The Diagnostic Power of Graphical Reports from Hematology Analyzers
Harold Tvedten Sweden
“A picture is worth a thousand words.” All too often, veterinarians do not receive the graphic displays of cells generated by the newer generation of hematology instruments. This decision, made by directors of referral laboratories, is because they do not believe veterinarians can interpret the cytograms and histograms. This unfortunate, yet common, decision reduces greatly the diagnostic utility of the report given to the submitting veterinarian.
Most of the comments and examples used in this presentation refer to the Bayer H-1 and Advia 120 systems because the author has worked most with these systems. Other examples are more universal in type and refer to impedance systems that produce very similar types of graphics that most veterinarians will see in practice. The Bayer systems use laser technology to measure the number, size, and internal complexity of erythrocytes and platelets. Impedance based systems use the most common counting method of most hematology analyzers. Impedance technology is also called the Coulter principle. Coulter hematology instruments used it for years so that the name “Coulter counters” was synonymous with “hematology analyzers.” Impedance measures the size and number of cells. The Abbott Cell Dyne uses impedance technology to measure size and number of platelets and erythrocytes and uses impedance and laser technology to count the number of leukocytes. Many smaller hematology analyzers use impedance technology to count all three cell types.
ERYTHROCYTE-PLATELET VOLUME HISTOGRAM
The erythrocyte-platelet volume histogram is the most common graphic seen by veterinarians. It is from impedance type cell counters. It should display two distinct peaks based on number of cells of a certain volume (y-axis) and cell volume (x-axis). The small peak to the left represents the platelets (smaller size and smaller number of cells) and a larger peak to the right represents erythrocytes. There should be distinct valley between the peaks to show that the instrument was capable of differentiating the two cell types. A common reason for the two peaks to merge is iron deficiency anemia. In iron deficiency anemia, the erythrocytes are smaller (microcytic) and there is often thrombocytosis with large platelets so the two cell types merge in size and may overlap. This results in small erythrocytes being counted as platelets and large platelets being counted as erythrocytes. Because platelets are fewer in number than erythrocytes, the platelet error is usually more noticeable. This change in the graphic (histogram) is both diagnostic and reveals a laboratory error. Notice that this graphic has two benefits (technical and diagnostic).
If an instrument does not display the erythrocytes and platelets graphically and if only numbers are reported, then erroneous results are not detected and the diagnosis may be missed or misinterpreted. Iron deficiency anemia is perhaps the best example of where instrument graphics improve diagnosis. Weiser revealed that 70% of five –week-old kittens had iron deficiency anemia but needed improved instrumentation to show what percentage of erythrocytes were smaller than normal. Most veterinarians get in their hematology reports only mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC). These mean (average) values are too insensitive to reveal iron deficiency in kittens as well as the early stages of iron deficiency anemia in dogs. Often there are too few microcytic erythrocytes to pull the mean values (MCV and/or MCHC) below the reference values. The platelet-erythrocyte histogram may show the change earlier. Bayer RBC cytograms and histograms are very sensitive in detecting iron deficiency anemia.
BAYER SYSTEMS GRAPHICS
The Bayer system measures both the volume and internal complexity (optical density) of erythrocytes, reticulocytes and platelets, unlike impedance counters that only measure volume of these cells. Therefore, the Bayer cytograms and histograms are the best for visualizing disease changes in these cells. It also is the best system for counting platelets because it can differentiate platelets from erythrocytes even if platelets are larger than erythrocytes.
Iron deficiency anemia is detected earliest by the Bayer system because very few microcytic hypochromic RBCs are needed in the patient’s blood to be seen on the RBC cytogram. The Bayer RBC cytogram displays every RBC in a sample, so it takes relatively few to be seen outside the normal area. It also is the only instrument to have a hemoglobin concentration histogram. Because the size and hemoglobin concentration of every RBC is measured, the percentage and absolute number of small subsets of atypical RBCs can be reported. Bayer Advia 120 can determine cell volume and cell hemoglobin of individual erythrocytes and reticulocytes which is helpful in the diagnosis of iron deficiency anemia in human blood and may be helpful with canine iron deficiency anemia evaluation.
