1、Veterinary Clinical Pathology,兽医临床病理学,主讲：唐兆新教授 Prof. Zhaoxin Tang,College of Veterinary Medicine, South China Agricultural University, Guangzhou, China , 510642,Preface,Veterinary Clinical Pathology: Veterinary Laboratory Medicine Include: 1 Clinical Hematology 2 Clinical biochemistry 3 Clinical cyt
2、ology 4 Clinical microbiology 5 Clinical parasitology 6 Clinical toxicology,Preface,General Laboratory conceptsVeterinarians have many choices regarding laboratory testing. Important factors include: -Need and usefulness -Practicality -Cost-effectiveness -Accuracy -Turnaround time,Complete Blood Cou
3、nt and Bone Marrow Examination:general comments and selected techniques,Complete blood count Quantitation techniques Blood smear analysis Other determinations Bone marrow examination Bone marrow biopsy and aspirate,Complete blood count (CBC),CBC is a profile of tests used to describe the quantity an
4、d quality of the cellular elements in blood and a few substances in plasma. CBC is a cost-effective screen the detects many abnormalities and disease conditions. Bone marrow examination is used in selected instances to answer questions the more readily available CBC cannot.,Quantitation Techniques,S
5、ample submission Microhemotcrit Hemoglobin concentration Cell counts Absolute nucleated RBC count Automated hematology cell counters,Blood Smear Analysis,Making the smear Stains Evaluating blood smears-platelet morphology-leukocyte morphology-leukocyte estimation-leukocyte differential count-erythro
6、cyte morphology,Bone Marrow Examination,Bone marrow is usually examined to answer certain question that arose from evaluating the CBC. Indications for bone marrow examination include:-nonregenerative anemia-Persistent neutropenia-Persistent thrombocytopenia-Unexplained polycythemia or thrombocytosis
7、-Atypical cells in blood,Erythrocytes,Basic concepts of erythrocyte function,metabolism, production and breakdown Heme synthesis Globin synthesis Iron metabolism,Erythrocyte metabolism,Embden-meyerhof pathway-Glycolysis generates ATP and NADH Pentose phosphate pathway-This pathway produces NADPH Met
8、hemoblobin reductase pathway-Methemoglobin(Fe3+) cannot transport oxygen Rapoport-luebering pathway-2,3 diphosphoglycerate(2,3 DPG),Red blood cellsThe fundamental stimulus for production of red blood cells (erythropoiesis) is erythropoietin(红细胞生成素）, a glycoprotein produced by the kidneys in response
9、 to renal tissue hypoxia. Other hormones, such as corticosteroids, thyroid hormone and androgens, stimulate the production or release of erythropoietin but have no intrinsic erythropoietic activity. The average lifespan of a circulating erythrocyte is 110-120 days in the dog and 68 days in the cat.
10、Aged or damaged red cells are removed primarily by macrophages in the liver, spleen and bone marrow.,Neutrophils The production of neutrophils, eosinophils and basophils is termed granulopoiesis. The neutrophils in the bloodstream either circulate freely (the circulating pool) or adhere to the vascu
11、lar endothelium (the marginal pool). In the dog the marginal pool and the circulating pool are approximately equal in size, whilst in the cat the marginal pool is two to three times larger than the circulating pool. There is a continual exchange of cells between these two pools. The half-life of cir
12、culating neutrophils is only 6-14 hours, after which time they leave the circulation and pass into the tissue pool. The circulating time is shortened during acute infections as neutrophils pass to the site of infection in the tissues. The main function of the neutrophil is the phagocytosis of pyogen
13、ic bacteria.,LymphocytesLymphoid primitive stem cells divide and differentiate into pre-B lymphocytes and pre-T lymphocytes in the bone marrow. Pre-T lymphocytes mature and proliferate into T cells in the thymus. Pre-B cells proliferate in the bone marrow and migrate to peripheral lymphoid organs (s
14、pleen and lymph nodes) where further proliferation takes place. Platelets Platelets are produced from the cytoplasm of megakaryocytes Once in the circulation, platelets survive for 8-12 days. Up to 20-30% of circulating platelets can be sequestered in the spleen; the figure may be a high as 90% if t
15、here is splenomegaly. Old or damaged platelets are removed from the circulation by the spleen, liver and bone marrow.,ROUTINE HAEMATOLOGY,The complete blood count is an integral part of the diagnostic investigation of any systemic disease process. It consists of two components: A quantitative examin
16、ation of the cells, including :packed cell volume (PCV)total red cell count (RBC) total white cell count(WBC) differential white cell countplatelet count mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), total plasma protein concent
17、ration. A qualitative examination of blood smears for changes in cellular morphology.,Table 1 Reference values for red cell indices,ROUTINE HAEMATOLOGY,RED BLOOD CELL INDICES,MCV(fl飞升)= PCV (L/L) 1000/ total red cells ( 1012/L) MCH (pg皮克) = total haemoglobin (g/dl) 10/ total red blood cells ( 1012/L
