CLP 410 Haematology Notes 2023 PDF

UNIVERSITY OF PRETORIA FACULTY OF VETERINARY SCIENCE HAEMATOLOGY CLINICAL PATHOLOGY 410 Department of Companion Animal Clinical Studies Copyright reserved 2023 1 INTRODUCTION  Haematology is the study of the haematopoietic sy...

UNIVERSITY OF PRETORIA FACULTY OF VETERINARY SCIENCE HAEMATOLOGY CLINICAL PATHOLOGY 410 Department of Companion Animal Clinical Studies Copyright reserved 2023 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 RED BLOOD CELLS The primary function of erythrocytes is to transport haemoglobin, which carries oxygen to the tissues. The deformable, permeable membrane that encloses the red cell components is made of lipids, proteins, and carbohydrates. Alterations in the lipid composition (primarily phospholipids and cholesterol) of the membrane may result in abnormal red cell shapes. Membrane proteins from the cytoskeleton of the membrane, and these proteins also play key roles in maintaining both cell shape and integrity. Abnormalities in membrane proteins have also been associated with abnormal red cell shapes. Normal erythrocyte morphology varies among different species. Briefly, the significant differences between species are size, shape, amount of central pallor, tendency to form rouleaux, presence of basophilic stippling in regenerative response to anaemia, and the presence of reticulocytes in response to anaemia Please refer to back to physiology (VPH 200, BVSc II) and the supplementary material on the CLP 410 ClickUP module page (non-examination purposes) for revision. A basic understanding of erythropoiesis and erythrocyte physiology is critical for this section. PATHOPHYSIOLOGY OF RED BLOOD CELLS Pathophysiological process of erythrocytes includes production and structural problems. Please refer the supplementary material on the CLP 410 ClickUP module page (non-examination purposes) for revision. 2 QUANTITATIVE HAEMATOLOGICAL METHODS For sample collection and stains used in haematology please refer to your IVD 300 Clinical Pathology (BVSc III) notes. Cell Counting Methods: To quantitate cells a haemocytometer (manual method) or an electronic cell-counter, i.e. automated haematology analyser can be used. Different methods are used in electronic cell counters. The methods most commonly used are: i) Impedance counting (e.g., Heska CBC-Diff Veterinary Hematology System): 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. Erythrocytes of some species, for example goats, sheep and some horses, are too small to be reliably detected. Also, large platelets (as commonly seen in cats) may be counted as erythrocytes instead of platelets. ii) Optical or laser flow cell cytometers (e.g., ADVIA): A dye is added to stain certain cells, and they are counted according to colour change. With 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 (RCC) 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. 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). 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. The Corrected WBC Most total WBC include all nucleated cells and therefore the nucleated erythroid precurors (old terminology – normoblasts) as well. If there is a high number of nucleated erythrocytes present, the WBC will be falsely elevated. In order to solve this problem, a corrected WBC is calculated. The number of nucleated erythrocytes is reported while the differential cell count is done. The total number of nucleated erythrocytes counted for every 100 white blood cells is reported as a % nucleated erythrocytes. Corrected WBC = [100 / (nucleated erythrocytes + 100)] × original WBC Differential Leukocyte Count 3 The differential leukocyte count is done to determine the relative proportions of each white cell type in circulation. The relative differential leukocyte count is expressed as the percentage of each type of leukocyte present on the blood smear. It is estimated by counting 100-200 leukocytes and classifying each cell. The percentage is then determined. The number of nucleated erythrocytes per 100 leukocytes must also be reported; this is used to calculate the corrected WBC (see previously – White cell count). The absolute differential leukocyte count is determined by multiplying the total WBC (automated result obtained on the haematology analyser) with the percentage of each white cell type (relative differential leukocyte count obtained during blood smear evaluation). Interpretations are done based on the absolute differential counts and not on the relative differential counts. The relative differential count is only a necessary instrument in the calculation of the absolute differential count. Method: In order to do an accurate differential cell count a good quality blood smear must be evaluated. Central venous blood is preferred because certain leukocytes sequestrate in capillary blood, e.g., immature neutrophils, eosinophils and active monocytes giving false high values for these cell types if capillary smears are evaluated. Blood in tubes must be mixed before smears are made and smears must not be too thick in order to identify cells properly. The blood should also not stand in the tubes for too long, otherwise in vitro changes can take place, which will make cell identification very difficult. Various counting patterns can be followed. The leukocyte subtypes are not randomly spread all over the smear and a technique that covers all the distribution areas must be employed. Neutrophils tend to be more on the edges of the smear, while lymphocytes occur mostly in the red cell area. Monocytes are spread all over the smear, but activated monocytes are concentrated in the feathered edge. Eosinophils are also more prevalent in the feathered edge. The “Battlement-method” is used most in human haematology laboratories. Figure 1: In the Onderstepoort Veterinary Academic Hospital we use a modified Battlement-method. Figure 2: At least 100 leukocytes should be counted to calculate the relative differential cell count, but if the leukocyte count is high, 200 cells should be counted. The accuracy of the differential cell count increases when a larger number of cells are counted. Modern electronic cell counters can do an absolute differential count, if this count is accurate for the species analysed, a relative differential count is not necessary. 