Follow the Indicies. It’s Not Always Cold!

One of my favorite things about working in Hematology is handling those “difficult” samples. You know the ones. The one that some techs put aside to work on “later,” or they might decide it’s time to take a break when they see them coming. I love investigating and working on these interesting but perhaps uncooperative samples. At times this involves running samples in different modes, making new slides or albumin smears, and diluting samples. At other times, we investigate a delta or unusual results by checking patient diagnosis and previous results or by calling the care provider for more information and clues to help us resolve the problem.
在血液学工作中，我最喜欢的事情之一就是处理那些“困难”的样本。你知道的。一些技术人员把“以后”放在一边工作，或者当他们看到他们来时，他们可能会决定是时候休息一下了。我喜欢调查和研究这些有趣但可能不合作的样本。有时，这涉及以不同模式运行样品、制作新的载玻片或白蛋白涂片以及稀释样品。在其他时候，我们会通过检查患者诊断和既往结果，或致电医务人员以获取更多信息和线索来帮助我们解决问题，从而调查 delta 或异常结果。

I’m sure you’ve all seen the sayings “Without the Lab, you’re only guessing” and “Laboratory Professionals get results.” Physicians rely on the lab every day for information used to help diagnose and treat patients. Therefore, our goal is to deliver to the care provider the best possible results in a timely manner. Which means that we don’t just report results because that’s the answer the instrument gave us. With today’s instruments and middleware, we get very accurate and precise results, and about 85% or more of hematology specimens autovalidate. This is important because it leaves us time to work on those specimens with flags, and discrepancies; the ones that need a little more time and attention.
我相信你们都看过“没有实验室，你只是在猜测”和“实验室专业人员获得结果”这句话。医生每天都依靠实验室获取用于帮助诊断和治疗患者的信息。因此，我们的目标是及时向护理提供者提供最佳结果。这意味着我们不只是报告结果，因为这是仪器给我们的答案。使用当今的仪器和中间件，我们获得了非常准确和精确的结果，大约 85% 或更多的血液学标本可以自动验证。这很重要，因为它让我们有时间处理那些带有标志和差异的标本;那些需要更多的时间和注意力。

When faced with unusual or conflicting results, we first need to ask ourselves if we are dealing with a spurious sample, interfering substances or true abnormal results. Many labs today use middleware that will give the operator alerts when a sample needs to be investigated. These alerts give us suggestions as to how to handle the specimen but are usually short phrases triggered by certain values or flags and cannot be all encompassing. Operator alerts cannot tell us all the steps we may need to follow to resolve, for example, deltas, platelet clumps, abnormal scattergrams or a possible cold agglutinin. The alerts are great guidelines but it is often necessary to do more. We may need to refer to procedure manuals for SOPS or check instrument manuals or technical bulletins to decide how to handle these specimens. Sometimes we need to be detectives to report the most accurate results. We must review results with a critical eye, use all that “stuff” we learned in school, and be able to make educated decisions based on this investigation.
当面对异常或相互矛盾的结果时，我们首先需要问问自己，我们是在处理虚假样品、干扰物质还是真正的异常结果。如今，许多实验室都使用中间件，当需要研究样品时，这些中间件会向操作员发出警报。这些警报为我们提供了有关如何处理标本的建议，但通常是由某些值或标志触发的简短短语，不能包罗万象。操作员警报无法告诉我们解决所有问题可能需要遵循的步骤，例如，deltas、血小板团块、异常散射图或可能的冷凝集素。警报是很好的指南，但通常需要做更多的事情。我们可能需要参考 SOPS 的程序手册或检查仪器手册或技术公告，以决定如何处理这些标本。有时我们需要成为侦探来报告最准确的结果。我们必须以批判的眼光审查结果，使用我们在学校学到的所有“东西”，并能够根据这项调查做出明智的决定。

