犬猫贫血的实验室诊断

1、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白细胞数据，但血小板的检测值可能被

2、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）的含量测定受血液样本

3、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<14%。 红细胞指数红细胞指数由红细胞的各项参数计算得出，它有效提供了红细胞的大小和血红蛋白浓度的数据。平均红细胞容积（MCV）表示单个红细胞的体积，MCV计算公式：PCV × 10RBC ×1012/lMCV记录单位是“毫微微升”（fl），当其数值上升时，预示“大红细胞症”，其数值下降时，预示“小红细胞症”。当血液样本被冷藏或邮寄至实验室时，MCV数值可能会被人为升高。大红细胞症也见于再生性贫血反应、叶酸缺乏、造血功能异常和部分FeLV（猫白血病病毒）感染病例。小红细胞症是某些犬种（如

5、日本秋田犬，沙皮犬，柴犬）的正常现象，也可能是缺铁反应或肝门静脉短路症。平均红细胞血红蛋白含量（MCH）是指单个红细胞中所含的血红蛋白量，单位是皮克（pg）。MCH计算公式：Hb × 10 RBC ×1012/lMCH值上升时，预示红细胞着色加深；其值下降时，预示红细胞着色变浅（最常见的原因是铁元素缺乏）。平均红细胞血红蛋白浓度（MCHC）指血红蛋白在总红细胞数量中所占的百分比（单位 g/dl）， MCHC计算公式：Hb × 100PCV仅当MCHC值下降时才具有临床意义，因为在生理学中并不存在MCHC值升高的现象。MCH值升高的原因是与红细胞破裂、脂血症或出现海

6、因茨小体有关的人为因素。 血涂片制备一张优质血涂片对于贫血的诊断非常有必要。 通常将一滴用EDTA处理的血液滴在载玻片上，用另一块载玻片连续而流畅地向前推动，涂片的尾部会出现“羽毛状的边缘”。血涂片经空气干燥后用diff-quik染色。在血涂片的羽毛状尾部，血细胞呈单层分布，这个区域适合在低倍和高倍镜下进行系统观测。所有细胞谱都应被评定。 红细胞形态学正常的红细胞呈“两面凹陷的圆盘状”，中间为浅着色区。犬红细胞直径大约为7mm，猫红细胞直径稍小（6 mm)。临床上可观察到一系列红细胞形态学变化，在评估血涂片的基础上对此鉴别很重要， 因为它们提供了必要的诊断信息。 钱串红细胞 在纤维蛋白原浓度升

7、高或球蛋白变性的高蛋白血浆中，红细胞常常堆砌在一起，形如一串钱币。这种现象在马的血液中属正常，在猫的血液中偶见。在犬上，钱串红细胞通常发生在炎症或肿瘤疾病中。在红细胞凝集（见下文）的情况下，鉴别钱串红细胞的结构很重要。 红细胞凝集红细胞凝集指红细胞的聚集（葡萄串状），这种情况也许可在EDTA处理的抗凝血中肉眼可见或通过镜检发现。红细胞凝集由抗体（通常为IgM）介导，并且强有力的指示了免疫介导的溶血性贫血。 红细胞大小不均等症红细胞大小不均等症简单说来就是出现了红细胞大小不一的变化，正常血细胞中混有大红细胞或小红细胞。大红细胞通常呈多染色性，并且可能属于网织红细胞。小红细胞是缺铁性贫血的特征，在

8、球形红细胞症中（见下文），红细胞也比正常时小。 红细胞多染色性红细胞多染色性是指红细胞染色性质的变异，通常出现浅蓝色的染色特征。这些细胞通常为大红细胞，并且可能是网织红细胞。红细胞多染色性伴随MCV值上升时，强烈提示再生性贫血反应。红细胞低染红细胞低染在镜下可见到红细胞中央的浅着色区扩大，这正如缺铁性贫血中可见的血红蛋白浓度降低。 异形红细胞症异形红细胞症简单说来就是红细胞的形状发生了异常变化。 球形红细胞症球形红细胞指小而圆，深染且中央浅着色区消失的红细胞。球形红细胞是被部分吞噬的红细胞：被抗体（和补体）结合的红细胞通过肝脏或脾脏时，附近的吞噬细胞移除了一部分的红细胞膜。红细胞能“自我修复”

9、，并且继续以体积变小的红细胞存在于循环血液当中。球形红细胞指示抗体的出现，但是却不能辨别免疫介导性溶血症是原发性，或是继发性。偶尔情况下，球形红细胞可能形成于非免疫介导性血管疾病。同时，球形红细胞在犬上易见。但猫的红细胞正常时体积偏小，所以不能确实分辨球形细胞。 裂红细胞裂红细胞是伴随着红细胞外伤性裂解产生的细胞碎片。这种情况等同于出现了血管内阻塞（例如寄生虫或血栓），这通常也是犬脾脏血管肉瘤的临床特征。裂红细胞也可见于血管内弥漫性凝血（DIC）、充血性心力衰竭、淋巴瘤或肾小球性肾炎的情况。棘红细胞棘红细胞表示红细胞表面不同位置出现不同长度的突起。棘红细胞可能形成于红细胞膜的胆固醇或磷脂异常，

