Interpreting of blood work in small ruminants (Proceedings)

A complete blood count can be an important extension of the physical examination in ruminants and may be used to suggest certain disease processes when exam findings are vague and is useful for establishing a prognosis in many cases.

Whole blood in EDTA is the preferred sample, with the vacuum volume of the tube fully replaced by the sample. An unstained, air-dried blood film should be made within 15 minutes of collection and submitted with the remaining sample in the tube for laboratory analysis if the time to analysis is greater than 2 hours. An accurate CBC can be achieved in properly stored samples in tubes up to 24 hours. If platelet counts are of particular importance, those counts should be made within 4-6 hours. If samples are to be shipped to an outside laboratory, they should be wrapped in packing material to protect from breakage and shipped on ice. Automated analyzers must be properly calibrated for the species of interest and stained smears should undergo manual examination.

Normal Complete Blood Count Values for Cattle, Sheep and Goats

Erythron

Red blood cell parameters

A complete evaluation of RBCs should include a PCV and/or hematocrit (HCT), RBC count, hemoglobin (Hgb), MCV, and MCHC, RBC morphology and parasites.

The PCV is determined by centrifugation of a microhematocrit tube and determination of the percentage of RBCs per volume of blood. This is easily performed in-clinic. The HCT is a calculated value based on RBC size and number

The MCV, MCH, and MCHC are characteristics of the RBCs, indicating average cell size, average cell hemoglobin content and average cell hemoglobin concentration, respectively. MCV may be calculated, but most instruments directly measure red cell volume, reporting the mean. A decreased MCV is termed microcytosis; increased MCV is macrocytosis and normal MCV is normocytosis. Note that many microcytes or macrocytes must be present to change the MCV since it is a mean value.

There are also two cellular hemoglobin measurements reported by many automated analyzers, MCHC and MCH. The MCHC is considered the more useful of the two cellular hemoglobin measurements, and is a calculated value. Descriptive terms relative to MCHC are normochromic, hypochromic or hyperchromic for normal, decreased and increased cellular hemoglobin concentrations, respectively. The most common cause of a slightly decreased MCHC is a strongly regenerative anemia, because immature red cells (polychromatophils) contain less than a full complement of hemoglobin. Iron deficiency anemia may also cause a decreased MCHC, and hypochromasia (pale red cells) may be visible on the blood film.

Red blood cell morphology

Normal ruminant RBCs are discoid, non-nucleated and lack the obvious central pallor seen in species with larger red cells (humans and canines). In contrast, camelids have ovoid RBCs. Morphologic abnormalities of red cells detected by evaluation of the blood film can be helpful in determining the cause of an anemia, and in some cases indicate specific disease processes.

     • Acanthocytes – have variable-length membrane projections and may be associated with hepatic disease; have also been recognized in normal young goats, and both anemic and non-anemic young calves.

     • Agglutination – grape-like aggregation, suggests immune-mediated hemolytic anemia (IMHA). Erythroparasites may be an underlying cause of agglutination and immune-mediated red cell destruction. Suspected agglutination should be confirmed with a saline agglutination test, in which a small amount of blood (< 1 drop) is mixed into saline on a slide and then examined microscopically. True agglutination will remain, while rouleaux will disperse.

     • Anisocytosis - refers to variation in RBC size. Mild to moderate anisocytosis is normal in ruminants. Marked anisocytosis is most often due to the presence of large polychromatophilic red cells, characteristic of a regenerative bone marrow response.

     • Basophilic stippling - common with regenerative anemias in ruminants. Basophilic stippling accompanied by many nRBCs with minimal or no anemia is suggestive of lead toxicity.

     • Dacryocytes are tear-drop shaped red cells most often seen in llamas with iron deficiency and have also been associated with phenylhydrazine-induced hemolytic anemia in calves.

     • Echinocytes - have evenly spaced, uniform membrane projections. They may be artifactual, caused by slow drying of the blood film (crenation), may form in vitro as an aging artifact, or be associated with electrolyte imbalances.

