Feature. Hematology Analyzer: From Workhorse to Thoroughbred. Ellen Sullivan

Transcription

1 Hematology Analyzer: From Workhorse to Thoroughbred Ellen Sullivan DOI: /TMQ6T4CBCG labmedicine.com May 2006 Volume 37 Number 5 LABMEDICINE 273

2 Hematology analyzers are the workhorses of the clinical laboratory. High-end, high-volume analyzers deliver reliable red blood cell counts, platelet counts, and 5-part differentials of white blood cells, identifying lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Nucleated red blood cell counts and immature granulocytes are emerging as sixth and seventh parameters. While electrical impedance still has a firm foothold in determining the overall number and size of cells, flow cytometry techniques have proven their worth in differentiating white blood cells and identifying abnormal cells. The increasing sophistication of flow techniques on the analyzer raises some interesting questions. What will be the dividing line between the hematology analyzer and the flow cytometer? Will new methods push some tests back to the hematology laboratory? Will that drive up costs for routine tests? Finally, are pathologists and laboratory professionals using the advanced technology appropriately, not just to increase efficiency, but also to improve medical-decision making? Evolution of the Analyzer The first automated cell counters came out in the 1950s based on Coulter s electrical impedance principle in which cells pulled through an aperture break an electric circuit, indicating both the presence of a cell and the size of the cell. Those were the prehistoric analyzers that just did counts and indices mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, said Kathleen Finnegan, MS, MT(ASCP)SH, Clinical Assistant Professor and Chair of the Clinical Laboratory Sciences Program at Stony Brook University, State University of New York. If you ve ever counted cells, you know it s very monotonous, and 2 technologists could never do it the same. So it has taken out all of that variability. In the 1970s, automated platelet counters, 7-parameter complete blood count (CBC) analyzers, and 3-part differential leukocyte counters (for lymphocytes, monocytes, and granulocytes) entered the market. For the first time, manual differentials were not the only way to analyze white blood cells. In the 1980s, a single instrument could produce a 10-parameter CBC. The 1990s brought further advancements in leukocyte differentials with the use of flow-cell techniques based on either electrical impedance or light scatter properties. Manufacturers often seek to separate their instruments from the pack by focusing on the particular package of technologies they use to differentiate white blood cells or to count platelets, but pathologists and laboratory professionals say it is hard to tell the difference between most analyzers. They all use similar technology, said Finnegan, who taught the 2005 ASCP continuing medical laboratory education (CMLE) course, Meeting Morphology Challenges: When Automated Analyzers Need Your Help. It s just that they add bells and whistles to tweak it differently. For instance, one analyzer may determine leukocyte differentials by inserting a fluorescent dye into the cell nucleus and measuring how strongly it fluoresces. One may alter the permeability of a cell and see how quickly it absorbs a dye. Another may measure enzyme activity in a cell placed in a particular substrate. Then there is the Volume Conductivity and Scatter (VCS) method that analyzes cells in their near-native state. The new technologies are great, said William G. Finn, MD, FASCP, Associate Professor of Pathology, Director of Hematopathology, and Associate Director of Clinical Pathology Laboratories at the University of Michigan. They re evolving toward flow cell-based technologies, where cells are interrogated cell by cell by optical systems that can then measure lots of things we never used to measure. The problem is that manufacturers appropriately want to take their own proprietary step to have their own identity. So they become very good at one thing, and maybe not as good at another. Bruce H. Davis, MD, of the Maine Medical Center Research Institute in Scarborough, ME, said all of the analyzers on the market are generally reliable. The differences are really minor in terms of bells and whistles that may appeal to one person or another, he said. The decision typically comes down to cost or someone is in a buying group so the decision has already been made. Ralph Taylor, Beckman Coulter s Vice President of Product Management for Cellular Analysis Clinical Business, said money did not used to be an issue. Now hematology is becoming a very competitive market, and sometimes pricing (rather than best available technology) does influence the acquisition of analyzers. The newest, high-volume analyzers can run from a single instrument in the neighborhood of $75,000 to a multiple-instrument and automation system in excess of $200,000 dollars, depending on the configuration. A complete laboratory automation system (that includes hematology, chemistry and immunochemistry analyzers with automated input, output, and refrigerated stockyards) can cost in the millions of dollars. Application of Flow Principles Barb Connell, MS, MT(ASCP)SH, Diagnostic Market Manager for Sysmex America, agrees that analyzers are comparable in certain areas, namely, total white and red blood cell counts, hemoglobin counts, and platelet counts. Yes, we count those normal, routine type samples pretty much equally for the most part, she said. But, are analyzers pretty much the same across the board? No, absolutely not. Some analyzers are primarily based on impedance principles, some use flow principles to perform laser light scatter, and others use fluorescent flow cytometry, she said. We re using some fluorescent