Platelet clumping can cause a falsely lowered platelet count on hematology instruments and can be difficult to resolve. With thrombocytopenia, physicians need an accurate count to diagnose, treat, or monitor patients. Clumping can be due to pre-analytic issues with specimen handling, can be caused by medications, or may be an in vitro phenomenon caused by anticoagulants. The clumping makes precise counting impossible and even estimates can be very tricky. If there are clumps, and recollection of the sample still yields platelet clumping, then many labs will have an alternate tube drawn or an alternative method to help resolve clumping.
血小板凝集可导致血液学仪器上的血小板计数假性降低，并且可能难以解决。对于血小板减少症，医生需要准确的计数来诊断、治疗或监测患者。结块可能是由于标本处理的分析前问题，可能是由药物引起的，也可能是由抗凝剂引起的体外现象。结块使得精确计数变得不可能，甚至估计也可能非常棘手。如果有团块，并且样本的回忆仍然产生血小板结块，那么许多实验室将抽取备用试管或替代方法来帮助解决团块。
Many of us have heard of using sodium citrate tubes for patients who have clumped platelets in EDTA. So, if you are having platelet clumping headaches, you can just order some sodium citrate tubes and start using those on your hematology analyzers, right? Not so fast. There are many published references of the use of sodium citrate tubes to resolve EDTA induced thrombocytopenia but we still see samples in which the clumping is not resolved with the sodium citrate tube. Published studies have shown that several other alternate methods have been helpful in resolving platelet clumping issues. These include drawing specimens in CTAD, ACD, or ‘ThromboExact’
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tubes, or adding amikacin or kanamycin to the EDTA after the specimen is drawn.
我们中的许多人都听说过将柠檬酸钠管用于EDTA血小板聚集的患者。所以，如果你有血小板聚集性头痛，你可以订购一些柠檬酸钠管，然后开始在血液分析仪上使用，对吧？没那么快。有许多已发表的参考文献使用柠檬酸钠管解决EDTA诱导的血小板减少症，但我们仍然看到使用柠檬酸钠管无法解决结块的样本。已发表的研究表明，其他几种替代方法有助于解决血小板聚集问题。这些方法包括在 CTAD、ACD 或“ThromboExact”
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管中抽取标本，或在抽取标本后将阿米卡星或卡那霉素添加到 EDTA 中。
So, why can’t we just order one of these other tubes and start reporting results? Hematology analyzers are only FDA approved for EDTA tubes. Before you can use any modified method, and before you can report any patient results, your laboratory must do validation or verification studies to prove that the method produces valid results.
那么，为什么我们不能只订购这些其他试管之一并开始报告结果呢？血液分析仪仅获得 FDA 批准用于 EDTA 管。在使用任何修改后的方法之前，在报告任何患者结果之前，您的实验室必须进行验证或验证研究，以证明该方法产生有效结果。
A validation provides objective evidence that a test performs as intended. A validation uses a defined process and is used when setting up and implementing a new test. One example is a laboratory developed test (LDT), which is a test performed by the clinical laboratory in which the test was developed. A LDT can be one that is neither FDA-cleared nor FDA-approved or can be one that is FDA cleared/approved but has been modified by the performing laboratory. The use of sample types or the use of collection devices not listed in manufacturer instructions constitute modifications, by this definition. In a validation, accuracy should be tested with at least 40 samples across the analytical measurement range (AMR). Correlations are then performed. Precision should be tested over approximately 20 days. A verification, on the other hand, uses an abbreviated process and is used when setting up and implementing new tests that are cleared or approved by FDA. Before reporting patient results, the laboratory must demonstrate that a test performs in agreement with prior claims and must demonstrate performance specifications are comparable to the manufacturer’s specifications. Verification therefore is a confirmation that a test method meets specified requirements and would be applied to a method which has already been validated. For a verification, a smaller sample size may be used, and precisions tested over 5 or more days.
验证提供了测试按预期执行的客观证据。验证使用定义的过程，并在设置和实现新测试时使用。一个例子是实验室开发的测试 （LDT），这是由开发测试的临床实验室执行的测试。LDT 可以是既未经 FDA 批准也未经 FDA 批准的 LDT，也可以是已获得 FDA 批准/批准但已被执行实验室修改的 LDT。根据此定义，使用样品类型或使用制造商说明中未列出的收集设备构成修改。在验证中，应在整个分析测量范围 （AMR） 内使用至少 40 个样品测试准确性。然后执行关联。精度应在大约 20 天内进行测试。另一方面，验证使用简化的过程，用于设置和实施经 FDA 批准或批准的新测试。在报告患者结果之前，实验室必须证明测试的性能与先前的声明一致，并且必须证明性能规格与制造商的规格相当。因此，验证是确认测试方法符合特定要求，并将应用于已经验证的方法。为了进行验证，可以使用较小的样本量，并在 5 天或更长时间内测试精度。
Table 1. Validations vs. Verifications
表 1.验证与验证
So, which would you do if you wanted to use an alternate method for reporting platelet counts? Hematology analyzers are only FDA approved for platelet counts on EDTA, but the by which the sample is analyzed does not change with an alternate tube, so it may be possible to do a limited validation or verification with a smaller sample size. A laboratory needs to prove correlation, accuracy, and precision. Follow your laboratory SOPs for validation/verification and consult with your accrediting agencies, if necessary. A plan needs to be written and signed off by laboratory director. Choose the alternative method you wish to investigate and run correlations for platelet counts on EDTA and the alternate anticoagulant. If your instrument has more than one platelet mode, it is important to run samples in the mode which you would normally use for thrombocytopenia or flagged platelet counts. Apply any dilutional factors and calculate correlations. This data will be Included in your report, which, along with a procedure needs to be signed by the laboratory director.
