Abstract

We already know from anatomy and physiology that the rabbit is not like other small mammals in many different ways. This trend also applies to the clinical pathology of the rabbit. The lecture will focus on how to interpret the rabbit complete blood count using comics as a learning aid. The rabbit immune system reacts to challenges very differently than other mammals and having a good understanding of the rabbit CBC will be a significant aid in providing excellent clinical care for these beloved pets.

Introduction

The task of successful science education in the age of multimedia, when attention spans continually decrease, is very challenging. In the author’s opinion, the importance of telling stories and providing visual aids to deliver content has unfortunately not reached the same popularity in the teacher’s world when compared to the preferences of their younger audiences. The use of stories to teach science is not a novel concept, and science education has been a subject of research for centuries.¹ The author is using comics to illustrate and highlight specific important points when interpreting the hematology results of the rabbit.

A Type of Teaching Technique

It is essential to realize that a compelling story cannot be told with just one picture. Therefore, it is crucial to recognize that the bloodwork of your patient is only one part of the story. Do not make the mistake of overinterpreting the status of the animal just based on one data set. Consider the bigger picture, examine the animal, look at the history, and look at the present and post data sets. However, sometimes the clinician is forced to limit the data collection to a single diagnostic test or procedure. To offer the best service, always recommend and document that you provided a variety of diagnostic tests that can provide the most information to evaluate the patient. Also, educate the owner that you might need to run repeated samples, for example, to monitor the response to a specific treatment. You have to eventually connect the dots to draw a picture, and it gets easier the more dots you have to work with.

For the sake of the lecture, we will focus on blood work as the data to evaluate. One of the most critical aspects of looking at bloodwork (especially chemistries) is to validate the reference one uses. Do go through the extra effort to figure out what is the population that was used to establish the reference range you are using. There are a large variety of ‘normal’ values published, which may differ significantly. The reader is encouraged to compare and contrast the sources of the published values.

The total volume of blood available for collection depends on the health status of the rabbit. If the rabbit is healthy, about 1% of the circulating blood volume can be considered available for collection. If the animal is diseased or if repeated bleeding events are needed, 0.5% might be safer. The circulating blood volume of the rabbit is approximately 55–70 mL/kg of body weight.² Common phlebotomy sites for the rabbit include the cephalic vein, saphenous vein, and the lateral ear vein. Because rabbit blood tends to clot quickly and the cells can lyse readily, the blood should be immediately placed in an anticoagulant. Slides need also be made immediately to avoid post-sample artifacts. Anticoagulants commonly used (EDTA and heparin) work well for rabbit blood.

Erythrocytes have a shorter lifespan (average ∼50–60 days) when compared to other domesticated mammals.³ Hence polychromasia is typical when compared to other larger mammals. So, the image that comes to mind when comparing the erythrocyte character on the blood smear of a rabbit might be a shopping mall where the erythrocytes are the people in the mall. Everyone can picture this and realizes that there is a significant age variation in the population when compared to a workplace, like a cubicle world of a larger company. The cubicle world could represent the erythrocyte population in a dog (much less polychromasia) as there is significantly less variation in the age of the employees when compared to the shopping mall attendees.

The morphology of rabbit lymphocytes is similar to that of other species. Lymphocytes have a large nucleus, that may be slightly indented, and a small amount of light blue cytoplasm. Although small lymphocytes predominate, large lymphocytes may be present. Reactive lymphocytes (immunocytes) are usually antigenically stimulated lymphocytes that are larger cells with a more intensely blue cytoplasm. In the comic world analogy, the lymphocyte would be represented by a “mall cop” who controls the area and looks for any abnormal activities.

Heterophils have a light purple, lobulated nucleus surrounded by cytoplasm containing diffuse, variably sized reddish granules. They are often misidentified as eosinophils by the untrained eye during the complete blood count. In the literature, they are sometimes referred to as “pseudo-eosinophils.” Heterophilic granules are generally smaller than those of eosinophils and may not occupy all of the cytoplasm. Heterophils range from 10 to 15 µm in diameter. Minor heterophil degranulation may accompany the use of rapid Romanowsky-type stains such as Diff-Quick. Stain-induced degranulation is presumed to be a sequel of short fixation time (5 to 10 seconds) during staining. Degranulated cells will still resemble heterophils, but vacuoles will be present where the granules previously were located. Stain-induced degranulation should not be confused with toxic change in which the cytoplasm has a blue cast. In the comic world analogy, the heterophil would be represented by a “special police unit” who would be called to the area, if any abnormal or significant violent activity would be reported by the “mall cop.”

In the monocyte, a few cytoplasmic vacuoles may be observed. Large, dark red granules have been described in the cytoplasm of some monocytes in association with nonspecific toxic changes of leucocytes. The monocytes usually appear in higher numbers if the initial immune response is insufficient to clear the problem. In the comic analogy, this would be illustrated by the “hostage negotiator” trying to deescalate the situation.

Eosinophils are slightly larger than heterophils and are 12 to 16 µm in diameter. The nucleus stains purple and often appears bilobed. Intensely acidophilic, round, cytoplasmic granules are present that are larger and more numerous than the granules in heterophils.

