Best method for osmotic fragility test

Expires: December 2, 2023

James Hoyer, M.D.Professor of Laboratory Medicine and PathologyDivision of Hematopathology

Mayo Clinic, Rochester, Minnesota

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Introduction

Interviewer: Well, I'm really excited to be bringing to everyone today a “Hot Topic” presentation. My name is Michaela Erickson. I am a product manager here at Mayo Clinic Laboratories. And today I'm discussing a rather interesting test and topic with one of our consultants in our metabolic hematology laboratory. Dr. James Hoyer is here with me today to discuss osmotic fragility testing. So, Dr. Hoyer, before we really get started and start talking about sort of the meat and potatoes of this testing, could you just give us a little background on yourself? 

Sure. Well, thank you for having me today. I am in the Division of Hematopathology. I've been on staff at Mayo Clinic for I think 27 years, and I've been, as Kala said, one of the directors of the Metabolic Hematology Laboratory at Mayo for over 20 years. 

Disclosure

I have nothing to disclose.

Case study

So I'd like to start by presenting a case. This is not made up, but this is an actual case that we had just probably about a couple of weeks ago. And unfortunately, this is a scenario that repeats itself several times. And in fact, people listening may have been in this particular scenario themselves. So we received a blood specimen for the osmotic fragility test on a four-day-old female. We didn't get any history, but more than likely it was because the baby was either anemic, where the baby was jaundiced and had to go under the bili lights. And as we request with osmotic fragility test, a control specimen was sent and received with the sample and labeled appropriately, and the following results were obtained. Now we'll talk about this more later, but you can see this test has four different sodium chloride solutions that we use. These are very hemodilute, and we'll talk about that a little bit later. And then what's listed here are the results of the test. It's the percent of the red blood cells that lyse or rupture. And you can see off to the right, that's the reference range. That's what a normal specimen would also still have some lyses. And in some you can see that the patient was very abnormal. If you look at the control compared to the rest of the claims the control was abnormal as well. 

Outcome

So what this meant unfortunately is because both the patient results and the control results were abnormal, the test had to be canceled and we could not release these results. We subsequently, as we often do in this scenario, we get a call back from the client, they were very frustrated, he said this was a very difficult draw, it was a very small baby, they thought they may not be able to get another specimen, and couldn't we release the results with a disclaimer saying that the control was abnormal. So that's going to be the topic today, is really the control specimen in this testing. 

That case scenario really hits home for me because there's nothing that any of us hate more than not being able to get an answer for their patient when that's what they're so desperately waiting for. And so I'm really just interested to talk with you about this testing today. 

What is osmotic fragility testing?

Interviewer: So can you give me a little background on what is the osmotic fragility test?

Sure, this test, the osmotic fragility test, is actually a very old test. It's probably one of the oldest tests that is still being performed. I would say it's been around for about 70 years. So for that reason, it's very well entrenched in the medical literature, and in the ordering patterns of many physicians, particularly the older ones, who have been ordering this test their entire careers. Basically, this test measures the ability of red blood cells to resist lysis, or rupture, when it's placed in a hypotonic or hemodilute solution. So that solution is less concentrated than the inside of the red blood cell. And so water enters the cell and causes them to burst. 

What is the test used for?

Interviewer: We've been doing this for a really long time, but what are we actually using this test for? 

This test, as far as I know, almost always is used as a confirmatory test when there's a clinical suspicion of hereditary spherocytosis. And first off, I want to emphasize that this test in and of itself is not diagnostic of hereditary spherocytosis. That is unfortunately a common misconception. You get an abnormal result that means absolutely the patient has hereditary spherocytosis. We don't have time to go into it today, but there are several other things that can produce an abnormal test. But this test does provide supportive information and the right clinical situation, meaning the right history, the right peripheral blood smear. This can very much support that suspicion. Hereditary spherocytosis is a disorder of the red blood cell cytoskeleton, or framework. And here's a really nice picture. And it shows some red blood cells there you can see — one of the interesting features is that it has no nucleus and it's a very pliable cell. This is basically demonstrating the red cell cytoskeleton, or the framework, of the red blood cell. This is a series of proteins that holds it together. It's kind of like, almost looks like chicken wire in a sense. If we get an abnormality in one of these proteins it makes that cytoskeleton weakened. So these red blood cells will have a tendency to rupture such as in our control situation when increased water enters the cell. And the final thing I'd like to say about this test is that it's very important to have an accurate diagnosis of hereditary spherocytosis, because the long established, and to this day, treatment for this disorder is to do a splenectomy. And it's actually very effective. So we want to be as sure about that diagnosis as possible because a splenectomy is not a benign procedure, it’s a major surgical procedure and we don't want to do it for an erroneous diagnosis. 

What's also interesting is that this test, which seems to be very common in the world of hematology, is just a piece of the entire puzzle as you put together that patient profile. And this isn’t just a very important piece, but it does need to be utilized appropriately in the right situation.

How is the test performed?