10、也可能是红细胞的机械性损伤（例如DIC或血管肉瘤），偶见于淋巴瘤、肝脏疾病、脾功能亢进或肾小球性肾炎。 锯齿状红细胞锯齿状红细胞表示红细胞表面出现大面积短而平滑的突起。通常是人为因素导致，预示EDTA过量（当采血量没有达到采血管的标注容量时），但也偶见于新陈代谢异常（例如丙酮激酸酶缺乏）或肾脏疾病。 嗜碱性彩斑嗜碱性彩斑指示有RNA的残留物，在牛的血液中属正常，偶见于猫的贫血病例。这也是犬铅中毒的特征（铅通过破坏亚铁血红素和球蛋白合成，从而妨碍红细胞生成） 豪威尔氏体豪威尔氏体是细胞核的残留物，在正常的猫血液中可见，或见于对贫血的应答反应中。豪威尔氏体也可在脾切除的动物血液中见到。 海因茨小体

11、海因茨小体是出现在红细胞膜表面的变性血红蛋白聚集物，最佳观测方式是配合使用活体染色剂观察（Leishmans染色或新亚甲基蓝染色）。正常猫血液中大约有5-10%的红细胞含有海因茨小体。当发生严重的氧化损伤时，可能会出现相关的偏心细胞。有核红细胞 有核红细胞并不存于正常血液中，但在再生障碍性贫血中常见，随着对红细胞需求的增加，可从髓内或髓外的造血机制中被释放出来。轻度骨髓基质损伤时也可能允许释放有核红细胞。健康的小型雪纳瑞犬和腊肠犬的循环血液中也可见一些有核红细胞。红细胞寄生虫在血涂片检查中，可能发现一系列红细胞相关的病原生物。在高寄生虫血症时，大体型犬巴贝斯虫尤其容易被发现。小体型犬巴贝斯虫和

12、表面支原体（血液支原体）通常难以发现，PCR实验对确诊此类感染具有高度敏感性。 血小板评估血涂片用于血小板评估比较准确，因为大部分的自动仪器会对血小板凝集效应的计数不准。在血涂片中（通常在边缘部分）可见血小板凝集，这也预示着假性机器低读数。猫的红细胞与血小板在体积上相似，这也加大了机器读数的误差。血小板减少症需要用血涂片计算平均血小板值来判定：在100倍镜下（油镜头，即最终放大率为1000倍），取10个视野计数的平均值。猫：平均血小板值×20=血小板数×109/l 犬：平均血小板值×15=血小板数×109/l 白细胞评估血涂片常用于手工白细胞分类计数，通

13、过测定一定数目白细胞内的各谱系细胞百分比来确定。通过血液分析仪或用血细胞计数板手工计数后，各白细胞百分比数值需转化为绝对值来表示结果(×109/l)。 白细胞计数近似值，也可以通过镜下评估血涂片得出。平均白细胞计数方法：40倍物镜下，取10个视野中的平均计数值（即在10倍目镜下获得400倍的放大率），乘以1500，等于白细胞数×109/l。白细胞形态学异常也要检查，同时要评估杆状或早期粒细胞所占比例。白细胞中的中毒颗粒物和空泡，以及非典型单核细胞的出现，这些也要熟悉判定。 网织红细胞计数网织红细胞计数对确诊再生性贫血反应是最为准确的。在犬猫临床上，网织红细胞过多症与红细胞多

14、染性存在普遍关联。网织红细胞仅能够通过活体染色法（例如新甲基蓝或亮甲酚蓝）来辨别，这时聚集的核糖体RNA显露。网织红细胞在形态学上分集结状或点状。犬仅存在集结状网织红细胞，但猫二者都有。在犬上，网织红细胞百分比（在红细胞中）需要表示为绝对值(×109/l)。网织红细胞计数>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

15、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

16、/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、

17、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 ANAEMIA INTRODUCTIONThe anaemic patient cannot be successfully diagnosed and managed without at least some level of laboratory testing. This may be undertaken in-practice or by sending EDTA anticoagulated blood to a commercial dia

18、gnostic laboratory. The minimum in-practice equipment required for basic haematological analysis would be a microhaematocrit centrifuge and tube reader, materials for preparation of a stained blood smear and a microscope for evaluation of the blood smear. A refractometer for assessment of plasma pro

19、teins is also valuable. Increasingly sophisticated instrumentation is now available for in-practice laboratories to undertake haematological analysis. Most of these machines produce acceptable data for red and white blood cells but may underestimate platelets. It is essential to examine a blood smea

20、r in parallel with assessing the readings from such equipment. This lecture reviews the erythrocyte parameters and cytological changes that may be seen on examination of the blood smear. ERYTHROCYTE PARAMETERSTotal red blood cell count, PCV and HbThe packed cell volume (PCV) is the percentage of blo

21、od 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. The haematocrit (Hct) is determined by the automated haematology analyser on the basis of cell size and number. The total RBC count is det