In my experience, one of the most common troublesome and perhaps misunderstood specimens I see is the one with a “hemoglobin (Hgb) interference” flag. An instrument flag “suspect, turbidity /Hgb interference?” is generally initiated when the MCHC is above a certain value. In our hematology lab, we see this flag when the MCHC is above 37.5 g/dL. What this is telling us is that turbidity may be present in the diluted and lysed sample. This turbidity can interfere with the Hgb detection light path and falsely increase the Hgb. Because the MCH and MCHC are calculated using the Hgb, these parameters are also affected. BUT, an MCHC >37.5 g/dL is not always something that can be or that needs to be corrected. With any parameter 95% of normal values will fall within 2SD of the mean. This means that 5% of normal healthy individuals have MCHC results <32 g/dL or >36 g/dL, and a few may have an MCHC over 37.5 g/dL. An MCHC >37.5 g/dL therefore can indicate a normal specimen, such as in a healthy young male with a Hgb at the high end of the reference range. High MCHCs can also be seen routinely in specimens from patients with spherocytosis or hemoglobinopathies such as Hgb SS, Hgb SC or Hgb C disease. In these conditions the RBCs are hyperdense due to altered surface volume and this leads to a high MCHC.
根据我的经验，我看到的最常见麻烦且可能被误解的标本之一是带有“血红蛋白（Hgb）干扰”标志的标本。当 MCHC 高于某个值时，通常会启动仪器标志“可疑，浊度/Hgb 干扰？”。在我们的血液学实验室中，当 MCHC 高于 37.5 g/dL 时，我们会看到这个标志。这告诉我们，稀释和裂解的样品中可能存在浑浊度。这种浊度会干扰 Hgb 检测光路并错误地增加 Hgb。由于 MCH 和 MCHC 是使用 Hgb 计算的，因此这些参数也会受到影响。但是，MCHC >37.5 g/dL并不总是可以或需要纠正的。使用任何参数，95% 的正常值将落在平均值的 2SD 以内。这意味着 5% 的正常健康个体的 MCHC 结果<32 g/dL 或 >36 g/dL，少数人的 MCHC 可能超过 37.5 g/dL。因此，MCHC >37.5 g/dL 可以表明样本正常，例如在 Hgb 处于参考范围上限的健康年轻男性中。在球形红细胞增多症或血红蛋白病（如 Hgb SS、Hgb SC 或 Hgb C 病）患者的标本中也经常可见高 MCHC。在这些条件下，由于表面体积的改变，红细胞密度过高，这导致高 MCHC。

