犬貓貧血的實驗室診斷

1、講座二 貧血的實驗室診斷原著 Professor Michael J. Day翻譯：劉睿 獸醫(yī)師（廣州），校對：戴庶 獸醫(yī)師（廣州） 前言如果要對貧血動物做出成功的診斷和管理，不可或缺一定水準的實驗室檢查。血液學檢查可在院內做；或保存為EDTA抗凝血后，送檢至商業(yè)診斷實驗室。要進行基本的血液分析，最低限度的實驗室設備應包括：微量血細胞比容法離心機和毛細管判讀器、制備染色血涂片的材料、用于評估血涂片所用的顯微鏡。準備一臺折射儀來測定血漿蛋白含量也是有價值的。如今，臨床實驗室已經(jīng)有條件使用日趨精密的檢測設備來進行血液分析。雖然大部分的檢測設備能夠獲得可信的紅細胞和白細胞數(shù)據(jù)，但血小板的檢測值可能被

2、低估。在使用儀器檢測血樣的同時，應配合血涂片鏡檢。本講座回顧了紅細胞參數(shù)的相關知識，也包括血涂片檢查中可能遇見的各種紅細胞形態(tài)學改變情況。 紅細胞參數(shù)紅細胞計數(shù)，PCV和Hb紅細胞壓積（PCV）是指紅細胞總數(shù)(RBCs)所占全血中的體積比。PCV測量：通過離心微量紅細胞壓積管后，人工判讀數(shù)據(jù)，并記錄為占血液總量百分比。血細胞壓積（HcT）通過自動血液分析儀在細胞大小和數(shù)量的基礎上判定。紅細胞總數(shù)可使用自動化儀器，或血細胞計數(shù)器在鏡下計數(shù)來獲得，用RBC ×1012/l 表示。血液中的血紅蛋白（Hb）含量同樣也可以使用自動化儀器判定，記錄為g/dl。Hb（血紅蛋白）的含量測定受血液樣本

3、中存在脂血癥、紅細胞溶解癥或膽紅素血的影響。一個檢驗結果精確性的簡單方法：3 ×Hb= Ht（血細胞壓積）。檢測到的數(shù)值要參照“正常范圍值”來比較，但要明確“正常范圍值”并不適用于所有情況。舉例來說，初生或者懷孕動物可能會呈現(xiàn)數(shù)值偏差，并且某些動物也存在品種效應（例如，視覺獵犬 (sight hounds)就呈現(xiàn)高PCV值和低嗜中性粒細胞計數(shù)）。PCV，RBC計數(shù)和HB都反映了紅細胞質量，并且這三個參數(shù)之間通常具有相關性，所以簡單的測定PCV值即是一個良好的臨床指征。PCV值通常被用于描述貧血的嚴重程度。犬PCV近似參考值：輕度貧血30-36%，中度貧血18-29%，重度貧血<

4、18%。貓PCV近似參考值：輕度貧血20-24%，中度貧血15-19%，重度貧血<14%。 紅細胞指數(shù)紅細胞指數(shù)由紅細胞的各項參數(shù)計算得出，它有效提供了紅細胞的大小和血紅蛋白濃度的數(shù)據(jù)。平均紅細胞容積（MCV）表示單個紅細胞的體積，MCV計算公式：PCV × 10RBC ×1012/lMCV記錄單位是“毫微微升”（fl），當其數(shù)值上升時，預示“大紅細胞癥”，其數(shù)值下降時，預示“小紅細胞癥”。當血液樣本被冷藏或郵寄至實驗室時，MCV數(shù)值可能會被人為升高。大紅細胞癥也見于再生性貧血反應、葉酸缺乏、造血功能異常和部分FeLV（貓白血病病毒）感染病例。小紅細胞癥是某些犬種（如

