Vet Times The website for the veterinary profession https://www.vettimes.co.uk Blood smear analysis in the emergency veterinary patient Author : Ashley Wemple Categories : RVNs Date : April 1, 2010 Ashley Wemple BS, RVN, explains how one extra drop of blood can be used to provide a fast, easy and reliable diagnostic tool IN all aspects of veterinary medicine, it is safe to say that being armed with information is a good thing. In an emergency situation, the ability to gain information accurately and quickly is of paramount importance. Blood smear analysis is a fast and easy way to obtain reliable information that can be vital to appropriate diagnosis and treatment of emergency patients. Although an in-house complete blood count (CBC) can be useful, some in-house analysers are less reliable than others and not all practices have them. Furthermore, it is possible to obtain some information from blood smear analysis that these analysers do not provide. Whether you are running a general health profile, pre-operative blood work or a minimum database, a single extra drop of blood is all that is required to facilitate the addition of blood smear analysis to that lab work. It is important to remember that blood settles, so smears must be made either at the time of blood sampling or after the anticoagulated sample has been mixed well for several minutes. Equipment required Microscope this does not have to be a top-of-the-range model, but staff must be trained on its proper use, and regular maintenance of the microscope is imperative ( Figure 1 ). Two clean, unused slides for each smear ( Figure 2 ). 1 / 9
Haematocrit/EDTA tube ( Figure 3 ). Romanowski stain stain pots should be emptied, cleaned and refilled weekly ( Figure 4 ). Immersion oil used for higher-magnification lenses only (50 and 100 ). Lenses must be wiped clean after use, with the appropriate lens-cleaning paper. Photos/books a reference source in your laboratory is an absolute must. Slide containers keeping interesting and unusual slides will allow you to discuss results with your colleagues. Maintaining a library of normal slides will give you a good reference source for training ( Figure 5 ). Blood smear preparation Classic method ( Figure 6 ). Wipe glass fragments from the edges of two new, clean slides. Apply a small drop of blood from a haematocrit tube about half an inch from the end of one slide ( Figure 7 ). Place the narrow edge of the other (spreader) slide in front of the blood drop. Pull the spreader slide backwards until the blood drop spreads evenly across the width of the slide. Angle the spreader slide at approximately 30 to 40. Push the spreader slide gently, but firmly, forward to the end of the blood smear slide. Like most aspects of veterinary nursing, producing good blood smears requires practice. If you find the classic method difficult, alternative techniques are available. Regardless of your technique, the goal is to create a single layer (monolayer) of cells on the slide. This will allow for an accurate assessment of cell morphology and number. The smear should have a feathered appearance at the edge, and should not quite reach the end of the slide. The most common mistake when making a blood smear is to apply too large a drop initially; a haematocrit tube allows for application of a smaller (less than an actual drop) amount of blood to the slide ( Figure 8 ). Staining 2 / 9
After allowing the smear to air dry, use a Romanowski stain, such as Rapi-Diff or Diff-Quik, to prepare it for examination. Examination Examination of a blood smear should go from low to high magnification. Begin on the lowest power (10 ) to achieve appropriate coarse focus without damaging the lenses. Use this opportunity to gain a general impression of cellular morphology, and the approximate number of nucleated cells. Check the feathered edge for large platelet clumps as these may contribute to inaccurate diagnosis of thrombocytopaenia ( Figure 9 ). Once satisfied with the examination on low power, increase to the larger magnification lenses. From this point on, use only the fine focus to examine the smear; use of the coarse focus on these lenses will damage the microscope. At 40 magnification, a better idea of individual cell morphology can be achieved. A manual white blood cell differential is possible, as well as determination of platelet and nucleated red blood cell numbers. At magnifications of 50 and 100, immersion oil is used. Place one drop of immersion oil on the slide in the area of examination. The lens should meet with the meniscus of the drop to form a seal. Once focused, these higher magnifications allow for further examination of cell structure, toxic change and identification of cellular inclusions. Terminology After examination of the smear, the next stage is interpretation of results ( Table 1 ). Terms commonly used in blood smear evaluation include: Microcytosis small red blood cells. Macrocytosis large red blood cells. Anisocytosis red blood cells of varying size. Hypochromic light red blood cells. Hyperchromic dark red blood cells. Polychromasia varying red blood cell colour. Polychromatophils immature red blood cells that are larger and darker in colour than normal red 3 / 9
blood cells. Poikilocytosis variation in red blood cell shape including acanthocytes, schistocytes and spherocytes. Acanthocytes red blood cells with rounded projections of variable length. Schistocytes cleaved or fragmented red blood cells. Spherocytes small, round, dense red blood cells with no central pallor. Nucleated red blood cells immature red blood cells the same size as mature red blood cells, but with a dense nucleus. Ghost cells red blood cell membrane. Crenation artefactual change in red blood cells consisting of short, evenly spaced surface projections. Heinz bodies small, refractile inclusions associated with denatured haemoglobin at the periphery or protruding from the red blood cells. Howell-Jolly bodies single, refractile bluish body located within red blood cells. Thrombocytopaenia decreased platelet count. Leukopaenia decreased white blood cell count. Neutropaenia decreased neutrophil count. Neutrophilia increased neutrophil count. Neutrophilic left shift presence of band neutrophils (can be degenerative or regenerative depending on neutrophil count). Toxic change evidence of rapid or abnormal neutrophil production. This is indicative of a severe inflammatory (but not necessarily infectious) response. Döhle bodies large blue to dark-purple cytoplasmic neutrophilic inclusions, which are evidence of toxic change. Red blood cells 4 / 9