18、) MCHC (g/dl) = total haemoglobin (g/dl)/PCV (L/L),RBC indices are helpful in the classification of certain anemias.,ROUTINE HAEMATOLOGY,Differential white cell counts The differential white cell count is performed by counting 200 leucocytes in a blood smear. The cells are counted along the long edg
19、e of the smear, using the battlement meander method: four high-power fields are counted in one direction, then four more in a direction at right angles to the first, and so on, following the shape of a battlement. The percentage of each type of cell is determined. This percentage is then multiplied
20、by the total white cell count to obtain an absolute count for each cell type.,ROUTINE HAEMATOLOGY,Plasma protein concentration (Reference range: 60-80 g/1 for the dog and cat) Total plasma protein (TPP) and PCV should be interpreted together. Qualitative examination of a blood smear A blood smear sh
21、ould always be evaluated when automated cell counts are made or when in-practice instrumentation is limited to a centrifuge for PCV Preparation of a blood smear A small drop of blood is placed on one end of a glass slide, using a capillary tube. A spreader slide (made by breaking off the comer of an
22、other slide, after scoring it with a glass cutter or diamond writer) is placed on to the slide holding the blood drop, in front of the drop and at an angle of 20-40.,ROUTINE HAEMATOLOGY,ANAEMIA Anaemia is characterized by an absolute decrease in red cell count, haemoglobin concentration and PCV. Acu
23、te haemorrhage Acute haemorrhage may be due to trauma or surgery, bleeding gastrointestinal ulcers or tumours, rupture of a vascular tumour (e.g. splenic haemangiosarcoma), or a coagulopathy (e.g. warfarin toxicity). Immediately following acute haemorrhage the red cell parameters, including PCV, are
24、 normal because both red cells and plasma have been lost in proportion. Compensatory mechanisms such as splenic contraction may further offset any fall in PCV. The PCV falls when blood volume is replaced by interstitial fluid and so does not indicate the full magnitude of blood loss for at least 24
25、hours after the onset of haemorrhage.,ROUTINE HAEMATOLOGY, Chronic haemorrhage Chronic external blood loss (e.g. chronic gastrointestinal haemorrhage, renal or bladder neoplasia) initially results in a regenerative anaemia but gradually the anaemia becomes non-regenerative as the iron stores become
26、depleted. Young animals become iron-deficient more bone marrow is already very active producing red cells quickly than adults following blood loss, partly because they have low iron stores and partly because their to match their growth rate and so has less capacity to increase its rate of haemopoies
27、is. Haemolytic anaemias Most cases of haemolytic anaemia are immune-mediated. In the dog most cases of immune-mediated is haemolytic anaemia (IHA) are primary (idiopathic) and are termed autoimmune haemolytic anaemia (AIHA). IHA may occur in association with: drugs(e.g. potentiated sulphonamides); l
28、ymphoreticular diseases (e.g. lymphoid leukaemia); systemic lupus erythematosus; or infections (e.g. Babesia, bacterial endocarditis).,ROUTINE HAEMATOLOGY,DISORDERS OF WHITE CELL NUMBER,Neutrophilia Figure 3.20 Causes of neutrophiliaPhysiological response (fear, excitement, exercise)Stress/corticost
29、eroid-inducedAcute inflammatory response: bacterial infection (localized or generalized), immune-mediated disease, necrosis,e.g.pancreatitis, neoplasia, especially with tumor necrosis.Chronic granulocytic leukaemiaNeutrophil dysfunctionParaneoplastic syndromes,Neutropenia The three main causes of ne
30、utropenia are: An overwhelming demand for neutrophils Reduced production of neutrophils in the bone marrow Defective neutrophil maturation in the bone marrow. An overwhelming demand for neutrophils may occur with peracute bacterial infections, especially Gram-negative sepsis and endotoxaemia. Other
31、possible causes include peritonitis, pyometra（子宫蓄脓）, aspiration pneumonia and canine parvovirus infection.,DISORDERS OF WHITE CELL NUMBER,Eosinophilia Eosinophils are distributed in the body among various pools in a similar way to neutrophils, although the bone marrow storage pool is minimal. Eosino
32、phils circulate in the bloodstream for only a few hours before entering the tissues, where they may live for several days. Their two main functions are to kill parasites and to regulate allergic and inflammatory reactions. Eosinopenia Eosinopenia in combination with lymphopenia occurs following stre
33、ss, administration of corticosteroids and in spontaneous hyperadrenocorticism (Cushings syndrome). Basophilia Basophils contain inflammatory mediators such as histamine and heparin and function in a similar manner to mast cells in hypersensitivity reactions.,DISORDERS OF WHITE CELL NUMBER,Lymphocyto
34、sis Causes of lymphocytosis1. Physiological lymphocytosis, with concomitant neutrophilia, in response to excitement (especially cats)2. Strong immune stimulation (e.g. in chronic infection, viraemia or immune-mediated disease)3. Chronic lymphocytic leukaemia4. Hypoadrenocortiscism (lymphocytosis may