     • Heinz bodies - denatured hemoglobin precipitates resulting from oxidative damage to the red cell membrane. They appear as small circular structures within or protruding from the red cell, and may be the same color of the cell or somewhat more pale. Heinz bodies can be difficult to appreciate on routine blood films, but stain dark blue with vital stains such as new methylene blue. Heinz bodies make the red cells more susceptible to both extravascular and intravascular hemolysis. In ruminants, Heinz body formation has been associated with deficiencies of phosphorus, selenium, and copper, ingestion of onions, red maple leaves, or plants from the Brassica family (kale, cabbage), and copper toxicity.

     • Howell-Jolly bodies - small, round, basophilic nuclear remnants that commonly accompany a regenerative anemia or splenic dysfunction. These can be difficult to distinguish from Anaplasma marginale.

     • Hypochromasia - refers to pallor of the RBC due to low hemoglobin content. If many hypochromatic cells are present the MCHC may be slightly decreased. Hypochromasia may be seen with iron or copper deficiency in ruminants.

     • Nucleated RBCs - immature RBCs that may be present in peripheral blood as part of a regenerative response to anemia, or due to bone marrow injury, splenic dysfunction, or lead toxicity, where they appear along with basophilic stippling without a marked anemia.

     • Poikilocytes - red cells of a variety of abnormal shapes. Their significance depends on the specific shape changes present. Poikilocytosis consisting of echinocytes, acanthocytes, and schistocytes has been reported in normal and anemic calves less up to 6 months of age, and poikilocytosis is common in young goats.

     • Polychromasia - pale blue color imparted to red cells by the presence of polyribosomes and mRNA in immature RBCs. Identification of polychromasia on the blood film should be followed by a reticulocyte count to quantitate the regenerative response.

     • Reticulocytes - immature RBCs which contain precipitated organelles when stained with a vital stain such as new methylene blue. Increased numbers of reticulocytes define a regenerative anemia. Reticulocyte count is the more sensitive indicator of a regenerative response than is polychromasia.

     • Rouleaux - stacking of RBCs (like a roll of coins), which may be enhanced by increases in plasma proteins such as immunoglobulins and fibrinogen that often occur with inflammatory processes. Rouleaux will disperse with the saline agglutination tests, and should not be mistaken for agglutination.

     • Schistocytes - RBC fragments that result from intravascular trauma. They have been associated with disseminated intravascular coagulation, in which fibrin strands physically shear the red cells, and conditions such as vasculitis, glomerulonephritis, and iron deficiency. They have been reported in calves with septic shock and drug-induced hemolysis.

     • Spherocytes - have lost a portion of their cell membrane, causing them to become small, dense spheres, but are extremely difficult to appreciate in ruminant species that normally have small red cells without much central pallor. They are typically seen in immune-mediated hemolytic anemia, and have been reported in cattle with anaplasmosis.

Red blood cell parasites

A variety of hemoparasites may be observed on the blood film. The rickettsia Anaplasma spp., bacteria Mycoplasma spp. (formerly Eperythrozoon), and three protozoal parasites, Babesia spp., Theileria spp., and Trypanosoma spp., may affect ruminants. A marginale (cattle) and A ovis (sheep and goats), appear as small, round basophilic inclusions on the periphery of RBCs. A significant hemolytic disease is seen in adult cattle infected by this parasite through tick transmission in the southeastern and western U.S.

Mycoplasma organisms, M wenyoni in cattle, M ovis in sheep and goats, and M haemolamae in camelids, may appear on the surface of RBCs or free in the background on the blood film. They typically cause anemia only in immunocompromised animals. A syndrome in heavily parasitized cattle has been described with signs including hind-limb edema and lymphadenopathy.

Anemia

Anemia is a common abnormality seen in the blood profiles of ruminants. Physical examination findings may include pale mucous membranes, weakness, exercise intolerance and mental depression or aggression. Anemia is subdivided into regenerative or non-regenerative anemia using RBC morphology and the reticulocyte count, limiting the list of differential diagnoses.

In ruminants, the presence of any polychromasia or reticulocytes indicates some degree of regeneration. Other common findings in a regenerative response include basophilic stippling, Howell-Jolly bodies, nRBCs, an increased MCV and decreased MCHC. In the absence of polychromasia and reticulocytosis, the anemia is considered non-regenerative; however the clinician must remember that ruminants typically take 2-4 days to mount a bone marrow response, with the peak response occurring at about 4-7 days.

