Ultramicroscopic Observation of Salvaged Blood During Craniotomy<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

梁　辉  首都医科大学2000级硕士研究生 
王保国  教  授
中国医学科学院首都医科大学附属北京天坛医院麻醉科, 北京100050
Hui Liang, MD and Baoguo Wang, MD
Department of Anesthesiology, Beijing Tiantan Hospital, Capital University of Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100050

ABSTRACT

Objectives: To investigate the surface morphological alterations of salvaged erythrocytes processed by different cell-saver during craniotomy and determine whether any exogenous impurity is mixed in the salvaged blood.
Methods: The shed blood of fifteen patients undergoing craniotomy was processed by three kinds of cell-saver systems, Ziti-2000 (group I, n=5), COBE BRAT-2 (group II, n=5) and Fresenius CATS (group III, n=5). Samples of the salvaged blood and three units of two-week-old banked packed red blood cell were observed using a scanning electron microscope. Quantitive assessment was made to determine the percentage of discocytes (DP) and the relative number of tiny particles, which was expressed as ratio of the number of particles to the counted erythrocytes (P/E), in every micrograph. The whole specimen was also scanned to search the particles bigger than erythrocytes.
Results: Before washing, some cell debris in groups ⅠandⅡ  were found, and DP was less than 40%. After washing, P/E significantly decreased (p<0.01), and DP significantly increased (p<0.01). The proportion of discocytes of the washed blood in three groups were similar. But some particles and even a neurocyte bigger than erythrocyte still could be found in the washed blood. Some salvaged erythrocytes had undergone marked morphological changes, including swell, echinocytes and stomatocytes. However, some cell debris and microaggregates were also found in the banked blood.
Conclusions: Comparing with two-week-old banked packed red blood cell, the quality of the washed erythrocytes processed by cell-saver systems during craniotomy is satisfactory. Neither of the tested systems could completely remove the particles mixed in the salvaged blood. The influence of the particles on the organ function is still remained further research.

　　术中血液回收（intraoperative blood salvage, IBS）是一种非常有效的血液保护方法，其根据对收集血液的处理方式不同可分为非清洗式（单纯过滤式）及清洗式（血液回收全程处理）。后者较为安全在临床应用广泛，尤其是对心血管外科、神经外科、骨科、整形外科及创伤急救等出血较多的手术有重要的价值。虽然已有大量临床应用经验及科学研究证实清洗式血液回收是一种安全、有效的自体输血方法，并且也有较多的对回收血液成分及质量的研究报道，但对于血液回收过程对回收红细胞超微形态的影响以及回收血液中混入杂质颗粒的情况的研究尚少。另外，手术种类对回收血液的质量有较大影响^[1]，相对其他手术而言，颅脑手术中的回收红细胞可能会受到更为严重的破坏，而且由于包括较多对骨及其它组织的操作，难免会有组织碎屑混入收集的血液中，而传统的离心式清洗并不能有效清除密度与红细胞接近的杂质颗粒^[2]，而目前尚无对颅脑手术中回收血液质量的研究。本研究利用扫描电子显微镜（scanning electron microscope，SEM）对颅脑手术中血液回收机清洗前、后的回收血液进行观察，通过对红细胞形态的定量分析，评价回收红细胞的质量，并观察血样中混有杂质颗粒的情况。