35、 be associated with an eosinophilia)5. Increased numbers of large reactive lymphocytes may occur transiently following vaccination6. Young animals have a higher lymphocyte count than adult animals,DISORDERS OF WHITE CELL NUMBER,LymphopeniaCauses of lymphopenia are listed.,Stress Glucocorticoid thera
36、py Hyperadrenocorticism Chylothorax (loss of lymphocytes into the pleural space) Lymphangiectasia (loss of lymphocytes into the gut) Acute phase of most viral infections (e.g. canine distemper, parvovirus, FeLV) Septicaemia/endotoxaemia,DISORDERS OF WHITE CELL NUMBER,Reference ranges for total and d
37、ifferential white blood cell counts,Table 2 shows the alterations in some of parameters in various diseases. Laboratory assessment Tests to assess primary haemostasis include:Platelet countBleeding timeClot retraction. Tests to assess secondary haemostasis include:Whole blood clotting time (WBCT)Act
38、ivated clotting time (ACT) Activated partial thromboplastin time (APPT)One-stage prothrombin time (OSPT)Thrombin time (TT),DISORDERS OF WHITE CELL NUMBER,Disseminated intravascular coagulation (DIC): This may be triggered by a wide variety of diseases, including endotoxaemia neoplasia (especially ha
39、emangiosarcoma 血管肉瘤) acute infections (e.g. infectious canine hepatitis) haemolytic anaemia pancreatitis heat stroke. The clinicopathological features of DIC are: Thrombocytopenia Increased OSPT/APTT Elevated FDPs Low fibrinogen Schistocytes in the blood film.,DISORDERS OF WHITE CELL NUMBER,兽医临床病理学,
40、College of Veterinary Medicine, SCAU, Guangzhou,China 510642,Veterinary Clinical Pathology,Clinical biochemistry,Introduction Serum proteinsTotal protein and albuminGlobulinsIndicators of renal functionUrea nitrogenCreatinine Markers of hepatic diseaseAlanine aminotransferaseAspartate aminotransfera
41、seAlkaline phosphataseGamma-glutamyi transferaseBilirubinBile acidsAmmonia Pancreatic diseaseAmylaseLipase,ElectrolytesSodium; Potassium; ChlorideMagnesium; Calcium; Phosphorus Muscle enzymesCreatine kinase Aspartate aminotransferase Carbohydrate metabolismGlucose Fructosamine Lipid metabolismCholes
42、terol Triglycerides Miscellaneous testsIronLeadZinc Copper Chemical profiles and test selection,SERUM PROTEINS,Total protein and albumin Physiology The circulating proteins are synthesized predominantly in the liver, although plasma cells also contribute to their production. Quantitatively the singl
43、e most important protein is albumin (35-50% of the total serum protein concentration). The other proteins are collectively known as globulins. The functions of proteins are many and varied but include maintenance of plasma osmotic pressure, transport of substances around the body (e.g. ferritin铁蛋白,
44、ceruloplasmin血浆铜蓝蛋白), humoral immunity, buffering and enzyme regulation. Indications for assay The measurement of proteins is generally included in an initial health screen in all patients but especially where intestinal, renal or hepatic disease or haemorrhage is suspected. Analysis Protein concent
45、rations can be estimated in serum, plasma, urine or body fluids with a refractometer or by spectrophotometry. Serum albumin levels are measured by bromocresol green dye溴甲酚绿 binding and the serum globulin is calculated by subtraction of the albumin concentration from the total protein concentration.,
46、Reference rangesNeonates and very young animals have lower concentrations of albumin and globulins (due to minimal quantities of immunoglobulins). As the animal gains immunocompetence the protein concentrations rise to reach adult values. Physiological decreases in albumin may be noted during pregna
47、ncy. Critical values Marked hypoalbuminaemia (15 g/L) is associated with the development of ascites and tissue oedema. Accumulation of peritoneal fluid may occur at higher albumin concentrations if there is concurrent portal vein hypertension, e.g. in chronic liver disease. Interfering phenomena Lip
48、aemia, haemolysis and hyperbilirubinaemia produce false increases in total protein concentrations. Drug effects Hormones have a marginal effect on plasma protein concentrations. Corticosteroids and anabolic steroids may increase the protein concentration due to their anabolic effects while the catab
49、olic effects of thyroxine can cause a decrease.,SERUM PROTEINS,Figure 4.3: Causes of hypoalbuminaemia. Increased lossGlomerular protein lossProtein-losing enteropathyCutaneous lesions, e.g. bumsExternal haemorrhageDecreased productionHepatic insufficiencyMalnutritionMaldigestionMalabsorptionSequestr
50、ationBody cavity effusion,SERUM PROTEINS,Globulins Analysis Serum protein electrophoresis (SPE) on cellulose acetate gels allows fractionation of the proteins, depending predominantly on their charge and size. After staining for protein, the cellulose acetate strip is scanned by a densitometer which
51、 converts the relative intensities of the protein bands to percentages and generates a graph that demonstrates the protein fractions (albumin, 1-globulin, 2-globulin, 1-globulin, 2-globulin, -globulin). Causes of hypoglobulinaemia The most common pathological causes are haemorrhage and protein-losing enteropathies.,
