犬猫贫血的实验室诊断(10页).doc

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1、-犬猫贫血的实验室诊断-第 10 页讲座二 贫血的实验室诊断原著 Professor Michael J. Day翻译：刘睿 兽医师（广州），校对：戴庶 兽医师（广州）前言如果要对贫血动物做出成功的诊断和管理，不可或缺一定水准的实验室检查。血液学检查可在院内做；或保存为EDTA抗凝血后，送检至商业诊断实验室。要进行基本的血液分析，最低限度的实验室设备应包括：微量血细胞比容法离心机和毛细管判读器、制备染色血涂片的材料、用于评估血涂片所用的显微镜。准备一台折射仪来测定血浆蛋白含量也是有价值的。如今，临床实验室已经有条件使用日趋精密的检测设备来进行血液分析。虽然大部分的检测设备能够获得可信的红细胞和

2、白细胞数据，但血小板的检测值可能被低估。在使用仪器检测血样的同时，应配合血涂片镜检。本讲座回顾了红细胞参数的相关知识，也包括血涂片检查中可能遇见的各种红细胞形态学改变情况。红细胞参数红细胞计数，PCV和Hb红细胞压积（PCV）是指红细胞总数(RBCs)所占全血中的体积比。PCV测量：通过离心微量红细胞压积管后，人工判读数据，并记录为占血液总量百分比。血细胞压积（HcT）通过自动血液分析仪在细胞大小和数量的基础上判定。红细胞总数可使用自动化仪器，或血细胞计数器在镜下计数来获得，用RBC 1012/l 表示。血液中的血红蛋白（Hb）含量同样也可以使用自动化仪器判定，记录为g/dl。Hb（血红蛋白）

3、的含量测定受血液样本中存在脂血症、红细胞溶解症或胆红素血的影响。一个检验结果精确性的简单方法：3 Hb= Ht（血细胞压积）。检测到的数值要参照“正常范围值”来比较，但要明确“正常范围值”并不适用于所有情况。举例来说，初生或者怀孕动物可能会呈现数值偏差，并且某些动物也存在品种效应（例如，视觉猎犬 (sight hounds)就呈现高PCV值和低嗜中性粒细胞计数）。PCV，RBC计数和HB都反映了红细胞质量，并且这三个参数之间通常具有相关性，所以简单的测定PCV值即是一个良好的临床指征。PCV值通常被用于描述贫血的严重程度。犬PCV近似参考值：轻度贫血30-36%，中度贫血18-29%，重度贫血

4、18%。猫PCV近似参考值：轻度贫血20-24%，中度贫血15-19%，重度贫血60 109/l是再生性贫血反应的象征，400 109/表示显著的再生。作为另外一个选择，网织红细胞生成指数（RPI）也可能需要确定。RPI对“校正”贫血程度的评估和网织红细胞寿命有重要价值。1）贫血程度校正校正值%（或计数值）=绝对值%（或计数值） PCV值/正常PCV值 (45)2）网织红细胞寿命校正RPI = 校正值 % 1/血液成熟时间血液成熟时间取决于PCV值：PCV 45% 1 天 35% 1.5 天 25% 2 天 15% 2.5 天RPI 2.5时，表示再生性贫血反应。 猫集结状网织红细胞最初从骨髓

5、中被释放，在经过10-12天的成熟期后，转变为点状网织红细胞。 相比之下，点状网织红细胞在循环血液中出现需要3-4周。在猫上，集结状网织红细胞计数可判定再生性贫血，它反映骨髓再生活跃程度，而点状红细胞计数代表累积再生贫血。集结状红细胞计数40 109/l表示再生性贫血，200 109/l时表示显著再生性贫血。骨髓评估对于非再生性贫血的病例，骨髓评估是诊断过程的一个重要步骤。可能需要采集两种样本：抽取骨髓内容物或用骨髓针穿刺活检。前一种样本给临床病理学专家分析，而后一种样本给组织病理学专家分析。骨髓穿刺针的优点是病理组织可得到更好的评估，但要获得优质的穿刺活检物却具有挑战性。将穿刺活检物放入福尔

6、马林溶液固定之前先做压片。通常在股骨干骨（猫）或髂骨嵴（犬）来获得样本。在大型犬，肋骨或胸骨节是骨髓抽取的备用位置。抽取的骨髓样本应放入EDTA或ACD（可从采血袋中获得）抗凝血剂中保存，同时至少要制备10张压片，迅速空气干燥后染色。主要的谱系细胞应该按照成熟程度、所占的相对比例、粒细胞和红细胞比例的顺序依次确定。同时应该检测异常细胞（肿瘤细胞）。LECTURE 2. LABORATORY DIAGNOSIS OF ANAEMIAINTRODUCTIONThe anaemic patient cannot be successfully diagnosed and managed withou

