RED BLOOD CELLS IMPORTANCE OF FILM EXAMINATION: PART ONE

Vet Times The website for the veterinary profession https://www.vettimes.co.uk RED BLOOD CELLS IMPORTANCE OF FILM EXAMINATION: PART ONE Author : Mark Richer Categories : Vets Date : March 25, 2013 Mark Richer warns practices to ignore the film comments on a haemotology report at their peril, since the most useful value may be found there Summary This article seeks to demonstrate the appearance of red blood cells (RBCs) on blood smears and to relate their appearance to potential disease processes. Morphological abnormalities of RBCs may help in identifying diseases processes that may be occult or only suspected on physical examination. Examination of RBC morphological changes can be a powerful diagnostic tool that is often underused. Key words red blood cells, haematology, reticulocytes, anaemia, neoplastic, cardiac LOOKING at haematology results many practitioners see normal figures and then ignore the haematologist s comment. In many instances, the film comment gives more information than the numbers, meaning practices that rely on in-house analysers without follow-up film examination may find themselves at a significant disadvantage. In particular, the comments can indicate whether an anaemia is regenerative, or even the presence of inflammatory, neoplastic, cardiac or other diseases. This article aims to help practitioners 1 / 6

interpret the comments to expand their differential diagnoses. The figures can be used to identify RBC morphological changes. Unless otherwise stated, all smears in the figures have been stained with Romanowsky stains. Staining your own blood smears Quick stains vs Giemsa Staining blood smears can be easily achieved with quick stains, such as Diff-Quik. Quick stains are not true Romanowsky stains. For such quick stains to give the best staining, the smear should be immersed in the first stain (the fixative) for two to three minutes, then 20 to 30 seconds in the eosin stain and then in the basophilic stain, regardless of any manufacturer s instructions to the contrary. Failure to fix for two to three minutes will lead to poor staining and potentially misleading interpretation. Better staining is achieved with Giemsa or other Romanowsky stains. Quick stains often poorly demonstrate polychromasia and spherocytes, therefore, despite the longer wait for staining, Giemsa is much more useful. Alternatives are Wright s stain or May- Grunwald-Giemsa. The methods for these stains are shown in Figure 1. Normal findings Red blood cells should be normocytic (normal sized) and normochromic (normal in colour); unless this is not the case it is normally not mentioned. A degree of polychromasia and anisocytosis will be seen in normal blood smears. Abnormal findings Abnormal RBC findings are complex and varied in their interpretation. Such findings are better delineated according to their interpretation. The author will explain the haematologist s comments in terms of their clinical relevance. Normal polychromasia in dogs is reported as 1+ or 2+ (not often greater than 1+) and anisocytosis up to 1+. In cats normal figures are up to 1+ polychromasia (often 0+) and 1+ anisocytosis. Figures greater than this normally indicate a response by the body to the anaemia. Low PCV/RBC count Regenerative versus nonregenerative anaemia When the RBC count is depressed it is most important to determine if the anaemia is regenerative. Anaemia only becomes regenerative after acute blood loss (haemorrhage or haemolysis) two to five days afterwards because of the time necessary for an adequate bone marrow response. 2 / 6