dyes that stain the unique characteristics of the cells to really separate them further, Connell said. By doing that, we ve been able to add additional parameters to our differential and RBCs (red blood cell counts), including a nucleated RBC count that s very sensitive on the low end, and quantitative immature granulocyte counts. New parameters for 2006 include Reticulated Hemoglobin Equivalent (RET-He), which is used to monitor erythropoiesis and the immature platelet fraction (IPF), she said. Taylor said the leaps in technology are starting to slow as the whole hematological platforms mature. Nonetheless, there are still plenty of refinements happening. Almost standard now is a CBC with a nucleated red blood count, he said. That s one of the newest changes. Also, the precision and accuracy of low platelet counts has improved considerably for most hematology analyzers. Manufacturers also are improving linearity and reportable range across systems. Often you can be linear over a certain set of counts but your reportable range can be higher, said Taylor. The range is getting wide because it stops people having to do dilutions to bring it back into the linear range. It s part of the effort to take out manual intervention. Another increasingly standard feature on the high-end analyzers is counting cells from body fluids, Taylor said. The ability 274 LABMEDICINE Volume 37 Number 5 May 2006 labmedicine.com

3 to perform a white count and red count on body fluid samples is a labor-intensive activity in the lab, he said. It s usually done manually on a hemocytometer, and is time-consuming and requires a skilled member of the laboratory staff to perform the count, and labs are looking to reduce manually performed tests. The next big step in hematology is the extended differential, Taylor said. While today s analyzers flag for suspect blast cells, suspect immature granulocytes and atypical lymphocytes, now the next step will be to count those parameters instead of just flagging. You will find a lot of analyzers today report them in some shape or form on an RUO parameter research use only. But it s not too far away. Most of the major companies are working on it. That s where their focus is going. Linda Sandhaus, MD, FASCP, Director, Core Laboratory for Hematology, University Hospitals of Cleveland, Cleveland, said today s analyzers deliver good quantitative but not qualitative information. They re good for counting particles and telling you what general category of particle it is: a red cell, a platelet, leukocyte, she said. They re less reliable in what I would call the qualitative determinations. So they can tell you it s a granulocyte, but they re not going to be as precise in telling you what stage of granulocyte maturation it is. Davis, who is also president of Trillium Diagnostics of Scarborough, ME, said the next generation of analyzers will better assess cell maturation, and that generation may be only 3 to 5 years away. My sense is that currently all the manufacturers have pretty well refined the technology surrounding the Coulter (impedance) principle and tweaked their software to the point where they can extract about as much information as they can, Davis said. What we re going to see in the coming years, and we ve seen some of it to date, is the attempt to introduce some new, novel technologies using particularly some cell functional as well as cell surface protein expression that indicates the stage of maturation or even the cell function. Where the Analyzer Ends and the Cytometer Begins Already some analyzers have started to integrate what many people might consider standard flow cytometry assays, said Davis. Antigen markers such as CD4 and CD8 are beginning to appear on some analyzers. Connell says Sysmex s instrument is as close to a flow cytometer as you can get without actually using antigen markers to actually tag those cells. We ve maybe reached our limit with what we can do currently on the analyzer. Connell sees analyzers continuing to develop the technology of flow cytometry, and flow cytometry becoming simpler. Eventually, there won t be a difference between the flow cytometer and the hematology analyzer, but it s going to take someone to see that advantage and continue to make advancements in hematology, she said Davis, too, sees the eventual integration of what may have been considered standard tests that went to flow cytometry coming back to hematology. For example, I will not be surprised if some of the instruments will be able to do fetal red cell counting to replace the Kleihauer Betke manual technique, he said. That s already done by flow cytometry in some labs, but I think bringing it back into the hematology lab will gain wider acceptance and will probably finally bring that assay from being a horrible assay from a precision perspective to something that s a little more in line with what we expect for diagnostic assays in the 21st century. The line between hematology analyzers and flow cytometers will probably continue to shift for the foreseeable future as technologies or methodologies evolve, Davis predicted. The reticulocyte count is a good example, he said. First it was manual, then it was done on a flow cytometer, then it swung back to the automated hematology instrument once the methodologies were made in an automated way. Patricia K. Kotylo, MD, FASCP, of Pathology Associates, Indianapolis, IN, thinks it would be possible to adapt some of the simpler tests to the hematology analyzer. Routine T-cell subsets, maybe eventually a very obvious, forthright chronic or acute leukemia where all the cells are uniform with a very clear phenotypic profile you could do as well, she said. If they can accurately identify where cells of interest are using scatter characteristics, you could adapt something to a CBC analyzer. Some more problematic cases