmentocytes (specific red blood cell abnormality) using morphological analysis of the peripheral blood smear is a final essential criterion for the correct diagnosis. Using a total lab automation solution (a full integration of cell counters, slide-maker-stainer, and digital microscopy systems in a one-track system), it is now feasible to quickly diagnose this medical emergency using well-defined criteria in a total lab automated fashion. Low hemoglobin and thrombocyte count are detected on the cell counters in combination with morphological detection of schistocytes/fragmentocytes using the integrated digital microscope.

This is only one example of the value of the technology, among many others. For instance, myelofibrosis can be quickly diagnosed using this integrated manner using cytopenia(s) as criterium/criteria in combination with the detection of teardrop cells. Remote review of both the cell counter results and the morphological pre-classification by the digital microscope can be performed by a laboratorian using a computer screen, integrating both results.

Looking ahead

Future applications of the digital imaging tools could involve a faster and more accurate detection of malaria in red blood cells. Moreover, novel scientific approaches could involve the analysis of morphological abnormal thrombocytes/platelets in the early detection and diagnosis of myeloproliferative diseases. All of the novel digital imaging applications will blossom with the integration of cell counter results.

Since the introduction of automated cell counters, these machines have evolved and proven themselves as reliable hematological diagnostic tools. Digital imaging and digital microscopes will undoubtedly do the same.