Introduction
The complete blood count and plasma chemistry profile are important diagnostic tools, with laboratory protocols and reference ranges well established in both human medicine and in veterinary medicine of domestic animals. Advances in zoo medicine have included the application of comparable CBC/Chemistry techniques adapted for many exotic animal species, including birds and reptiles, providing the veterinarian with a valuable tool for health assessment of newly-acquired quarantine animals, routine physical examinations, and for clinically ill animals. With the exception of some speciesspecific differences in analyte ranges, clinical chemistry instruments developed for human and veterinary hospitals are readily used for fish specimens. However, as we continue development to hematology methods for fish species, we face challenges similar to those encountered with the early applications of CBC techniques for birds and reptiles. Like their terrestrial non-mammalian counterparts, fish erythrocytes are nucleated, and a number of the leukocytes also show similar morphology on Romanowsky-type stained blood films: thrombocytes, monocytes, lymphocytes, and basophils.
A common source of confusion in fish cell nomenclature is in the varied names given to the granulocytes, which can differ by species and do not have a clear mammalian or avian/reptilian counterpart.
In addition, the general term “fish” encompasses a very large and diverse group of aquatic animals, including primitive cartilaginous fish, such as the holocephalans (ratfish), the modern elasmobranchs (sharks, skates and rays), and teleosts (fish with true bones). The goal of this contribution is to provide an overview of clinical chemistry testing and a summary of the literature as a basis for future work to establish standard classification of fish leukocytes.
Clinical Chemistry
Blood samples are typically collected by venipuncture of the caudal vein, which lies just ventral to the spinal cord, either by ventral or lateral approach. The blood is then immediately transferred to either a serum separator tube or a tube coated with heparin. Serum or heparin plasma are both acceptable for routine blood chemistry profiles; using heparin tubes allows separation of the plasma from erythrocytes without delay. Specimen stability for fish species is comparable to that of other vertebrates.
The primary issue of analyzing fish blood with a commercial blood chemistry analyzer is the group/species-specific range of linearity for some analytes. The following are a few examples that pertain to elasmobranchs (sharks, skates and stingrays)
- Sodium and chloride typically run ~ 300 mmol/L, requiring a 1:2 or 1:3 dilution, while potassium values are comparable to other vertebrates
- Urea is a major contributor to blood osmolality, typically ~ 800-1,000 mOsm/Kg. For bench-top dry slide analyzers, a 1:10 dilution is usually sufficient, while floor model commercial analyzers may be pre-set to automatically dilute at a different ration. If the specimen is analyzed using a rotor-based instrument, this test cannot be performed because the necessary dilution alters the sample matrix to the degree that it cannot be recognized.
- Albumin is typically below the lower limit of detection
While these examples are generalities relating to a large group of fish (elasmobranchs), they illustrate the importance of establishing reference intervals. Further, studies have shown that it is necessary to determine reference intervals for the group of animals being evaluated or involved in a research study; the data from one group may not be applicable to the species overall20. The Quality Assurance in Laboratory Standards committee of the American Society for Veterinary Clinical Pathology has recently published guidelines for establishing reference intervals; this information can be found in the journal Veterinary Clinical Pathology as well as free access on their website www.asvcp.org/Publications
Hematology of Fish
Hematopoiesis
Literature shows that interest in understanding the blood cells of fish dates back to the mid 1800s; Leydig described the lymphomyeloid structures in elasmobranchs as a source of granulocytopoiesis (organ of Leydig) in 1857. Fange, et al. further studied hematopoiesis in a wide variety of elasmobranch species and described the following tissues: the organ of Leydig, a white mass located in the dorsal and ventral wall of the esophagus (abundant granulocytes and lymphocytes); the epigonal organ, associated with the gonads (abundant granulocytes and undistinguished blast-type cells); the spleen (white pulp primarily lymphocytes and red pulp primarily erythrocytes); and the thymus (lymphoid only). In the more primitive holocephalans, the site of granulocytopoiesis is found in the tissues within the cranium. In teleosts, the anterior portion of the kidney, referred to as the head kidney, is a major organ of hematopoiesis, with minor sites including the spleen, liver, and thymus.
Description of Fish Blood Cell Morphology (Romanowsky-type Stained Blood Smears)
Erythrocytes:
Fish erythrocytes resemble their avian/reptilian counterpart; they are oval in shape with abundant smooth eosinophilic cytoplasm and a central, oval-shaped condensed nucleus. Cell size varies greatly with species; this was extensively demonstrated by Dorothy Chapman Saunders’ work in the 1960s where she measured mature erythrocytes from over 600 specimens of marine fish of Puerto Rico and, in a second study, over 200 fish from the Red Sea. Both studies show the significant size difference found in the erythrocytes of elasmobranchs (~20 x 15 μ) and teleosts (variable, ~ 10 x 5 μ). A small percentage of polychromatophilic and immature erythrocytes are common.