that have mixed cell populations or really small populations of cells with unusual or more aberrant phenotypic profiles might be more difficult. Now hematology is becoming a very competitive market, and sometimes pricing (rather than best available technology) does influence the acquisition of analyzers. Finnegan does not look forward to the day hematology analyzers are serving as flow cytometers. We don t need that level, she said. We re not going to be flowing every patient. A CBC costs about $1.50 to $2. To flow a patient is more like $50 to $60. I don t know that the doctors are looking for that sophistication. Hematology is a routine test. There should be routine things that you look at. If a patient does get flagged and it is abnormal, then we have other tests, but a basic hospital and your doctor s office are not going to want to do all that other stuff. Davis, however, said the more advanced tests would be run separately, so they would not increase the cost of routine CBCs. I don t envision that a complicated acute leukemia workup or large panels that are used in flow cytometry for diagnostic workups will rapidly come back to the hematology lab, he said. Kotylo, who taught Clinical Applications of Flow Cytometry in the 2006 ASCP s CLME course, Diagnostic Hematopathology said there s no getting around the fact that flow cytometry is expensive, but there are ways to reduce costs by combining reagents in different ways. Another factor may slow the transition of flow tests to the hematology analyzer, and that is the loss of revenue. Some people don t want to lose flow business because they feel that it s already been hit with the decreased reimbursement, she said. Reliability and reproducibility of flow results are other important considerations for the hematology laboratory. The impedance-based analyzers are workhorses for high-volume labs that are reporting many CBCs in a busy hospital setting, said Kotylo. They have to be reliable. They have to be fast. You have to make sure it works and is cost-effective. We know that their accuracy and reproducibility are its strength. As flow cytometry applications continue to emerge, she added, they will have to be proven and established. Because of the type of technology that flow is, you have to have good quality labmedicine.com May 2006 Volume 37 Number 5 LABMEDICINE 275

4 control and standardization of your instrument and your reagents, Kotylo said. If you don t do that, you can get some erroneous results. You have to have someone who is trained, knows what they are doing, knows what they are looking at. It s pretty sophisticated. Your best techs are the ones working with this technology. At Trillium, Davis is working on an assay based on CD64 expression on neutrophils to detect infection or sepsis that can be integrated into a high-end hematology analyzer. Within 3 to 5 years we re going to see a new set of parameters that s going to somewhat revolutionize lab hematology or at least wake us up out of the doldrums which in my mind has been going on for 10 or 15 years with little change, he said. Those instruments that already have an ability to measure fluorescence are in a much better situation in terms of being amenable to add-ons or assays adapted for that. Not to say that somebody couldn t get clever with just using light scatter principles, but my bias is that fluorescence affords a better degree of sensitivity and specificity. Middleware, Rules, and Automation While the forecasters look to the future, instrument manufacturers today still have to angle for an advantage over their competitors. In addition to highlighting differences in technology, companies distinguish their products by adding data management software that gives the instruments the ability to auto-verify normal cells based on each laboratory s set of rules, significantly reducing review rates and leaving medical technologists more time to focus on abnormal cells. (See the upcoming July 2006 issue of LABMEDICINE for more on middleware and other new software applications for the laboratory.) At the level of the analyzer, it is difficult to distinguish the advantages of different products, said Bruce A. Friedman, professor of pathology at the University of Michigan Medical School and Director of Pathology Data Systems at the UM Medical Center in Ann Arbor. So to a certain extent, by producing a (middleware) device that will perform certain key functions after a result has been generated, they Instrument manufacturers are also promoting automation systems to high-volume laboratories to help with staff shortages. have a way of distinguishing their product in the market. First and foremost, (the in vitro diagnostic companies) are into the middleware market as a way of protecting their key business, but they feel they have to be in information management to survive in the market. Maria C. Grana, BS, MT(ASCP)SH, Technical Manager in charge of Hematology, Blood Bank, and Flow Cytometry at Quest Diagnostics in Miami, says computer software has improved significantly from one generation of analyzers to the next. They have updated the computer 100% and they have much better discrimination for manual differentials, she said. The ability to reduce the microscopic review rate is very important. If you have an instrument that is accurate, then you can actually look at the ones you need to look at the ones with problems which is a better use of our technologists. And these instruments can do that. That s what I think labs are looking for: ease of use, efficiency, and reduced microscopic review rate. Finn is concerned that some pathologists and laboratory professionals may focus too much on getting the best technology rather than optimizing the technology to improve medical decision-making. You can buy the fanciest analyzer in the world, but if you are repeating every