FISH HEMATOLOGY G. William Klontz, M.S., D.V.M. I. INTRODUCTION Hematology is the science of studying the anatomical, physiological, and pathological aspects of blood. Blood is a fluid tissue contained within the cardiovascular system. The fluid element of blood is plasma and the formed elements of blood are the erythrocytes, leukocytes and thrombocytes. The primary functions of blood are (1) oxygenation of tissues; (2) nutrition of tissues; (3) maintenance of acid-base balance; and (4) removal of metabolic waste products from tissues. Thus, any dysfunctions of blood can have severe effects on the physiological activities of the entire body. Also, certain physiological dysfunctions in the body are reflected as alterations in blood constituents, which can be used as diagnostic indicators. The history of applying hematological methods as diagnostic aids in episodes of noninfectious and infectious diseases in confined and free-living populations of fish is quite meager (Blaxhall, 1972). The major reason for the lack of utility, as compared with mammalian medicine, is the variability of data. Fish are subjected to many environmental influences which alter the healthy hemogram; i.e., the baseline data for cellular and plasma components. Such is not the case in mammals. Thus, when hematological methods are used as aids in fish disease diagnostics, the following variables must be taken into account: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Species of fish Sex of fish Origin (gene pool) of fish Water temperature Water chemical nature, especially alkalinity, pH, and hardness Diet Physiological age of fish Sampling techniques Cell counting techniques Staining techniques Personnel II. SAMPLE COLLECTION Site: 1. 2. 3. 4. 5. 6. 7. 8. Anesthetics: 1. 2. 3. 4. 5. 6. 7. 8. Cardiac puncture Venipuncture Arteriopuncture Caudal severance Ventral aorta severance Kidney Liver Spleen Tricaine methanesulfonate Benzocaine Methyl pentynol Propoxate Etomidate Carbon dioxide Chilling Physical stunning Anticoagulants: 1. Heparin 2. Dipotassium ethylenediaminetetraacetic acid Collection Instruments: 1. Capillary tube 2. Vial 3. Syringe and needle 4. Slide III. HEMATOCRIT A. General Considerations: 1. This technique measures the volume of packed cells (erythrocytes, leukocytes and thrombocytes) as % of a volume of blood. 2. B. It is a useful indicator of anemia, hypoproteinemia, and leukocytosis. Technique: 1. The appropriate sample of blood may be obtained directly via an hematocrit tube or indirectly via a sample containing an anticoagulant. 2. The direct method of collection is usually via the severance of the caudal peduncle as follows: a. The fish is anesthetized either by a sharp blow to the cranium or by one of the chemical anesthetics. To prevent rapid coagulation of the blood, care must be taken not to overly stress the fish. b. Sever the caudal peduncle with a sharp knife (not scissors) a few millimeters posterior to the adipose fin. c. Fill two hematocrit capillary tubes simultaneously. The tubes should not be more than 3/4 filled. NOTE: if using tubes with painted ends, fill the tubes from the painted end. One may use either heparinized or unheparinized tubes. d. Seal the vacant end of the tube with Critoseal (a special clay) or by flame. If the tubes are to be flame-sealed, take care not to overheat the tubes. If a bulb is created, the sample should be discarded as it most certainly will break during the centrifugation phase, or, if it does not, it will create an error in determining the packed cell volume. 3. The indirect method of collection is as follows: a. Resuspend the cellular elements in the blood sample using a glass stirring rod or by gentle inversion. b. Fill two non-heparinized hematocrit capillary tubes individually. The tubes should not be more than 3/4 filled. NOTE: If using tubes with painted ends, fill the tubes from the painted end. c. Seal the vacant end of the tubes as in Step 2.d. 4. Each tube is placed into the special centrifuge with the sealed end facing outboard. Paired samples should be placed opposite each other. The cover plate is "finger-tightened" and the lid put into the Down and Lock position. The centrifuge is set to run for 5 minutes. 5. The percent packed cell volume, following centrifugation, is determined using either the Graphic Reader or the Circular Reader. C. Interpretation: 1. Hematocrit values have been recorded for clinically healthy members of the following species of fish: Species Range (%) Lamprey Common carp Dogfish Yellow perch Rainbow trout 14-20 26-39 16-37 24-43 13-42 Mean (%) 23.5 31.3 23.3 35.3 27.2 2. The hematocrit is subject to a great deal of variability caused by environmental conditions and physical handling during the blood sampling process. Thus, baseline values for a population must be obtained from clinically healthy animals if the test is to be used as a valid diagnostic aid. VIII. MORPHOLOGICAL HEMATOLOGY A. General Considerations: 1. An understanding of the relative numbers of circulating leukocytes; i.e., granulocytes, lymphocytes (small, medium and large) and macrophages is a useful diagnostic aid in cases of systemic diseases. 