Interviewer: Real quickly, moving on to my next question, how is this test performed in your laboratory? 

Yeah, good question. Thank you. And I think it's really important just to go over this briefly because it will relate to what we next talk about, the control. So the osmotic fragility test, it's really actually a very simple procedure. It is labor intensive as we'll get into. So what we do is we take an aliquot of the patient sample and we place it in this series of dilute sodium chloride solutions. And so by increasing hypotonicity, or concentration. And you can see here, this is just an example of how we would run the test. We do these actually on microtiter plates and the reason for that is we can use an auto pipetter system then to aliquot out these the sample. So you can see on this plate there are two patients, their labeled P4 and P5, and their accompanying controls are C4 and C5. At the very top you see the different sodium chloride solutions that are up there, and you'll notice that each patient and each control is done twice. That's a lot of pipetting if you would do this manually, and actually people have gotten wrist problems, they’ve gotten carpal tunnel syndrome, from doing too much pipetting. So when we place it in these hypotonic, there's going to be a certain number of red blood cells that are going to burst. And when they do that they release hemoglobin into this solution. And that's what's giving the red color of the samples here. Particularly for example, patient 4 there, you can see almost everything is red. Off to the right you can see these are the sodium chloride concentrations. As I mentioned before, even a normal people, you'll get a certain number of red blood cells. The very top solution is the most dilute one and they're getting a little bit more concentrated as we go. So it makes sense that the most dilute ones have the most cells that will lyse down to the most concentrated. One thing I want to not forget to mention is that the last three tubes are actually incubated. And it's been long known that when you incubate these overnight, it enhances that lyses, particularly in hereditary spherocytosis patients. So you can see that in the control there's some degree of lyses that's going on. So what we can do, is we have this color thing, that we can do an absorbance with a spectrophotometer. And we have a calculation that we use that's going to give us a calculation that gives us back approximately how many red blood cells have lysed in that particular tube. And so you can see down below, I imagined this must be patient 4 because of the abnormal results. This is the results that we would display. And you can see, particularly in the most dilute one, 90% of the red blood cells lice. And even in the most concentrated, there is still a very large number of red blood cells that lyse, so this is extremely abnormal results. This is just the kind of result that a patient with hereditary spherocytosis would get. Now off to the left you can see the results for the control. This is just within the lab, the control resulted I do not believe are reported out, but everything is normal there. And we'll talk about why that's important in just a second. 

So what's really interesting as I listen to you talk is just hearing all of the work that goes into who these patients with not only the incubation and all of the different dilutions, but also making sure that you're running that control appropriately as well. 

Why is the Control Tube so Important to this Particular Testing?

Interviewer: And so my next question really is, why are the control tubes so important?

Yes. Thank you. And that's really not the whole purpose I think of this Hot Topic, is this control. Because over the years, the years that I've been here, there seems to be some misunderstanding of why do you need a control. So to start, we are a reference laboratory in Rochester, Minnesota, and the vast majority of the specimens that we receive come from outside of Rochester and must be shipped in. So in the shipping of the sample to our laboratory, something could happen to that specimen and shipment, it could be compromised or damaged in some way. Most commonly what we know is if it gets too hot or too cold as it's being shipped, this could damage the specimen. And unfortunately, the manifestation of that damage is that these cells either re-lyse or are very much more prone to lyse because of their damage. So what happens then is that simple compromise of the sample will create a positive result on the osmotic fragility test. So the control is really meant to be a shipping control and it is labeled that way, but people maybe don't see that a lot. So it should be a normal person, say someone else that works in the lab, the phlebotomy lab, that’s drawing the specimen that is known to be healthy, is not known to have any kind of problem and is drawn at the exact same time as the patient. Both of those tubes are then put in the same container, travel together to our laboratory, and then they're both run at the same time and in exactly the same way. So as an example that I just presented, if the shipping control gives normal results, and yet the patient's results are abnormal, we can conclude as best we can that there was nothing that happened to that specimen in transit and therefore those results in the patient are valid results. On the other hand, if the control is abnormal as well as the patient, we are very concerned something happened to that specimen in transit and therefore we cannot release those results. We are not a 100% sure that those results are valid; in fact, they are probably not valid results. 

Interviewer: So if I'm hearing you correctly, what this is really about is, like you said, that's an environmental control, but it ensures the fact that a positive patient result really, truly is positive and we aren’t giving them a result that may point them in a direction that is not beneficial for our patient. Really what I'm hearing is it's a double-check to make sure that we're giving those correct answers and we're doing best by our patient. 

Exactly right. And I would hate for us to release the results that we were not sure are right because as I even said before, there's this misconception if the osmotic fragility is abnormal, the patient has hereditary spherocytosis. They get a splenectomy. And we've seen there is almost a knee jerk in some people and we cannot have that. That’s why we are so adamant about this particular test. We have to be because the result of a misdiagnosis. 