abnormal result, you have instantly negated the power of your technology, he said. That assumes that abnormal results are innately more likely to be incorrect than normal results, which isn t true actually. I ve heard of laboratories auto-validating only normal results, and there s no logic to that whatsoever. Finnegan said that every laboratory has to determine the criteria they will use for what gets reviewed and what gets scanned and generates a manual differential. In her laboratory, there s no doubt that the overall number of manual differentials has decreased. And that has taken out the drudge, she said. So you can spend time with an abnormal differential and not feel that you re rushing through it because you ve got 17 million other slides that you ve got to do behind it. Middleware allows laboratories to set up rules for auto-validation and suspect flags based on sample location or patient population. For example, if a laboratory handles a lot of samples from cancer patients, the system can be set up to auto-verify abnormal red cells on those samples. The important thing is not just to auto-validate normal samples, but to reduce the number of false positive flagging, said Taylor. The manual differential is the most technically demanding in the lab, he said. It s the most labor intensive. The bottom line is reducing the amount of time someone goes to the microscope. They only want to go to the microscope when it is abnormal, and not when it s normal and you flagged it incorrectly. Instrument manufacturers are also promoting automation systems to high-volume laboratories to help with staff shortages. With an automation system, a technologist places the samples on the automation line. The system then routes tubes to the analyzer, from the analyzer to an outlet for additional testing, or into a temperature-controlled stockyard where they may be recalled quickly for add-on tests. Automated slidemakers and stainers further reduce hands-on staff time. Middleware can be considered a component of an automation system, but it can also be purchased separately. Automation will continue to grow in the hematology laboratory as the number of technologists continues to decrease, said Sysmex s Connell. You need to have sophisticated systems where you can put your samples on and go do other work and only be available to look at those truly abnormal samples. Beckman Coulter s Taylor agreed. I think the state of the art of hematology analyzers is moving in the direction of less interaction with the hematology sample, he said. The ability to automate for walk-away systems to improve productivity is a big area. Most automation systems are customized for each laboratory setup. In some cases, standardized configurations are available. Grana, who is a member of the ASCP Board of Registry Hematology Examination Committee, said her hematology laboratory at Quest Diagnostics has a proprietary laboratory information system with flagging algorithms for specimen management and reflexing. This optimizes the technologists time as well, she said. Finn cautioned against automating for the sake of automating. I heard about a lab that undertook a major robotic deployment project and built in an up-front automation lab, high-cost, hightech, sounds like a wonderful bit of progress, and they repeated 276 LABMEDICINE Volume 37 Number 5 May 2006 labmedicine.com

5 Automated Blood Cell Counting Specimen: Whole blood Method Description Most automated blood cell counters measure or calculate the following parameters: hemoglobin content of RBCs, hematocrit, RBC count, mean corpuscular volume (MCV) of RBCs, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, mean platelet volume, and WBC count with differential. Hemoglobin is measured directly from the whole blood specimen using a method based on cyanomethemoglobin formation. Enumeration of the RBCs, the WBCs, and the platelets can be achieved by several methods. Many cell counters employ an electrical impedance method. This method relies on the change in conductance evoked by cells passing through a small aperture. The aperture size differs for RBC, WBC, and platelets. The change in conductance results in an electric impulse that can be detected and recorded. This method also allows for the measurement of the cell volume. Analysis of the WBCs requires the lysis of the erythrocytes. After performing red cell lysis, the different populations of the WBCs are identified by flow cytometry within the cell counter. Reprinted from: Laposata M. Laboratory Medicine: Clinical Pathology in the Practice of Medicine. ASCP every CBC that isn t normal, he said. You have just negated that investment by one silly lapse in medical decision-making. Optimizing the Automated Analyzer Finn said there is too much emphasis on how good the analyzers are getting, and not enough on optimizing the use of automated and manual technologies. Part of the problem is that pathologists in charge of hematology laboratories trained as residents in anatomic pathology and did not learn about laboratory medicine. There is not a culture of training residents to be medical decision-makers in the clinical laboratory, he said. That s where you have to start. A lot of pathologists are performing validation functions when they should be performing interpretive functions. The concepts aren t firm with them. There are 2 functions in the laboratory: One is to stand by the numbers you release, and the other is to interpret the results you release. The next step is to practice evidence-based medicine, says Finn. If you look at the last 10,000 cases that you released and see no evidence that they could not have been auto-verified with the exact same results, then you should be auto-verifying, he said. If you look at the last 10,000 blood smears that a pathologist reviewed and there was no medical value