CLP 410 Haematology NOTES 2022 PDF

UNIVERSITY OF PRETORIA FACULTY OF VETERINARY SCIENCE HAEMATOLOGY CLINICAL PATHOLOGY 410 Department of Companion Animal Clinical Studies Copyright reserved 2022 1 INTRODUCTION Haematology is the study of the haematopoietic s...

UNIVERSITY OF PRETORIA FACULTY OF VETERINARY SCIENCE HAEMATOLOGY CLINICAL PATHOLOGY 410 Department of Companion Animal Clinical Studies Copyright reserved 2022 1 INTRODUCTION Haematology is the study of the haematopoietic system. It includes the study of the erythron, leukon as well as haemostasis. Blood consists of plasma, red blood cells (erythrocytes), white blood cells (leukocytes) and platelets in the vasculature of the body. The haematopoietic system is a very large “organ”, distributed throughout the body and it includes the bone marrow, the lymphoid organs and in certain instances the liver. It functions as a transport system; it is part of the host defence system and it plays a vital role in body homeostasis. Therefore, it often reflects processes taking place elsewhere in the body. For this reason, the Complete Blood Count (CBC) is the single laboratory test requested most frequently, even if primary involvement of the haematopoietic system is not suspected. The Erythron is the red cell mass in the body. This includes circulating red cells, erythroid precursors and stem cells in the bone marrow. The main function of the erythron is oxygen transport. The Leukon is the total number of white blood cells in the body. The main function of the white blood cells is to act as the defence mechanism of the body. Haemostasis is the interaction between blood vessels, platelets and clotting factors in the blood to form and dissolve clots when necessary. Clotting is the process forming insoluble fibrin and fibrinolysis is the breakdown of the insoluble fibrin clot. PHYSIOLOGY AND DEVELOPMENT OF THE RED BLOOD CELLS All blood cells take origin from a “pluripotent” stem cell (these cells look fairly inconspicuous, resembling large lymphocytes). These stem cells respond to a number of “priming” compounds (poetins and interleukins and other cytokines) as well as the “local” environment, to “specialise” and become dedicated toward the production of a generation of cells of a specific cell line. Note that the last nucleated form, called a “meta-rubricyte” (see below) does not replicate/divide. At the metarubricyte stage the haemoglobin (Hgb) content has reached about 75 to 80% of that found in the reticulocyte and mature cell. The other feature that is important to note is the progressive reduction in cell size with each division. Terminology used in identifying the different stages of erythroid development. There is a fairly standardised nomenclature in use for describing cells derived from the bone marrow. This is illustrated in the figure below. Fortunately, there is also a very “simplified” system in which all red cell precursors (with a nucleus) are called normoblasts and this terminology is widely used in clinical discussions when it is not essential to specify individual maturation stages. If one looks at the right-hand columns (figure below), the names are broken up to emphasise what these names actually stand for. 2 (Illustration: Vander’s Human Physiology. Tenth Edition, 2004) Erythrocyte maturation series Probably one of the most important concepts to realise here, and often forgotten in case discussions, is that each of these stages takes time. All in all it takes some 3 days, from the time of the first stimulus for increased production (such as a sudden loss of erythrocytes) to give effect to a wave of young erythrocytes (in the form of reticulocytes, principally, and meta- rubricytes) to enter circulation. If the loss/lack of erythrocytes is very severe and/or prolonged, then polychromatophilic rubricytes, and even basophilic rubricytes, are released. This has an implication in assessing the degree of marrow response (see later in these notes). 3 The figure on the right illustrates a “nest” of normoblasts in the bone marrow showing the different stages of maturation from the rubriblast, at the centre, to the prorubricyte below it and the various stages of rubricytes surrounding the blast. Erythrocyte membrane + The erythrocyte membrane has the following structural/chemical features that are important in understanding the pathophysiology of diseases affecting the erythrocyte: A bilayer phospholipid membrane. o The lipids in this membrane are in dynamic equilibrium with those in the plasma (and exchange readily if the equilibrium is disturbed). This means that if the plasma lipid profile alters, the membrane profile will mimic this. Membrane fluidity and flexibility is, to a large extent, determined by the lipid profile. o Interspersed in this membrane are cholesterol molecules. o Normally, some 40% of these are esterified, the proportion affecting membrane fluidity. o Some of the lipids bear glucose polymers on the external surface (see sialic acid, below) Trans-membrane proteins that may be receptors, ion channels or integral (structural as in band 3). 4  o Many of these trans-membrane proteins have glucose chains attached (on the outside), loosely referred to as sialic acid. These represent receptors (viral, chemical) and blood group determinants. o The integral proteins are attached, as anchoring posts, to a network of contractile proteins by a protein called ankyrin (2.1 in the figure). The sub-membrane contractile proteins, actin and spectrin, are calcium-activated, like their muscle counterparts, actin and myosin. o The intracellular concentration of calcium is 4-orders of magnitude lower than plasma (this is achieved by Na-Ca-ATPase membrane pumps). o The calcium-activated contractile proteins served a purpose during the meta- rubricyte/reticulocyte stage to allow the cells to leave the marrow by pseudopodal movement. The erythrocyte membrane of the dog (with the exception of the Japanese Akita breed) and cat contains essentially NO sodium-potassium-ATPase, the classical membrane pump responsible for maintaining a low intracellular sodium and high potassium. Consequently, dog and cat erythrocytes are low potassium cells that tend to rely on membrane expansion and contraction to help in maintaining osmotic integrity. Metabolism 5 The erythrocyte of most species, including the dog and cat, uses a substantial amount of glucose. It is often assumed that this is used in the maintenance of Na-K-ATP-ase [via the Embden-Meyerhof (EM)-pathway, using, amongst other enzymes, pyruvate kinase] to maintain osmotic integrity. To some extent this is true, as the Na-Ca-ATP-ase pumps have to be supplied with ATP, but a significant proportion of the glucose is NOT channelled to the EM pathway, but to the Pentose Phosphate-shunt (PP-S) pathway instead. This pathway (PP-S) generates, via Glucose-6-Phosphate Dehydrogenase (G-6-PDH), the compound NADPH, and this potent reducing agent plays a vital role in reversing the effects of oxidant damage to the intracellular and membrane organic molecules. It does so by reducing oxidised Glutathione that is used as a “sacrificial lamb”, becoming oxidised more easily than the other organic molecules i.e. haemoglobin. Without this essential mechanism the erythrocyte and its contents (including the haemoglobin) would not survive very long. There is, it must be added, a small anti-oxidant contribution from the EM-pathway’s production of NADH that is used by Met-haemoglobin Reductase (NADH-MR) to reverse the oxidation of the iron-containing pocket in the haemoglobin molecule, keeping the iron in the active “ferric” (Fe2+) state. In this context it is important to note that the erythrocyte has a fairly long life expectancy. In the dog this is approximately 110 days; cat 70 days; horse 150 days; and cattle 150 days. During this period, because there is no nucleus to code for protein synthesis, all enzymes, structural proteins, and haemoglobin (as well as lipids in the membrane) that are in the erythrocyte just after the reticulocyte stage, have to survive for that period of time WITHOUT renewal in the face of a highly oxidative environment. Obviously, this is where the demand for a highly effective anti-oxidant system comes from and explains