Thrombocytes:
Typical fish thrombocytes closely resemble those found in avian and reptilian species; they are small oval cells with clear, colorless cytoplasm and a central oval, condensed nucleus. In peripheral blood smears, as in the hemacytometer, fish thrombocytes can appear in the typical oval shape, as spindle-shaped, or round. The variations in shape may be due to cell maturation or degree of activation. Thrombocytes are often confused with the similarly-sized lymphocyte; this similarity contributes to error in the total white cell count and on the differential. On the blood smear, the key features that distinguish the thrombocytes from lymphocytes are the cytoplasm color (colorless vs. light blue, respectively) and the N:C ratio (higher in the lymphocyte). Fish thrombocytes are an important part of the clotting process: they convert prothrombin to thrombin. Multiple authors have described a second population of thrombocytes in some elasmobranch species that is identical to the typical thrombocyte with the exception that the cytoplasm is filled with slender rod-shaped eosinophilic granules. This cell is the G4, of Mainwaring and Rowley’s commonly cited G1-4 granulocyte series, and the clinical significance of this cell is not yet understood.
Lymphocytes:
Similar to those found in birds and reptiles, fish lymphocytes are small round cells with a high N:C ratio and a rim of smooth light blue cytoplasm around the large oval-round condensed nucleus. The lymphocytes of some fish, especially elasmobranch species, often appear misshapen with “blebs” or outpocketings of the cell membrane. Some authors further classify the size as small and large, although the functional difference is not yet understood.
Monocytes:
As with lymphocytes, the morphology of fish monocytes is comparable to those found in mammals, birds, and reptiles. The monocytes are large, usually round cells with abundant blue cytoplasm often containing vacuoles. The nucleus can be round, oval, or lobed with loosely compact chromatin.
Basophils:
The morphology of fish basophils is similar to that of terrestrial animals. They are extremely rare in most teleosts but are commonly found in low numbers on peripheral blood smears from stingray species (Arnold, unpublished data). On Wright’s stained smears, the granules stain blue-black, while the granules may appear as empty vacuoles on commonly used 3-step quick Romanowsky-type stains. The nucleus is usually difficult to observe due to the abundance of granules.
Neutrophils:
Teleost neutrophils are large round cells with abundant clear cytoplasm and an eccentric, condensed nucleus that is either round or multi-lobed. The cytoplasm is often light gray and, in some cells, contains very fine azurophilic granules. This cell type is sometimes reported as heterophil in the literature, but to those familiar with avian and reptilian blood the term heterophil indicates a different cell type (one with rod-shaped red cytoplasmic granules). The functional distinction for these two cell morphology types in fish is not yet well understood14. Neutrophils in elasmobranchs appear as large cells with clear colorless cytoplasm containing a round or multi-lobed condensed nucleus, often both found on the same blood smear. Mainwaring and Rowley termed this cell as the G2 of the G1-4 series.
Eosinphilic Granulocytes:
The literature varies in the nomenclature used to describe the variety of fish cells containing eosinophilic granules. Teleost cells often are described using the classification for birds and reptiles, where elasmobranch cells are described in a variety of nomenclature systems.
- Heterophil/Fine Eosinophilic Granulocyte/G3/Granuloblast: Large round cell with clear to pale blue-gray cytoplasm filled with slender rod-shaped eosinophilic granules. The nucleus can be either round or multi-lobed.
- Eosinophil/Coarse Eosinophilic Granululocyte/G1: Large round cell with pale blue cytoplasm filled with large round to oval eosinophilic granules. The color of the granules varies with species, from bright red or orange to pale pink. The nucleus can be either round or multi-lobed.
A clear understanding of the blood cell morphology is essential for each species of fish in order to establish CBC reference ranges. It is encouraging to see recent progress in this effort. Hrubec, et al., reported hematology reference intervals for three important aquaculture species: the sunshine bass (reciprocal hybrid striped bass cross of female Morone chrysops x male Morone saxatilis), the palmetto bass (female Morone saxatilis x male Morone chrysops), and tilapia Oreochromis hybrid. And Tripathi, et al., reported hematology reference intervals for the ornamental strain of the common carp, koi (Cyprinus carpio). These studies show that it is possible to work through the challenges presented in CBC techniques for fish species and serve as good examples to inspire similar studies of other species.
August 2013