added to that, then you shouldn t be doing that. At the same time, if you released 10,000 results that could have had medical value added, you should have been reviewing those. We are very poor on evidence-based practice. Another challenge is to help the technologists understand the information coming off the hematology analyzer. Sandhaus, who teaches ASCP CMLE courses on preventing errors in the hematology laboratory and on diagnostic hematopathology, said that technologists understanding is variable. Most of the techs in my lab really don t have that much of an understanding of the technology of the instrument, she said. Also, their understanding of the graphic displays is very limited, so as part of our continuing education in the lab, we try to emphasize correlating the graphic displays with their morphologic findings, so they can get more out of that information. Even the CBC batteries have become overly complex, said Sandhaus. There s just a ton of data that s produced on every CBC analysis, she said. We ve got all these numbers that are produced, all these indices, graphic displays, and interpretive comments. All of that information in some way needs to be integrated into that laboratory s algorithm for which results they re going to review and which ones they re going to auto-validate. If you make that process more complex, you re going to be slowing down your lab considerably. You have to weigh the benefits of more information against the additional complexity that it introduces into your whole operation. To be fair, Finn said, it would take a biophysicist to understand everything the analyzers are doing to cells. We can let the manufacturers be the biophysicists, he added. But we are all smart enough to run the instruments right if we get the right medical attitude to doing it. Medical decision-making really has to take hold in the application of high-throughput laboratory hematology. The first step is to have a medical director who is devoted to the practice of laboratory medicine and is not just a spectator. Finn said medical directors should use the technology to free up technologists to do more interpretive work of cells that are interrogated by methods other than microscopes, to teach them how to read the flow-based histogram data and the distributions on the cells that have been manipulated with different reagents. The potential is basically accumulating behind this wave of technology, he said. The technology is advancing rapidly, and labmedicine.com May 2006 Volume 37 Number 5 LABMEDICINE 277

6 Flow Cytometry Specimen: Whole blood, any body fluid, dispersed bone marrow aspirate, disrupted tissue Method Description Flow cytometry is a method in which a single cell can be characterized by size, shape, and biochemical or antigenic composition. The first flow cytometry figure illustrates the basic principles of flow cytometry. The cells flow in a single stream through a cuvette, where they are exposed to a beam of intense light. A single cell scatters the light in all directions. Forward scatter, resulting from diffraction, correlates with cell volume. Side scatter (right angle) is a result of refraction and approximates internal cellular granularity. Populations of cells such as neutrophils and lymphocytes, which differ by size and/or granularity, can be identified using forward and side scatter data. The second flow cytometry figure depicts an example of the use of fluorescence to detect different cell populations. Monoclonal antibodies used in identifying cytoplasmic and cell surface antigens most commonly are labeled with fluorescent compounds. Fluorescent compounds, like fluorescein or R-phycoerythrin, have different emission spectra, allowing for the identification of cells based on the color of the emitted light. A cell suspension is incubated with 2 monoclonal antibodies, each labeled with a different fluorochrome. As cells with bound antibodies pass through the cuvette, laser light at 488 nm excites the fluorescent compounds causing them to fluoresce at specific wavelengths. A system of lenses and filters detects the emitted light and translates it into an electric signal that can be analyzed by the computer. The side scatter and forward scatter and the intensity of emitted light at specific wavelengths characterize each cell type. The data collected from thousands of events are gathered, analyzed, and plotted on a histogram. Flow cytometry is highly useful in the diagnosis of leukemias and lymphomas. The use of different antibody markers allows for the precise identification of cells. Reprinted from: Laposata M. Laboratory Medicine: Clinical Pathology in the Practice of Medicine. American Society for Clinical Pathology, Chicago: we should be grateful for that and encouraged by that, but behind this wave, there is accumulating so much potential for the appropriate utilization of the technology, and I just don t see the mindset of clinical pathology evolving as quickly as the technology, and that s unfortunate. The rate-limiting steps are really the attitudes toward medical practice in the clinical laboratory. People have to start leading and defining how we practice medicine with tools that include automated platforms. There are smart ways to use smart technology and we re not always using them. A B That s not to say that hematology laboratories should not embrace the technological advances. It doesn t have to be either/or, Finn said. You can get new technology and improve your attitude about medical practice. In the end, said Kotylo, emerging technology is going to come no matter what. You can t keep hanging on to the way it was, she said. I think what you ll see in the future is that flow will be less isolated, and it s going to be more a part of morphology and molecular diagnostics. LM 278 LABMEDICINE Volume 37 Number 5 May 2006 labmedicine.com