the utilization of the PP-S pathway as a major route of glycolysis. Senescence of the erythrocyte and its removal from circulation at the end of its 3 to 5 month lifespan is brought on by the ravages of age, which are principally expressed through the inability to keep reversing or repairing oxidant damage. The oxidation causes cross-linking of the sub-membrane proteins (Actin and Spectrin), rendering a more rigid cell that cannot escape the flexibility demands of the splenic sinusoidal fenestrations. One might argue that the cat should then be able to tolerate “old” cells better because it has a non-fenestrating spleen, and should therefore have cells that outlive those of other species. Alas, the cat has developed this splenic anomaly in order to cope with a unique instability of its haemoglobin (4 times as many cross-linkable SH groups (numbering 8) as in the dog) causing it to have a constant Heinz Body (see below) challenge, which probably actually accounts for the shorter red cell lifespan in cats. At the end of its lifespan, the erythrocyte is removed by splenic macrophages as it struggles to pass through the fenestrations quickly enough to escape their attention. There is also evidence (in man) that the membrane lipids and/or sialic acid groups become altered by oxidation or other time-dependent processes and this then makes the cell appear “foreign” to these splenic macrophages. It is important to note that this normal (turn-over) removal process is very efficient at conservation of the intracellular constituents such as iron and the amino acids (AA) derived from the haemoglobin proteins. The AA’s are recycled into the body AA pool. The iron, released from haem, is initially in the ferric (oxidised) form due to the PP-S- pathway dependent phagolysosome “digestion”. For this iron to cross the splenic macrophage membrane, it needs to be returned to the ferrous (Fe3+), reduced form and then, once externalized, oxidized again to the ferric state in order to be “picked-up” by transferrin. The tetrapyrole ring (the haem without iron) is converted into protoporphyrin, decarboxylated to 6 biliverdin and subsequently into bilirubin (unconjugated). This compound is very poorly water- soluble and is carried from the spleen to the liver on serum proteins (principally albumin). Some of the senescent erythrocytes may become too fragile to withstand the osmotic challenge of existing in plasma or become disrupted by mechanical means (e.g. heart valve turbulence), having lost their flexibility. The haemoglobin released from these cells is “mopped” up by plasma Haptoglobin (Hpt) and the Hgb-Hpt complex is taken up from the plasma by the Kupffer cells to be further degraded by the hepatocytes. Note that this process does NOT require bilirubin to be transported in the plasma although splenic macrophages may also pick up the complex. Manufacture of haemoglobin Probably the single most important ingredient is the iron required for the formation of Hgb. Extremely little of this, under normal circumstances, is derived from the diet as the recirculation of iron from “old” cells is normally very efficient. However, some iron is “added” and this is diet derived. The important issue here is that this iron must be readily assimilable as either haem iron from meat or non-bound iron (tannates and phosphates tend to bind iron and render it unavailable, and in alkaline medium iron tends to precipitate out as insoluble hydroxide). Intestinal mucous is thought to attach the free iron and present it as membrane integrins, which make it available to the intracellular shuttle/transport protein. This protein, mobilferrin, requires to be synthesized anew each time it is used rendering the transport system “saturable or exhaustible”. If excess iron is taken up, it is stored as ferritin to render it harmless (free iron is a potent oxidant). Plasma transferrin collects the iron from the basement membrane side of the enterocytes and, with the help of ceruloplasmin (a copper metallo-protein), which renders the iron into the ferric form, accepts the iron and transports it to the marrow. In the marrow, the iron, derived principally from splenic conservation and some from the diet, but all transported by transferrin, is presented to the rubricytes and membrane receptors take up the transferrin-iron complex, internalise it by a process similar to pinocytosis and then releases the iron inside an endosome due to a lowering of the pH inside this vesicle. The 7 manufacture of the tetrapyrole porphyrin ring and the introduction of iron into this to form haem are illustrated below. An important point to note is that the very first step in the synthesis of porphyrin requires the presence of adequate Vit B6. The binding of iron to transferrin and the release of iron from transferrin are dependent on Vit B6 and Ceruloplasmin (Cu-metallo-enzyme). The normoblasts have very typically got intensely blue-staining cytoplasm. This is associated with the large number of RNA-containing ribosomes needed to manufacture all the protein that the cell will require for its lifetime (principally haemoglobin). The residual RNA that is left over in the cytoplasm of the reticulocyte after the metarubricyte has “pitted/expelled” its nucleus, stains blue with a number of stains, including the Romanowski stains (see later), and this creates the typical staining reaction of the reticulocyte. This RNA (and hence the colour) is removed by the second day by the enzyme Pyrimidine-5-nucleotidase in the normal course of maturation. If, however, the cell is released early, this will take a little longer. 8 PATHOPHYSIOLOGY OF THE ERYTHROCYTES 1. Production problems Interference with cell proliferation Ablation of pluripotent stem cells occurs in myelofibrosis, a condition that may follow bone marrow injury as in necrosis, vascular pathology, inflammatory states and neoplasia (including pre-neoplastic myeloproliferative disorders, primary neoplasms and secondary, metastatic neoplasms), as well as in drug induced (oestrogens, phenylbutazone, potentiated sulphonamides, albendazole, griseofulvin and cancer chemotherapeutics), viral (CPV, FeLV), bacterial (chronic ehrlichiosis) and immune-induced (autoimmune stem-cell aplasia) and radiation-induced eryhtroid hypo- or aplasia. Anaemia of inflammatory disease (AID): Inflammatory disease affects erythropoiesis by two principal groups of mechanisms, namely cytokine-induced inhibition and iron sequestration. The recycling of iron from senescent cells “stripped” by splenic macrophages is severely reduced due to the strong oxidative state in the RE system which does not allow the iron to be reduced to the ferric form for trans-membrane export to transferrin. Lack of erythropoietin (EPO) production, associated with renal disease and inhibition of EPO production or function by IL-1 (again an inflammatory side-effect) also plays a role. The former is especially prevalent in older dogs. Interference with maturation Lack of Folic acid and/or Vit B12 (pernicious anaemia) is relatively rare in domestic animals, but seen in captive wild animals (zoos and breeding colonies), and produces a pathophysiologically interesting form of anaemia. These “nutrients” are required in the maturation of nuclei but not at the rate at which Hgb is produced. Consequently, the late- stage rubricytes (polychromatophilic) reach the cytoplasmic Hgb concentration which “switches off” replication before they can replicate to form metarubricytes. Consequently, larger “normoblasts” with strange, immature and bizarre nuclei are released into circulation, which become reticulocytes and mature into erythrocytes without ever going through the metarubricyte stage. These cells are, consequently, larger and hence the technical description of “Megaloblastic anaemia”. An inability to produce haemoglobin at the tempo normally required produces an equally interesting form of anaemia, - a sort of “inversion” of the above. In this case, the proliferation rate is normal but the rate of haemoglobin production is slow. This leads to the cells reaching the metarubricyte stage well before the cytoplasmic Hgb reaches the mitosis “switch off” concentration. This allows the metarubricytes to undergo one (or even occasionally two) extra mitoses. As the cell size diminishes with each mitosis, the resulting reticulocytes and red cells are smaller than usual. Furthermore, the cytoplasmic Hgb levels never quite get up to “normal” and consequently the cells are also pale. The “under filling” of the cell also reflects in increased “flexibility” and may lead to targeting (vide infra). 