S1 E2: The Mystery of the Dripping Blood

Holly: Welcome to Tales from the Lab, where an ever optimistic veterinarian and slightly salty technician entertain listeners with true stories and tall tales revolving around laboratory diagnostics.

Jessica: Names have been changed to protect the innocent, but the lab work is real.

Holly: You can listen on your lunch break, on your commute, or when you’re hiding from your kids in the bathroom.

Jessica: Each episode we hope to leave you a little smarter, a little brighter, and feeling more empowered in the lab.

Holly: Here’s today’s tale from the lab, the mystery of the Dripping blood.

Jessica: Oh, no.

Holly: Okay. Oh, it’s recording. Yeah. Okay, here we go.

Jessica: Here we go. This is podcast episode number one. I’m super duper excited. We are currently in our little podcast recording space, AKA the closet. We have thought about doing this podcast for a very long time.

Holly: We.

Jessica: It’s. I love podcasts. I absolutely love them,

and I think it’s such a good way to learn things. And so what a cool opportunity to be able to do this for Clinpath. And I’m just really excited to be.

Holly: Doing this with you and to be taking our passion for education that historically we’ve had to travel to do in front of live audiences and to be able to put into a different format to reach even further.

Yeah.

Jessica: I’m Jessica. I’m the slightly salty technician part of this duo.

I am a certified veterinary technician and also have a BTS in small animal internal medicine.

And I have worked at the same practice for 15 years. I’ve been a technician for 17 years. Nine of those years I got to spend working with Holly at the animal hospital,

splitting my time between clinical pathology and internal medicine, which is not really splitting your time, it’s doing same thing. Because Clinpath is intertwined in internal medicine.

And so the time that I got to spend with her and learning about Clinpath really helped me become a better technician because I could better communicate with my doctors and the clients and really help people understand why we’re doing the diagnostics that we’re doing, which is really made me feel very empowered as a technician.

We also really love to educate, so this podcast is just another form of that love of education because we’re able to give information that we’re learning that we’re using in our day to day at the animal hospital and spread that across to the profession, both technicians and doctors, to help them do better at their jobs.

So it’s a really cool opportunity.

Holly: And I’m Holly. I’m a clinical pathologist, and I have had a unique position working in a hospital setting. So being an on staff, clinical pathologist at a general referral and emergency hospital.

So we see a wide variety of cases and it’s super rewarding for me to get to contribute my expertise. Patient side. Right. Being that close to the patients and looking through the blood work, getting to trend the data like very sensitively alongside the patients, seeing how they respond, looking at other cytologic specimens,

fluid specimens, learning how to maximize that information we can get in particular at a point of care has been something that Jessica and I have really focused on, I think especially in the emergency setting, where really we need to get that information at hand asap and using that information to deliver value.

Right. To the clinicians on the case and to the owners as well.

Jessica: Yeah. And I think adding you into the animal hospital really changed the dynamic. It changed how everybody practiced medicine. The doctors, the technicians like it completely altered kind of how they did diagnostics.

And I think that was what a huge impact you had on have on the animal hospital.

Holly: Yeah. And what a reward for me. Right. So I still consider myself really a clinician at heart. So being that close to the cases and being on the animal care team in that respect.

Right. Working together with those attending or the emergency veterinarians, along with the staff, our laboratory staff, the other nursing staff. I think in general, a big mission of ours was just to raise awareness around the testing and the results so that everyone was on board with an understanding, a better understanding of the case.

I feel like the technicians got more reward when they knew what was going on with their patients, better communication with the clients. And in general, the more informed our clients were, I think the more reward they felt.

Right, right. The better satisfaction through the care they were getting at the hospital and really allowed us to get them involved in that diagnostic process. Right. Having the owner being an important part of that medical care team for their patient as well.

Jessica: Yep. Yeah.

Holly: So then our passion of education, you know, gets to extend further. Right. Beyond the paired talks that we have done, you know, at conferences throughout the country. I’m getting to do this through podcasts where we take some of our favorite cases from the hospital and present them to our listeners,

carry them along the diagnostic journey.

Jessica: Sally, a two year old female beagle, presented to the animal hospital for dripping blood from the back end. That was her little description on the check in sheet. Technician goes in to get the history.

Dad is kind of a gruff, older gentleman.

He reports that Sally runs freely on his property. He has multiple beagles in the household because he uses them as hunting dogs. She is known to get into Things he says she’s eating and drinking.

Everything else looks normal. Normal. He did report that he saw blood dripping two times. Once when she was out kind of running around, he saw a drop of blood. And then when she came inside after, you know, being out going to the bathroom, he did notice one little drop of blood that appeared to be coming from somewhere on her back end.

Holly: So on physical exam, the first thing the clinician on the case checks is of course, make sure she’s not in heat. Right. That would be maybe the most obvious rule out here in a two year old intact dog.