Causes of regenerative anemia

     • Hemorrhage/ Whole Blood Loss – low PCV with proportionately low TPP

          o Internal – Whole blood into a body cavity

               • Hemoabdomen, hemothorax – Erosion of vessel by abscess or neoplasia, rupture of middle uterine artery

          o External – Whole blood exits the body

               • Abomasal ulcers

               • Parasites – Haemonchus, sucking lice

               • Hemorrhagic enteritis

               • Caudal vena cava syndrome

               • External trauma

          o * Abnormalities in the PCV and TPP are not noted after acute blood loss for 24-48 hours and it may take 2-4 weeks after the insult has ended for the PCV to return to its original level.

     • Hemolysis – low PCV, normal to increased TPP

          o RBC parasites: Anaplasma spp., others

          o Infectious: Clostridium novyi, Leptospira spp.

          o Toxins: Copper, onion, Brassica spp, red maple leaves, water intoxication

Causes of non-regenerative anemia

     • Anemia of Inflammatory (Chronic) Disease – normocytic, normochromic

          o Gastrointestinal disorders

               • Lymphosarcoma

               • Chronic BVDV infection

               • Johne's disease

          o Chronic abscess

          o Hepatic diseases

               • Liver abscesses

          o Endocrine diseases

     • Anemia of chronic renal failure – normocytic, normochromic

     • Chronic Nutrient Deficiencies

          o Iron – microcytic, hypochromic anemia

          o Copper – microcytic, hypochromic anemia

          o Cobalt- normocytic, normochromic

     • Intrinsic bone marrow disease – often have neutropenia, thrombocytopenia

          o Bracken fern toxicity

          o Myelofibrosis

          o Myelophthisis

Polycythemia/erythrocytosis

Polycythemia, or increased PCV, is another frequent finding on the ruminant CBC. The most common is a relative polycythemia, caused by dehydration. This can be confirmed clinically by detecting prolonged skin turgor, tacky mucous membranes, recession of the globe, and an elevated TPP and albumin.

Absolute polycythemias, in which the red cell mass is truly increased, rarely occur in ruminants, but may result from systemic hypoxia seen in animals living at high altitudes, chronic pulmonary disease, or right-to-left cardiovascular shunting. Blood gas analysis and consideration of the animal's geographic origin confirm this etiology.

Leukon

A differential count by cell type is more important than is the total white blood cell count, as increases and decreases in individual cell types may occur simultaneously, leaving the total WBC count unchanged.

Neutrophils

Neutrophils function by migration into damaged tissue within 2 hours of an insult for phagocytosis of foreign material and bacteria. Neutrophils are the dominant WBC in young ruminants, but with age, the lymphocyte becomes the dominant WBC, with a normal neutrophil:lymphocyte ratio of 1:2 in adult animals. Goats may have equal or slightly increased neutrophil numbers when compared to lymphocytes.

Two types of neutrophils exist, mature, segmented ruminant neutrophils and immature, band neutrophils. Bands are frequently released into circulation with acute inflammation. Toxic changes may be noted on manual differential counts and indicate changes to the neutrophil morphology from severe inflammation, usually associated with Gram negative organisms and septic shock. Toxic changes occur in the bone marrow and include Döhle bodies in the cytoplasm, toxic granulation, diffuse cytoplasmic basophilia, bizarre giant forms, and cytoplasmic vacuolation or foamy cytoplasm. It is important to note that cytoplasmic vacuolation may be artifactual from prolonged exposure to EDTA which can be avoided by prompt blood film preparation. Hypersegmentation of neutrophils may occur as an artifact of aged EDTA samples and as a result of corticosteroid treatment.

Neutrophilia is primarily caused by the presence of mild to moderate inflammation or during recovery from more severe inflammatory conditions, such as infectious processes, tissue injury, neoplastic diseases, and non-inflammatory conditions. Bovine Leukocyte Adhesion Deficiency (BLAD) is a significant genetic cause of neutrophilia in Holstein cattle, which, with a monocytosis, results in WBC counts in excess of 40,000 cells/μL. With this condition, leukocytes fail to express adhesion receptors on their surface that are required for movement from the vasculature to tissue sites of inflammation. Affected calves may have recurrent bacterial infections, diarrhea, pneumonia, lymphadenopathy, dermatosis and stunted growth.