7、t at least some level of laboratory testing. This may be undertaken in-practice or by sending EDTA anticoagulated blood to a commercial diagnostic laboratory. The minimum in-practice equipment required for basic haematological analysis would be a microhaematocrit centrifuge and tube reader, material

8、s for preparation of a stained blood smear and a microscope for evaluation of the blood smear. A refractometer for assessment of plasma proteins is also valuable. Increasingly sophisticated instrumentation is now available for in-practice laboratories to undertake haematological analysis. Most of th

9、ese machines produce acceptable data for red and white blood cells but may underestimate platelets. It is essential to examine a blood smear in parallel with assessing the readings from such equipment. This lecture reviews the erythrocyte parameters and cytological changes that may be seen on examin

10、ation of the blood smear.ERYTHROCYTE PARAMETERSTotal red blood cell count, PCV and HbThe packed cell volume (PCV) is the percentage of blood volume made up of red blood cells (RBCs). It is determined manually by centrifugation of a microhaematocrit tube and recorded as a percentage of blood volume.

11、The haematocrit (Hct) is determined by the automated haematology analyser on the basis of cell size and number. The total RBC count is determined in automated fashion or may be measured manually using a haemocytometer chamber and microscope. This parameter is recorded as RBC 1012/l. The concentratio

12、n of haemoglobin (Hb) in the blood is also determined in automated fashion and reported as g/dl. Determination of Hb concentration will be affected by the presence of lipaemia, haemolysis or bilirubin in the blood sample. A simple check for accuracy is that the Hb 3 should equal the haematocrit. The

13、se values are assessed relative to a normal reference range but it should be remembered that these reference ranges may not apply to all situations. For example very young animals or pregnant animals may have outlying values and there are some breed-related effects (e.g. sight hounds have relatively

14、 high PCV and lower neutrophil count). The PCV, RBC count and Hb all reflect the red cell mass and these three parameters generally correlate, so simple PCV measurement is a good index to use. The PCV may be used to describe the severity of anaemia. As an approximation a dog has a mild anaemia with

15、a PCV of 30 36%, moderate anaemia at 18 29% and severe anaemia at 18%. A cat has mild anaemia at 20 24%, moderate anaemia at 15 19% and severe anaemia at 14%. Erythrocyte indicesThe erythrocyte indices are derived from the parameters above and provide a useful assessment of the size and haemoglobin

16、concentration of the erythrocytes. The mean corpuscular (cellular) volume (MCV) indicates the size of individual RBCs and is calculated by the formula:PCV 10RBC 1012/lThe MCV is recorded as femtolitres (fl) and an elevation indicates macrocytosis whilst reduction indicates microcytosis. The MCV may

17、artefactually elevate when a blood sample is stored or when samples are sent to a laboratory by post. Macrocytosis also occurs with a regenerative response or in folate deficiency, dyserythropoiesis and some cases of FeLV infection. Microcytosis may be a normal feature in some canine breeds (akita,

18、shar pei, shiba inu) or reflect iron deficiency or portosystemic shunt. The mean corpuscular haemoglobin (MCH) indicates the mass of Hb in each individual RBC in picograms (pg). MCH is calculated by the formula:Hb 10 RBC 1012/lAn increase in MCH indicates hyperchromasia and a decrease indicates hypo

19、chromasia (most often due to iron deficiency). The mean corpuscular haemoglobin concentration (MCHC) indicates the percentage of the entire RBC mass that is composed of Hb (in g/dl) and is calculated using the formula:Hb 100PCVOnly a reduced MCHC is of meaning as it is physiologically impossible for

20、 there to be an elevation in MCHC. Elevated MCHC is an artifact due to haemolysis, lipaemia or the presence of Heinz bodies.THE BLOOD SMEARPreparation of a good blood smear is essential to diagnostic haematology. A spreader slide is used to smoothly smear a drop of EDTA blood such that the resultant

21、 smear has a good feather edge. The smear is air-dried and stained by Diff-Quik. The monolayer of the smear, behind the feather edge should be evaluated at both low and high magnifications in a systematic fashion. All lineages should be assessed.ERYTHROCYTE MORPHOLOGYThe normal RBC is a biconcave di