But actually in discussing with the owner, he was well aware of her estrous cycle, she was not in heat,

and she had no vaginal discharge. Actually, anatomically, everything looked normal to the clinician. He did a rectal exam. There were no abnormalities palpated, There was no blood or abnormal feces noted on the glove finger, no problems with her **** glands, and no overt lacerations or changes to the skin to explain where the bleeding could be coming from.

So as a result of a otherwise normal physical exam and understanding getting a little more description on what where this blood may have been coming from, the thought was maybe this was hematuria.

So the clinician on the case collected urine and indeed it was dark red pigmented urine.

Jessica: So I get the lab sample, so I get her urine and I see that it is red in color. So the assumption is that we have hematuria. And so the first thing we do is to spin the urine down.

So we’re going to create our sediment slide from that. And when we spun out Sally’s urine, it did not clear. So in hematuria, when we have blood in our urine, we will get that nice clearing of the urine because the red blood cells will go to the bottom and create that little pellet and then the supernatant will clear out.

And in Sally’s case, we didn’t have.

So now I have to start thinking, is this something else?

And the rest of her urinalysis, our specific gravity was greater than 1060. When I looked at the sediment underneath the microscope, it confirmed what I was already seeing within the tube, that there were not any red blood cells present on the urine sediment.

Holly: So this is really curious. Right? So already the case is taking sort of a 180 from what they thought, which was pretty presenting for hematuria. And indeed when we looked at the urine, grossly thought she did have hematuria.

But when we spun down the sample, and in particular not only to make the sediment, but probably to Run the chemistry strips as well. Right. So with the color metric changes on the chemistry strip for the urinalysis, you know, if it was heavily pigmented, then we’re not going to get an accurate read on that.

Right. So often the technique in the lab, of course, is to spin it down and use a supernatant for the biochemical analysis. But as Jessica said, it didn’t clear out.

Right. The red cells weren’t able to pellet at the bottom, leaving a more clear urine to use for that purpose. So we went from believing we had hematuria to realizing those are not red blood cells in there.

Confirmed on the sediment analysis. Right. And so instead we’re dealing with now we should say pigmented urine or pigmenturia. Right. We’re overstating. So that’s sort of one of our errors there that we tend to do.

Right. Is jump to presumptive diagnoses without doing the lab work. Right. So we find these cases to be really enlightening because it’s always fun when it takes like a 180 and realize that we were off course with our presumptions.

So Ali has pigmented urine that, since it didn’t clear, was not because of hematuria or intact red blood cells. That pigmented urine could either be hemoglobinuria from ruptured red blood cells.

Right. Or myoglobinuria. We have a next test that we can do, and that is that we can spin down her blood in a microhelumatic tube. So just like we’re going to do routinely for a PCV and total solids measurement, we can spin down her blood in the microharmatic tube.

And if the plasma doesn’t clear, if that remains pink, it’s hemoglobinemia or excess hemoglobin. Right. So that hemoglobinemia, it pigments the plasma pink. Right. So just like we’re used to seeing when it’s a result of a challenging phlebotomy.

Right. Causing hemolysis of the sample. So if we rupture red blood cells, we get release of free hemoglobin, we get that pink plasma. But in her case, we know that the plasma is pink not because of something that has to do with her blood draw, but we know that this is more pathologic because it was able to concentrate from the blood through the kidneys into the urine to make that dark red pigmented urine.

Jessica: This is a really important point. This is like our little tip for you guys when you are presented with pigmented urine. So we’re going to Take our urine, and we’re going to spin it down because it’s really hard to get the chemistry strips and also the urine specific gravity can be a little bit difficult when we have really pigmented urine.

So we’re going to spin it down. If it spins out, meaning we have a clear supernatant and a little red blood cell pellet at the bottom, or large red blood cell pellet, depending on how much red blood cells are in there, then we have hematuria.

If it does not spin out, then we’re kind of at a crossroad where we need to determine is it hemoglobin or is it myoglobin? Correct.

So what we can do, we can take our little micro hematocrit tube. If that clears out, then we have myoglobin. If it remains pink, then we have hemoglobin. Correct.

Holly: Yeah.

Jessica: And then we can transfer that information into our. Helping us determine what’s going on with our urine.

Holly: Great. So we realize that, as we do in Sally’s case, have pink plasma, then we have support for her having hemoglobinemia, resulting in her hemoglobin area. And we have to figure out why the excess hemoglobin in the blood, what’s happening to her red blood cells.

Right. So now we’re going to move to the cbc. Right. So that’s an area of passion for us, I think, because there are so many subtle nuances throughout the CBC that give us great clinical reward when we figure it out diagnostically.

So as we looked at Sally’s cbc, we do see that she is indeed anemic. Right. Which makes sense because something’s happening to her red blood cells to release that free hemoglobin.

