HEMATOLOGY: BLOOD SMEAR REVIEW IN THE EMERGENCY SETTING

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1 HEMATOLOGY: BLOOD SMEAR REVIEW IN THE EMERGENCY SETTING Deanna Schaefer, DVM, MS, MT(ASCP), Dipl ACVP Assistant Professor, Veterinary Clinical Pathology, University of Tennessee The main objective of this lecture is to review the basics of blood smear evaluation. We will evaluate cases examples from dogs and cats to emphasize several important pieces of information that can be obtained by a quick microscopic review of a blood smear in the emergency setting. Learning Objectives: 1. Describe the pros and cons of performing an abbreviated blood smear review versus a complete blood smear review on critical patients. 2. Explain the technique for an abbreviated low power and high power evaluation of blood smears. 3. Using blood smears from case examples: a. Differentiate between a regenerative and non-regenerative anemia b. Identify specific causes of hemolytic anemia c. Recognize severe thrombocytopenia d. Identify signs of inflammation e. Identify abnormal cells (e.g. big blue cells ) Abbreviations CBC Complete blood count EDTA Ethylene diamine tetraacetic acid (lavender-top tube anticoagulant) IMHA Immune-mediated hemolytic anemia RBC Red blood cell WBC White blood cell Importance of blood smear review Microscopic blood smear review is a critical part of the routine CBC. Even the most sophisticated hematology analyzer will be unable to definitively identify: RBC morphologic abnormalities Neutrophilic left shift or toxic change Neoplastic cells in circulation Infectious agents in circulation Platelet clumps Additionally, analyzers may or may not be able to enumerate reticulocytes to allow classification of anemia as regenerative or non-regenerative. Lastly, blood smear review can serve as a quality control measure to verify results of the automated analyzer (e.g. does the automated WBC differential look accurate?). Blood smear preparation Characteristics of a high-quality blood smear: Prepared from EDTA-anticoagulated blood within 2-3 hours of sample collection Results in a blood smear that is approximately 1 to 1.5 inches long Contains a complete feathered edge and a thin monolayer, as in the following image

2 EDTA is the preferred anticoagulant for blood smears in dogs and cats. Heparin yields suboptimal smears because it affects the staining properties of cells and can allow platelet clumping. The blood smear should be prepared within 2-3 hours of collection of the blood sample in order to avoid artifacts of sample aging such as nuclear swelling in neutrophils that mimics a left-shift, RBC crenation or lysis, platelet clumping, and detachment of blood parasites. The goal is to create a blood smear that ends with a thin monolayer of cells followed by a feathered edge. Most high-quality blood smears are about 1 to 1.5 inches long. In the feathered edge, cell morphology is often distorted, impeding evaluation of cellular detail. However, it is a good area in which to look for things such as platelet clumps and microfilaria, because large objects tend to preferentially locate there. We spend the majority of time evaluating the cells in the monolayer, because this is where cell morphology is optimal. The monolayer is typically located 1-2 low power fields (10x objective) back from the feathered edge. In a non-anemic animal, the RBCs in the monolayer should be almost touching, but not overlapping. Hints for staining blood smears with a quick-dip stain: Make sure the smear is completely dried before it is stained. Dry the slide rapidly without heat (heat fixation may interfere with sample quality). This can be accomplished simply by waving the slide back and forth quickly in the air. If the slide dries too slowly, you can get artifacts that cause the RBCs to have irregularly-shaped, shiny, refractile areas that make it difficult to assess RBC morphology. Ensure the fixative is fresh. Moisture in the fixative is another reason you can see refractile artifacts in the RBCs. Ensure stain reagents are fresh and well-filtered. Precipitates will form and can mimic RBC parasites. Stains will also fatigue with time and alter staining quality. A well-stained blood smear should have RBCs that are pink or orange-pink. Quick blood smear review technique It is essential to perform a complete and thorough blood smear evaluation, because that will give you the maximum amount of information from your sample. However, I recognize that in emergency situations you may not always have more than a couple minutes for the initial blood smear review. If that is the case, an abbreviated blood smear review may be the best you can do. The goal of this quick evaluation is to look for any big, obvious abnormalities that might be present on the blood smear. Subtle abnormalities will likely be missed without a more