4 Calculation of Absolute Differential Counts: To calculate the absolute differential count, the relative count (%) is multiplied with the total white cell count (WBC). Example 1: Total WBC: 50.0 ×109/L Relative Count: Neutrophils: 30% Absolute Count: Neutrophils: 15.0 ×109/L Band neuts: 2% Band neuts: 1.0 ×109/L Lymphocytes:60% Lymphocytes:30.0 ×109/L Monocytes: 7% Monocytes: 3.5 ×109/L Eosinophils: 1% Eosinophils: 0.5 ×109/L Basophils: None Basophils: None 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: n.nn × (106/L ×106 = n.nn × 1012/L. The important issue being that the n.nn remains the same. 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. ii) The electronic cell counting methods are far more accurate and have better precision. 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 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, in the red cell area, 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. 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 5  this number by the white cell count (WBC). The following values are used to classify platelet counts: Classification Actual count No Plat per nr RC No per field Normal: ≥200 ×109/L 1 per 20 RC 8 to 10 Thrombocytopenia: 80 g/L for foals and >100 g/L for calves and crias. A level of 400 mg/dL is usually adequate for a newborn which has had a normal birth, is generally in good health, and is kept in a relatively clean environment. Fresh frozen plasma transfusion has been the classic treatment for foals and calves with FPT discovered after gut closure. In horses, care must be taken to check the plasma prior to administration for anti-erythrocyte antibodies. 20 mL/kg of plasma administered to a foal or calve intravenously has been shown to raise IgG levels into the minimum "protective" range of 400-800 mg/dL. Up to 40 ml/kg can be administered to foals with complete FPT. 54 HAEMATOLOGICAL RESPONSES AND DISORDERS POLYCYTHAEMIA Polycythaemia refers to an increase in the concentration of erythrocytes in the blood as evidenced by an increased packed cell volume (PCV; or haematocrit), red-blood-cell count, or haemoglobin concentration. Because the term polycythaemia implies that all blood cells, including leukocytes, are increased in concentration, the term erythrocytosis is sometimes preferred; in domestic animals with true polycythaemia, usually only the erythrocytes are increased in concentration. Polycythaemia may be either relative or absolute.  In absolute polycythaemia the total red cell mass is increased due to increased production of erythrocytes.  In relative polycythaemia the Hct, RBC and Hgb concentrations are increased, but total red cell mass remains normal. Causes of Polycythaemia Relative Polycythaemia: 1. Dehydration: This is due to a decrease in the plasma volume, and this also results in an increase in plasma protein concentration. A physical examination and history will help determine if the animal is dehydrated. Causes of dehydration:  External water loss due to vomiting, diarrhoea, diuresis, water deprivation, sweating, fever.  The daily fluctuation of Hct between 2% and 5% in diseased animals can be ascribed to changes in hydration status. 2. Haemoconcentration: Due to a decrease in plasma volume (excl. dehydration), however, plasma proteins are often normal or decreased. Examples include internal fluid loss via increased vascular permeability as seen with shock and “Red biliary”, and haemorrhagic gastro-enteritis (HGE). 2. Redistribution of red cell: The epinephrine released during fear and excitement causes splenic contraction with release of high Hct blood into circulation. This is commonly seen in horses and cats. Absolute Polycythaemia: 1. Primary Absolute Polycythaemia (i.e., polycythaemia vera) is a rare myeloproliferative condition of the stem cells in the bone marrow. It can be associated with thrombocytosis and leukocytosis. Erythropoietin levels are normal or decreased and PO2 is normal. The diagnosis is normally made by exclusion of other causes of polycythaemia. The Hct remains between 70% and 80% in spite of fluid therapy. 2. Secondary Absolute Polycythaemia:  Appropriate secondary polycythaemia develops due to compensatory erythropoietin (Epo) secretion due to chronic hypoxia. Causes include: i) Living at high altitudes ii) Cardiovascular abnormalities iii) Chronic pulmonary disease 55  iv) Pickwickean-like syndrome (overweight)  Inappropriate secondary polycythaemia: there is no hypoxia (pO2 normal), but Epo levels are increased. This happens in certain renal lesions (usually tumours that induce localised renal hypoxia) such as hydronephrosis, renal cysts, renal carcinoma and tumours such as embryonal nephroblastomas that secrete Epo, and certain endocrinopathies. Diagnostic approach When PCV is increased, one should consider if the patient is excited or dehydrated and then perform a second complete blood count to confirm that the finding is repeatable. If the total protein concentration also is increased, the polycythaemia likely is relative, secondary to dehydration and decreased plasma volume. If relative polycythaemia is excluded, secondary absolute polycythaemia due to hypoxaemia from congenital heart disease or pulmonary disease