Neutropenia is caused by acute, severe inflammatory diseases in cattle including gram negative sepsis, metritis, mastitis, peritonitis, pneumonia, Salmonella infection and many others. It may also be seen with bone marrow injury such as that caused by bracken fern poisoning, often accompanied by non-regenerative anemia and thrombocytopenia.

Lymphocytes

Lymphocytes are predominantly B cells or T cells, functioning in antibody and cell mediated immunity, respectively. Normal and reactive large lymphocytes in the bovine may have atypical features often associated with malignancy in other species, and should be interpreted by experienced pathologists.

Pathologic lymphocytosis is unusual in ruminants, but may be associated with chronic viral infections, chronic pyogenic conditions or autoimmune diseases. Approximately 30% of cattle infected with bovine leukemia virus (BLV) will exhibit a benign persistent lymphocytosis of B cells [34]. This does not indicate the presence of a tumor; however BLV-infected cattle with persistent lymphocytosis are at increased risk for developing a lymphoid malignancy.

Lymphopenia may be seen with stress or corticosteroid administration, acute viral or bacterial infections, endotoxemia, BVD virus infection, and rare immunodeficiencies.

Eosinophils

Eosinophils function in the immune response to parasites, allergens, and other inflammatory processes. Cattle normally have higher numbers of eosinophils than other species, but eosinophilia may result from parasite migration, atypical interstitial pneumonia, acute bovine pulmonary emphysema and milk autoantibody formation in dairy cattle.

Basophils

Basophils are present in very small numbers in normal ruminants, functioning in allergic and inflammatory processes by releasing heparin, histamine and other inflammatory mediators in immediate hypersensitivity reactions. Their numbers may be increased with allergic dermatoses and hypersensitivity reactions.

Monocytes

Monocytes enter the tissues from the circulation to become macrophages, capable of phagocytizing infectious organisms, particulates, and cell debris. Their numbers are quite variable in cattle and are not sensitive indicators disease processes. Increases may accompany chronic inflammation, tissue necrosis, hemolysis, or a stress response. Low monocyte numbers have been associated with endotoxemia and viremia.

Inflammatory leukogram

Inflammatory diseases occur commonly in ruminants and it is important to note the changes that occur in the leukogram with inflammation at various stages of the disease process. The lack of a significant segmented (mature) neutrophil bone marrow storage pool in adult cattle results in an initial neutropenia for 24-48 hours in the face of severe inflammation, decreasing the neutrophil:lymphocyte ratio. Stress associated with the disease process may also cause lymphopenia and eosinopenia. A left shift, signified by the appearance of numerous immature neutrophils (bands or earlier forms) in circulation, typically appears within 24 hours with acute inflammation. A degenerative left shift refers to the situation when immature forms outnumber segmented neutrophils, or to a left shift with concurrent neutropenia. While this is considered a poor prognostic sign if persistent, it is not unusual in ruminants due to their initial neutropenic response to severe inflammation. A regenerative left shift occurs as the bone marrow responds to the inflammation with increased production, resulting in increased mature neutrophil numbers in addition to bands, with mature neutrophils outnumbering immature forms.

The bone marrow is normally able to replenish the bone marrow pool of neutrophils in 4-5 days, returning counts to normal if the inflammation is resolved. A neutrophilia with or without a left shift may appear if the inflammation is ongoing. As inflammation becomes chronic, lasting for a few days to weeks, the WBC and differential counts will often return to normal, making the diagnosis of chronic inflammation difficult in ruminants. In some cases, there may be slight increases in the neutrophil, lymphocyte and monocyte numbers.

Stress leukogram

The stress leukogram results from glucocorticoid administration or release, generally from a non-inflammatory disease, such as a displaced abomasum or indigestion. Typically, there is a normal or increased total WBC count, mature neutrophilia, lymphopenia, eosinopenia, and mild monocytosis. The neutrophil:lymphocyte ratio may be 2-3:1, but there is no left shift.

Physiologic leukocytosis

Physiologic leukocytosis results from epinephrine release during excitation or exercise. An increase in blood pressure and splenic contraction leads to a transient, mild leukocytosis, mature neutrophilia and a lymphocytosis. This phenomenon is not as prevalent in ruminants as in other species.