3 thorough assessment, so a complete evaluation of the smear should be performed later, as soon as time permits. Below is a summary of one approach you can use for a brief evaluation of a smear. You may alter what areas of the evaluation you emphasize depending on the clinical findings in the patient, e.g. if the main issue is anemia, then focus more time on evaluation of RBCs, and if the main issue is inappropriate bleeding, then focus more time on platelets. With some practice, you may be able to complete this quick slide evaluation in about 2-3 minutes. The blood smear is first reviewed using the 10x objective. 1. Scan the entire feathered edge to look for platelet clumps, microfilaria, or large atypical cells (e.g. mast cells, big blue cells ) 2. Locate the monolayer, and evaluate a few sweeps to look for large atypical cells. If any are noted, evaluate them further using a high power objective (see later discussion). Then review an area of the monolayer using the 100x oil objective. * 3. Review the WBCs to determine if there are: a. Frequent band neutrophils or toxic neutrophils ( qualitative differential ) b. Large atypical cells 4. Review the smear for platelet clumps, focusing on identifying any large clumps at the feathered edge. If you do not have a platelet count from an analyzer, then perform a platelet estimate. This step is most important in patients with inappropriate bleeding. 5. Review the RBCs, particularly if the animal is anemic a. Determine if there are increased numbers of polychromatophils (i.e. reticulocytes). You can use the following chart, but keep in mind that these are only general guidelines. In order to determine definitively if the anemia is regenerative or not, you need an automated reticulocyte count. Number of polychromatophils per 100x field Interpretation DOG <2 Likely non-regenerative anemia 2-4 Unclear (need automated reticulocyte count to accurately classify anemia) >4 Likely regenerative anemia CAT <1 Likely non-regenerative anemia 1-2 Unclear (need automated reticulocyte count to accurately classify anemia) >2 Likely regenerative anemia * You can evaluate the WBCs using a 40x objective instead of 100x if you would like, but RBCs and platelets should always be evaluated using the 100x objective. Note: If you use a 40x objective, you will need to coverslip the slide in order for the focus and resolution to be optimal. This does not need to be a permanently-mounted coverslip, rather you can just put a couple of drops of immersion oil onto the slide, then place the coverslip on top of the oil to create a temporary coverslip. Coverslipping is not necessary if you use the 100x objective.

4 b. If there are increased numbers of polychromatophils/reticulocytes (based either on blood smear review or automated reticulocyte count), then evaluate for evidence of hemolytic disease, e.g. spherocytes, Heinz bodies, eccentrocytes, or RBC parasites. Interpretation of blood smear results The results of your brief blood smear examination can be used to answer the following five questions in your assessment of the critical patient: 1. Is the anemia regenerative? The main method we use to classify anemia in dogs and cats is to determine if it is regenerative or non-regenerative, based on whether the reticulocyte count is above the reference interval or not. Assessment of the degree of polychromasia on the blood smear can be used to estimate reticulocytosis when your hematology analyzer does not provide a reticulocyte count. If the anemia appears regenerative, the anemia is likely due to hemorrhage or hemolysis. If the anemia is non-regenerative, consider disorders that can suppress erythropoiesis in the marrow. 2. If the anemia is regenerative, is there morphologic evidence of hemolytic disease? You will more commonly identify RBC morphologic abnormalities that indicate a specific cause of the anemia in cases of hemolytic diseases, e.g. IMHA, oxidant-induced hemolytic anemia, or RBC parasites. Therefore, you will want to emphasize your evaluation of RBC morphology more if the anemia appears regenerative. Examples of a few of the important morphologic indicators of hemolytic disease are summarized in the following chart: Morphologic abnormality Pathophysiologic cause Spherocytes Immune-mediated RBC destruction Heinz bodies Oxidant injury to hemoglobin Eccentrocytes Oxidant injury to RBC membrane RBC parasites Most common associated diseases Primary IMHA, Secondary IMHA Onion/garlic toxicity, Zinc toxicity, Acetaminophen toxicity, Skunk musk, Endogenous oxidants (inflammation, diabetes mellitus, hyperthyroidism, lymphoma) Hemotropic mycoplasmas, Babesia species, Cytauxzoon You shouldn t need to spend a lot of time looking for spherocytes, Heinz bodies, or eccentrocytes during your blood smear review. In general, if there are clinically relevant numbers of them, then you should be able to find them quickly (i.e. they ll be in every microscope field). For RBC parasites, the amount of time it takes to find them varies depending on the degree of parasitemia. If there is a high-level parasitemia, you should be able to find organisms quickly, often in every microscope field. If you don t find parasites quickly, you can t rule them out (i.e. there may be a low-level parasitemia), but it is often not rewarding to spend a long time