9 Severe hypochromia: Leptocytes on left and various degrees of central pallor, up to leptocytes on the right To identify the possible causes of hypochromia one simply has to consider what goes into the making of Hgb. Haemoglobin is composed of haem and 4 globulin chains (2 alpha and 2 beta in late foetal and mature companion animals). Haem consists of iron and porphyrin. Therefore, this type of anaemia (microcytic, hypochromic) is seen in iron deficiency (uncommon but may be seen in very young, suckling animals), iron sequestration (see AID, above) and iron loss (principally through blood loss as in GIT bleeding). 2. Structure problems Lipids Bearing in mind that plasma lipids are in dynamic equilibrium with membrane lipids, any condition or disease that influences the lipid profile can be expected to have an effect on the erythrocyte membrane appearance. The liver is not only the principal organ involved in lipid metabolism but is also the organ responsible for the synthesis of apolipoprotein molecules (the carrier proteins). Consequently, diseases of the liver are among those that can produce fairly profound membrane alterations. Two of these are targeting (codocytes) and acanthocyte formation. Targeting occurs when the membrane becomes excessive in surface area in relation to the cell content. This leads to a very flexible cell that distorts excessively when passing through the capillary bed where there is a pronounced lamination of flow rate (high friction retardation at the endothelial surface and much less so in the centre of the capillary lumen). This tends to force the erythrocytes into a “thimble” or “helmet” or “bullet/rocket” shape while in the capillary. Upon emerging, the cell tries to flatten out but often leaves the rocket nose cone in the middle, filled with Hgb. A useful rule-of-thumb for the interpretation of marked targeting is as follows: “Is the hypochromia marked? If not, think liver, otherwise (i.e. if hypochromic), think Hgb. Codocyte (target cell) formation 10 The liver is responsible for the esterification of cholesterol, so when there is significant loss of functional hepatic mass, the proportion of esterified cholesterol changes. As most of the membrane cholesterol is normally in the esterified form, this has a profound effect on the fluidity of the membrane, in the form of polyp-like (or club-shaped) projections of the erythrocyte membrane, known as acanthocytosis. Loss of apolipoproteins, as is seen in protein-losing nephropathies, is also going to influence the lipid profile. This is reported to result in a different membrane abnormality, known as burr cell (ecchynocyte) formation. Probably the best description of these cells would be “cookie cutter” cells. Spiculated erythrocytes (red blood cells with membrane projections) can be divided into two groups, i.e. ecchinocytes and acanthocytes. Ecchinocytes o Type I – crenated erythrocytes (in vitro artifact) o Type II & III – Burr cells (“cookie cutter cells”) Acanthocytes Acanthocytes Ecchinocytes (Burr cells) Transmembrane proteins and sialic acid residues – immune reactions against the erythrocyte Untoward development of immune response to these compounds leads to Immune Mediated Haemolytic Anaemia (IMHA). IMHA is caused by antibody-mediated attack directed against antigenic determinants in/on the erythrocyte membrane. The antibodies involved are usually IgM and more rarely IgG (IgA has also been identified in rare cases) and the complement system often plays a significant role. The antibodies may be directed against auto-antigens on the erythrocyte (autoimmune haemolytic anaemia (AIHA)), or it may be alloantibodies directed against transfused erythrocytes or the antibodies transferred from the mother reacting against the foetal erythrocytes (Neonatal Isoerythrolysis). The antibodies may also be directed against exogenous agents, especially drugs that have an antigen in common with the erythrocytes or that attach to the erythrocyte membrane (hapten). Certain infectious agents may also elaborate antigenic compounds that attach to the membrane (and act as haptens). It is believed that babesiosis of the various domestic species, equine piroplasmosis (equine theileriosis), as well as feline infectious anaemia cause secondary IMHA in this manner. In very rare instances the antibodies may only be activated at temperatures lower than 37ºC, causing cold-agglutinin disease. There may be underlying medical disorders associated with IMHA, such as autoimmune disease, haematological malignancy, medication, recent blood transfusion or recent infections. Mechanisms of erythrocyte destruction can be due to either erythrophagocytosis (extravascular haemolysis) or intravascular haemolysis. 11 Extravascular haemolysis: Macrophages have receptors for both the Fc component of antibody as well as compliment (C3b), and removal of erythrocytes by macrophages occurs in multiple organs, including the spleen, bone marrow and liver (conceptually, these organs are considered to be “outside” the vasculature, although that is patently not the case in reality). Rarely, monocytes that have phagocytosed erythrocytes may be observed on blood smears (this is a fairly common finding in canine babesiosis). The macrophage receptors (Fc and C3b) facilitate the recognition and attachment of macrophages to erythrocyte membranes that are coated with antibody and/or C3b. Affected erythrocytes are completely or partially phagocytosed. Spherocytes, the hallmark of IMHA, are formed by partial phagocytosis with subsequent resealing of the erythrocyte’s membrane. Because more membrane is removed than cell content, spherocytes appear small (although their volume is normal), and because they are sphere-shaped, lack central pallor and appear to be dense. They have a shortened half-life because they are less deformable as normal biconcave disk-shaped erythrocytes. Because this process (the disposal of large amounts of haemoglobin from the phagocytosed cells) yields a large amount of unconjugated bilirubin that has to be transported from the spleen to the liver (on albumin), it is often associated with bilirubinaemia with clinically significant icterus. Intravascular haemolysis: In severe cases, with high levels of antibody attachment and complement fixation resulting in a transmembrane pore (membrane attack complex), membranes may be severely damaged resulting in extravascular water leaking into the erythrocyte cytoplasm, causing rupture of the cell in circulation (intravascular haemolysis). Erythrocytes are destroyed within the circulation, releasing haemoglobin into the plasma (haemoglobinaemia) where it is “mopped up” by plasma haptoglobin to be transported to the liver for removal. Severe haemolysis will quickly saturate haptoglobin’s capacity leading to haemoglobin being filtered by the kidneys (haemoglobinuria). Ghost erythrocytes can occasionally be observed on blood smears. Usually the antibody is attached to erythrocyte membrane glycoproteins. If IgM is involved, agglutination of erythrocytes can usually be observed on the blood smear, and may be grossly evident in the blood tube. A typical set of haemolysed and icteric serum samples during peak babesiosis season at the OVAH 12 High percentage spherocytosis in a case of Low percentage spherocytosis in IMHA (small, dark RBC) microangiopathy (arrows) The erythrocyte and oxidative challenge The erythrocyte and its contents are under constant challenge from oxidation.There is constant intracellular production of oxidative agents. These oxidise haem as well as the intracellular proteins (structural, enzymes and haemoglobin). Under normal circumstances glutathion (regenerated via Glutathion peroxidase and Glutathion reductase using NADPH, from the PP-P as a proton source) and methaemoglobin reductase (using protons from both NADH and NADPH) provide sufficient reductive capacity. If the oxidative challenge is increased, as in nitrite, onion, procaine and acetaminophen poisoning, these systems may be unable to cope and this is expressed in the form of Heinz bodies (oxidised Hb) and/or Methaemoglobin (Met-Hb). The degree to which one or the other of these oxidation products predominates is oddly unpredictable. Less obvious and seldom considered is the fact that other substrates will also become oxidised – sub-membrane proteins (which would lead to cellular ridgidity and spherocyte formation) and membrane lipids (which could lead to lipid peroxidation and membrane disruption). Even in the face of normal oxidant challenge, certain species develop Heinz bodies and exhibit exquisitive sensitivity to relatively innocuous oxidants owing to “unusual” molecular structure (as in feline Hb – it would appear that several felid species are so afflicted) or relatively low levels of antioxidant intermediates. The Perissodactyla (Horses and Rhinoceri) are also frequently reported to develop Heinz bodies in the face of apparently normal oxidant challenge. 