ADVANCED HAEMATOLOGY BATTLE OF THE BANDS. Dennis B. DeNicola, DVM, PhD, DACVP IDEXX Laboratories, Inc. Westbrook, Maine, USA BACKGROUND

Transcription

1 ADVANCED HAEMATOLOGY BATTLE OF THE BANDS Dennis B. DeNicola, DVM, PhD, DACVP IDEXX Laboratories, Inc. Westbrook, Maine, USA BACKGROUND The identification of immature neutrophils (bands, metamyelocytes, myelocytes, etc.) and toxic neutrophil forms are critical for characterizing not only the presence of inflammation but also for characterizing the severity of the inflammatory process itself. In addition, following serial complete blood counts (CBCs) and monitoring the increasing or decreasing presence of immature and/or toxic neutrophils once an inflammatory response is identified allows the veterinarian to accurately determine if the inflammatory process is worsening or improving, respectively. In the past this has only been accomplished through the microscopic identification of these cells on a peripheral blood film. This task has been no simple accomplishment due to the difficultly in accurately and consistently identifying these cells and because of the nature of the process of manually identifying these cells on a blood film, this is an extremely imprecise process. Advent of newer haematology technology is now changing the manner in which inflammatory disease is characterized in veterinary medicine. THE VALUE OF DETECTING IMMATURE AND/OR TOXIC NEUTROPHILS The characterizing of leukograms requires accurate identification and differentiation between leukocytes. A five-part leukocyte differential has become a standard for this characterization. Among the most common leukogram changes seen in veterinary medicine, inflammatory, stress (glucocorticoid-induced) and excitement (epinephrine-induced) leukograms predominate. All three of these processes commonly have a neutrophilia, a change that many use as an indicator of inflammation if immature and/or toxic neutrophils are not identified. The only differentiation between these three common conditions is finding the presence of immature and or toxic neutrophils. See figure 1 below. Legend for figure 1: up arrow = increased, down arrow = decreased, N = normal

2 Not only is the identification of immature and/or toxic neutrophils essential for identifying the presence of inflammatory disease in most cases, but the number of these cells can assist in characterizing the severity of the inflammatory process. See figure 2 below. Legend for figure 2: up arrow = increased, down arrow = decreased, N = normal PROBLEMS WITH MICROSCOPIC IDENTIFICATION OF IMMATURE AND/OR TOXIC NEUTROPHILS There are multiple factors related to the difficulty in accurate identification of immature and/or toxic neutrophils. Varying definitions for the band neutrophil, the most commonly seen immature neutrophil form during inflammatory disease, is one contributing factor. One definition includes the presence of neutrophils with a parallel-sided nucleus often taking a horse shoe shape; however, this classic form is rarely observed and many have slightly modified the definition to include nuclei with slight indentations. This modification clearly leaves too much to interpretation. Another definition is that if the thinnest portion of the neutrophil nucleus is thicker than one third the thickness at the widest portion of the nucleus, the cell may be called a band. Again, there is much subjectivity to this definition leaving much to the interpretation since actual measurements are rarely performed. An additional complication for the identification of band neutrophils is the fact that the nucleus is commonly twisted or folded on top of itself, which makes it difficult to impossible to accurately determine the thickness of the nucleus in different regions. See figure 3 below.