5 searching because even if you do find a few organisms, your confidence level in your diagnosis will usually be pretty low in that case. If you re not absolutely sure of what you re seeing after a quick review, send a blood smear to a lab to be reviewed by a pathologist. 3. Is there evidence of severe thrombocytopenia that may predispose the patient to inappropriate bleeding? The rules of thumb are that when there is an average of <2 platelets/100x field, then the patient will likely have spontaneous clinical bleeding. If there are 3-5 platelet/100x field, then there may be inappropriate bleeding after trauma or venipuncture. CAVEAT: This only applies if there are no platelet clumps identified during slide review. Keep in mind that these are just guidelines. Some patients with platelet counts higher than these guidelines may bleed inappropriately if they have decreased platelet function or if they have problems with secondary hemostasis. 4. Is there evidence of severe inflammatory disease? A quick blood smear review without a complete WBC differential will be unlikely to detect a mild inflammatory leukogram. However, in the critical patient you are likely to be more concerned about detecting severe inflammation that warrants immediate clinical intervention (e.g. septic peritonitis). In these cases, you are pretty likely to detect evidence of an inflammatory leukogram during a quick blood smear review, such as a marked left shift and severe toxic change. 5. Are there any large atypical cells that may indicate hematopoietic neoplasia? The most common atypical cells that you may see during blood smear review are big blue cells, which include lymphoma cells, acute leukemia cells, and non-neoplastic reactive lymphocytes. These cells are generally as big or slightly bigger than a neutrophil, with a high nuclear:cytoplasmic ratio, and a small amount of dark blue cytoplasm. It can be difficult to distinguish between neoplastic and non-neoplastic big blue cells. If you see more than a couple during the low-power scan portion of your brief smear review, it is probably best to send a sample to a clinical pathology laboratory for review by a pathologist. Rarely, you may see circulating mast cells in dogs and cats with mast cell neoplasia. Importantly, some dogs with intense inflammatory disease can also have low numbers of mast cells in blood. So seeing occasional mast cells on a blood smear from a dog does not necessarily mean the dog has a mast cell tumor.

6 CYTOLOGY OF INFECTIOUS DISEASE Deanna Schaefer, DVM, MS, MT(ASCP), Dipl ACVP Assistant Professor, Veterinary Clinical Pathology, University of Tennessee The main objective of this lecture is to review the microscopic appearance of a broad range of infectious agents in cytology smears from dogs and cats. We will focus mainly on those organisms that are more commonly seen in the cytology specimens we examine from animals in this geographic region, but examples of less common agents will also be provided. A review of sample collection and handling will also be included. Learning Objectives: 1. Describe the optimal method to prepare cytology samples from solid tissue or body fluid. 2. Explain the appropriate technique for low power and high power microscopic evaluation of cytology samples. 3. Review the cytologic appearance of the following infectious agents: a. Bacteria, including higher bacteria (Nocardia, Actinomyces, Mycobacterium) b. Blastomyces c. Histoplasma d. Sporothrix e. Cryptococcus Solid tissue cytology: collection and handling: Cytology samples can be collected from solid lesions by several techniques including fine needle techniques and exfoliative (impression smears) cytology. After collecting a sample by a fine needle technique, slides are prepared using a squash prep method (see Figure 1). To prepare a good squash prep slide, quickly remove the syringe from the needle and fill it with about 2-3cc of air. Reattach the air-filled syringe and expel the material from the needle onto a clean glass slide, about 1 cm from the frosted end of the slide with the needle close to the slide and bevel pointed down toward the slide. Place a second clean glass slide on top of the first slide, directly over where you expelled the material onto the slide. Apply minimal pressure to this spreader slide, as the weight of the slide is often sufficient to spread the tissue material. Quickly draw the spreader slide across the length of the bottom slide, without any significant downward pressure. The aspirated material should spread across the slide. Body fluid cytology: collection and handling Smears from body fluids can be made using 1 drop of fluid and the same squash prep technique described above. If the sample is turbid, it is likely of high cellularity, so make direct smears. If the sample is fairly clear, it may be of low cellularity, so it is best to concentrate the cells before making smears. Centrifuge a portion of the sample using the same centrifuge settings as for