13 Heinz bodies in feline red cells (paracetamol Supravital staining with NMB reveals the toxicity) inclusions The erythrocyte and enzyme deficiency Enzymes critical to glycolysis (Pyruvate kinase in the EMP and Glucose-6-Phosphate Dehydrogenase in the PPP) are recognised in humans and animals as being related to episodes of spontaneous haemolysis and in inducing sensitivity to mildly oxidant drugs such as antimalarials. There is considerable evidence that selenium deficiency, leading to low Glutathion peroxidase activity, is also associated with an increased sensitivity to oxidant challenge. Slow removal of ribosomal residues in reticulocytes In severe anaemia, with very strong regeneration, reticulocytes are released a little earlier than usual. This appears to present pyrimidine-5-nucleotidase with a challenge it cannot meet and allows time/opportunity for ribosomes to aggregate into clusters of polyribosomes, seen as basophilic stippling. Lead poisoning (particularly chronic) inhibits numerous enzymes (such as ALA-synthase and Haem-synthase) leading to acquired porphyrias as well as pyrimidine-5- nucleotidase. This results in basophilic stippling. A “rule of thumb” for differentiating these two different causes is that lead poisoning does not cause severe anaemia whereas the basophillic stippling associated with early reticulocyte release does. 14 METHODS IN HAEMATOLOGY SAMPLE COLLECTION For a large number of applications, blood collected from the patient should not be allowed to clot and should retain the level of metabolites and cellular appearance/size that were present when the sample was taken, for as long as possible. To this end, a variety of anticoagulants/preservatives are used at blood collection, each with its own strengths and weaknesses for different applications. As a general rule anticoagulants: render calcium unavailable or stimulate an anti-clotting factor or inhibit clotting factor function. Anticoagulants and collection tubes used routinely in the Clinical Pathology laboratory: 1. EDTA (purple stopper) Mechanisms of action: forms insoluble Calcium salts. Advantage: i) Excellent preserving of cellular elements (very slow lysis) ii) Recommended for routine haematology Disadvantage: i) Interferes with most chemical assays 2. Heparin (green stopper) Mechanisms of action: antithrombin and antithromboplastin effect. Advantages: i) Least effect on size of erythrocytes ii) Least effect on haemolysis and leukocyte lysis. Disadvantages: i) Unsuitable for staining of smears ii) Expensive iii) Clotting not prevented for more than 8 hours iv) Interferes with some chemical reactions 3. Sodium Citrate (light blue stopper) Mechanisms of action: forms insoluble Calcium salts Advantage: i) Preferred sample for coagulation tests (PT; PTT) Disadvantages: i) Interferes with many chemical tests ii) Prevents clotting only for a few hours 4. Sodium Fluoride (grey stopper) Mechanism of action: forms a weakly dissociated Calcium component Advantages: i) Both an anticoagulant and preservative ii) Excellent for blood glucose determination, as it inhibits glycolysis Disadvantages: i) Haemolysis and leukocyte lysis happen quickly ii) Interferes with urease in urea determination 15 5. Citrate Phosphate Dextrose/Glucose (anti-coagulant in transfusion bags) Citrate mechanism as in 3 above, and in addition, buffers the sample and supplies energy substrate. Advantages: i) Both an anticoagulant and preservative ii) Preserves cellular energy well and therefore ideal for blood transfusions Disadvantage: i) Platelet function not preserved Heparin (green stopper) EDTA (purple stopper) Citrate (light blue stopper) Fluoride (grey stopper) Serum tube – no anticoagulant (red stopper) Serum tube with clot seperator (yellow stopper) Blood collection It is important to keep conditions as uniform as possible when collecting blood in order to be able to compare results from occasion to occasion in a single patient or when comparing a patient’s result with a referral interval. The sample should also be collected in such a way that it is an accurate reflection of the conditions in the patient. Collection sites: Capillary (ear): usually done in dogs and cats to make blood smears. Capillary (tail): used to make blood smears in bovines, not often done in dogs. Venous (cephalic vein): used for sample collection in tubes, especially large breed dogs. Venous (jugular): used for sample collection in tubes in dogs (smaller breeds, puppies), cats, horses and smaller production animals. Venous (tail): used for sample collection in tubes in bovines. Femoral artery: used to collect arterial blood for blood-gas analysis. Factors affecting blood sampling: 1. Patient factors: i) Excitement (catecholamine release) ii) Exercise (flushing of the muscle capillary bed) iii) Tissue contaminants (often seen when blood smears made directly from patient) 2. Method: i) Collection by syringe and then transferring to anticoagulant (clotting common) 16  ii) Gravity feed (needle in vein and allowing blood to drip into tube (clotting common and the blood will not be sterile leading to poor keeping qualities) iii) Collection “set” using evacuated tubes (vacuum may cause vein to collapse – esp. in cats and small dogs). a. Anything that delays contact with anticoagulant will allow platelets to aggregate and even small clots to form. Slow bleeding (often due to vein collapse) in cats is very commonly associated with this problem. b. “Blind” stabbing in an attempt to find the vein will introduce tissue fluids, which have a potent procoagulator effect. Bleeding kit and needles Steps and precautions to be taken when collecting blood for a haemostasis profile: Sodium citrate (blue stopper) is used to collect blood for evaluation of haemostasis and EDTA for platelet count evaluation. The samples must not be contaminated with tissue fluid. To achieve this a atraumatic venipuncture is necessary. This can only be done if the animal is properly restrained and a vein is clearly palpable. It is also advisable to collect blood in the serum tube first before collecting blood in the citrate and EDTA tubes (in this order) in order to remove all the tissue fluid first. The blood and anticoagulant must be in the exact proportions (9 part blood: 1 part citrate) to be able to interpret results meaningfully. The collection tube must therefore be filled. Collecting blood into a syringe without anticoagulant and then transferring the sample to a citrate tube is not recommended, because coagulation may begin during venipuncture and progress far enough to alter haemostasis test results prior to mixing with the citrate. The haemostasis tests must be done as soon as possible after the sample has been collected. If the sample cannot be analysed immediately, it must be centrifuged and the plasma must be removed from the red blood cells. The plasma must be transferred to a plastic or silicone coated glass tube. The plasma must be frozen as fast as possible and then stored at -20ºC or less. When it is sent to the laboratory, it must be kept frozen by packing it in ice or dry ice. A control sample from a normal animal must always accompany