3 Legend for figure 3: These neutrophil images show neutrophils with poorly lobed nuclei; however, because the nucleus is bent or twisted on itself, accurate characterization and band neutrophil identification is impossible. Even if the microscopic detection of immature neutrophils was simple and accurate, there is a problem related to the precision of the manual leukocyte differential. The presence of only low numbers immature neutrophils can be critical for the identification of an inflammatory process. In situations where there is only a 10,000 /µl total leukocyte count, as few as 3-5% immature neutrophils supports the presence of an inflammatory process. If even a 200 leukocyte differential is performed, the 95% confidence interval limits for a 3% and 5% relative count is 1-7% and 2-10%, respectively. Toxic neutrophil forms have one common feature and that is that the cytoplasm has more RNA compared to the mature neutrophil, which normally has minimal to no RNA since it is an end-stage cell with little to no capability of cell division or protein synthesis. The greater the amount of RNA in the cytoplasm, the greater the degree of toxicity is documented. Just like the increasing numbers and severity of a left shift (presence of immature neutrophils in circulation) is directly related to the severity of the inflammatory process, so is the degree of toxicity. Microscopically, the darker blue staining of the cytoplasm, the greater the amount of RNA is present in the neutrophil. The problem with consistently determining the degree of blue staining is the variability in the staining outcome from routine quick haematology stains typically used in a veterinary practice. NEW TECHNOLOGY CAN HELP OBJECTIVELY CHARACTERIZE INFLAMMATORY DISEASE With the introduction of recently developed software for a veterinary practice haematology analyzer, the IDEXX ProCyte Dx (PDx), objectivity in identifying immature and/or toxic neutrophils is now possible. This is the first haematology analyzer in either human or veterinary medicine to accomplish this. With the PDx, leukocyte differentials are accomplished with a combination of technologies. With the use of laser flow cytometry and optical fluorescence with a fluorescent dye specific for nucleic acids, detailed definition and characterization of the different leukocyte types is possible. This detailed characterization is demonstrated graphically in the WBC Dot Plot presented with the haematology results. In figure 4 below, the dot plot is composed of the optical fluorescent measurements on the y-axis and a flow cytometric measurement of cytoplasmic complexity or granularity on the x-axis. One of the key features in identifying immature and/or toxic neutrophils is the cytoplasmic content of RNA (optical fluorescence) on the y-axis. In the dot plot, each dot represents the digitized signal of a single cell. The greater the amount of cytoplasmic RNA, the higher on the y-axis, the dot/cell will be located. The common feature to immature and/or toxic neutrophils is the presence of increased amounts of cytoplasmic RNA compared to a normal mature neutrophil.

4 Figure 4: Normal canine WBC Dot Plot with neutrophils colored purple, eosinophils colored green, basophils colored light blue, lymphocytes colored dark blue and monocytes colored red. In figure 5 below, note the difference between a canine blood sample with a mild increase in the number of immature neutrophils (9% Bands) and a sample with no bands. The cluster of digitized events colored in purple (neutrophils) has a raised profile with digitized events extending upward from their normal location on the y-axis (RNA content). A normal canine WBC Dot Plot is shown to the right of the sample with the left shift to serve as a reference. Figure 5: Canine WBC Dot Plot with a mild left shift (left) and a normal canine WBC Dot Plot (right) The changes associated with the presence of immature and/or toxic neutrophils in circulation are accompanied with appropriate highly sensitive and specific messaging to alert the end user to their presence. Unlike the subjectivity associated with identifying these leukocyte changes morphologically with the microscopic review of the blood film, the graphic display of RNA content within the cells proves to be much more objective. In addition to identifying the presence of immature and/or toxic neutrophils using the new technology, the serial evaluation of the graphic displays provides an excellent means of following the progression or regression of an inflammatory process. As the inflammatory process is resolving, the number of immature and/or toxic neutrophils decreases and associated with these changes, the cluster of digitized events representing the

5 neutrophil population progressively decreases along the y-axis until the normal dot plot profile returns. This is best demonstrated by example as is shown in figure 6 below. The series of dot plots are from a dog during resolution of a severe septic pleuritis. Figure 6: Series of WBC Dot Plots from a dog recovering from severe septic pleuritis. Note the gradual transition from abnormal with a prominent left shift to normal. CONCLUSION The identification of immature and/or toxic neutrophils is essential for accurately characterizing the inflammatory process. Since most CBCs in the veterinary practices do not have an associated blood film microscopic evaluation and even if this is performed, the accurate characterization of these leukocytes is both lacks both accuracy and precision. Newly available in-house haematology analyzer software can assist the veterinarian with this important hematologic characterization.