"With LaserCyte's expanded differential and WBC range, we can work up cases faster to determine appropriate treatment or further diagnostics."

James F. Young, DVM, Hospital Director
Atlantic St. Veterinary Hospital and
Pet Emergency Center
Roseville, California

Reticulocyte Characterization

The characterization of reticulocytes in the peripheral blood provides the best objective information regarding the ability of the bone marrow to respond to a peripheral demand for red blood cells. Reticulocytes are nonnucleated red blood cell precursors that typically do most of their maturation within the hematopoietic tissue of the bone marrow. Extremely low numbers of reticulocytes are released into the peripheral blood. In the healthy dog and cat, much less then 1% of all red blood cells are reticulocytes, and in some species, even fewer or extremely rarely seen reticulocytes (horse) are present in the blood.

Increased numbers of reticulocytes, which is called a reticulocytosis, indicate that the bone marrow is adequately responding to a peripheral demand. In most cases, this is associated with anemia; however, non-anemic animals may have a reticulocytosis also. Animals with shortened red blood cell lifespan (immune-mediated destruction, underlying metabolic disorders, selected red blood cell enzyme deficiencies, animals recently relocated to high altitudes where oxygen tension is lower, etc.) may have a red blood cell count within the reference range if the bone marrow has the ability to compensate for the need and maintain a higher than normal rate of red blood cell production.

Reticulocyte counts are valuable in determining if an anemia is regenerative (adequate bone marrow response) or nonregenerative (inadequate bone marrow response), as well as in identifying underlying occult compensated hemolytic processes.

Reticulocytes are identified in a peripheral blood sample by staining with new methylene blue (NMB) stain. These "young" red blood cells have the distinctive feature of containing significant amounts of ribosomal RNA, which is lost during the maturation process. The RNA is needed to allow the cell to produce hemoglobin; a reticulocyte does not have the full complement of hemoglobin that a mature red blood cell has. The NMB stain precipitates the RNA in the cell, allowing it to be easily identified (figure 1) because of the presence of aggregates of dark blue precipitated RNA in the cytoplasm. Reticulocytes with sufficient RNA in the cytoplasm can be identified in a peripheral blood film stained with a standard Romanovsky stain, including most quick stains used in practice. These immature cells are identified as polychromatophilic nonnucleated red blood cells with a pale blue cytoplasm (figure 2) compared to the normal orange-red staining of the mature red blood cell with its full complement of hemoglobin. Relatively "mature" reticulocytes with only small amounts of RNA cannot be accurately identified in Romanovsky-stained peripheral blood films.

The primary difficulty most people have regarding reticulocyte counts is related to the interpretation, which can be relatively confusing. Some reference laboratories actually report reticulocyte analyses in potentially four different ways, which are outlined below. Most believe that the easiest way to assess reticulocyte values is to look at absolute reticulocyte counts per microliter of blood.

Reticulocyte Analysis

Reticulocyte Count—This value is most typically determined by identifying the percentage of reticulocytes by evaluating 1,000 nonnucleated red blood cells stained with NMB stain. The primary difficulty in interpreting this value lies in the fact that the number is a "relative" number that does not take into consideration changes in red blood cell mass during anemia or potential dilution or concentrating effects of abnormal water balance.

Corrected Reticulocyte Count—The corrected reticulocyte count is calculated by multiplying the patient reticulocyte count by the value calculated from the patient PCV (%) by a dog or cat's "normal" PCV (45% for the dog and 30% for the cat). This "correction" takes into consideration the change in red blood cell mass associated with anemia or changes in hydration status (dehydration—concentrating effect; over-hydration—diluting effect).

Reticulocyte Production Index (RPI)—This value attempts to correct for the fact that during a situation of severe demand for red blood cell production by the marrow and marked reticulocytosis, reticulocytes are released from the bone marrow at an earlier than normal stage of maturation. Since they are "less mature," they take a longer time to mature within the peripheral blood. Normally, reticulocytes take approximately one day to mature when released from the marrow. In the dog, it is estimated that when the hematocrit is 35%, reticulocytes take approximately 1.5 days to mature, approximately two days to mature with a hematocrit of 25%, and 2.5 days to mature with a hematocrit of 15%. The RPI is calculated by dividing the corrected reticulocyte count by the approximate maturation time. It should be noted that this "correction" can only be performed with the dog. Cat reticulocytes are more unpredictable in relation to maturation times.

Absolute Reticulocyte Count—As was mentioned above, this is the least confusing way to evaluate reticulocyte responses. By reporting the number of reticulocytes per microliter, the issue of correcting for red blood cell mass changes is not needed. Maturation times still come to play with the bottom line interpretation, but degrees of response have been categorized to limit the need to be concerned about changes in maturation times. In most laboratories, this value is determined first by manually counting the relative number of reticulocytes among the red blood cell population, and multiplying this number by the measured number of red blood cells per microliter (RBC count). There are few instruments used in reference laboratories that directly measure the number of reticulocytes in the blood sample. The LaserCyte® Hematology Analyzer performs a direct count of reticulocytes with special stained aliquots of the blood sample. Direct measurements of reticulocyte counts are a much more precise and accurate method of determining an absolute reticulocyte count.