the patient sample where species-specific reference ranges are not available. Interpretation: If species- specific reference ranges are not available, haemostasis is seen to be affected if the patient sample coagulation time is >20% longer than that of the control sample. Stains used in haematology Romanowski-type stains: For the routine evaluation of blood smears Romanowski-type stains are used. Romanowski- type stains include Giemsa, Leishman’s, Wright’s and Diff Quick or Cam’s Quick. Diff Quick and Cam’s Quick are preferred, because the staining process takes only about two minutes. Parasites are stained well and stain deposits do not cause problems with these stains. Diff 17 Quick stain consists of three components, i.e. a fixative, a red (eosin-based) stain and a blue (haematoxylin-based) stain. A blood smear is made, air-dried and fixed in the fixative (thin smears fix instantaneously). Thereafter it is stained in the red stain for ± 8-10 seconds and then in the blue stain for 30-40 seconds. The smear is then washed with cold running tap water and dried. DO NOT BLOT DRY!!!!! If the material being stained could be contaminated with bacteria, then it is advisable to stain the smears on a staining rack instead of “dipping” in stain jars. Supravital stains: In certain instances, supravital stains are necessary to visualise specific cellular structures. When supravital stains are used, the cells are not fixed with a fixative. Examples are: New Methylene Blue (NMB) Brilliant Cresyl Blue Uses: i) Reticulocyte counting. ii) Demonstration of Heinz bodies. iii) To see nucleolar detail. iv) To stain certain cytoplasmic granules, for example mast cell granules. Cell Counting Methods: To quantitate cells a haemocytometer (hand method) or an electronic cell-counter can be used. Different methods are used in electronic cell counters. The methods most commonly used are: i) Impedance counting: Blood is diluted in a medium that conducts electricity. A measured volume is passed through a small orifice, between two electrodes. Cells are poor conductors of electricity and cause resistance as they pass between the electrodes. This is registered as a voltage reading, proportional to the cell size. The apparatus can be set to only count cells within a certain size range. ii) Flow cytochemistry: A dye is added to stain certain cells, and they are counted according to colour change. These analysers are used for human haematology and animal cells do not always show the same reaction with the dyes as the human cells (leukocytes from domestic animals do not have the same cytoplasmic enzyme activity as in humans), which can cause inaccuracies. iii) Laser light scattering: Cells are classified and counted according to the way that they reflect, refract and scatter laser light. This is an accurate method, but because cells differ in their light-scattering properties in the different species the instrument has to be pre-set for the specific species. Red Corpuscle Count or Red Blood Cell Count (RBC): This is a measure of the erythron and reports the total number of erythrocytes per unit volume (litre) of blood. The RBC is expressed as n.nn × 1012/L, e.g. 4.55 x 1012/L (SI unit). The old unit was expressed as n.nn × 106/L (L = mm3). To convert the old unit to the SI unit: n.nn × (106/L ×106 = n.nn × 1012/L (The important issue being that the n.nn remains the same). In conversational use (colloquial veterinary use) we talk of: a RBC of: n.nn (e.g. 5.43) OR a RBC of: n.nn million (e.g. 5.43 million) 18 White Blood Cell (WBC) count: The white blood cell count is the total number of leukocytes per unit volume (litre) of blood. The WBC is expressed as n.nn × 109/L (SI Unit), e.g. 7.54 x 109/L. The old unit was expressed as n.nn × 103/L (L = mm3), using the OLD unit system. To convert the old unit to the SI unit: n.nn × (103/L × 106 = n.nn × 109/L (The important issue being that the n.nn remains the same). In conversational use (colloquial veterinary use) we talk of: a WBC of: n.nn (e.g. 7.54) OR a WBC of: n.nn thousand (e.g. 7.54 thousand) The Corrected WBC Most total WBC include all nucleated cells and therefore the normoblasts as well. If there is a high number of normoblasts present, the WBC will be falsely elevated. In order to solve this problem, a corrected WBC is calculated. The number of normoblasts is reported while the differential cell count is done. The total number of normoblasts counted for every 100 white blood cells is reported as a % normoblasts. Corrected WBC = [100 / (Normoblasts + 100)] × original WBC Platelet Count: The platelet count is the total number of platelets per unit volume (litre) of blood. The platelet count is expressed as nnn ×109/L (SI Unit), e.g. 324 ×109/L. The old unit was expressed as nnn ×103/L (L = mm3). To convert the old unit to the SI unit: nnn × (103/L × 106 = nnn ×109/L. (The important issue being that the nnn remains the same). In conversational use (colloquial veterinary use) we talk of: a platelet count of: nnn (e.g. 324) OR a platelet count of: nnn thousand (e.g. 324 thousand) The platelet count can be conducted with a haemocytometer or an electronic cell counter. It can also be estimated on a blood smear. Methods used for platelet counting: i) The haemocytometer method is rarely used, because it is a time consuming method and it has poor precision (with a coefficient of variation of 20-25%). A special diluent (ammonium-oxalate) is also required. The single factor in its favour is that, in experienced hands, the count represents platelets and nothing else – this is not the situation with machine counts. ii) The electronic cell counting methods are far more accurate and have better precision (coefficient of variation ± 5%). However, there are some problems experienced in running animal samples from some species on instruments designed for humans. In cats especially inaccurate counts can be obtained because of excessive clumping of platelets, giving false low counts. The electronic counters making use of the impedance method also experience problems in species where the platelets are relatively large and/or the red cells small i.e. cats, goats, sheep and cattle. As a rule, if the species has small red cells (MCV < 45 fl), then most instruments will confuse small red cells for platelets and large platelets for small red cells. The reason for this is that the identification of a platelet, on these instruments, is based solely on the size of the particle. A sound principle is to 19  examine a blood smear whenever the machine platelet count is very low or very high, to make sure the machine is not getting confused. iii) Electronic cell counter-derived platelet counts, in particular, and haemocytometer counts to a lesser extent, should always be verified by comparison with the platelet numbers seen on a blood smear. There are three approaches to “smear counting” of platelets: a. On a normal blood smear, examined under 1000 x magnification (10 × 100), there should be 8-10 platelets per field. 