Anaemia may only become apparent 12 (often as long as 24) hours or more after haemorrhage (not haemolysis) because of fluid shifts within the body. In particular, it should be remembered sight hounds normally have a high PCV (less than 50 per cent) and, as a result, in such breeds a PCV less than 45 per cent should suggest anaemia. Where there is low-grade chronic bleeding or haemolysis, the regenerative response may be poor or absent and the PCV may still remain normal. If anaemia is regenerative, there should be significant polychromasia (red-blue cells on film examination) and anisocytosis (variation in cell size due to macrocytes, which may or may not be polychromatic). In either species, polychromasia is the most indicative of regeneration. Dogs with regenerative anaemias commonly have target cells and other leptocytes. In regenerative anaemias, in particular in cats, relatively large spherical blue bodies, called Howell-Jolly bodies, may be seen within RBCs these are nuclear remnants indicating a regenerative response. In markedly regenerative anaemias nucleated RBCs may be seen. Occasionally, basophilic stippling of RBCs may be noted (this is common in cattle with regenerative anaemias, but in small animals is more often seen with lead poisoning). The greater the regenerative response the more profound all these changes will be. The presence of Howell Jolly bodies and/or nucleated RBCs in the absence of anaemia or with a non-regenerative anaemia may indicate bone marrow diseases. Nucleated RBCs and basophilic stippling may be seen with lead poisoning where there is minimal or no anaemia. Nucleated RBCs may also be seen in other non-anaemic diseases, such as cardiac disease, hyperadrenocorticism or inflammatory conditions. Non-regenerative anaemias lack significant polychromasia and may be normocytic, microcytic (for example, iron deficiency) or macrocytic (for example, FeLV) and hypochromic or normochromic. Reticulocytes Reticulocytes are immature anucleate RBCs that contain residual RNA and mitochondria, aggregated into a reticular pattern when stained with supravital stains (such as New Methylene Blue note that Methylene Blue and New Methylene Blue are not the same). Reticulocytes can either be termed aggregate or punctate dogs produce only aggregate reticulocytes, cats produce both aggregate and punctate reticulocytes. The aggregate reticulocyte is larger appearing and macrocytic on a smear, thereby increasing the degree of anisocytosis and the RBC distribution width (known as RDW), and stains blue-ish with Romanowsky stains. Aggregate reticulocytes are referred to as polychromatic RBCs when seen on Romanowsky-stained smears and their presence on blood smears is termed polychromasia. The degree of polychromasia on a blood smear is at least equivalent to the degree of aggregate reticulocytosis, because all polychromatic cells are reticulocytes, but not all reticulocytes are 3 / 6

polychromatic. Where there is profound polychromasia it can usually be assured that the anaemia is regenerative. The reticulocyte count is of particular use in clarifying when the degree of polychromasia is low (differentiating an early regenerative response from a non-regenerative anaemia), but when there is profound polychromasia the reticulocyte counts often adds no additional useful information because values will always be elevated. It should be remembered the degree of polychromasia (or reticulocytosis) must be appropriate to the severity of the anaemia; the more severe the anaemia the greater the degree of polychromasia (or reticulocytosis) that must be present to indicate a significant regenerative response. Performing a reticulocyte count allows the appropriateness of the regenerative response to be clarified clearly. Automated reticulocyte counts Some analysers give automated reticulocyte counts. These analysers will count reticulocytes, RBCs with basophilic stippling, Howell-Jolly bodies, parasites and nucleated RBCs as reticulocytes, thereby overestimating the reticulocyte count. The degree to which individual analysers do this varies with the analyser type. Manual methods are more accurate. Performing the reticulocyte count Reticulocytes can be demonstrated with New Methylene Blue stain. Equal volumes of ethylenediaminetetraacetic acid (EDTA), blood and stain are incubated in a test tube at room temperature for 15 minutes and then a blood smear is made from the solution (do not stain a smear). The percentage of reticulocytes (RP) should be calculated by counting 1,000 RBCs. In dogs and cats only aggregate reticulocytes are counted (but see below). The degree of reticulocytosis can be compared to the severity of the anaemia to determine whether the regenerative response is adequate for the degree of anaemia and so indicative of true regeneration. Several mathematical models are used to assess the appropriateness of the reticulocyte response. Because the RP may overestimate the reticulocyte response, either the absolute reticulocyte count (ARC) is calculated or the corrected reticulocyte percentage (CRP) is calculated: ARC = RP (expressed as a fraction) RBC count CRP = (RP patient PCV) / Average PCV for species* *37 per cent for cats, 45 per cent for dogs (sight hounds 55 per cent). 4 / 6