should be considered. Hypoxaemia is best diagnosed by performing an arterial blood gas analysis to determine the arterial partial pressure of oxygen (PaO2) and oxygen saturation. If the PaO2 is less than 60 mm Hg, then hypoxaemia likely is the cause of polycythaemia. If hypoxaemia is excluded, secondary absolute polycythaemia caused by increased erythropoietin production should be considered. Tumours of the kidney are the most common cause of increased erythropoietin production. Serum erythropoietin concentration usually is increased in animals with hypoxemia or inappropriate erythropoietin production and is normal to decreased in animals with primary polycythaemia. If secondary polycythaemia caused by inappropriate erythropoietin production is excluded, then the likely diagnosis is polycythaemia vera. 56 Schematic: Diagnostic Approach of Polycythaemia Polycythaemia (PCV 60-80%; Hct 0.60-0.80l/l)  Repeat PCV   PCV remains high PCV returns to normal   Relative (splenic contraction)   Fluid therapy   Remains Polycythaemic Returns to normal   Absolute Polycythaemia Relative (hypovolaemia)  Blood gas analysis     Normal PO2 Hypoxia (PO2) Secondary polycythaemia   Appropriate response  Erythropoietin levels      Normal or decreased  Increased   Polycythaemia vera Secondary inappropriate polycythaemia 57 Treatment of Polycythaemia Relative Polycythaemia: If dehydration is the cause, then the dehydration must be corrected with fluid therapy. There is no need to treat the polycythaemia if it is due to the redistribution of red cells. Absolute Polycythaemia:  Polycythaemia vera: The treatment of this condition is repeated phlebotomy to decrease the hyperviscosity of the blood. Injectable iron may need to be given to avoid iron- deficiency anaemia. Chemotherapy to decrease red cell production also may be used; oral hydroxyurea is the most common such treatment. Dose and frequency are variable, depending on the response. Alternately, radioactive phosphorus has been used with success in some cases. Hypercoagulabilty is also treated to prevent thrombotic events. Aspirin is used as an antithrombotic drug.  Appropriate secondary absolute polycythaemia: Treat the underlying cause.  Inappropriate secondary absolute polycythaemia: The same treatment protocol as for Polycythaemia vera can be used, but the underlying cause should also be treated. The prognosis in these cases is guarded. 58 HAEMATOLOGICAL RESPONSES AND DISORDERS NEOPLASIA OF BLOOD CELLS Proliferative disorder is a nonspecific term for a hematopoietic cell neoplasm that is distributed in blood, bone marrow, other tissues, or a combination of these and other sites. Proliferative disorders are classified into lymphoproliferative and myeloproliferative categories. The distinction between the lymphoid and bone marrow stem cell systems is somewhat artificial, but these two classes of proliferative disorders have different biologic behaviour and case management prognosis. Haemopoietic tumours can be divided in 2 main groups:  Lymphoid origin (lymphoid cells and plasma cells)  Bone marrow origin (myeloid cells) Lymphoproliferative Disorders Lymphoproliferative disorders are neoplastic processes with lymphoid cell differentiation. If the neoplasm is confined to solid tissues, it is termed lymphosarcoma or lymphoma. If it involves blood and/or bone marrow, it is termed lymphocytic leukaemia. A specific form with plasma cell differentiation is termed myeloma, which is usually associated with production of a monoclonal immunoglobulin that may be detected in blood. Thus, it is a group of neoplastic conditions of the lymphoid cells:  Lymphomas  Lymphoid leukaemias.  Plasma cell myelomas. Organisation and general terminology for lymphoproliferative disorders Thrall – Veterinary Hematology and Clinical Chemistry Lymphoma is a neoplasm of lymphoid cells that originates in lymphoid organs excluding the bone marrow (old terminology: lymphosarcoma). Leukaemia refers to the presence of neoplastic cells in the circulation. The neoplastic cell type that is present designates more specifically the classification of the leukaemia present. The classification may be determined by a combination of cell population morphologic differentiation features seen on the blood film, surface marker cytometry panels, and immunocytochemistry 59 reactions. Lymphoid leukaemias are relatively common in cats and dogs. Plasma cell myeloma (also known as multiple Myeloma) is a neoplasm of the plasma cells with a single clone of cells, which produces immunoglobulins in excess. It occurs focally in the bone and causes focal osteolytic lesions. The main clinical signs are caused by the hyperglobulinaemia. Myeloproliferative Disorders Myeloproliferative disorders are a collective noun for neoplasia of the non-lymphoid haemopoetic cells originating in the bone marrow. A leukaemic blood picture is common, and solid masses are not typical of these neoplasms. Cats with myeloproliferative conditions are usually FeLV positive. Organisation and general terminology for myeloproliferative disorders. The top box shows general differentiation pathways based on morphologically recognized cell lineages. The bottom box shows historical and commonly applied terminology for the myeloproliferative disorders based on morphologic identity Thrall – Veterinary Hematology and Clinical Chemistry 60 Diagnosis of Haemopoetic Neoplasia Sensitivity of diagnostic Blood WCC Lymphnode Bone marrow methods smear aspirate aspirate LYMPHOPROLIFERATIVE Lymphoma     Lymphoid leukaemia     Plasma cell myeloma +/-    MYELOPROLIFERATIVE Acute myeloid  -   leukaemia Erythraemic myelosis     Myelodisplastic  all cells    syndrome Eosinophilic leukaemia     difficult Chronic myelocytic     leukaemia Polycythaemia Vera     61