Basophils have a light purple, lobulated nucleus and dark purple to purple-black cytoplasmic granules. They are approximately the same size as heterophils. Rabbits usually have more basophils than other mammals. Up to 30% of leukocytes in healthy rabbits has been seen. However, the significance of this observation is unclear.⁴

Because these last two cell lines play a minor role in the evaluation of the rabbit CBC, there is no significant role assigned to any of these in the comic strip.

Additional Evaluation

One of the main observations in the complete blood count (CBC) of a sick rabbit is a decrease in the hematocrit (HCT).⁴ When this observation is translated to our comic world, it would be depicted as a mall where there is a fire or something seriously going on, and most of the attendees (RBC) are leaving the area while some of the mall cops (lymphocytes) are coming to investigate and trying to clear the problem.

The lecture will use a few clinical cases and real data to illustrate the analogy with the comic strip “shopping mall” image to highlight the most important aspects of interpreting the changes in the rabbit CBC.

The “Cliff Notes” in the appendix at the end of this document are designed to be printed out and laminated to have as a reference when reviewing a blood smear in your clinic.

Example Cases Discussed in Lecture

Case 1. Normal rabbit

Case 2. Stress leukogram

Case 3. Diseased rabbit

Case 4. Septic rabbit

Case 5. Break-down phase of the immune system

Case 6. Thymic lymphoma

Case 7. Leukemia

Fig

Rabbit Hematology Cliff Notes

by Dr. Joerg Mayer

General points:

• See the bigger picture (always correlate findings to physical exam and other tests).
• Run repeat samples (connect the dots to see the trend).
• Always doubt (if the result does not make sense call the lab to verify).
• Pick reliable reference source and stick with that (reference values can vary significantly).
• For phlebotomy 1% of bwt can be safely taken (10 ml from a 1 kg animal).

Rabbit blood	Reference values
Clots very fast, fill EDTA first in case you fill more. If serum is needed, clot will be mature in about 30 minutes. The lymphocyte is the most common leukocyte in the blood of young animals.	WBC=6,300–10,060 cells/µl Heterophils=1,490–3,210 cells/µl Lymphocytes=3,360–7,000 cells/µl Monocytes=50–450 cells/µl Eosinophils=100–150 cells/µl Basophils=60–360 cells/µl
Changes in the sick rabbit	CBC in the septic rabbit
Many sick rabbits have hemoglobin and PCV much lower than normal count. Rabbits with a bacterial infection will not have leukocytosis but have increased heterophil count (absolute) and decrease in lymphocyte count (relative lymphopenia).	General hematology
			Results
	WBC		4.240 x 103/µl <L>
	RBC		3.74 x 106/µl <L>
	Hemoglobin		7.6 g/dl <L>
	Hematocrit		24%
	MCV		62.2 fl
	MCH		20.3 pg
	MCHC		31.7 g/dl
	RDU		14.1%
Neutropenia	Differential
Occurs when consumption is higher than production. Often in acute inflammation or with marked severity of disease.		Results
		Absolute		Relative
	Seg N	3.731 x 103/µl		88%<L>
	Lymphocytes	0.466 x 103/µl		11% <L>
	Monocytes	0.042 x 103/µl		1% <L>
Summary
One of the main effects of disease is to reduce the total blood cellularity, particularly with respect to the red cell values but also to a lesser extent with the white cell counts. The hematocrit might be one of the best indicators to differentiate between sick and healthy. The least important variable was the basophil count, followed by the monocyte and eosinophil counts.
Lymphocyte
Reactive lymphocytes (immunocytes) are antigenically-stimulated lymphocytes that are larger cells with a more intensely blue cytoplasm. The morphology of the rabbit lymphocytes is similar to that of other species. Lymphocytes have a large nucleus, that may be slightly indented, and a small amount of light blue cytoplasm. Although small lymphocytes predominate, large lymphocytes may be present.	Fig
Heterophil
They have a light purple, lobulated nucleus surrounded by cytoplasm containing diffuse, variably sized reddish granules. Often misidentified as eosinophils. Heterophilic granules are generally smaller than those of eosinophils and may not occupy all of the cytoplasm. Heterophils range from 10 to 15 µm in diameter.	Fig
Eosinophil
Eosinophils are slightly larger than heterophils and are 12 to 16 µm in diameter. The nucleus stains purple and often appears bilobed. Intensely acidophilic, round, cytoplasmic granules are present that are larger and more numerous than the granules in heterophils.	Fig
Monocyte
Monocytes are the largest circulating leukocytes in health and measure 15 to 18 µm in diameter. Monocytes have a large, variably shaped nucleus with chromatin that appears less condensed than that of heterophils. The cytoplasm is abundant and stains gray to blue-gray. A few cytoplasmic vacuoles may be observed. Large, dark red granules have been described in the cytoplasm of some monocytes in association with nonspecific toxicity.	Fig
Basophil
Basophils have a light purple, lobulated nucleus and dark purple to purple-black cytoplasmic granules. They are approximately the same size as heterophils.	Fig

Images courtesy of UGA.

References

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2.  Suckow MA, Schroeder V. The Laboratory Rabbit. 2nd ed. Boca Raton, FL: CRC Press/Taylor & Francis; 2010.

3.  Marshall KL. Rabbit hematology. Vet Clin North Am Exot Anim Pract. 2008;11:551–567, vii.

4.  Hinton M, Jones DR, Festing MF. Haematological findings in healthy and diseased rabbits, a multivariate analysis. Lab Anim. 1982;16:123–129.

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