Applications of the Blood Smear in Emergency Medicine
Josep Pastor, DVM, PhD
Patología Médica, Facultad de Veterinaria, Universidad Autónoma de Barcelona
Evaluation of blood cells morphology in a blood smear renders important information to the clinician. Although, it is an underutilized technique due to the fact that it is time consuming and experience is needed for cell morphology interpretation. Adequate blood film preparation is critical to blood cell evaluation. A blood film is prepared by placing a small drop of blood on the slide. The end of a second slide is then placed against the surface of the first slide at 45 degrees and drawn back into the blood drop. When the blood has spread along most of the width of the spreader slide, it is pushed forward with a steady and even rapid motion. A properly prepared blood film is thin and has an even distribution of cells. After air-drying, the film should be stained with a Romanovsky type stain. Diff-quick gives acceptable results.
The main goal of blood smear evaluation is to obtain diagnostic information; it is also a valuable tool as an internal quality control of blood cell analyzers. The blood film should be inspected under low-power magnification (10x) to note cell distribution and the presence of microfilaria, then a 40x objective can be used to estimate leukocyte number and to scan the feathered edge for platelets clumps or large abnormal cells. Afterwards, the observer should use the oil immersion objective (100x) to estimate platelet numbers and evaluate the morphology of erythrocytes, leukocytes and platelets.
An estimation of the leukocyte number can be done using the 40x objective. This technique is only accurate when looking at a monolayer blood film and the leukocytes have an even distribution with no aggregation. The leukocytes present in 10 fields (40x) at the featheredge are counted. Then, the mean count of the leukocytes obtained in the 10 fields is calculated and multiplied by 1500; the result is an estimate of leukocytes per /µl. A differential leukocyte count can be obtained by using the oil immersion objective. At this high power magnification, the individual leukocyte morphology can be studied and inherited or acquired morphologic abnormalities noted:
Pelger-Huet anomaly. Characterized by the presence of immature shaped nuclei (band or myelocyte form) with a coarse mature chromatin. Normal segmented neutrophils can be seen in heterozygotes animals. The neutrophil's function is normal in all the affected animals.
Birman cat neutrophil granulation anomaly. This is an autosomal recessive inherited disease characterized by the presence of neutrophils with fine eosinophilic or magenta cytoplasmic granules. The neutrophil's function is normal.
Mucopolysaccharidoses (MPS). The MPS are a group of inheritable lysosomal storage disorders caused by a deficiency of lysosomal enzymes needed for the degradation of GAG. Neutrophils and lymphocytes may have metachromatic granules.
Chediak-Higashi syndrome. Described in cats. Neutrophils have large fused 2 µm pink or eosinophilic granules within the cytoplasm. Not all neutrophils are affected and their function is normal. However, these cats usually have a tendency to bleed due to platelet dysfunction.
Neutrophilic nuclear hyposegmentation. This nuclear pattern usually reflects an early release of bands or immature neutrophils. Immature and band granulocytes indicate an inflammatory process. Guarded prognosis must be considered when either the total number of immature cells including bands are greater than that of mature segmented ones or when 10% of the circulating neutrophils are immature in an animal with neutropenia. Both of these conditions are described as a degenerative left shift.
Neutrophilic nuclear hypersegmentation. Neutrophils with 5 or more lobes are considered hypersegmented. This is usually due to aging of neutrophils in vivo or in vitro. In vivo, this can be caused by endogenous or exogenous steroids, which prolong the half-life of circulating neutrophils. Hypersegmentation can also be seen in animals with either marked neutrophilia associated to chronic inflammatory disease or shortly after recovery from inflammation.
Neutrophilic toxic changes. Toxic changes are an outcome of aberrant granulopoiesis and indicate a systemic inflammatory effect on the bone marrow. The most severe changes can be seen in animals with sepsis, endotoxemia or tissue necrosis. The severity of the toxic changes is considered at least as significant as the actual proportion of cells involved. Toxic changes can be (from less to more toxic): Doehle bodies, cytoplasmic basophilia, foamy cytoplasmic vacuolization, toxic granulation, giant neutrophils and ring-shaped nuclei in neutrophils. These changes are less significant in cats.
Reactive lymphocytes. These are immune-stimulated T and B cells. They are seen as lymphocytes with a deep basophilic cytoplasm with a pale, perinuclear Golgi region near the nuclei. Reactive lymphocytes in peripheral blood suggest active immune stimulation.
Mast cells. Peripheral mast cells can be seen in animals with systemic mastocytosis or in dogs with a severe inflammatory condition.
WBC inclusions. Inclusions within the WBC indicate phagocytic material, including other cells, cell debris and infectious organisms. Canine distemper virus inclusion bodies, rickettsial morulae, Hepatozoon canis or Leishmania sp can bee seen within the WBC.