5、日本秋田犬，沙皮犬，柴犬）的正?，F(xiàn)象，也可能是缺鐵反應或肝門靜脈短路癥。平均紅細胞血紅蛋白含量（MCH）是指單個紅細胞中所含的血紅蛋白量，單位是皮克（pg）。MCH計算公式：Hb × 10 RBC ×1012/lMCH值上升時，預示紅細胞著色加深；其值下降時，預示紅細胞著色變淺（最常見的原因是鐵元素缺乏）。平均紅細胞血紅蛋白濃度（MCHC）指血紅蛋白在總紅細胞數(shù)量中所占的百分比（單位 g/dl）， MCHC計算公式：Hb × 100PCV僅當MCHC值下降時才具有臨床意義，因為在生理學中并不存在MCHC值升高的現(xiàn)象。MCH值升高的原因是與紅細胞破裂、脂血癥或出現(xiàn)海

6、因茨小體有關的人為因素。 血涂片制備一張優(yōu)質血涂片對于貧血的診斷非常有必要。 通常將一滴用EDTA處理的血液滴在載玻片上，用另一塊載玻片連續(xù)而流暢地向前推動，涂片的尾部會出現(xiàn)“羽毛狀的邊緣”。血涂片經(jīng)空氣干燥后用diff-quik染色。在血涂片的羽毛狀尾部，血細胞呈單層分布，這個區(qū)域適合在低倍和高倍鏡下進行系統(tǒng)觀測。所有細胞譜都應被評定。 紅細胞形態(tài)學正常的紅細胞呈“兩面凹陷的圓盤狀”，中間為淺著色區(qū)。犬紅細胞直徑大約為7mm，貓紅細胞直徑稍?。? mm)。臨床上可觀察到一系列紅細胞形態(tài)學變化，在評估血涂片的基礎上對此鑒別很重要， 因為它們提供了必要的診斷信息。 錢串紅細胞 在纖維蛋白原濃度升

7、高或球蛋白變性的高蛋白血漿中，紅細胞常常堆砌在一起，形如一串錢幣。這種現(xiàn)象在馬的血液中屬正常，在貓的血液中偶見。在犬上，錢串紅細胞通常發(fā)生在炎癥或腫瘤疾病中。在紅細胞凝集（見下文）的情況下，鑒別錢串紅細胞的結構很重要。 紅細胞凝集紅細胞凝集指紅細胞的聚集（葡萄串狀），這種情況也許可在EDTA處理的抗凝血中肉眼可見或通過鏡檢發(fā)現(xiàn)。紅細胞凝集由抗體（通常為IgM）介導，并且強有力的指示了免疫介導的溶血性貧血。 紅細胞大小不均等癥紅細胞大小不均等癥簡單說來就是出現(xiàn)了紅細胞大小不一的變化，正常血細胞中混有大紅細胞或小紅細胞。大紅細胞通常呈多染色性，并且可能屬于網(wǎng)織紅細胞。小紅細胞是缺鐵性貧血的特征，在

8、球形紅細胞癥中（見下文），紅細胞也比正常時小。 紅細胞多染色性紅細胞多染色性是指紅細胞染色性質的變異，通常出現(xiàn)淺藍色的染色特征。這些細胞通常為大紅細胞，并且可能是網(wǎng)織紅細胞。紅細胞多染色性伴隨MCV值上升時，強烈提示再生性貧血反應。紅細胞低染紅細胞低染在鏡下可見到紅細胞中央的淺著色區(qū)擴大，這正如缺鐵性貧血中可見的血紅蛋白濃度降低。 異形紅細胞癥異形紅細胞癥簡單說來就是紅細胞的形狀發(fā)生了異常變化。 球形紅細胞癥球形紅細胞指小而圓，深染且中央淺著色區(qū)消失的紅細胞。球形紅細胞是被部分吞噬的紅細胞：被抗體（和補體）結合的紅細胞通過肝臟或脾臟時，附近的吞噬細胞移除了一部分的紅細胞膜。紅細胞能“自我修復”