On our instrument, an MCHC >37.5 g/dL will cause a Hgb/Turbidity flag. An asterisk (*) will appear next to the Hgb, MCH and MCHC. The middleware triggers an operator alert that says “MCHC >37.5. Incubate at 37C for 30 mins. Evaluate for lipemia, icterus, hemolysis, Plasma replacement if indicated, rerun”. So, what’s the first thing to do?? Incubate? Hold on…not so fast. This is one of those instances where hematology is not just black and white. This operator alert is giving us suggestions of how to handle a specimen, but techs need to evaluate the specimen before jumping on the ‘cold’ wagon. Incubating will usually help resolve a cold agglutinin, but won’t help with a sickle cell specimen, or resolve one that’s icteric or lipemic. A grossly hemolyzed sample can give a spurious high MCHC result and, if so, needs to be recollected, not warmed. Putting a specimen that’s hemolyzed or lipemic or icteric in the heating block for 30 or more minutes would only delay reporting of results. My first case example involves a 45 year old female. The MCHC on initial run was 38.1 and the specimen gave a Hgb turbidity flag. The sample was incubated and rerun several times. After 1 hour of incubation, the MCHC was reported as 37.1 with a comment “repeated after warming for 1 hour at 37C”. In this case the patient was a known sickle cell patient. Previous results show that this patient’s MCHC is typically high and previously reported results ranged from 36.1- 37.8 g/dL. When evaluating a specimen with a high MCHC it is important to check the pattern of results. In this case the MCHC was high but the MCV was low. This does not fit the pattern for a cold agglutinin. As noted above, super dense RBCs in sickle cell patients may cause a high MCHC. This specimen was warmed, and even though the MCHC was a bit lower after warming, it would have been acceptable to report the original run MCHC. Checking patient history and previous results, and reviewing the smear for morphology would have allowed these results to be reported in a timely fashion. The operator alert does say “incubate the specimen” but it also says to evaluate. Be sure to check the MCV and MCHC along with patient history before warming specimens that don’t fit the pattern of a cold agglutinin.
在我们的仪器上，MCHC >37.5 g/dL 将导致 Hgb/浊度标志。星号 （*） 将出现在 Hgb、MCH 和 MCHC 旁边。中间件会触发操作员警报，显示“MCHC >37.5.在 37C 下孵育 30 分钟。评估脂血症、黄疸、溶血、血浆置换（如有指征），重新运行“。那么，首先要做的是什么？？孵化？坚持。。。没那么快。这是血液学不仅仅是非黑即白的例子之一。这个操作员警报为我们提供了如何处理标本的建议，但技术人员需要在跳上“冷”货车之前评估标本。孵育通常有助于解析冷凝集素，但对镰状细胞标本或黄疸或脂血症标本无济于事。严重溶血的样本可能会产生虚假的高 MCHC 结果，如果是这样，则需要重新收集，而不是加热。将溶血、贫血或黄疸的标本放入加热块中 30 分钟或更长时间只会延迟结果报告。我的第一个案例涉及一名 45 岁的女性。初次运行时的MCHC为38.1，标本给出了Hgb浊度标志。将样品孵育并重新运行数次。孵育 1 小时后，MCHC 报告为 37.1，并附有“在 37C 下加热 1 小时后重复”的评论。在本例中，患者是已知的镰状细胞患者。既往结果显示，该患者的 MCHC 通常较高，先前报告的结果范围为 36.1-37.8 g/dL。在评估MCHC高的标本时，检查结果模式很重要。在这种情况下，MCHC较高，但MCV较低。这不符合冷凝集素的模式。如上所述，镰状细胞患者的超致密红细胞可能导致高 MCHC。这个标本是加热的，即使MCHC在加热后有点低，报告原始运行的MCHC也是可以接受的。检查患者病史和既往结果，并检查涂片的形态学，可以及时报告这些结果。操作员警报确实说“孵育标本”，但它也说要评估。在加热不符合冷凝集素模式的标本之前，请务必检查 MCV 和 MCHC 以及患者病史。

The second example is from a 75 year old male. The CBC flagged Hgb turbidity with an MCHC of 45.8 g/dL. The MCHC >37.5 operator alert triggered Checking the pattern of results for the indicies, the MCHC was very high and the MCV was low. In a specimen with a low or normal MCV and a high MCHC, lipemia, icterus, abnormal proteins or severe leukocytosis can be affecting the Hgb. On evaluation, this sample’s Hgb and Hct did not meet the ‘rule of 3’. The rules of 3 are now generally recognized to be valid only for samples when the RBCs are normal, but the * here is telling us that there is an interference affecting the Hgb. In these cases it is valuable to know what the interference is so we know how to handle the specimen. By spinning down a small aliquot, (or asking chemistry!) we can investigate for lipemia or icterus. The specimen was found to be grossly lipemic. Flagging guidelines for lipemic specimens suggest diluting the specimen 1:5 and rerunning. Alternately, with severely lipemic or icteric samples, plasma replacement procedure may be necessary to correct the results. In this case, a plasma replacement was performed. After a plasma replacement, the WBC, RBC, Hct, MCV and platelet count are reported from the original run. The Hgb interference is what was causing the problem. Thus, when you correct the Hgb you must always correct any indicies that are calculated with the Hgb. The Hgb from the plasma replacement sample is used and the MCH and MCHC are recalculated. Notice that the new lower Hgb value now matches the Hct.
第二个例子来自一名75岁的男性。CBC 标记的 Hgb 浊度为 45.8 g/dL。MCHC >37.5 操作员警报触发 检查指标的结果模式，MCHC 非常高，MCV 很低。在MCV低或正常以及MCHC高的标本中，脂血症、黄疸、异常蛋白或严重的白细胞增多可能影响Hgb。经评估，该样品的 Hgb 和 Hct 不符合“3 法则”。现在，人们普遍认为 3 规则仅对红细胞正常时的样品有效，但这里的 * 告诉我们存在影响 Hgb 的干扰。在这些情况下，了解干扰是什么很有价值，这样我们才能知道如何处理试样。通过旋转一个小等分试样（或询问化学！），我们可以研究脂肪血症或黄疸。该标本被发现是严重的脂血症。脂血症标本的标记指南建议将标本以 1：5 的比例稀释并重新运行。或者，对于严重的脂血症或黄疸样本，可能需要血浆置换程序来纠正结果。在这种情况下，进行了血浆置换。血浆置换后，从原始运行中报告 WBC、RBC、Hct、MCV 和血小板计数。Hgb干扰是导致问题的原因。因此，当您校正 Hgb 时，您必须始终校正使用 Hgb 计算的任何指标。使用血浆替代样品中的Hgb，并重新计算MCH和MCHC。请注意，新的较低 Hgb 值现在与 Hct 匹配。