Platelets

Platelets form the initial hemostatic plug to damaged vasculature and maintain vascular integrity. Petechia and mucosal bleeding tendencies are clinical signs that warrant evaluation of platelets. Thrombocytosis is usually secondary (reactive thrombocytosis), and may occur with exercise, stress, or inflammatory conditions. In ruminants, a false elevation in the platelet count can occur due to the counting of small RBCs as platelets by some automated analyzers.

Thrombocytopenia is defined as a platelet count less than 100,000 cells/μL, with prolonged bleeding occurring when platelets number less than 40,000 cells/μL, and spontaneous bleeding at less than 10,000 cells/μL. Extensive hemorrhage may result in mild thrombocytopenia due to platelet loss in whole blood. Specific causes of thrombocytopenia in ruminants include septicemia, endotoxemia, vasculitis, bracken fern and trichloroethylene toxicities, Salmonella infection, mastitis, metritis, neoplasia, disseminated intravascular coagulation, bovine viral diarrhea virus infection and bluetongue virus infection in sheep.

Plasma proteins

Plasma of cattle should appear grossly clear and colorless, but may have a yellow cast if animals are fed green feeds. Pathologic yellow plasma can occur with icterus, suggesting hemolysis (intravascular or extravascular) or liver disease. A red discoloration indicates hemolysis, either in vitro or in vivo (intravascular), which must be differentiated. This may be done by repeat sampling with strict attention to sample handling.

A TPP level can be achieved in house by use of a handheld refractometer. In order to interpret alterations in the TPP, one should consider the differential values of albumin and globulins. This will require additional testing than what is typically included on a CBC. If TPP is elevated, it must be determined if albumin or globulins are elevated, or both (panhyperproteinemia). Conversely, decreases in TPP may be due to low albumin, low globulins or both (panhypoproteinemia). The normal albumin:globulin (A:G) ratio is 0.84-0.94 in cattle.

     • Panhyperproteinemia

          o Dehydration

     • Panhypoproteinemia

          o Nematode parasitism, Johne's disease, salmonellosis

          o Hemorrhage

     • Albumin

          o Hyperalbuminemia

               • Dehydration

          o Hypoalbuminemia

               • Inadequate production: Severe, chronic liver disease, poor intake

               • Loss: Renal, GI disease, hemorrhage, exudation

     • Globulins

          o Hyperglobulinemia

               • Chronic inflammatory disease: traumatic reticuloperitonitis, liver abscess, chronic pneumonia

               • Hepatic disease

          o Hypoglobulinemia

     • Not common alone in mature ruminants

     • Neonates with failure of passive transfer

Fibrinogen is an acute phase protein, readily analyzed by most laboratories. Fibrinogen increases over a period of two days after initiation of inflammation of bacterial, viral or chemical origin, or trauma. Dehydration may cause a relative hyperfibrinogenemia in addition to a relative hyperproteinemia. To correct an increased fibrinogen for hydration status a TPP:Fibrinogen ratio should be considered. If the ratio is <10:1, there is an absolute increase in fibrinogen, indicating inflammation. A potential problem with this analysis is that inflammation may also increase globulins, causing an absolute increase in the TPP, so clinical hydration status should always be considered when interpreting plasma proteins. A decreased fibrinogen is unusual, but may be caused by liver disease and disseminated intravascular coagulation.

Suggested readings

Cole DJ, Roussel AJ, Whitney MS. Interpreting a bovine CBC: Collecting and sample and evaluating the erythron. Vet Med 1997; 92(5):460-468.

Cole DJ, Roussel AJ, Whitney MA. Interpreting a bovine CBC: Evaluating the leukon and acute-phase proteins. Vet Med 1997; 92(5):470-478.

Harvey JW. Atlas of veterinary hematology: blood and bone marrow of domestic animals. Philadelphia: WB Saunders; 2001.

Jones ML, Allison RW. Evaluation of the ruminant complete blood cell count. Vet Clin N Am Food Animal Pract 2007;23(3):377-402.

Roussel AJ, Whitney MS, Cole DJ. Interpreting a bovine serum chemistry profile: part I. VetMed 1997;92:553–8.

Roussel AJ, Whitney MS, Cole DJ. Interpreting a bovine serum chemistry profile: part II.Vet Med 1997;92(6):559–66.

Russell KE, Roussel AJ. Evaluation of the ruminant serum chemistry profile. Vet Clin N Am Food Animal Pract 2007;23(3):403-426.