9、，并且繼續(xù)以體積變小的紅細胞存在于循環(huán)血液當中。球形紅細胞指示抗體的出現(xiàn)，但是卻不能辨別免疫介導性溶血癥是原發(fā)性，或是繼發(fā)性。偶爾情況下，球形紅細胞可能形成于非免疫介導性血管疾病。同時，球形紅細胞在犬上易見。但貓的紅細胞正常時體積偏小，所以不能確實分辨球形細胞。 裂紅細胞裂紅細胞是伴隨著紅細胞外傷性裂解產(chǎn)生的細胞碎片。這種情況等同于出現(xiàn)了血管內阻塞（例如寄生蟲或血栓），這通常也是犬脾臟血管肉瘤的臨床特征。裂紅細胞也可見于血管內彌漫性凝血（DIC）、充血性心力衰竭、淋巴瘤或腎小球性腎炎的情況。棘紅細胞棘紅細胞表示紅細胞表面不同位置出現(xiàn)不同長度的突起。棘紅細胞可能形成于紅細胞膜的膽固醇或磷脂異常，

10、也可能是紅細胞的機械性損傷（例如DIC或血管肉瘤），偶見于淋巴瘤、肝臟疾病、脾功能亢進或腎小球性腎炎。 鋸齒狀紅細胞鋸齒狀紅細胞表示紅細胞表面出現(xiàn)大面積短而平滑的突起。通常是人為因素導致，預示EDTA過量（當采血量沒有達到采血管的標注容量時），但也偶見于新陳代謝異常（例如丙酮激酸酶缺乏）或腎臟疾病。 嗜堿性彩斑嗜堿性彩斑指示有RNA的殘留物，在牛的血液中屬正常，偶見于貓的貧血病例。這也是犬鉛中毒的特征（鉛通過破壞亞鐵血紅素和球蛋白合成，從而妨礙紅細胞生成） 豪威爾氏體豪威爾氏體是細胞核的殘留物，在正常的貓血液中可見，或見于對貧血的應答反應中。豪威爾氏體也可在脾切除的動物血液中見到。 海因茨小體

11、海因茨小體是出現(xiàn)在紅細胞膜表面的變性血紅蛋白聚集物，最佳觀測方式是配合使用活體染色劑觀察（Leishmans染色或新亞甲基藍染色）。正常貓血液中大約有5-10%的紅細胞含有海因茨小體。當發(fā)生嚴重的氧化損傷時，可能會出現(xiàn)相關的偏心細胞。有核紅細胞 有核紅細胞并不存于正常血液中，但在再生障礙性貧血中常見，隨著對紅細胞需求的增加，可從髓內或髓外的造血機制中被釋放出來。輕度骨髓基質損傷時也可能允許釋放有核紅細胞。健康的小型雪納瑞犬和臘腸犬的循環(huán)血液中也可見一些有核紅細胞。紅細胞寄生蟲在血涂片檢查中，可能發(fā)現(xiàn)一系列紅細胞相關的病原生物。在高寄生蟲血癥時，大體型犬巴貝斯蟲尤其容易被發(fā)現(xiàn)。小體型犬巴貝斯蟲和

12、表面支原體（血液支原體）通常難以發(fā)現(xiàn)，PCR實驗對確診此類感染具有高度敏感性。 血小板評估血涂片用于血小板評估比較準確，因為大部分的自動儀器會對血小板凝集效應的計數(shù)不準。在血涂片中（通常在邊緣部分）可見血小板凝集，這也預示著假性機器低讀數(shù)。貓的紅細胞與血小板在體積上相似，這也加大了機器讀數(shù)的誤差。血小板減少癥需要用血涂片計算平均血小板值來判定：在100倍鏡下（油鏡頭，即最終放大率為1000倍），取10個視野計數(shù)的平均值。貓：平均血小板值×20=血小板數(shù)×109/l 犬：平均血小板值×15=血小板數(shù)×109/l 白細胞評估血涂片常用于手工白細胞分類計數(shù)，通