Case 3 is a sample from an 80 year old woman. This was an interesting sample because there were multiple things going on here. This patient had an initial result with a high MCHC and MCH, with decreased RBC and Hct. In this patient the initial WBC was 0.64 and the RBC was 0.31. The Hgb of 9.1 /dL was less than the Hct of 3.1 %. MCV was 116 fl and the MCHC was 293.5 g/dL! In specimens with a high MCV and high MCHC we can suspect a cold agglutinin. When the MCV is very high it is because the RBCs are going through the aperture as one big bunch and this is measured as the size of one RBC. Often the Hct is less than the Hgb. Sometimes the RBC and Hct are so low that it causes the MCV to be appear within normal range. On our instrument, a RBC count of <0.5 x10 6 /μL will give a flag “abnormal RBC scattergram” but no other indicies related flags are generated, so we didn’t even get an operator alert to evaluate the MCHC. But, it’s clear there is something very wrong with these results. Warming the sample is used to loosen clumping of RBCs, which lowers the MCV and allows the RBCs to be counted. Make a smear to examine for RBC clumping and look at the sample tube. Many cold agglutinin samples will appear to be ‘grainy’ or have agglutination along the side of the tube. This is the time when we want to incubate the sample. To resolve a cold agglutinin, warming the sample is necessary. Sometime 30 minutes is enough, sometime they need to be incubated longer. Some cold agglutinins are so strong that after incubation a dilution or plasma replacement still needs to be done. Warming this sample did not lower the MCHC. After incubating, I diluted this sample, and also did a plasma replacement to see how results would compare. The new results matched. This sample took a bit more time than others but the cold agglutinin was resolved and we were able to report valid results.
病例 3 是一名 80 岁女性的样本。这是一个有趣的示例，因为这里发生了很多事情。该患者的初步结果为高 MCHC 和 MCH，RBC 和 Hct 降低。在该患者中，初始白细胞为 0.64，红细胞为 0.31。Hgb 为 9.1 /dL，低于 Hct 的 3.1 %。MCV 为 116 fl，MCHC 为 293.5 g/dL！在MCV和MCHC高的标本中，我们可以怀疑冷凝集素。当 MCV 非常高时，这是因为 RBC 作为一个大束穿过孔径，这被测量为一个 RBC 的大小。通常 Hct 小于 Hgb。有时 RBC 和 Hct 非常低，以至于导致 MCV 出现在正常范围内。在我们的仪器上，红细胞计数为 <0.5 x10 6 /μL 将给出“异常红细胞散点图”标志，但没有生成其他与指示相关的标志，因此我们甚至没有收到操作员警报来评估 MCHC。但是，很明显，这些结果存在严重问题。加热样品用于松动红细胞的结块，从而降低 MCV 并允许计数红细胞。涂片以检查红细胞结块并查看样品管。许多冷凝集素样品会呈现“颗粒状”或沿试管侧面凝集。这是我们要孵育样品的时候。为了分离冷凝集素，需要加热样品。有时 30 分钟就足够了，有时它们需要孵化更长时间。一些冷凝集素非常强，以至于在孵育后仍需要进行稀释或血浆替代。加热该样本并没有降低母婴健康。孵育后，我稀释了这个样品，并进行了血浆置换，看看结果如何比较。新结果匹配。这个样本比其他样本花费的时间要长一些，但冷凝集素得到了解决，我们能够报告有效的结果。