2. An understanding of the relative numbers of circulating erythrocytes by maturation stage is a useful diagnostic aid in certain viral diseases and nutritional diseases. 3. The following descriptions of the developing stages of erythrocytes, leukocytes and thrombocytes in rainbow trout are based upon the reports of Downey (1909), Jordan and Speidel (1930; 1931), Catton (1951), and Klontz (1959), plus numerous other texts on avian, reptilian, amphibian and fish hematology. 4. The sites of hematopoiesis in the teleost are, in order of activity: the head (anterior) kidney; the midkidney; the posterior (tail) kidney; the spleen; and the liver. The spleen functions mainly as an erythrocyte maturation and storage area. B. Preparation and Staining of Blood Smears and Imprints: 1. Blood smear preparation: The microscope slides should be of the frosted-end type and stored in 95% ethanol. Dry and label the appropriate number of slides. A drop of blood (with or without coagulant) is placed on the unfrosted end of the slide. A clean slide, held at a 45 angle, is touched to the slide with the drop and gently moved to the drop of blood. When the blood spreads across the edge of the held slide, with a direct, non-stop movement move the held slide towards the frosted end of the slide. This should produce an even smear over the base slide, which is allowed to air dry. When the smear is dry, it is placed into a Coplin jar (or some other suitable container) filled with absolute methanol. This fixes the cells. After at least 5 minutes (and up to hours), the slide is removed from the methanol and stored in a dust-free fashion until being stained. 2. Tissue imprint preparation: Dry and label the appropriate number of slides. Using a scalpel blade, cut a small piece of tissue from the spleen, liver, and/or kidney. Holding the cut piece of tissue in a pair of fine-pointed forceps, gently press the cut surface of the organ repeatedly across the slide. When the imprints are dry, place the slide into absolute methanol for at least 5 minutes. If staining is to be delayed, store the slides in a dust-free container until staining. 3. Leishman-Giemsa staining technique: Overlay each slide with 1.0 ml Leishman stain After 1-2 minutes overlay each slide, with the Leishman stain still present, an equal volume of Giemsa working stain. Take care to evenly distribute the Giemsa solution throughout the length of the slide. After a few seconds, a metallic sheen should appear on the surface of the stain mixture. This indicates a proper staining mixture. The staining time is variable - usually 14-20 minutes - at the end of which the stain is flushed from the slide with distilled water from a plastic squeeze bottle. The underside of the slide is wiped with an absorbent towel and the slide placed on edge to drain dry. C. Morphological Descriptions: Stem Cells Hemocytoblast: This is a totipotential cell giving rise to the three series of blood cells; i.e., erythrocytes, leukocytes and thrombocytes. Its origin is a reticuloendothelial cell in the anterior and middle kidney hematopoietic tissues. It is epithelioid; i.e., resembles a squamous epithelial cell, 10-20 um in diameter, round in shape with a central or sometimes slightly eccentric nucleus, and has a cytoplasmic:nuclear ratio of 1.5-1.6. When stained by the Leishman-Giemsa technique, the cytoplasm is deep blue with a homogenous distribution of particulate material. The nucleus stains basophilic and the chromatin material is not too prominent. Lymphoid hemoblast: There are two types of lymphoid hemoblasts: small and large. Both originate from the hemocytoblast and both are pluripotential. The small lymphoid hemoblast gives rise to the erythrocytic, thrombocytic and the lymphocytic series, which includes the small, medium and large lymphocytes and the plasma cells. The large lymphoid hemoblast gives rise to the granulocytes; i.e., the neutrophils, basophils and the eosinophils. Current thinking also is that this cell gives rise to the circulating macrophages, although there are disagreements over this contention. The small lymphoid hemoblast is 10-15 nm in diameter with a fairly homogeneous, dusty, azure blue cytoplasm. The spheroid nucleus is eccentric, has a think nuclear membrane, and contains relatively fine chromatin material. The cytoplasmic:nuclear ratio is 1.25-12.5:1. In anterior kidney imprints it is quite commonly seen surrounded by blast cells destined to be in either the erythrocytic or lymphocytic series. The large lymphoid hemoblast is spherical to ovoid and 12-17 nm in diameter. The cytoplasm is fairly homogenous and stains a dusty, azure blue and does not have granulation. The fairly centrally-located, round nucleus has prominent nucleoli. Mitotic figures are quite commonly noted. The cytoplasmic:nuclear ratio