那么，如果您想使用另一种方法来报告血小板计数，您会怎么做？血液分析仪仅获得 FDA 批准用于 EDTA 的血小板计数，但分析样品的依据不会因备用试管而改变，因此可以使用较小的样本量进行有限的验证或验证。实验室需要证明相关性、准确性和精密度。请遵循您的实验室 SOP 进行验证/验证，并在必要时咨询您的认证机构。计划需要由实验室主任编写并签字。选择您希望研究的替代方法，并运行血小板计数与EDTA和替代抗凝剂的相关性。如果您的仪器具有多种血小板模式，请务必以通常用于血小板减少症或标记血小板计数的模式运行样品。应用任何稀释因子并计算相关性。这些数据将包含在您的报告中，该报告以及程序需要由实验室主任签署。
The most important thing is to write a plan and a follow-up report according to your SOPs and to make sure any requirements of accrediting agencies are included. There can be some differences in interpretation of standards, so it is the laboratory’s responsibility to make sure what you have done meets the standards that apply to your lab.
最重要的是根据您的 SOP 编写计划和后续报告，并确保包括认证机构的任何要求。在对标准的解释上可能存在一些差异，因此实验室有责任确保您所做的工作符合适用于您实验室的标准。
The use of alternate tubes for platelet counts has been well reviewed in literature. Sodium citrate tubes are the most common, likely because they are the easiest to use and the most cost effective. Remember though that sodium citrate and other methods cannot resolve all case s of pseudothrombocytopenia. There are several special notes to consider. Counts from sodium citrate tubes are known to be stable for approximately 3 hours, after which counts decrease. As well, it has been shown in literature that sodium citrate tubes do show a negative bias. It has been reported that the 10% dilutional factor may be too low. Some studies have been done to determine dilution factors that correlate more closely with EDTA tubes, and researchers have suggested factor of 17%-25%. If your laboratory wishes to determine its own dilutional factor for sodium citrate or other tubes, this will also have to be included in your platelet studies. Lastly, CBCs are calibrated for EDTA, so only the platelet count should be reported from an alternative anticoagulant.
使用备用试管进行血小板计数已在文献中得到充分评价。柠檬酸钠管是最常见的，可能是因为它们最容易使用且最具成本效益。请记住，柠檬酸钠和其他方法并不能解决所有假性血小板减少症的病例。有几点需要特别注意。已知柠檬酸钠管的计数稳定约 3 小时，之后计数减少。同样，文献表明，柠檬酸钠管确实显示出负面偏倚。据报道，10%的稀释因子可能太低。已经进行了一些研究来确定与EDTA管更密切相关的稀释因子，研究人员建议因子为17%-25%。如果您的实验室希望确定柠檬酸钠或其他试管的稀释因子，这也必须包含在您的血小板研究中。最后，全血细胞计数应针对 EDTA 进行校准，因此仅应报告替代抗凝剂的血小板计数。
The end of another busy and challenging year is upon us, and at this time of year we can find ourselves rushed to finish ‘end of year’ tasks such as competencies and continuing education requirements. and a response to Sysmex’s recent webinar “Those Sticky, Tricky Platelets – Solving the Puzzle of Platelet Clumping” (Oct.20,2021). After the webinar I had many questions from techs asking, “Do we need to validate our alternative method?” and “How do we go about doing that?” The webinar discusses pseudothrombocytopenia and its causes in more detail than my earlier blog from Oct 2019, “Hematology Case Study: The Story of the Platelet Clump: EDTA-Induced Thrombocytopenia”. The webinar can be found at https://webinars.sysmex.com/webinars/11ae743e-ac99-47e7-acb7-2b24cedc1a1a and is available for CEU, free of charge.
又一个忙碌而充满挑战的一年即将结束，在每年的这个时候，我们会发现自己急于完成“年终”任务，例如能力和继续教育要求。以及对希森美康最近的网络研讨会“那些粘稠、棘手的血小板——解决血小板结块的难题”（2021 年 10 月 20 日）的回应。网络研讨会结束后，技术人员向我提出了许多问题，他们问：“我们是否需要验证我们的替代方法？”以及“我们如何去做？”网络研讨会比我之前在 2019 年 10 月的博客“血液学案例研究：血小板团块的故事：EDTA 诱导的血小板减少症”更详细地讨论了假性血小板减少症及其原因。该网络研讨会可在 https://webinars.sysmex.com/webinars/11ae743e-ac99-47e7-acb7-2b24cedc1a1a 上找到，并免费提供给 CEU。
References
引用
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-Becky Socha, MS, MLS(ASCP)
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graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years. She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.
-Becky Socha，MS，MLS（ASCP）
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毕业于马萨诸塞州安多弗的梅里马克学院，获得医学技术学士学位，并在马萨诸塞大学洛厄尔分校完成了临床实验室科学硕士学位。她担任医疗技术专家已有 40 多年，并在梅里马克学院、马萨诸塞大学洛厄尔分校和史蒂文森大学担任兼职教员 20 多年。她曾在临床实验室的所有领域工作过，但对血液学和血库特别感兴趣。她目前在马里兰州巴尔的摩的 Mercy 医疗中心工作。当她不忙于成为一名疯狂的科学家时，她可以在外面骑自行车。