13、過測定一定數(shù)目白細胞內的各譜系細胞百分比來確定。通過血液分析儀或用血細胞計數(shù)板手工計數(shù)后，各白細胞百分比數(shù)值需轉化為絕對值來表示結果(×109/l)。 白細胞計數(shù)近似值，也可以通過鏡下評估血涂片得出。平均白細胞計數(shù)方法：40倍物鏡下，取10個視野中的平均計數(shù)值（即在10倍目鏡下獲得400倍的放大率），乘以1500，等于白細胞數(shù)×109/l。白細胞形態(tài)學異常也要檢查，同時要評估桿狀或早期粒細胞所占比例。白細胞中的中毒顆粒物和空泡，以及非典型單核細胞的出現(xiàn)，這些也要熟悉判定。 網(wǎng)織紅細胞計數(shù)網(wǎng)織紅細胞計數(shù)對確診再生性貧血反應是最為準確的。在犬貓臨床上，網(wǎng)織紅細胞過多癥與紅細胞多

14、染性存在普遍關聯(lián)。網(wǎng)織紅細胞僅能夠通過活體染色法（例如新甲基藍或亮甲酚藍）來辨別，這時聚集的核糖體RNA顯露。網(wǎng)織紅細胞在形態(tài)學上分集結狀或點狀。犬僅存在集結狀網(wǎng)織紅細胞，但貓二者都有。在犬上，網(wǎng)織紅細胞百分比（在紅細胞中）需要表示為絕對值(×109/l)。網(wǎng)織紅細胞計數(shù)>60 ×109/l是再生性貧血反應的象征，>400 ×109/表示顯著的再生。作為另外一個選擇，網(wǎng)織紅細胞生成指數(shù)（RPI）也可能需要確定。RPI對“校正”貧血程度的評估和網(wǎng)織紅細胞壽命有重要價值。1）貧血程度校正校正值%（或計數(shù)值）=絕對值%（或計數(shù)值）× PCV值/正常

15、PCV值 (45)2）網(wǎng)織紅細胞壽命校正RPI = 校正值 % × 1/血液成熟時間血液成熟時間取決于PCV值：PCV 45% 1 天 35% 1.5 天 25% 2 天 15% 2.5 天RPI >2.5時，表示再生性貧血反應。 貓集結狀網(wǎng)織紅細胞最初從骨髓中被釋放，在經(jīng)過10-12天的成熟期后，轉變?yōu)辄c狀網(wǎng)織紅細胞。 相比之下，點狀網(wǎng)織紅細胞在循環(huán)血液中出現(xiàn)需要3-4周。在貓上，集結狀網(wǎng)織紅細胞計數(shù)可判定再生性貧血，它反映骨髓再生活躍程度，而點狀紅細胞計數(shù)代表累積再生貧血。集結狀紅細胞計數(shù)>40 ×109/l表示再生性貧血，>200 ×109

16、/l時表示顯著再生性貧血。 骨髓評估對于非再生性貧血的病例，骨髓評估是診斷過程的一個重要步驟。可能需要采集兩種樣本：抽取骨髓內容物或用骨髓針穿刺活檢。前一種樣本給臨床病理學專家分析，而后一種樣本給組織病理學專家分析。骨髓穿刺針的優(yōu)點是病理組織可得到更好的評估，但要獲得優(yōu)質的穿刺活檢物卻具有挑戰(zhàn)性。將穿刺活檢物放入福爾馬林溶液固定之前先做壓片。通常在股骨干骨（貓）或髂骨嵴（犬）來獲得樣本。在大型犬，肋骨或胸骨節(jié)是骨髓抽取的備用位置。抽取的骨髓樣本應放入EDTA或ACD（可從采血袋中獲得）抗凝血劑中保存，同時至少要制備10張壓片，迅速空氣干燥后染色。主要的譜系細胞應該按照成熟程度、所占的相對比例、

17、粒細胞和紅細胞比例的順序依次確定。同時應該檢測異常細胞（腫瘤細胞）。 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