is 1.5-2.0:1. Erythrocytic Series Proerythroblast: This is a spherical cell, 7-12 nm in diameter with an unevenly stained royal blue to azure blue cytoplasm. The uneven staining is considered to be due to the hemoglobin precursor pigments. The nucleus is round, centrally located, contains prominent chromatin material, frequently nucleoli, and is surrounded by the relatively thick nuclear membrane. The cytoplasmic:nuclear ratio is 1.25-1.5:1. Mitotic figures are quite common. Erythroblast: This is a spheroid cell, 7-12 nm in diameter. The cytoplasm stains a rather even smokey-blue. The central nucleus is enclosed in a prominent nuclear membrane and stains a deep rose-purple. The chromatin material is quite prominent and irregularly arranged. The cytoplasmic:nuclear ratio is 1.5-1.75:1. Early Polychromatocyte: This cell corresponds to the mammalian polychrome normoblast. It is spheroid, 10-12 nm in diameter. The cytoplasm stain a homogeneous light blue-grey. The circular nucleus is centrally located with a fairly prominent nuclear membrane, and contains coarse chromatin beginning to become radially arranged. The cytoplasmic:nuclear ratio is 1.51.75:1. Mitoses apparently do not occur; only maturation. Mid-Polychromatocyte: This cell corresponds to the mammalian normoblast. It is spheroid, 7-12 nm in diameter. The cytoplasm stains somewhat lighter than its predecessor, the early polychromatocyte. The central nucleus is circular, has a think nuclear membrane, and contains chromatin materials which is quite compacted into thick, radially arranged threads. The cytoplasmic:nuclear ratio is approximately 2.0:1. On rare occasions this cell might be noted in peripheral blood smears. Late Polychromatocyte: This cell corresponds to the mammalian orthochrome normoblast and is the least mature cell to appear in the peripheral vascular system in any numbers. It is ovoid, 10-15 nm long by 7-10 nm wide. The cytoplasm is a homogeneous grey tinged with yellow (precursor hemoglobin). The central nucleus is ovoid containing densely staining material. The nuclear membrane is not visible. The cytoplasmic:nuclear ratio is 2.0-2.5:1. Reticulocyte: This cell without supravital staining resembles a mature erythrocyte. The supravital staining accents the cytoplasmic reticulum. The cytoplasm stains an even yellow. The cell is ovoid to elongate as is the central nucleus. The cytoplasmic:nuclear ratio is 2.9-3.3:1. Erythrocyte: This is the fully matured cell in the series. It is elongate, measuring 12-16 nm long by 7-9 nm wide, with a homogenous yellow cytoplasm. The central nucleus is elongate and compact. The degree of compactness increases with the age of the cell. The cytoplasmic:nuclear ratio is 3.5-4.0:1. Lymphocytic Series Lymphoblast: This cell differentiates from a small lymphoid hemoblast and is similar to the proerythroblast. The main distinguishing feature is its evenly staining cytoplasm. Prolymphocyte: This is a spheroid cell measuring 7-15 nm in diameter and contains a clear azure-staining cytoplasm without azurephil granulation. The eccentric nucleus is spherical but may have a slight indentation on the side not touching the cell membrane. The nuclear membrane, chromatin material and nucleoli are quite prominent. The cell is not usually phagocytic but is quite active mitotically. Its appearance in the peripheral vascular system is of pathological significance. The cytoplasmic:nuclear ratio is 1.25-2.0:1. Lymphocyte: This is the functional, mature cell in the series. It occurs as two major "races" or types: large and small; although there have been reports of a "medium" designation. The differences are not only in cytoplasmic:nuclear ratios but also in function. The small lymphocyte is a spheroid cell 7-10 nm in diameter. The eccentric nucleus is circular and nearly fills the cell leaving only a small portion of clear, blue cytoplasm showing. The nuclear material is quite compact and homogeneous. The cytoplasmic:nuclear ratio is slightly more than 1.0:1. Current thinking about the function of this cell is that it actively produces globulins. The large lymphocyte is an ovoid cell 10-15 nm in greatest dimension. The eccentric nucleus is often irregular the typical "kidney-bean" nucleus. The cytoplasm stains a clear, pale blue and may contain "dusty" particles. It is a phagocytically active cell. The cytoplasmic:nuclear ratio is 1.5-1.7:1. Both types of lymphocytes are considered to be pluripotential in that they may differentiate into other cell types; e.g., the small lymphocyte differentiates into the plasma cell and the large lymphocyte differentiates into the circulating macrophage. Plasma Cell: This is a small, ovoid to pear-shaped cell 7-14 nm in greatest dimension. The markedly eccentric nucleus contains irregular chromatin material. The cytoplasm is termed "muddy" grey to blue-grey. There is often a clear paranuclear space, the "hof." This cell is not a usual inhabitant of the peripheral vascular system but in certain stages of