7 urine sediments. Pour off the supernatant into a clean collection tube and resuspend the cell pellet in the residual fluid that remains in the centrifuged tube. Squash prep smears can be prepared from this fluid. If you submit the fluid to a lab for cytologic evaluation, submit an aliquot of uncentrifuged fluid, and ALWAYS make smears while the sample is still fresh and submit these as well (indicate if the submitted slides are from unconcentrated or concentrated fluid). This helps us recognize artifacts from sample-aging during transport (e.g. bacterial overgrowth, cell degeneration/lysis, or erythrophagocytosis). Characteristics of a high-quality cytology slide Adequately cellular Intact cells Well-spread monolayer of cells All three characteristics have to be present for optimal diagnostic yield. Samples that are of low cellularity, that consist primarily of disrupted cells, or in which cells are poorly spread will generally be non-diagnostic. Hints for making good quality cytology slides: Don t take the squash in squash prep literally. Use minimal pressure on the spreader slide or the cells may rupture. Make the smear quickly once you expel the sample onto the slide, or it will dry and either rupture or not spread well. Make multiple slides. Usually 3-5 slides is optimal. Aspirate solid areas of cystic masses or masses with soft fluctuant areas (fluid filled areas will often only contain poorly cellular fluid, blood or necrotic debris). Avoid preparation of thick smears, because cells and infectious agents may be impossible to identify if they are not well spread. Use only a small drop of sample on each slide. Hints for submitting slides to a clinical pathology laboratory: Provide a concise, relevant history and lesion description. Adequate labelling: Every slide should contain a clear patient identifier (e.g., full name or medical record number) and the specific site that was sampled. Cross-contamination: Take care to use a slide only for one lesion. If there is any chance a given slide might contain material from a previous aspirate (even if you cannot visualize anything on the unstained slide), dispose of that slide. Send the best/most highly cellular slides to the pathologist. Diagnosis will be delayed if you retain the only diagnostic slide in your clinic. Quick-dip one slide to assess sample quality before sending them out. However, avoid staining all slides a good pathologist can generally work with a well-stained quick-dip slide, but they will give you the best information using the stain they employ in their lab. Do NOT refrigerate slides: Condensation may form on refrigerated slides, which can result in lysis of the unfixed cells and a non-diagnostic sample. If you are uncertain about what samples to submit or how to prepare/store them, call your diagnostic laboratory. We want to help you optimize sample quality and the chance of getting a diagnosis for you and your clients.