There are other factors that can affect the Hct or Hgb and cause a high MCHC. Icteric specimens act much like lipemic ones and the Hgb can be corrected with dilution or a plasma replacement. An electrolyte balance can affect the Hct. Abnormal proteins and severe leukocytosis can affect the Hgb. Grossly hemolyzed samples can have a high MCHC. It is important to evaluate the indicies in these samples and correlate the values with previous results and patient history. What concerns me is that I have seen samples being warmed that do not match the indicies patterns for cold agglutinins. I have seen samples from sickle cell patients signed out with a comment “warmed at 37C. Possible cold agglutinin.” I have seen lipemic or icteric samples that are reported out with high MCHCs, erroneously high Hgb or parameters that are not reported at all. While warming these samples may actually lower the MCHC a bit, it still usually remains on the high side and does not give us the clean results that dilution or plasma replacement will. A little extra time looking at the indicies can give us important clues as to how to handle these samples. Doctors use our results every day to make patient care decisions. We need to make sure that we are making decisions every day to give them the best possible results so that patients can get the best care possible.
还有其他因素会影响 Hct 或 Hgb 并导致高 MCHC。黄疸标本的作用与脂血症标本非常相似，Hgb可以通过稀释或血浆替代来校正。电解质平衡会影响 Hct。异常蛋白质和严重的白细胞增多会影响 Hgb。严重溶血的样本可能具有高 MCHC。评估这些样本中的指标并将这些值与先前的结果和患者病史相关联非常重要。令我担心的是，我看到加热的样品与冷凝集素的指示模式不匹配。我看到镰状细胞患者的样本在评论上签字“在 37C 下加热。可能是冷凝集素。我见过脂血症或黄疸样本报告的母婴健康体高、Hgb 错误高或根本没有报告的参数。虽然加热这些样品实际上可能会降低 MCHC，但它通常仍然偏高，并且不会给我们稀释或血浆置换带来的干净结果。多花一点时间查看这些指标可以为我们提供有关如何处理这些样本的重要线索。医生每天都会使用我们的结果来做出患者护理决策。我们需要确保我们每天都在做出决定，为他们提供最好的结果，以便患者能够得到最好的护理。

References 引用

1. Costa, B. M. B., Vellés, M. C., Viana, M. M. F. B., & Rebelo, C. I. M. (2018). Interference of cold agglutinin autoantibodies in erythrogram interpretation: a case report and literature review. Jornal Brasileiro De Patologia e MedicinaLaboratorial , 54 (4). doi: 10.5935/1676-2444.20180043
   Costa，BMB，Vellés，MC，Viana，MMFB和Rebelo，CIM（2018）。冷凝集素自身抗体对红细胞造影解释的干扰：病例报告和文献综述。Jornal Brasileiro De Patologia e MedicinaLaboratorial，54（4）。doi：10.5935/1676-2444.20180043

2. Sysmex USA. XN-Series Flagging Interpretation Guide. Document Number: 1166-LSS, Rev. 6, March 2021
   希森美康美国。XN 系列标记解释指南。文件编号：1166-LSS，修订版6,2021年3月

3. It’s not Black and White: Unraveling the puzzles of Hematology. Becky Socha MS, BB, MLS(ASCP) Mercy Medical Center, Baltimore, MD
   这不是非黑即白的：解开血液学的谜题。Becky Socha MS、BB、MLS（ASCP） 慈善医疗中心，马里兰州巴尔的摩

-Becky Socha, MS, MLS(ASCP) CM