And whenever we have an anemia, we want to assess whether or not it’s regenerative. Right. So I’m always thinking about these low cell counts. I sort of want to bucket the question, like, is it a production problem?

Is something happening in the marrow? Is something happening in circulation, or are we losing them from the body? In Ali’s case, initially we thought she was bleeding, losing it from the body.

Right. Which could be the cause for her anemia. But then we found out that wasn’t red blood cells. Right. That’s free hemoglobin. So actually, something’s happening to the red cells in circulation.

So we ruled out that it was a production problem because we could look at the young red blood cells. So some automated analyzers will report those as reticulocytes, or we could look at a blood film and look for polychromatophils.

So the same cell type, both looking at young red blood cells to say the bone marrow response to the anemia is appropriate. And in her case, red cell lysis was our top differential.

So we made a blood film to try and understand what’s happening to her red blood cells. So we first did a routinely stained blood film looking for causes of hemolysis.

So trying to rule out whether she had spherocytes. Right. Potentially to support imha, if she had any red cell parasites or any other red cell fragmentation.

So as we look closer and we see that these red cells have these little noses or protuberance off the red cell membrane. These are Heinz bodies. So capital H E I N Z.

Heinz bodies. And Heinz bodies are caused from an oxidative damage that causes cross linking of that red cell membrane that makes that little nose protuberance.

Jessica: Who was the genius that named Hines bodies and Howell Jolly Body. Like why did they have both have to have H’s?

Holly: Why were those two research scientists named with H’s?

Jessica: Makes it hard for us. Well, hard for me.

Holly: It is hard. I think it actually is commonly confused. I totally agree. Yeah.

Jessica: So how are we going to find those Heinz bodies? We can use a special stain called new methylene blue. So we’re going to use this for Heinz body identification and also for reticulocyte count.

So Holly talked about the, the importance of having a retic count for kind of classifying your anemia. Is it regenerative or is it non regenerative? And so this is a stain that we would use to highlight those reticulocytes.

The instructions, super duper easy. Take three drops of blood, three drops of your stain, put it in. We use our Eppendorf tubes, mixy, mixy it and then let it sit for about 15 minutes.

And so what that’s going to allow to happen is you’re going to get RNA aggregation and also binding of your hemoglobin. So it’s going to really make those little noses pop out.

Holly: I just think it’s neat to think about the difference in the stains. Right. So for me, for us, maybe two people. How about your mom?

Jessica: Yeah, my mom.

Holly: So because when we do our diff cooks stain or another three part Romanowski type stain, those are stains that we put on the fixed cells. Right. So that first Copeland jar, that blue light blue liquid that we put the slide in, that’s going to fix the cells.

And then we put the pink stain on top and the blue stain on top. But new Methylene blue is called a supra vital stain, meaning it goes on top of, like, the living cells, and it’s going to intercalate within the cells and cause some changes within the cell.

So that way we get to see some of these distinguishing features really prominently. So it stains hemoglobin, Right, the new methylene blue stain, but it stains it differently in the denatured hemoglobin of the Heinz body than it does in the rest of the red cell.

So it’s a very pale blue stain on top of most of the red cells. But where the Heinz body is, where that nose protuberance is, it’s a very deep blue.

So it really stands out when you’re looking at it under the microscope as well. The RNA aggregation that happens within our young red blood cells that still have residual rna, it’s dense aggregates of blue material in there, of clumped blue material within the cells.

So, again, reticulocytes or young red blood cells really stand out as well. So it’s really an excellent stain to highlight those two things and a great one to try in faces of regenerative anemias, where you don’t know the underlying cause.

Jessica: Correct. Once you have let your sample sit. So that mixture of your new methylene blue stain and your red blood cells, you just make a normal blood film. So the same motion you would make for a blood film, that’s what you’re gonna do for here, for this.

And you’re not going to stain it because you’ve already introduced the stain in there. And I think that’s where people really struggle. They want to diff quick this on top of it.

On top of it, which I totally get, because that’s what we do with blood films.

Holly: And it’s so pale.

Jessica: Yeah, it is. It is very, very pale.

Holly: So when we look at it microscopically, what we’ll see is that differential stay staining of the Heinz bodies. You’ll have that blue nose, really show a lot more prominently on that red cell membrane.

And sometimes you may have more than one that becomes evident. And as well, you’ll see that reticulated RNA that’s highlighted within the young red blood cell population.

So it’s pretty exciting for us to find a Heinz body anemia, because we don’t see them that commonly. So when we were able to confirm that and enumerate them better with the new methylene blue stain, we go to talk to the clinician, he stops by the lab and we said,

oh, Sally’s got a Heinz body. Anemia, this is pretty cool. And he said, well, that’s great, but what causes that? Right. So we think about it, it’s some oxidative damage, right.