5-7 per field is indicative of a thrombocytopenia and less than 3-4 per field is consistent with a severe thrombocytopenia. This method is strongly dependent of the thickness of the blood smear and, with some of the older and/or cheaper microscopes, dependent on the size of the field (controlled principally by the quality of the ocular lens). b. The proportion (ratio) of platelets to red cells can also be used in estimating platelet counts. Less than 1 platelet per 20 red cells indicates a thrombocytopenia. However, the red cell count (as reflected by the haematocrit, see below) has a profound effect on this ratio. If the patient is markedly anaemic (say down to half the normal RBC) the above ratio changes to one platelet per ten red cells. c. The number of platelets counted for every white blood cell seen, can also be used. The calculation of the platelet count is then simply derived by multiplying this number by the white cell count (WBC). The following values are used to classify platelet counts: Classification Actual count No Plat per nnn RC No per field Normal: ≥200 ×109/L 1 per 20 RC 8 to 10 Thrombocytopenia: 50.0109/L) which resembles a granulocytic leukaemia, because of the presence of large numbers of immature granulocytic precursors (metamyelocytes and myelocytes) in circulation. It carries a poor prognosis, because there is a massive tissue demand, but the neutrophilia appears to be ineffective. Toxic changes: Morphological abnormalities seen in neutrophils in severe inflammatory disease, especially severe bacterial infections. These changes occur in the bone marrow prior to release and are associated with accelerated neutrophil production and reduced maturation time, as a result of intense stimulation of granulopoiesis. Toxic granulation: Characterised by the presence of purple-grey cytoplasmic granules in the neutrophils. These are the primary granules that retain their staining ability. It indicates severe toxic changes and is usually seen in horses and cats. Döhle bodies: Blue-grey, angular intracytoplasmic inclusions, they are retained aggregates of rough endoplasmic reticulum. Often seen in cats. Cytoplasmic basophilia and vacuolisation: Diffuse blue colour from retained ribosomes. Cytoplasmic vacuolisation (foamy appearance) is a more severe manifestation of toxic change than basophilic cytoplasm. Usually seen in bacteraemias and general infections, but it is not specific for infections. Giant neutrophils, nuclear swelling & doughnut shaped nuclei Lymphocyte reactions: Reactive lymphocytes: Immunologically stimulated lymphocytes with dark blue cytoplasm and their nuclei are larger and less compact. Monocyte reactions: Active monocytes: Monocytes with large vacuolar cytoplasm. Their presence in circulation indicates phagocytic activity. They occur in the diseases caused by blood parasites (e.g. babesiosis, ehrlichiosis) and in immune mediated haemolytic anaemias. 39 HAEMATOLOGICAL RESPONSES AND DISORDERS ANAEMIA Anaemia is an absolute decrease in erythrocyte numbers in the body, reflected in a decrease in Hct, [Hgb] and RBC count. A relative anaemia occurs with an increase in plasma volume (over hydration). Clinical signs associated with anaemia Weak animal, with decreased exercise tolerance. Pale mucous membranes. Water hammer pulse (fast, weak pulse due to tachycardia). Increased respiration rate (tachypnoea). Heart murmur due to change in blood viscosity. Icterus, haemoglobinuria, fever or bleeding - depending on cause of anaemia. Shock if one third of the blood volume is lost in a short time period. When the anaemia develops suddenly, the body does not have time to adjust and a relatively small decrease in red cell mass will result in severe clinical signs. When the anaemia develops over a longer time period, the body has time to adjust and can cope better with the anaemia. One of the most important mechanisms in the adaptation to anaemia in the dog is the increase in 2,3-DPG levels. This causes the haemoglobin dissociation curve to shift to the right and causes oxygen to be released more readily at the tissue level. CLASSIFICATION OF ANAEMIAS The cause of the anaemia should be identified where possible because anaemia per se does not constitute a diagnosis. Anaemia can be classified: a. According to size (MCV) and Hgb concentration (MCHC) of the erythrocyte MCV: normocytic, macrocytic or microcytic MCHC: normochromic or hypochromic b. According to the bone marrow response Regenerative Non-regenerative c. According to major pathophysiologic mechanism Blood loss (haemorrhagic anaemia) Accelerated erythrocyte destruction (haemolytic) Reduced or defective erythropoiesis 40 1. HAEMORRHAGIC ANAEMIAS Causes of blood loss Acute Haemorrhage Chronic Haemorrhage Trauma Parasitism Surgery Ancylostomiasis Gastro-intestinal ulcers Coccidiosis Haemostasis defects Fleas, ticks, lice Thrombocytopenia Hemonchosis DIC Strongylosis Rodenticide toxicosis Gastro-intestinal ulcers Factor X deficiency in pups Haematuria Haemophilia A and B Neoplasia Bracken fern toxicosis Gastro-intestinal neoplasms Sweet clover toxicosis Vascular neoplasms Neoplasia Haemophilia Splenic haemangiosarcoma Thrombocytopenia Vitamin K deficiency Laboratory findings with acute and chronic haemorrhage Acute haemorrhage Hct is normal initially because all blood components (i.e. cells and plasma = whole blood) are lost in similar proportions, but the animal can be in hypovolaemic shock. Splenic contraction can even result in a slight increase in the Hct. The blood volume begins to stabilise 2-3 hours after initial bleeding by the addition of interstitial fluid and this continues for the next 48-72 hours, causing a drop in Hct and plasma protein concentration. Platelets increase during the first few hours. A sustained increase in platelets indicates a continuing blood loss. A neutrophilia is seen ± 3 hours post bleeding. Signs of increased erythrocyte production (e.g. polychromasia, reticulocytosis) are visible 48-72 hours after bleeding and reach a maximum at 7 days. Plasma protein concentration begins to increase within 2-3 days and returns to normal before the haemogram. The haemogram returns to normal within 1-2 weeks after a single haemorrhagic episode. If the reticulocytes stay increased for more than 2-3 weeks, sustained haemorrhage should be suspected. Chronic haemorrhage: Regeneration is less intense. Chronic haemorrhage is usually accompanied by a hypoproteinaemia. Persistent thrombocytosis is present. Iron deficiency anaemia (i.e., microcytosis, hypochromasia) may develop. External vs. Internal haemorrhage External: Components such as Fe and protein are lost. Hypochromic, microcytic, anaemia with hypoproteinaemia can develop. 41 Internal: Re-absorption of red cells and their components take place. Fe and protein can be reused and the anaemia is less severe. 2. HAEMOLYTIC ANAEMIAS Mechanisms of erythrocyte destruction can be due to either erythrophagocytosis (extravascular haemolysis) or intravascular haemolysis. i) Immune-Mediated Haemolytic Anaemia: Immune-Mediated Haemolytic anaemia (IMHA) is one of the most common causes of anaemia in small animals and is also one of the most prevalent immune-mediated diseases. IMHA can be subdivided into two main types: 1. Primary, idiopathic or autoimmune haemolytic anaemia (AIHA) – Autoimmune disorder characterised by immune system dysregulation, antibody production against unaltered red cells, and absence of an underlying cause. 2. Secondary immune-mediated haemolytic anaemia – IMHA can also occur secondary to a wide range of pathological processes. Although complete proof of causation is usually lacking, many different infectious, allergic, inflammatory and neoplastic causes of secondary IMHA have been suspected in domestic animals. Certain drugs may also trigger IMHA. Distinguishing between primary and secondary IMHA is important for treatment to be effective. Secondary IMHA will not respond well to treatment unless the underlying cause is eliminated and may recur. Pathophysiology (also see “pathophysiology of the RBCs”): IMHA is caused by antibody-mediated cytotoxic (type II hypersensitivity) destruction of the erythrocytes in circulation. The antibodies involved are IgM, IgG (IgA may also be implicated) and the complement system. The antibodies may be directed against auto-antigens in the red cells (autoimmune haemolytic anaemia (AIHA)), or it may be alloantibodies directed against transfused red cells or transferred from the mother and attacking the foetal red cells (Neonatal Isoerythrolysis). The antibodies may also be directed against exogenous agents, especially drugs that have an antigen in common with the erythrocytes (secondary IMHA). In very rare instances the antibodies may only be activated at temperatures lower than 37ºC, causing cold-agglutinin disease. RBC destruction due to antibody attachment is triggered by several mechanisms: In severe cases, with high levels of antibody attachment and complement fixation resulting in a transmembrane pore (membrane attack complex), membranes may be severely damaged resulting in extravascular water leaking into the red cell cytoplasm, causing rupture of the cell in circulation (intravascular haemolysis). This is seen more in IgM-mediated IMHA since IgM is better than IgG at complement fixation. Less severe cases, antibody attachment and membrane damage results in accelerated destruction of affected red cells by tissue macrophages within the mononuclear