22、ermined in automated fashion or may be measured manually using a haemocytometer chamber and microscope. This parameter is recorded as RBC ×1012/l. The concentration of haemoglobin (Hb) in the blood is also determined in automated fashion and reported as g/dl. Determination of Hb concentration w

23、ill 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. These values are assessed relative to a normal reference range but it should be remembered that these reference ranges may not a

24、pply 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 high PCV and lower neutrophil count). The PCV, RBC count and Hb all reflect the red cell mass and these three parameters gen

25、erally 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 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

26、 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 concentration of the erythrocytes. The mean corpuscular (cellular) volume (MCV) indicates the size of individual RBCs

27、 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 artefactually elevate when a blood sample is stored or when samples are sent to a laboratory by post. Ma

28、crocytosis 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, shar pei, shiba inu) or reflect iron deficiency or portosystemic shunt. The mean corpuscular haemoglobin

29、 (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 hypochromasia (most often due to iron deficiency). The mean corpuscular haemoglobin concentrati

30、on (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 there to be an elevation in MCHC. Elevated MCHC is an artifact due to haemolysis, l

31、ipaemia 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 smear has a good feather edge. The smear is air-dried and stained by Diff-Quik. Th

32、e 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 disc with a zone of central pallor. Canine RBCs are approximately 7 mm in diameter a

33、nd 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 hyperproteinaemic plasma with elevated concentration of fibrinogen or altered nature o

34、f 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 inflammatory or neoplastic disease. It is important to distinguish rouleaux formation from

35、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 antibody (generally IgM) and is strongly indicative of an immune-mediated haemo

36、lytic 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 be reticulocytes. Microcytic cells characterize iron deficiency anaemia and sp

37、herocytes (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 also often macrocytic and are likely to represent reticulocytes. Polychromasia wi

38、th 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 deficiency anaemia. PoikilocytosisPoikilocytosis simply means a variation in the s

39、hape 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 complement) coated RBC passes through the spleen or liver and a nearby macr

40、ophage 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 discriminate between a primary and secondary immune-mediated haemolysis. Occasiona

41、lly 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 reliably define these cells in the cat. SchistocytosisA schistocyte is a red c

42、ell 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. Schistocytes may also occur in disseminated intravascular coagulation (DI

43、C) 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 phospholipid of the cell membrane or where there is mechanical damage (e.

44、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 indicate an excess of EDTA (when the blood tube was not optimally filled

45、) 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 cats. It is also a feature of lead toxicity in the dog (lead interferes w

46、ith 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 animals. Heinz bodiesHeinz bodies are aggregates of denatured haemoglobin th

47、at 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 severe oxidation. Nucleated erythrocytesNucleated erythrocytes are not s

48、een 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

49、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 if there is high parasitaemia. Small form Babesia and the surface Mycoplasma (haemoplasmas)

50、 are often difficult to discern and PCR testing remains the most sensitive means of confirming these infections. Evaluation of plateletsThe smear should always be assessed for platelets as most automated machines do not provide accurate platelet counts when these cells aggregate. Aggregated platelet

51、s may be seen in the smear (often towards the edges) and will indicate whether there is a falsely low machine reading. In cats, there is overlap in size of platelets and red cells providing further inaccuracy. Thrombocytopenia may be assessed from the blood smear by counting the mean number of plate

52、lets in ten ×100 (oil immersion) fields (i.e. final magnification of ×1000). For cats the mean number × 20 = platelets ×109/l and for dogs the mean number × 15 = platelets ×109/l. Evaluation of leucocytesThe blood smear may be used to manually calculate the differential

53、 leucocyte count by determining the percentage of cells of each lineage after counting a set number of leucocytes. These percentages should always be converted to absolute numbers using the total white cell count (×109/l) as determined by the haematology analyser or manually with a haemocytomet

54、er chamber. An approximation of leucocyte number may also be determined by evaluation of the blood smear. The mean number of leucocytes in ten fields counted with the x40 objective of the microscope (i.e. magnification of ×400 with ×10 eyepiece) multiplied by 1500 gives the total WBC in &#

55、215;109/l. Morphological abnormalities in leucocytes should also be assessed. The proportion of band or earlier granulocytes should be assessed. The presence of toxic granulation or vacuolation of granulocytes or the presence of atypical mononuclear cells is readily determined. THE RETICULOCYTE COUN

56、TA reticulocyte count is the most accurate means of determining the presence of a regenerative response. There is a good general correlation between reticulocytosis and polychromasia in the dog and cat. Reticulocytes can only be identified by supravital staining (e.g. new methylene blue or brilliant

57、 cresyl green) to demonstrate aggregated ribosomal RNA. Reticulocytes may aggregate or punctate in morphology. In the dog only aggregate reticulocytes occur but both forms are recognized in the cat. In the dog the percentage reticulocytes (of red cells) should be expressed as an absolute number (

58、15;109/l). A reticulocyte count of >60 ×109/l is indicative of a regenerative response and a count of >400 ×109/l indicates marked regeneration. Alternatively, the reticulocyte production index (RPI) may be determined. The RPI corrects the count for the degree of anaemia and the lifespan of the reticulocyte. The first correction is for degree of anaemia that is