8 Cytology low power (10x) evaluation cellularity and smear quality Initial scanning of a smear should be done at low magnification so that the entire smear can be reviewed. At low power you should: Estimate the overall cellularity and quality of the smear. Locate well-stained, well-spread areas with intact cells for closer examination. Identify any large infectious agents or other structures that may be found in low numbers and missed if only a portion of the slide is examined. Cytology high power (40x, 50x, 100x) evaluation cell and organism identification Once you complete the low power smear review and locate a few good areas, move in for a closer look. Evaluate quite a few fields to get an impression of what types of inflammatory cells and/or non-inflammatory cells are present. You are more likely to find infectious agents if there is inflammation on the slide, but certain infectious agents (e.g. Cryptococcus) may not elicit a significant inflammatory response. Inflammation may also be mild or absent if the animal is immunosuppressed. A short description of non-inflammatory cells is included below, but is not the focus of this lecture. Inflammatory cells: o Estimate the relative proportions of different inflammatory cells (neutrophils, macrophages, lymphocytes, eosinophils etc.) o Classify the type of inflammation and look for a cause. The type of inflammation may help you focus on which infectious agents would be most likely, e.g. fungi if granulomatous, or parasites if eosinophilic). However, this is not always the case, and we have seen fungal infections with a significant eosinophilic component to the inflammation, and parasites with predominantly suppurative inflammation. o Note: Often there is collection-related blood contamination on cytology smears and the cytologist must subjectively determine if the number of leukocytes is higher than the expected contribution from the blood in the sample. Non-inflammatory cells: o Epithelial: cohesive o Mesenchymal cells: individualized, spindle to stellate o Discrete/round cells: individualized, generally round o Determine if this is the expected cell type for the area sampled. o Assess for criteria of malignancy. Tips for identification of specific infectious agents Nocardia and Actinomyces We most commonly see these organisms in skin lesions or pyothorax. They are important to distinguish from other bacteria because they usually require prolonged and aggressive treatment. They often cause pyogranulomatous inflammation and are identified by their fairly distinctive morphology, i.e. the bacteria are in long filamentous chains and are beaded (light blue with frequently repeating small pink areas). Certain anaerobic bacteria, e.g. some forms of Fusobacterium, can have a similar morphology, so culture is recommended for confirmation. However, Actinomyces and Nocardia can be difficult to culture, so you should perform both aerobic and anaerobic cultures, and inform the bacteriology laboratory that there is a suspicion of Actinomyces or Nocardia. Even if the organisms do not grow in culture, I would not rule out that they are part of the infection if you see chains of filamentous beaded rods on the cytology.

9 Mycobacterium We diagnose mycobacterial infections rarely in our lab, usually from skin lesions or lymph nodes. The infection is typically associated with granulomatous or pyogranulomatous inflammation. The organisms have an unusual cytologic appearance in that they do not stain with routine Wrights or quick-dip stain, and appear as small, regularly-shaped, negatively-staining (i.e. white) bacteria that are often present within macrophages. In my experience, these bacteria can sometimes have a refractile appearance on routine cytology slides. Mycobacteria stain with acid-fast staining, which can be performed by most clinical pathology or histology labs. Blastomyces This is a fairly common systemic fungal disease in this area. We diagnose the organisms almost exclusively in dogs, and only very rarely in cats. The most common sites are lymph nodes and skin lesions. They can be identified by the alliterative phrase Blasto = Big and Blue with Broad-Based Buds. They can be easy to overlook on cytology smears, though, because they are often not present in very high numbers, and can be so dark blue that they can be confused for debris or poorly-spread cells. Histoplasma and Sporothrix Histoplasma appears to be more common in our area than Sporothrix. In our lab, we have seen Histoplasma most commonly in cats, in a wide range of sites including skin lesions, lung, spleen, liver, and bone marrow. The organisms are commonly (but not always) present in fairly large numbers. They can be extracellular, but are also frequently found within macrophages, or rarely within neutrophils. They are small (about 2-4 microns in diameter), pale blue, and round to oval, with small eccentric cresent-shaped nucleus, and a thin clear halo. Sporothrix most commonly causes skin lesions, and in cats the organisms are often present in fairly large numbers. Some of the organisms look morphologically indistinguishable from Histoplasma, but with Sporothrix you should be able to find at least some that are quite elongated (up to about 9 microns long) and are often described as cigar-shaped. Remember that there is zoonotic potential when handling cats infected with Sporothrix, particularly for immunocompromised people. Cryptococcus This is not extremely common in our area, but we do see occasional cases, predominantly in nasal lesions from cats, but also rarely in CSF or other sites from dogs. The distinguishing feature of Cryptococcus is the very thick clear capsule that is usually present. However, there are poorly-encapsulated types of Cryptococcus which can look very similar to Histoplasma. There may or may not be much associated inflammation, and the type of inflammation can be quite variable ranging from pyogranulomatous, suppurative, or occasionally having a significant eosinophilic component.