Leukemias. Atypical or bizarre cells or a disordered collection of immature cells on a peripheral blood smear suggest leukemia.
A platelet count can be estimated from a monolayer blood smear, if there are not platelet clumps along the feathered edge. Under 100x oil immersion magnification, platelets on each of 10 fields are counted, averaged, and multiplied by 15.000 in order to get the estimated number per /µl. The presence of platelet clumps suggest that their number is at least adequate for hemostasis (> 50.000 PLT/µl). Platelet anisocytosis suggest a rapid turn over between production and consumption. Enlarged or giant platelets suggest active thrombopoiesis or, less often, abnormal thrombopoiesis associated with myeloproliferative conditions or myelofibrosis. Inclusion bodies of canine distemper or E. platys can also be seen.
Erythrocyte morphology is often an important aid in the diagnosis of anemia, and it can be helpful in the diagnosis of other disorders. Morphologic changes of RBC can be categorized according to cell arrangement in a blood smear, color, size, shape and structures inside or on the surface of the erythrocytes. Figure 1 summarizes the most common morphologic abnormalities that can be diagnostically useful.
Rouleaux formation. It is the spontaneous association of RBC in linear stacks. A slight amount of rouleaux formation is normal in the cat. However, in the dog and the cat rouleaux is enhanced when concentration of plasma proteins (fibrinogen, immunoglobulins) are increased.
Agglutination. It is the irregular spherical clumps of RBCs as result of antibody-related bridging of RBCs. Agglutination is strongly suggestive of immune mediated hemolytic anemia. To confirm that the agglutination is real, a drop of blood is mixed with 1-2 drops of saline solution and if the agglutination persists, it is immune mediated.
Polychromasia. Polychromatic cells are usually large and more blue-colored than mature RBC. They are young erythrocytes (reticulocytes). A high percentage of polychromatic cells suggest a regenerative anemia.
Hypochromia. Hypochromic RBC have an increased central pallor area as a result of decreased hemoglobin concentration due to iron deficiency or porto-systemic shunt.
Microcytes. Microcytes are smaller than normal RBC. They can be observed with iron deficiency anemia or with B12 deficiency. They can also be due to RBC fragmentation.
Macrocytes. Macrocytes are larger than normal RBC. They are often immature RBC, and they are usually polychromatic. Other causes of macrocytosis are the Poodle macrocytosis or the inhered stomatocytosis of the Alaska malamute or miniature Schnauzer.
Nucleated RBC. They are usually metarubricytes. They appear in cases of regenerative anemia, lead poisoning, bone marrow diseases, bone fractures or in animals with a non-functional spleen.
Basophilic stippling. The basophilic granules are aggregates of ribosomes, usually associated with immature RBC. They can be observed in animals with marked regenerative anemias (rare in dogs and cats) or lead poisoning.
Spherocytes. They are small, darkly staining RBC lacking central pallor. They are not easily identified in species other than the dog. Their presence suggests an immunomediated hemolytic anemia.
Echinocytes. They are speculated cells with numerous short, evenly spaced, blunt to sharp surface projections, which are quite uniform in size and shape. They can be an artifactual result of pH change during slow drying. They have also been associated with renal diseases, lymphoma or rattlesnake envenomation in dogs.
Acanthocytes. They are irregular speculated RBC with few, unevenly distributed surface projections of variable length and diameter. They are thought to result from changes in the cholesterol concentration of the RBC membrane. They are observed with liver diseases, altered lipid metabolism, and hemangiosarcoma.
Schistocytes. They are fragmented RBC due to shearing by intravascular trauma. They can be observed with DIC, vascular neoplasms (hemangiosarcoma) or iron deficiency.
Heinz bodies and eccentrocytes. They are produced by the oxidation of hemoglobin. Cats are more susceptible than other species. The oxidative denaturalization of the hemoglobin reduces the RBC deformability, making them more susceptible to intravascular or extravascular hemolysis. The oxidative denaturalization of the Hb can be caused by drugs, chemicals (onions, naphthalene,..) or certain diseases in cats such as diabetes mellitus, lymphoma or hyperthyroidism.
Howell-Jolly bodies. They are nuclear remnants. Increased numbers are associated with regenerative anemia, splenectomy and suppressed splenic function.
Red blood cell parasites. Sometimes it is possible to see parasites such as: Haemobartonella felis, Babesia canis, Babesia gibsoni or Cytouxzoon felis.
Viral inclusions. Distemper inclusions can be seen within the RBC as 1-2µm faint blue or magenta bodies.
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Josep Pastor, DVM, PhD