血小板 (Blood platelet)是哺乳动物血液中的有形成分之一，是从骨髓成熟的巨核细胞胞质裂解脱落下来的具有生物活性的小块胞质。正常情况下，血小板以分散的状态在血管内运行，当血管损伤、血液流变学改变或受化学物质刺激时，血小板之间会发生变形、黏附、聚集、释放等变化，进而发生交联，促进血栓形成。血小板在血栓形成过程中起关键作用，制备有活性的血小板或洗涤血小板是研究抗血栓药物对血小板功能的影响及作用机制的实验基础。
富血小板血浆 (PRP)制备液又称富含血小板血浆分离液，能制备具有活性的血小板，可将血小板与其他血细胞成分分开，但不与血浆分开，操作简单，该试剂未经过滤除菌，100ml该溶液可分离900ml新鲜血液。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、新鲜全血
2、微量吸管、离心管或试管、离心机、细胞计数板 (备选)、显微镜 (备选)
操作步骤(仅供参考)：
1、取2周内未应用过阿司匹林等抑制血小板功能药物的新鲜血液，迅速按新鲜血液: 富血小板血浆 (PRP)制备液=9: 1加入离心管或试管中混匀。
2、1000rpm/min离心10min，如果效果不佳可延长至15min。
3、取上层溶液，即为富血小板血浆，注意不要吸取中间层。
4、如需计数可用血小板稀释液进行稀释后，置于显微镜高倍镜下依次计数中央大方格内四角和中央共五个中方格内血小板数：血小板数/L=5个中方格内血小板数×10⁹/L
5、一般22℃或4℃保存，如长期保存应加入6%DMSO，血小板在体外活性维持时间较短，一般在6h以内完成下游实验。
结果分析：
制备的血小板血浆已经将血小板与其他血细胞成分分开，但不与血浆分开。
注意事项：
1、针刺深度应稍深，拭去第一滴血后，首先采血做血小板计数。操作应迅速，防止血小板聚集。
2、该试剂可将血小板与其他血细胞成分分开但不与血浆分开，操作简单易行。
3、血小板充入计数池后一定要静置10-15min，室温过高时应注意保持计数池周围的湿度，防止水分蒸发；计数时光线应适中，应注意有折光性的血小板和杂质相区别；用相位显微镜计数，效果更佳，更准确。
4、试剂开封后请尽快使用，以防影响后续实验效果。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Platelets are one of the formed elements in the blood of mammals. They are small, biologically active cytoplasmic fragments shed from the cytoplasm of mature megakaryocytes in the bone marrow. Under normal physiological conditions, platelets circulate in a dispersed state within blood vessels. When blood vessels are damaged, blood rheology changes, or platelets are stimulated by chemical substances, they undergo a series of changes including deformation, adhesion, aggregation, and release, followed by cross-linking, which promotes thrombosis.
Platelets play a key role in the process of thrombosis. Preparing functional platelets or washed platelets serves as the experimental basis for investigating the effects and mechanisms of antithrombotic drugs on platelet function.
Platelet-Rich Plasma (PRP) Preparation Solution, also known as Platelet-Rich Plasma Isolation Solution, is designed to prepare functional platelets. It can separate platelets from other blood cell components while retaining them in the plasma, with simple and convenient operation. The reagent is not sterilized by filtration; 100 ml of this solution can be used to process 900 ml of fresh blood. This reagent is for research use only and not intended for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. Fresh whole blood
2. Micropipette, centrifuge tubes or test tubes, centrifuge, hemocytometer (optional), microscope (optional)
Operating Procedures (For Reference Only)
1. Collect fresh blood from donors who have not taken platelet function inhibitors (e.g., aspirin) within 2 weeks. Promptly mix the fresh blood with PRP Preparation Solution at a ratio of 9:1 in a centrifuge tube or test tube.
2. Centrifuge the mixture at 1000 rpm for 10 minutes. If the separation effect is unsatisfactory, extend the centrifugation time to 15 minutes.
3. Collect the supernatant, which is the platelet-rich plasma. Avoid aspirating the middle layer during collection.
4. For platelet counting (if needed), dilute the PRP with platelet diluent. Then place the diluted sample on a hemocytometer and count the platelets in five medium-sized squares (the four corner squares and the central square) under the high-power lens of a microscope.
5. Calculation formula: Platelet count (per L) = Number of platelets in 5 medium-sized squares × 10⁹/L
6. Store the prepared PRP at 22°C (room temperature) or 4°C generally. For long-term storage, add 6% DMSO. Note that platelets maintain their biological activity for a short period in vitro; it is recommended to complete subsequent downstream experiments within 6 hours.
Result Analysis
The prepared platelet-rich plasma achieves the separation of platelets from other blood cell components, while keeping platelets combined with plasma rather than isolating them.
Precautions
1. When collecting blood, the needle insertion depth should be slightly deeper. Wipe off the first drop of blood, then collect the blood sample for platelet counting immediately. Perform all operations rapidly to prevent platelet aggregation.
2. This reagent separates platelets from other blood cell components without separating them from plasma, featuring simple and feasible operation.
3. After loading the platelet suspension into the counting chamber, allow it to stand undisturbed for 10-15 minutes. If the room temperature is too high, maintain humidity around the counting chamber to prevent water evaporation. Use moderate light intensity during counting, and distinguish between refractive platelets and impurities. Counting under a phase-contrast microscope yields better and more accurate results.
4. Use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
5. For your safety and health, wear a lab coat and disposable gloves during operation.
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