In dogs the reticulocyte production index (RPI) is sometimes calculated (below). This can be useful in severe anaemias as the ARC and CRP may further overestimate the bone marrow response in such circumstances because the reticulocytes released from the bone marrow are more immature and take time to mature in the peripheral blood. RPI = CRP/RMT The RMT is the reticulocyte maturation time. RMT varies with the severity of the anaemia ( Table 3 ). The RPI is not valid for any species other than dogs and remains controversial for dogs. Interpretation of each of these results is shown in Table 1. Feline punctate reticulocytes Feline punctate reticulocytes result from maturation of aggregate reticulocytes and persist longer in the circulation. The aggregate and punctate reticulocyte responses of cats are shown in Table 2. Although rarely used, the punctuate reticulocyte count in cats can sometimes be useful. Because of the shorter persistence in the circulation of aggregate reticulocytes after acute blood loss, the presence of aggregate reticulocytes reflects recent blood loss. Enumeration of punctuate reticulocytes in cats can help identify blood loss which occurred two to four weeks previously ( Table 2 ). Regenerative anaemia Immune mediated haemolytic anaemia The regenerative response with haemolytic anaemias is usually greater than with haemorrhage as recycled iron is more available for erythropoiesis than is stored iron. Certain RBC morphological abnormalities are associated with immune-mediated haemolytic anaemia (IMHA), none specific. Leptocytes, target cells; spherocytes, ghost cells and eccentrocytes may all be seen. The presence of other poikilocytes may help to differentiate primary and secondary IMHA. These changes all reflect changes in the RBC membrane either directly or indirectly as the result of macrophage damage or interference in the production of the RBC membrane. The presence of RBC parasites should also be noted. Agglutination of RBCs may be seen in the film. This should not be confused with rouleaux formation. The serum may be haemolysed or icteric. If there is marked spherocytosis then this is almost pathognomonic for IMHA. The classical change of spherocytosis must be clearly identified to be relevant. Such cells must be smaller and uniformly darker than normal RBCs, without central pallor on film examination, and, if there is any doubt, the film should be examined further back from the feathered edge to ensure such features are reproduced. In cats, this can be almost impossible. Conclusive identification of spherocytes may be difficult with Diff-Quik due to overall darker staining of RBCs. Where there is any doubt 5 / 6

Powered by TCPDF (www.tcpdf.org) spherocytosis should be rebutted. Spherocytes, target cells, leptocytes, ghost cells and eccentrocytes may be seen in other conditions. In some cases, IMHA may appear poorly or non-regenerative due to destruction of RBC precursors within the bone marrow itself. A positive slide agglutination test (either macroscopic or on inspection of agglutination microscopically best seen at the edge of the film on a wet preparation under the microscope) should be considered conclusive evidence of IMHA (whether primary or secondary) and a Coombs test adds no further information. A Coombs test is only useful when these tests are negative and it should be noted that a Coombs test may be positive in many other conditions particularly where inflammation is present. A Coombs test should be considered as only weak evidence of IMHA due to its unreliability, unless the titre is high (>1: 16, preferably >1: 32 or higher). Oxidative injury In addition, Heinz bodies may be seen in certain circumstances. Small numbers are normal in cats. These are circular protrusions at the edge of the RBC membrane, which stain either the same colour as, or paler than the RBC with Romanowsky stains, or stain blue with New Methylene Blue. Heinz bodies are the result of oxidative damage, commonly from onion poisoning or drug toxicity in dogs and cats. Haemorrhage As mentioned, haemorrhage is not apparent for 12 to 24 hours after acute haemorrhage, due to the need for fluid shifts to alter the PCV. Internal and external haemorrhage cannot be differentiated but this is usually apparent from the history. The presence of various poikilocytes may help narrow the source of the haemorrhage. Haemorrhage, particularly external, which includes into the gastrointestinal and urinary tracts, is usually accompanied by a concomitant fall in plasma proteins. Internal haemorrhage into body cavities leads to rapid recycling of plasma proteins and, therefore, these are not always reduced, particularly if the haemorrhage is not recent. 6 / 6