phagocytic system (MPS) (extravascular haemolysis) mostly present in the spleen and liver. Red cell destruction by the MPS is mediated by fragment crystallisable (Fc)- receptors on the macrophage surface, which bind the Fc-component of the antibodies attached to the red cell membranes. With high levels of antibodies, many individual antibodies can each bind to two different RBC, resulting in clumping of the RBCs (agglutination). Cases with significant 42  agglutination will have increased extravascular haemolysis, because clumping facilitates RBC removal by the MPS. Typically, IMHA is caused by antibodies directed against circulating mature erythrocytes, with the bone marrow mounting a healthy regenerative response. In some cases, antibodies may also be directed against red cell precursors in the bone marrow at any stage in their development, resulting in a haemolytic anaemia with an inappropriately poor regenerative response. By contrast, if antibodies are directed against membrane components that are present only on red cell precursors in the bone marrow and not on mature red cells, non- regenerative anaemia will develop without peripheral haemolysis. Pure red cell aplasia, in which all stages of red cell precursors in the bone marrow are noticeably reduced or absent, is the most extreme form of this process. Patient presentation: Although primary IMHA can occur in dogs of any breed, age or sex it has been reported more commonly in middle-aged dogs (rare in dogs 60×109/L). Usually increased numbers of nucleated erythrocytes (normoblasts) are also present. Reticulocyte counts can sometimes be inappropriately low either because the anaemia is peracute (takes 43 up to 5 days for the bone marrow to mount a strong regenerative response) or because antibodies are directed against red cell precursors. Moderate to marked leukocytosis, usually as a result of a pronounced neutrophilia with a left shift, is a common haematologic finding (66-98% of cases). This is most likely in response to both the non-specific marrow stimulation and the inflammatory process associated with the haemolysis. Platelet counts are usually normal unless the patient also suffers from immune-mediated thrombocytopenia (IMT). Evan’s syndrome (concurrent IMHA and IMT) may affect up to 25-70% of dogs affected with IMHA. Careful examination of the blood smear may yield important information regarding the cause of the IMHA, such as blood-borne parasites and abnormal erythrocyte morphology (i.e. spherocytes). High numbers of spherocytes strongly suggest a diagnosis of IMHA. Spherocytosis may not be present because spherocytes may not accumulate in blood if their rate of formation is less than the rate of their removal. Because of the smaller erythrocyte size in species other than the dog, spherocytes may be more difficult to identify. Blood smear examination may show microscopic evidence of red cell clumping (autoagglutination). Sometimes the agglutination can form large rafts of cells that, on close inspection of the vacutainer tube, are visible to the naked eye as multiple red speckles. However, similar speckles can be created by rouleaux formation. Therefore, to differentiate rouleaux from true autoagglutination an in-saline agglutination (ISA) test must be performed. A positive test result is highly suggestive of IMHA and also indicates that the condition is acute and severe; however, a negative test does not exclude the possibility of IMHA since many patients may have non-agglutinating antibodies. Automated haematology analysers sometimes register a clump of agglutinated red cells as a single cell, resulting in a artifactually high MCV and/or lower calculated Hct if the clumped cells are not recognised as red cells. Since the haemoglobin within all red cells is still measured by the analyser, this leads to an erroneously high estimation of MCHC. This is also the case where intravascular haemolysis is present and free haemoglobin is present in the plasma. When agglutination is suspected to be the cause of a lower than expected Hct, packed cell volume (PCV), which is not affected by cell clumping, should be monitored by microhaematocrit tube centrifugation rather than an automated analyser. In an attempt to support a diagnosis of IMHA specific immunological tests, such as the Coomb’s test (also known as the direct antiglobulin tests (DAT)), can be used which detects antibodies and/or complement bound to RBC membranes. If agglutination and large numbers of spherocytes are present, a Coomb’s test is not necessary. A standard Coomb’s test typically uses a mix of species-specific antiserum containing antibodies directed against IgG, IgM and complement, and is performed on washed erythrocytes at body temperature. Results are reported as the highest dilution of antiserum at which autoagglutination is observed. Modifications of the test that may increase its diagnostic value include running the test at different temperatures (i.e. 4°C). Although, strictly speaking, a positive Coomb’s does support a diagnosis of IMHA, both false positive and false negative results do occur relatively commonly. Factors that have been implicated in false negative Coomb’s results include low level of membrane-bound immunoglobulin or complement, low binding affinity or high dissociation constant of membrane-bound antibody (e.g. improper antisera to antibody ratio, improper washing or dilution of cell preparations). A diagnosis of IMHA should therefore always be based on clinical judgement as well. Flow cytometric detection of anti-RBC antibodies is more sensitive and specific for diagnosis of canine IMHA; however, not yet widely available. Measurement of serum antinuclear antibody (ANA) is only indicated where IMHA is suspected to be a component of systemic lupus erythematosus (SLE), therefore indicated in patients displaying involvement of more than one body system, such as IMT, 44 glomerulonephritis, polyarthritis etc. ANA is not indicated (and is usually negative) in those patients suspected of having uncomplicated IMHA. Because IMHA is usually secondary, confirmation of IMHA is not the end of the diagnostic trial. Primary or AIHA can only be diagnosed with absolute certainty once potential hidden underlying causes have been excluded after thorough investigation. A list of conditions that are reported to predispose to IMHA appear below in a table. Standard screening tests for underlying diseases should include a complete blood count (with careful examination of the blood smear), serum biochemistry, urinalysis, thoracic and abdominal radiographs and testing for infectious diseases such as viruses in cats (specifically FeLV) and blood-borne parasites in dogs. Further tests, particularly in elderly animals where neoplasia is suspected, include abdominal ultrasonography, lymph node aspiration cytology. Bone marrow analysis (aspiration cytology and/or core biopsy histopathology) is indicated in all patients with suspected non-regenerative forms of IMHA to diagnose pure red cell aplasia. Bone marrow examination may also reveal macrophages phagocytosing RBCs or RBC precursors. Techniques, such as immunofluorescent or immunoperoxidase staining of bone marrow samples may confirm the presence of antibodies directed against RBC precursors. As IMHA closely resembles South African canine babesiosis (B. rossi infection), it is useful to note that virtually all cases of babesiosis are markedly to severely thrombocytopenic whereas most cases of IMHA have a normal thrombocyte count. ii) Haemolytic Anaemias due to Erythroparasitic Organisms: In South Africa blood-borne parasites, such as babesiosis, mycoplasmosis, theileriosis and anaplasmosis, are common causes of haemolytic anaemias in in key domestic species. Dogs with B. rossi also often have a secondary IMHA (see previous point). iii) Haemolysis due to Oxidative Injury to Erythrocytes: There are three forms of oxidative injury: oxidation of the heme iron resulting in methaemoglobinaemia denaturation of haemoglobin resulting in Heinz body formation oxidative cross-linking of membrane proteins In dogs the most common causes of Heinz body formation are as a result of ingestion of onions (most common) and garlic. The Hgb molecule of felids has a