Erythrocytes, commonly known as red blood cells, are the oxygencarrying cells within the blood. When these cells are low in number, the patient is anaemic; this can be determined by measuring packed cell volume (PCV) or haematocrit. Once diagnosed, it must then be determined whether the anaemia is regenerative or non-regenerative. Indications of regenerative anaemia: anisocytosis; polychromasia (polychromatophils); and nucleated red blood cells. ( Figures 10-12 ). Indications of a non-regenerative anaemia: microcytosis; hypochromasia; and lack of polychromatophils and nucleated red blood cells in the face of extreme anaemia. Regeneration can be more accurately quantified by performing a reticulocyte count using a supravital stain, such as new methylene blue, but this is often not an option in an emergency situation. Care must be taken not to over-interpret artefacts. Also remember that feline red blood cells are smaller than canine ones, so keeping normal smears of both species for reference is recommended. Platelets An example of platelets can be seen in Figure 13. Platelet counts from blood analysers can be inaccurate, especially in cats. A manual assessment is, therefore, highly recommended in general, and vital in any patient with a suspected coagulopathy. The feathered edge of the smear will often harbour some platelet clumps, which is one reason for mechanical misdiagnosis of thrombocytopaenia. A normal platelet count equates to approximately eight to 15 platelets per highpowered (100 ) field. White blood cells When evaluating leukocytes, or white blood cells, it is important to remember that you are assessing both quantitative and qualitative properties. With experience, and frequent reading of 5 / 9
normal smears, the ability to make a subjective assessment of increased or decreased white blood cell numbers will develop. A manual differential adds even more information and can confirm any machine values obtained. Leukopenia/neutropenia White blood cells may appear low on the smear due to: reduced production for example, immunosuppressive therapy, parvovirus infection; sequestration severe infection or inflammation; and laboratory error sample not fresh or mixed properly. Neutrophils Neutrophils ( Figure 14 ) comprise approximately 50-70 per cent of white blood cells, forming a first defence against infection and inflammation. They show signs of toxic change when the body is forced to produce and distribute them rapidly. Toxic change Characteristics of toxic change ( Figure 15 ) include: basophilia (bluish granulation) of the cytoplasm; foamy appearance of the cytoplasm; swelling of the nucleus; and Döhle bodies ( Figure 16 ). Band neutrophils Characteristics of band neutrophils ( Figure 17 ) include: immature neutrophils; an unsegmented, horseshoe-shaped nucleus; released before fully mature due to increased demand; 6 / 9
their presence in blood smear is termed left shift ; can be regenerative or degenerative: regenerative = neutrophilia in which mature neutrophils are present in greater numbers than band neutrophils; degenerative = a low to normal total neutrophil number with greater than 10 per cent band neutrophils, generally a poor prognostic indicator. Eosinophils Eosinophils ( Figure 13 ) have the following characteristics: slightly larger than a neutrophil; contain coarse, dark-staining, cytoplasmic granules; and can be elevated in the blood for a variety of reasons, including allergic reactions, parasitism and paraneoplastic conditions. Basophils Characteristics of basophils include: larger than a neutrophil; long, ribbon-like nucleus; contain lavender to dark-purple granules against a bluish cytoplasm; and basophilia tends to echo eosinophilia, commonly seen with allergic reactions or parasitism. Monocytes Monocytes ( Figure 14 ) have the following characteristics: appear larger than a neutrophil; variable-shaped nucleus; Abundant cytoplasm with ground glass appearance and occasional pink granules; and 7 / 9
can be associated with chronic inflammation. Lymphocytes Lymphocytes ( Figures 13 and 18 ) exhibit the following characteristics: variable in size (be careful not to mistake small ones for nucleated RBCs and vice versa); densely staining round/oval nucleus; minimal cytoplasm (appears to extend only part-way around the nucleus); and reactive lymphocytes are large with increased amounts of basophilic cytoplasm. Guidelines Guidelines for cases in which blood smear evaluation may be useful are listed below. However, it is recommended that until you are comfortable reading blood smears, you should evaluate slides from both healthy and sick patients. It is impossible to recognise what is abnormal without first knowing what is normal. Blood smear evaluation should be performed on all patients: with abnormal results from a haematology analyser; diagnosed as anaemic; suspected of having coagulation disorders; suspected of having inflammatory or infectious conditions; and suspected of having immune-mediated thrombocytopaenia or immune-mediated haemolytic anaemia. It is understandable that we are not expected to be clinical pathologists, but we do have the ability to greatly increase our knowledge in this area. With training and regular practice, a smear can be made, stained and briefly read while waiting for other test results. The information gained far outweighs the cost of time and materials. Making blood smear evaluation a matter of routine is an easy way to increase the standard of care in your practice. You can read more on this topic and take part in forums and discussions by visiting the website www.vetecc.com 8 / 9
Powered by TCPDF (www.tcpdf.org) References Boag A (2009). Interpretation of blood smears in ECC, Vets Now Emergency and Critical Care Congress 2009. Boag A (2009). An Introduction to Emergency and Critical Care, Vets Now induction course. Day M, Mackin A and Littlewood J (2000). BSAVA Manual of Canine and Feline Haematology and Transfusion Medicine, BSAVA, Gloucester. Doxey D L and Nathan M B F (1989). Manual of Laboratory Techniques, Wiley. IDEXX Laboratories (2000). Haematology Practical Notes. Jasani S (2009). The Emergency Database, vetecc.com (clinical resources). Villiers E and Blackwood L (2005). BSAVA Manual of Canine and Feline Clinical Pathology, BSAVA, Gloucester. 9 / 9