Written by Dennis B. DeNicola, DVM, PhD, Diplomate ACVP, Chief Veterinary Educator, IDEXX Laboratories.

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1000^th LaserCyte^® Customer!

Photographed from left to right are Shawna Morris (holding Fancy), Emily Ackerman (kneeling), Dr. Susan Ridinger, and Stephanie Triay.

This November, almost exactly one year after the launch of the LaserCyte hematology system, the 1000^th LaserCyte was installed at Briarcliff Animal Hospital in Jacksonville, Florida.

Dr. Susan Ridinger, principle veterinarian at Briarcliff, was looking for an in-house hematology analyzer that could provide an additional level of information than was previously available in-house.

"We were looking for an analyzer that could help us with diagnosing the more problematic cases that we see daily. The QBC® VetAutoread™ worked very well for our preanesthetic workups, but we needed a system that could provide more information for dogs with cancer and sick cats—specifically eosinophil counts."

Travis White, the local IDEXX diagnostic product consultant, Brad Brazell, Senior Marketing Manager for Hematology and Harold Flynn, Hematology Director, were all present for the celebration. Congratulations to all at Briarcliff Animal Hospital.

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At the North American Veterinary Conference, Orlando, Florida

Lunch will be provided at the above session. To register, send us an e-mail with your name, practice, location and how best to contact you. We will send you more specific location information. Seating is limited, so sign up early!

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"Can you please explain left shift? I never quite got this. Is it just the presence of bands; or is there a lot more to it?"

A left shift is a term used to describe the presence of immature neutrophils in the peripheral blood. This indicates the release of immature neutrophils from the bone marrow in response to active inflammatory disease involving neutrophils. Band neutrophils are normally present in very low numbers (typically less than 300/microliter), and represent the most common immature form found in the peripheral blood during inflammatory disease. During severe and sometimes overwhelming inflammation, earlier stages of maturation, including metamyelocytes and myelocytes, may be seen in the peripheral blood.

Some technicians or pathologists will actually report the presence of significant numbers of hyposegmented neutrophils on a peripheral blood film. These are cells between a textbook band and mature segmented neutrophil; the process of maturation between these cell stages is very gradual. The identification of significant numbers of hyposegmented neutrophils in circulation is of similar value as the identification of true band neutrophil forms.

The recognition of a left shift in the neutrophil series is highly valuable in the identification of inflammatory disease, the grading of the severity of the inflammatory process, and for following progression or regression of inflammatory disease. It should be noted that no hematology analyzer currently used in reference laboratories or available for the veterinary practitioner has the ability to recognize a left shift. A microscopic evaluation of a freshly prepared peripheral blood film is an essential component to the numerical characterization of blood by a hematology analyzer.

Figure 3 is a composite image from a case of a dog with severe inflammatory disease and a marked left shift. All cells are from the same blood film.

Figure 3 click to enlarge the image

Would you like to submit a hematology question for our clinical pathologists to answer in an upcoming issue of Diagnostic Edge?

Responding to this month's question is Dennis B. DeNicola, DVM, PhD, Diplomate ACVP, Chief Veterinary Educator, IDEXX Laboratories.

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Reducing platelet clumping—The most common cause for platelet clumping is related to sample collection and quality. Platelet clumping can occur if there is a delay in transferring the collected sample into the EDTA anticoagulant or difficulty in collecting a sample as a result of small blood vessel size in a young or small animal or patient excitement during the collection process. During difficult sample collection various tissue factors are introduced into the specimen, which causes in-vitro platelet clumping. When platelet clumping is identified, collection of a fresh sample for accurate assessment of platelet numbers is recommended.

We recommend using the Vacutainer® method for drawing the blood. This utilizes the natural blood pressure of the animal for the blood to transfer into the EDTA tube. When a syringe is used, the plunger pulls the blood from the vein and is then transferred into an EDTA tube. These extra steps and the added "pressure" from the person drawing the blood can increase the likelihood of platelet clumping. The preferred technique to prevent clumping is for the blood to go from the vein directly into the EDTA tube.

Note: This is just a recommendation and will NOT prevent all samples from clumping.

One of the many benefits of the LaserCyte's laser-flow cytometry is that it will not falsely count clumped platelets as white blood cells. However, if there is platelet clumping, a low platelet count may be reported, indicating thrombocytopenia, as is the case with hematology analyzers used in reference laboratories. This number must be interpreted as a "minimal" platelet count and caution should be taken when interpreting these results. Collection of another sample, preferably with the Vacutainer® method, is recommended to obtain an accurate platelet count. It should be noted that this is true for both the LaserCyte and reference laboratory platelet enumerations. While feline samples are more likely to exhibit platelet clumping, problems can also occur with canine and other species. The value of a brief microscopic evaluation of a well-made peripheral blood film cannot be understated.

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... and we want you to hear from us! Pass the following link along to your friends and colleagues. They can register for the Diagnostic Edge to learn more about hematology, stay informed of the latest IDEXX hematology products and services, and respond to various customer and market surveys that we will soon offer. Registration is easy at www.idexx.com/diagnosticedge.

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