22、sc with a zone of central pallor. Canine RBCs are approximately 7 mm in diameter and feline RBCs are smaller (6 mm). A range of morphological changes is recognized and it is important to identify these on evaluation of the blood smear as they provide essential diagnostic information.RouleauxIn hyper

23、proteinaemic plasma with elevated concentration of fibrinogen or altered nature of globulins the erythrocytes often stack together like a role of coins. This can be a normal feature of equine blood and may occasionally be seen in normal cats. In the dog, rouleaux formation often occurs in inflammato

24、ry or neoplastic disease. It is important to distinguish rouleaux formation from agglutination (see below).AgglutinationAgglutination is aggregation of erythrocytes (like a bunch of grapes) that may be observed macroscopically in the EDTA blood sample or microscopically. Agglutination is mediated by

25、 antibody (generally IgM) and is strongly indicative of an immune-mediated haemolytic anaemia.AnisocytosisAnisocytosis simply means variation in the size of erythrocytes such that there is a mixture of normal cells with microcytes or macrocytes. Macrocytic cells are often polychromatic and likely to

26、 be reticulocytes. Microcytic cells characterize iron deficiency anaemia and spherocytes (see below) are also smaller than normal.PolychromasiaPolychromasia is variation in the colouration of erythrocytes but generally refers to the presence of cells with a pale blue colouration. These cells are als

27、o often macrocytic and are likely to represent reticulocytes. Polychromasia with elevated MCV is strongly suggestive of a regenerative response.HypochromasiaHypochromasia is seen as an increase in the zone of central pallor and indicates a reduction in Hb concentration as would be seen in iron defic

28、iency anaemia.PoikilocytosisPoikilocytosis simply means a variation in the shape of the red cells.SpherocytosisA spherocyte is a smaller, rounder, more darkly stained red cell that lacks a central zone of pallor. A spherocyte is a partially phagocytosed red cell that is formed when an antibody (and

29、complement) coated RBC passes through the spleen or liver and a nearby macrophage removes a portion of the cell membrane. The cell is able to repair itself and continue in the circulating blood as an erythrocyte of reduced volume. Spherocytosis indicates the presence of antibody but does not discrim

30、inate between a primary and secondary immune-mediated haemolysis. Occasionally some spherocytes may form with vascular disease due to non-immune-mediated damage. Whilst spherocytes may be readily detected in the dog, the normally small size of feline erythrocytes means that it is not possible to rel

31、iably define these cells in the cat.SchistocytosisA schistocyte is a red cell fragment that appears following traumatic disruption of the red cell. This often equates to the presence of intravascular obstructions (e.g. parasites or thrombi) and is a common feature in canine splenic haemangiosarcoma.

32、 Schistocytes may also occur in disseminated intravascular coagulation (DIC) or in congestive heart failure, lymphoma or glomerulonephritis.AcanthocytesAre spiculated red cells with membrane projections of varying length and position. These cells may form when there is abnormality of cholesterol or

33、phospholipid of the cell membrane or where there is mechanical damage (e.g. DIC, haemangiosarcoma) or occasionally with lymphoma, liver disease, hypersplenism or glomerulonephritis.EchinocytesAre spiculated cells with short and evenly spaced membrane projections. These are generally an artifact and

34、indicate an excess of EDTA (when the blood tube was not optimally filled) but may occasionally occur in metabolic (e.g. PK deficiency) or renal disease.Basophilic stipplingThis indicates the presence of residual RNA and is a relatively common feature in cattle and occasionally observed in anaemic ca

35、ts. It is also a feature of lead toxicity in the dog (lead interferes with erythropoiesis by disrupting heme and globin synthesis).Howell-Jolly bodiesHowell-Jolly bodies are nuclear remnants that may be normal in the cat or occur in response to anaemia. They may also be seen in splenectomized animal

36、s. Heinz bodiesHeinz bodies are aggregates of denatured haemoglobin that associate with the cell membrane and are best demonstrated with supravital stains (Leishmans stain or new methylene blue). Up to 5 10% of normal feline red cells may have Heinz bodies. The related eccentrocyte may occur with se

37、vere oxidation.Nucleated erythrocytesNucleated erythrocytes are not seen in normal blood but in markedly regenerative anaemias with demand for erythrocytes may be released from the bone marrow or sites or extramedullary haematopoiesis. Mild bone marrow stromal damage may also permit release of nucleated RBCs. Healthy miniature schnauzers and dachshunds may also have some nucleated RBCs in the circulation.Erythrocyte parasitesA range of red cell-associated organisms may be detected on evaluation of the blood smear. In particular the large form Babesia is readily detected