Some answered questions

Interviewer: Even though this test has been around for a really long time, we do still get a lot of questions about it. So we actually talked with your laboratory team in metabolic hematology that got a list of some of the most commonly asked questions. One of the first questions that typically comes up is, why can't Mayo use its own controls that we've potentially pulled from a patient here in house? 

Yeah, I think this is probably the one we get more than most other ones. And I think it relates to this misunderstanding that the purpose of the control is like maybe controls in other laboratories, like a chemistry laboratory that, you know, that it's a standard or something to make sure your assay is working correctly. And there are actually other controls. We have in-lab controls that have that purpose. So as we've really outlined this, this is not that kind of control. This is a shipping control to ensure the integrity of the specimen. So we can't use some other specimen or say we can't draw someone in our laboratory to service that control — it has to be with a patient coming at the same time, that's just what's really essential. 

This reminds me about another misconception that we have seen. That the laboratories know that what we say is — you have to have a control. So in an effort, maybe they've forgotten to run a control and the patient's gone, so they just take a CBC tube and just put a label on that says control, and that's what they send in. And we know this because when we peel back the label, we see other labels underneath. One might have a patient name on it. So that maybe just some tube that was sitting has been already tested, they grab it, and it’s the control. A lot of times what happens with that then is that that's an older specimen. Older specimens also are prone to lyse more than fresh specimens. So the control would be abnormal even though the patient might be okay or might have abnormal results. We'd have to cancel that test. 

Interviewer: So the next question that your lab said was really common is why is that SCTRL label for the control not enough to identify the tube as that patient's control sample?

Over the years we've gotten clients that this is a repeat problem for, that it keeps happening again and again. And so they will actually work with them to make sure that it isn't happening. The things they find out is that they're drawing the patient into both the osmotic fragility tube and the control tube, they're filling both with the patient. We require that they hand write an identifier under the control so they ensure us, this is a separate person; it is not the patient's in the control tube as well. The really bad thing about that is if that's a misconception, they drawn both and the patient does have hereditary spherocytosis, what we will see then is abnormal control and abnormal patient because it's basically just duplicate results. But as far as we know, we have to cancel that. And even if we hear back, well, but that's a patient tube, can't you release that? Well, no, we can't because now we don't know because it's the same person that’s serving as the shipping control tube. That's a very unfortunate situation if that would happen. 

Interviewer: So really what that piece comes down to is making sure that the phlebotomist and your client team is appropriately informed that those have to be separated and they have to be on two different people, but drawn at the same time. And that's why we need that second sign off and identifier.

Exactly right. 

Interviewer: The next question that the lab told me to throw at you was why can’t the results be released with the disclaimer?

It happens to us on virtually all of our tests that we will get if we have that, we have our strict guidelines as to how old a specimen can be, it's validation limits before it's too old and we don't feel confident about our results, that we say, well, can't you just release those results with the disclaimer? And there are some situations where we try, for example, weekends often cause a problem where maybe we're not testing on the daily, and it’s just barely beyond our stability limit. We will release that, or we feel confident based on the nature of the tests, some tests are not as critical, we feel that they're valid. Say for example, hemoglobin electrophoresis is one, it might be beyond stability, but as we look at the results, we say, we think these results are okay. We’ll need to go do that release for the disclaimer or just release it. But this test, as we talked about with the control, we can't do that. We cannot say something the control was abnormal, so interpret with caution because my feeling when you say something with a disclaimer, you've got to realize they're going to take those results just as if there were no disclaimer and they're going to take those results as valid. And in this particular case, we can’t have that happen. 

Interviewer: So the last question that the lab gave me to ask you is, how come sometimes when a control is not sent, they still are getting results and other times the test is canceled?

In the past what we have done is, you must have a control. It would not make it to our laboratory if there was no control. It got stopped right at accessioning and it really didn't get any further than that. And then we started, I guess, to feel bad particularly in these little kids where they drew it, and so we changed in an effort to be more accommodating, we would say, okay, we'll run it knowing that we may have to cancel it. We will run it without a control and if the patient’s results are normal then we will release it, because it's never happened that the patient is abnormal in shipping it to us and it becomes normal. That's not ever the way it goes. So if it comes to us normal without a control we will release that; however, if it comes to us abnormal and no control we can't, that's the situation. So it's always consistently, normal ones can get released; abnormal ones have to be canceled because no control was released. It gets back to what you were saying. There's a patient behind this, right. And it isn't the patient's fault that maybe somebody misunderstood or forgot to do this. So we will try our best to maybe still be able to release those results even though no control was sent. 

Interviewer: No, that makes total sense to me, a normal is a true normal, but when we have a potential positive, we need to be ultimately very sure. Because again, the decisions that we could be affecting really do have a very big impact on your patient's life down the road and how their disease is going to be treated. 

Thank you

Interviewer: Well, I really appreciate you talking with us today, Dr. Hoyer. I find this test just super interesting. And again, a lot of the clarifications in your discussion today really helped me to understand sort of the ins and outs and the nuances of this particular test. And I appreciate your time.