the immune response it is not unusual - between 1-2% of the leukocyte population. Macrophage: This circulating macrophage is of hematogenous origin; i.e., differentiated in the hematopoietic tissues. The "resting" macrophage is of histogenous origin; i.e., differentiating in the loose connective tissues: e.g., the lamina propria of the intestinal tract. The circulating macrophage is a relatively large cell (15-19 nm) with an often irregular, eccentric nucleus. The cytoplasm contains many granules and has a dusty, grey staining characteristic. This cell is the primary phagocytic cell in the peripheral vascular system. It is not uncommon to see these cells in various states of degeneration in smears of circulating blood. Some investigators, particularly the immunologists, designate these cells as monocytes. While the teleost macrophage and the typical mammalian monocyte appear quite similar, their roles in the host response are quite different. The macrophage appears quite early in the inflammatory response; whereas the monocyte appears quite late in the regenerative phase of the inflammatory response and is of favorable prognostic significance. Granulocytic Series Granuloblast: This cell develops from the large lymphoid hemoblast. It is spheroid to ovoid, 10-15 nm in diameter, and has a granular medium to light blue cytoplasm. The cell is mitotically active. The nucleus is central to slightly eccentric. The chromatin material is fairly fine and loosely arranged. Nucleoli are often present. The cytoplasmic:nuclear ratio is 1.52.0:1. Progranulocyte: This cell corresponds to the mammalian promyelocyte; however, the prefix "myelo-" refers to marrow, of which the teleost has none. Thus, the term would be inappropriate for fish hematological designations. The cell is spherical to ovoid with a granular light blue cytoplasm. The nucleus is often eccentric and is indented. The cell is mitotically active. The cytoplasmic:nuclear ratio is 1.5-1.75:1. Granulocyte: This cell corresponds to the mammalian myelocyte. It is spherical to ovoid measuring 10-15 nm in diameter. The pale blue cytoplasm contains many rather prominent dark-staining granules with the greatest aggregation being in the region of the nuclear indentation. The eccentric nucleus is round with an indentation on the central aspect. The chromatin material is quite coarse, loosely arranged, and quite darkly staining. There are nucleoli. The cytoplasmic:nuclear ratio is 1.4-1.6:1. If this cell gives rise to all three types of granulocytes; i.e., the neutrophil, eosinophil and the basophil, is quite open to speculation. In the salmonid the eosinophilic granulocyte is histogenous in origin and the basophilic granulocyte is not known to occur, although mast cells do occur. In the cyprinids all three types of granulocytes occur in the peripheral vascular system. Juvenile neutrophil: This cell corresponds to the mammalian metamyelocyte and occurs in relatively few numbers in the peripheral vascular system. The preceding developmental stages are confined to the hematopoietic centers and their occurrence outside these centers would be of pathological significance. The cell is spherical to ovoid and 10-15 nm in diameter. The cytoplasm is pale and contains a very fine granulation. The nucleus is slightly eccentric and has a marked indentation or cleft on the central aspect. The chromatin material is variable but frequently quite compacted. The cytoplasmic:nuclear ratio approaches 2.0:1. Band neutrophil: This cell corresponds to the similar mammalian counterpart. It is very similar to the juvenile form except that the nuclear cleft has enlarged to the point that the nucleus would be termed elongate and curved. The nuclear material is quite compacted. The cytoplasm does not take up stain well and contains few minute granules. The cytoplasmic:nuclear ratio is 1.3-1.5:1. Segmented neutrophil: This is the fully differentiated cell in the series. The nuclear has become lobed (2-3) with fine strands connecting each lobe. The cytoplasm is agranular and stains very poorly. The cell membrane is often quite indistinct. Phagocytic activity for this cell has not been unequivocally reported, although it does appear in some numbers in foci of active inflammation. Marked increases in the peripheral vascular system during the early stages of a systemic bacterial disease have been reported. The cytoplasmic:nuclear ratio is 2.0-4.0:1. D. Summary: Application of hematological examinations to investigating clinical episodes of infectious and noninfectious diseases of fish must be viewed with some degree of caution. There are no "normal" values because of the responsiveness of the blood vascular system to external stimuli. The most appropriate approach is to establish the baseline data for the fish in a specific situation and monitor the population for changes in the hemogram. If good techniques are applied and interpretations are guarded, hematological methods have value in fish health management.