unique structure and it is very sensitive to oxidative injury. Normal cats have a low (sometimes even high) percentage of Heinz bodies in circulation. A number of chemical agents and drugs can cause Heinz body formation and haemolysis. A common cause of this in South Africa is paracetamol toxicity and ingestion of onions. ivii) Haemolytic Disease Associated with Inherited Metabolic Disorders: Rare red cell enzyme deficiencies, i.e. Pyruvate kinase deficiency and Phosphofructokinase deficiency can occur in some dog breeds. Congenital erythropoietic porphyria is a very rare disorder in cats that may or may not cause haemolysis. v) Neonatal isoerythrolysis Neonatal isoerythrolysis is an important cause of anaemia in newborn foals, mules and kittens and can be life-threatening in severe cases. The prevalence varies among horse breeds but 45 has been reported to be 1% in Thoroughbreds and 2% in Standardbred. In mules (donkey sire/horse dam) the case rate is as high as 10% due to the unique donkey erythrocyte antigen. In cats it is very common when a type B queen is mated with a type A tom (type A or AB kittens), due to the high anti-A titres found in type B cats. Pathogenesis: Neonatal isoerythrolysis is caused by maternal allo-antibodies directed against specific surface blood-group antigens on the affected foal or kitten’s erythrocytes. Cats are born with allo-antibodies, however, in horses allo-antibodies are produced after sensitisation of the dam with incompatible blood group erythrocytes (i.e., leakage of blood across the placenta during pregnancy or at delivery, transfusion of mismatched blood). Allo-antibody production in the mare may persist for years. Neonatal isoerythrolysis is an acquired immunologic haemolytic disease. IgG allo-antibodies do not cross the placenta but are acquired by ingesting colostrum and are absorbed intact into the foal or kitten’s circulation the first hours after birth. If the foal or kitten inherits the erythrocyte antigens (from the sire or tom) that the mare or queen lacks, the foal or kitten is at risk to develop neonatal isoerythrolysis. The maternal allo-antibodies bind to neonatal erythrocytes, causing primarily haemolysis and also haemagglutination with subsequent extra- or intravascular haemolysis. Diagnosis: Neonatal isoerythrolysis should be considered in a newborn foal or kitten (“fading kitten syndrome”) presenting with weakness and icterus in the first few days of its life. Typical clinical and clinicopathologic findings include anaemia (PCV 4.5kg (males) Up to 9 years of age They should be healthy based on history, a clinical examination and PCV determination (35%) prior to donation, as well as annual/bi-annual laboratory evaluation that includes a complete blood count and general biochemistry profile. No pregnant queens – previous pregnancies do not exclude queens as potential donors Free of infectious diseases (e.g. M. haemofelis, B. felis, FeLV, FIV). Serologic or PCR screening for such diseases is recommended. One feline unit = ± 10 mL/kg (40-60 mL); once every 4-6 weeks. Equine donor requirements: Healthy, young gelding weighing at least 500 kg. Vaccinations must be up-to-date Donors should not have had any previous blood transfusion. Mares that have had foals should not be donors. 52 A healthy horse can donate approximately 20% of its total blood volume (40-45 L) every 30 days. When more than 15% is collected, volume replacement with intravenous fluid is recommended. As a general rule horses can donate 1-2 L per 100 kg. CROSS MATCHING Cross matching is done to identify existing incompatibilities; a compatible result does not mean that future incompatibilities cannot develop after a compatible transfusion. The “major” cross match is done to detect recipient antibodies against donor red cells, and the “minor” cross match is done to detect donor antibodies against recipient red blood cells. It is probably not necessary to do it for first transfusions in dogs, but it is definitely necessary in cats if blood typing cannot be performed as well as horses. It is advisable to do cross matching in all cases with IMHA. Donors showing the lowest incompatibility should be chosen. If universal donors are used it is not necessary to do cross matching in cases of multiple transfusions. Method: Samples: EDTA and serum tubes from both recipient and donor(s) Reagents: Physiologic saline Equipment: Incubator, centrifuge, slides, microscope, test tubes, pipettes Procedure: Label 2 test tubes, one “DONOR” and one “RECIPIENT”. Place 6 drops of EDTA blood in each of the respective tubes and fill with saline. Mix and centrifuge for 1 minute at 1000 G (+ 2000 rpm on the average bench centrifuge). Remove supernatant saline by pipette and re-suspend cells in new saline. Repeat twice. Prepare a suspension of red cells in saline (2 drops in 1 ml saline) -appears cherry red when held up to light source. Allow serum tubes to stand for + 1 hour, then centrifuge the serum tubes for 10 minutes and remove serum. Label 3 test tubes, one “MAJOR”, one “MINOR” and a “CONTROL” In the “MAJOR” tube place 2 drops of recipient’s serum and 2 drops of the donor red cell suspension. In the “MINOR” tube place 2 drops of donor’s serum and 2 drops of the recipient’s red cell suspension. In the “CONTROL” tube place 2 drops of recipient’s serum and 2 drops of the recipient’s red cell suspension. Incubate 30 minutes at 37ºC. Centrifuge tubes at 1000G for 3 minutes. Examine supernatant for the presence of haemolysis and note if present. Mix the tube gently by tapping to detect grossly visible agglutination. Transfer a small drop to a glass slide and examine under low power (10x10) of the microscope for microscopic agglutination. Report as incompatible if any haemolysis or agglutination occurred. Note: in horses severe rouleaux may be difficult to distinguish from agglutination. BLOOD COLLECTION AND STORAGE 53 The jugular vein is the preferred site for blood collection. The hair should be clipped and the area must be prepared aseptically. If chemical restrained is necessary in donor dogs, benzodiazepines (Valium) or low dose medetomidine (Domitor) should be used instead of acetylpromazine. In cats a combination of ketamine (10 mg/kg) and midazolam (Dormicum; 0.2 mg/kg) IM. If exsanguination precedes euthanasia, a short acting barbiturate should be used. Venipuncture must be rapid to prevent activation of clotting factors and platelets. Whole blood is collected into commercially available, sterile, airtight plastic bags containing an anticoagulant, that allows for subsequent processing and storage of components without exposure to the environment (closed system). Plastic bags are preferred because platelet and factor XII activation does not readily occur and blood can easily be separated into the different components. An open system has one or more additional sites of entry and possible bacterial contamination. Whole blood, or blood components, collected into an open system are not intended for storage. Open systems are commonly used to collect blood from cats. Blood may be collected into the collection bags aided only by gravity and donor blood pressure, in which case the bag should be continuously and gently rocked by an assistant during collection. Alternatively, light suction, using a custom-made vacuum chamber may also be used. When colleting blood from a donor cat via a syringe and butterfly needle (open system), only gentle suction is applied to avoid collapsing of the vein or causing haemolysis. During collection the well-being of the donor should be continuously monitored i.e. mucous membrane colour, pulse rate and strength, respiratory rate and habitus. Anticoagulants and Preservatives for Canine Blood Products: Anticoagulant Amount Storage time at 0-6oC Heparin 625 U/50 ml blood 2 days 0.9% citrate 1 ml/9 ml blood 2 days Acid citrate dextrose 1 ml/5 ml blood 21 days (ACD) Citrate phosphate 1 ml/7 ml blood 21 days dextrose adenine (CPDA 1) 100 ml/250 ml pRBC 42 days Additive solution Feline whole blood can be stored for 28 days in ACD or CPDA 1 at 0-6oC, if collected with a closed system. 54  Blood Components: Storage, indications, dose and rates: DOSE/ADMINISTRATION COMPONENT STORAGE INDICATIONS RATES Fresh whole