Regenerative anemias are best diagnosed with Bayer systems. It is easy to determine the percentage of macrocytic hypochromic RBCs from the RBC volume and hemoglobin concentration histograms and the percentages directly available from the instrument. I believe the number of macrocytic hypochromic RBCs better illustrates the regenerative function of the bone marrow than reticulocyte numbers especially in later stages (10–15 days) of response to anemia in dogs and early stages (4–7 days) in cats. Canine reticulocyte numbers decrease after the peak seen 4–7 days after onset of an anemia. Thus, the number of reticulocytes at 10–15 days may seem insufficiently responsive for the severity of the anemia. At this stage, the number of macrocytic hypochromic cells remains high to better reflect an active bone marrow. Aggregate reticulocytes in the cat are the type seen early (4–7 days) in a regenerative response but can be relatively low (5%) compared to a strong canine reticulocyte response (25–33%). Thus, there are often too few aggregate reticulocytes to affect the MCV and MCHC, leading to misleading conclusions (normocytic normochromic or macrocytic hypochromic) anemias. The Bayer RBC cytogram clearly shows the presence of macrocytic hypochromic RBCs in these cats though the MCV and MCHC often do not reveal the process.
PLATELET CYTOGRAM AND HISTOGRAM
The Bayer platelet cytogram is unique in displaying platelets by both size and optical density. The ability to count platelets up to 60 fl is a clear advantage of the system but does increase the risk that large particles other than platelets can enter the counting area. The platelet cytogram can reveal other particles such as erythrocyte ghost cells. Ghost cells can be a sample problem in platelet counting in stored equine blood or lipemic canine blood. We found that equine blood samples that were one to two days old could have significant numbers of ghost cells that could be miscounted as platelets. Examining the platelet cytogram easily demonstrated the distinct population of ghost cells. Bayer has a software adjustment for this ghost cell problem in equine blood. Lipid droplets may be counted as platelets in systems that have a very low size threshold for counting platelets. These small particles were also demonstrated by the platelet volume histogram of the Bayer H-1 system. A major utility of graphics is to display visually what is being counted and to demonstrate laboratory errors that went undetected by earlier systems.
LEUKOCYTE CYTOGRAMS AND HISTOGRAMS
Leukocyte graphics usually reveal whether the automated differential leukocyte count was likely accurate. No hematology system provides a consistently accurate leukocyte differential count, or is sensitive enough to detect most left shifts or toxic changes in neutrophils. No system detects canine or feline basophils well or at all. Monocyte counts are usually different from manual monocyte counts. Some veterinary systems do an adequate job of performing a differential leukocyte count on normal horses, dogs and even cats but often have increasing error in differential leukocyte counts in ill animals. Thus one should at least scan blood smears for left shifts, toxic change, unusual cell types, RBC morphology and platelet clumps on animal blood, especially ill animals.
An exception to the preceding is toxic left shifts in horses as detected by the Bayer leukocyte cytograms. I believe that the basophil cytogram of the Bayer system illustrates toxic left shifts in horses better than most people that look at equine blood smears. Part of this is due to the equine neutrophil normally appearing so much more mature than neutrophils in other species. Thus, mild changes in the nucleus is often undetected by the human eye but detected by decreased optical density of the nucleus as seen by the laser system. The human eye is better, however, in detecting left shifts and toxic change in canine and feline neutrophils than the automated systems.
The automated differential leukocyte counts are helpful to those doing a manual differential leukocyte count but the manual differential count is more sensitive in diagnosis. This especially true of systems that give only a two or three cell differential count.
The diagnosis of leukemia is aided by graphics. Blast cells are well demonstrated on the Bayer basophil and peroxidase cytogram. Even occasional patients with many reactive lymphocytes and lymphoblasts will have some cells demonstrated on graphic displays. Specific diagnosis of types of leukemia requires blood smear evaluation.
Graphic displays of hematology analyzers visualize changes in blood cells well. These pictures reveal changes due to disease and laboratory error better than numbers or older graphics do. The graphics should be available to veterinarians who submit the blood or clinical pathologists in the laboratories need to read the graphics and interpret the diagnostic features for the submitting veterinarian. Laboratory personnel need to detect laboratory errors revealed by these graphic displays.
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