资料和方法<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

病例选择 
　　15例择期颅脑手术病人（ASAⅠ～Ⅱ），术前无血液病史，估计术中出血量超过500ml，无IBS的禁忌证（非恶性肿瘤病人、无败血症、手术区无污染），随机分为自体-2000组（I组，5例）、COBE BRAT-2组（II组，5例）及Fresenius C.A.T.S. 组（Ⅲ组，5例）。
血液回收方法  
　　手术开始后，三组分别用国产自体-2000型(北京京精医疗设备有限公司，北京)、COBE BRAT-2型(COBE Cardiovascular Inc., Division of Sorin Biomedica Arvada Co., USA) 及Fresenius C.A.T.S. 型 (Fresenius AG, Bad homburg, Germany) 血液回收机的配套一次性双腔收集管路，以0.02MPa的吸引负压，用相同口径的吸引器收集术中出血。用肝素浓度为50IU/ml的乳酸钠林格液作为抗凝剂，从吸引管路的副腔中注入以与收集的血液混合。术中根据出血情况随时调整抗凝剂滴速，使之保持以与出血体积比为1:5的速度注入。收集的抗凝血液经配套储血罐内的滤芯（I、II、III三组的滤芯孔径分别为40μm、30μm及40μm）过滤后，进入储血罐内储存，并每隔10分钟轻轻摇动储血罐以尽量使收集血液与抗凝剂充分混合。待估计收集血量达到500ml时，记录进血前血液收集的总时间、储血罐内的血水总量及抗凝剂和术野冲洗液的用量，并启动血液回收机的自动程序（automatic）开始对收集的血液进行处理。其中I组选择自动慢速处理程序（血泵速度300ml/min，离心速度5600rpm），II组将泵速调至300ml/min, 离心速度5600rpm。待回收机自动转入清洗程序（wash）的即刻（离心杯中已充满回收血液，清洗前），启动回血程序在回血的过程中在进血管路中采血0.5ml（清洗前），然后启动进血程序重新充满离心杯，两组均用乳酸钠林格液1500ml进行清洗。Ⅲ组使用标准清洗程序（standard）清洗。清洗完毕后，回收的红细胞悬液自动泵入输血袋内储存，依手术进程适时通过PallSQ40S型微聚体滤器（40μm孔径，Pall corporation, East Hills, New York, USA）回输。三组均于清洗后在输血袋内采血0.5ml（清洗后），留检。由于Fresenius C.A.T.S. 型回收机的进血、离心分离、清洗及排空同步进行，因此Ⅲ组只采集清洗后的血样。另外，分别从3单位库存两周的红细胞悬液（packed red blood cell，PRBC）中采血0.5ml，留检。
标本制备
　　采血后立即将各血样分别滴1-2滴在玻片上，不再对玻片上的血滴施加外力（不推片）。在4℃的条件下，分别制备扫描电子显微镜标本。具体制备方法如下: （1）固定：将滴有血滴的玻片浸入2.5％戊二醛（化学纯）中固定2小时。（2）缓冲: 将固定后的标本浸入pH值为7.2的磷酸缓冲液（化学纯）中，每隔10分钟换液一次，共换3次缓冲液。（3）1%饿酸后固定2小时。（4）双蒸水冲洗；标本浸入双蒸水中，每隔10分钟换液一次，共换3次。（5）梯度乙醇溶液脱水；用浓度为50%，70%，90%，100%的乙醇溶液分别浸泡标本10分钟。（6）置换：先后用比例为1:1,3:7以及1:9的乙醇、醋酸异酯溶液中，每隔10分钟换液一次，共换液2次。（7）二氧化碳临界点干燥。（8）离子喷金镀膜：用金铂合金喷镀标本。
观察方法 
　　用扫描电子显微镜（model S-520, HITACHI, Japan）进行超微形态学检验。形态学检验方法: 在400倍的放大率下（工作电压15KV），浏览整个玻片，寻找杂质颗粒，观察血样中杂质微粒的大小及数量，发现直径超过红细胞直径（达到或超过10 μm）者即拍照，再随机选取5个细胞疏密程度适中的放大1000倍的视野拍照。形态学分析方法：统计各1000倍视野中，红细胞总数、形态正常的红细胞的数量以及微小杂质颗粒（主要为细胞碎片及微小组织碎屑，直径明显小于红细胞）的数量。并计算各视野中红细胞形态正常率以及微小杂质颗粒的相对数量（微小杂质颗粒数量与同视野内红细胞总数之比，P/E）。正常形态红细胞的标准[3,4]: 细胞仍保持典型的“双凹圆盘形”，细胞膜光整，表面无突起，中央凹陷区约占细胞单侧表面积的1/3-2/5。观察时只统计整个胞体均在视野内，且单面胞体完全可见的细胞（未被其他细胞遮挡）。 
统计学处理  
　　所得数据以均值±标准差表示，用SPSS 10.0统计软件包进行统计学处理。Ⅰ、Ⅱ两组组内比较采用自身前、后配对t检验，三组间清洗后红细胞形态正常率以及P/E的比较采用单因素方差分析。P<0.05认为有显著性差异。

结　果<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

         三组血液收集时间，抗凝剂及术野冲洗液用量相近（表1）。扫描电子显微镜观察发现: 在血液回收过程中，部分红细胞发生了形态改变，其中以不同程度的肿胀为最普遍的现象(图1.A)，也可发现部分棘形(echinocyte, 图1.C)及口形红细胞(stomatocyte, 图1. E)。清洗前，Ⅰ、Ⅱ两组红细胞形态正常率均小于40％(图1. A, C)，但清洗后均明显升高（p<0.01）(图1. B, D)，各组清洗后红细胞形态正常率均在(55-60)%的范围内(表 2)。许多库存两周的红细胞也发生了明显的形态改变，其中可发现较多不同级别的棘形红细胞。其红细胞形态正常率为(31.5±9.7)%, 与清洗前的回收血液较为接近(图1. F)。回收血液清洗前可发现较多微小杂质颗粒（主要包括细胞碎片、微小组织碎屑及其它微小杂质）(图1. A, C)。清洗后，微小杂质颗粒明显减少，P/E明显下降（p<0.01）,III组的P/E整体上优于I、II组; 但三组均偶见微聚体及组织碎屑（最大直径均在10-40μm左右）（图2. A, B），甚至还偶见神经细胞（图2. C）。库存两周的红细胞悬液中也可发现细胞碎片及少量直径大于红细胞的微聚体（图2. D）。

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