So the body’s been exposed to an oxidant. It gets within the red cells, it causes a cross linking of the red cell membrane. And so what was again a biconcave disk, the red cell that had excess membrane, we cross linked some of that membrane that made that nose protuberance,

but also turns the rest of the cell into like a, a sphere. Right. A more taut bag of fluid and hemoglobin. Right. Than it was before. And so as a consequence, when these go through circulation, the reason red cells have that excess membrane to cytoplasm contents is so that they’re malleable and they can bend and fold and move through the microvasculature much more readily.

But now that it’s this sort of taut bag of hemoglobin, because of that nose protuberance, they’re more likely to lyse in circulation as well. So that leads to that hemolysis, the release of the free hemoglobin and the hemoglobinemia that pigments the plasma, then concentrates through the kidneys in the urine as the hemoglobinuria,

which was her red pigmented urine.

So we have to figure out what oxidant was she exposed to. So our top differentials, we think about onion and garlic exposure, so the brassica species of plants. And we talked to the owner and he does not feed her any onions and garlic.

He does not cook with them. As far as he knew, she didn’t have access to like a compost bin or garbage to get into them. Acetaminophen or Tylenol, we see that even more commonly in cats as a problem.

And again, something that he did not believe she was exposed to. We talked to him about zinc, the possibility.

We sort of stopped there with saying, as we commonly do, saying, can we take one lateral radiograph to see if we could appreciate any coin ingestion because they’ll be so radio opaque and really obvious.

Jessica: We take a lateral radiograph and there are coins. And I think we, one of our emergency veterinarians said to us what he had learned at another practice was when he had a highly regenerative anemia in a, in an animal to just grab a quick lateral radiograph as one of the first things to check for was coins.

Holly: Yeah, for exactly this reason. And before you jump to thinking that you have an imha, which is something I think we have a tendency to do. And there’s that confirmation bias.

Right? You think you have regenerative anemia. That might be imha. You look under the microscope and on first look you might think that these are spherocytes. Right? Because a lot of the red cells who are affected by the Heinz body formation are.

Have appeared of a smaller diameter. They’re denser, they’re spheroid, they stain a little denser. But indeed, the new methyl and blue highlights that those have noses on them. So rather than sphere site formation from immune mediated attack, it’s Heinz body formation from this oxidant exposure.

Jessica: We took Sally first to endoscopy to try to get those coins out. The clinician felt that he should at least try endoscopy. I feel like it’s such an art, it’s such a skill.

This doctor is actually really good at it. But he was unable to.

I think I remember him saying that coins oftentimes get stuck like suctioned almost to the side of the stomach. And so we ended up needing to take her to surgery and we indeed removed the several coins from Ms.

Sally.

Holly: Whenever we’re presenting this live, I always ask people like, what’s wrong with this picture? So there’s three pennies and a nickel. And indeed the nickel has nothing to do with her story.

Right. So it appears that these would be maybe the three implicated pennies. Right. Pennies having pennies minted after 1983 that have a high zinc content on the inside. Right. When they get ingested, there’s erosion of the outer surface of the penny by the gastric acids.

Right. The stomach acids that exposes a zinc on the interior. That’s a real irritant to the GI tract. You get GI ulceration often. That bleeding actually more readily allows that translocation of the zinc that acts as an oxidant that creates the Heinz body formation in the red cells, which leads to a lot of red cell lysis or hemolysis and the leakage of that free hemoglobin that then concentrated through the kidneys into the urine.

But in this case, actually the three pennies that we removed were not eroded at all. They were actually. Were in great shape. We rinsed them and they actually were all minted before 1983.

So they didn’t even have high zinc content inside them, even if they were to have been eroded by the stomach acid.

So turns out we actually didn’t need to remove these at surgery. But how can they not be part of her story? Right. She has a Heinz body Anemia. And she has known COIN ingestion and three of which are pennies.

We talked to the owner a little more thoroughly and it turns out he did actually report she had vomited twice over the weekend that he had seen. So she may have vomited more.

Right. She definitely had some gastroenteritis as a result of this. She must have ingested more than these three pennies. Right. That she did ingest somewhere in this process.

One penny at the high zinc content on the inside that sat in the GI tract long enough to break down the surface, ulcerate her GI tract, allow that translocation of the zinc and the oxidative exposure.

And so maybe she had vomited up the implicated pennies, maybe she had pooped them out, I don’t know. But case solved, I think.

Jessica: And Sally went home, you know, the day after surgery, supportive care. She did really well and recovered just fine from this incident.

Holly: So Sally’s the case of the dripping blood, turns out wasn’t blood. Right. So just to recap, it was pigmented urine. Right. So pigmenturia, when you have that dark pigmented urine, you don’t want to.

I presume that that is blood. We spin it down in the lab. The red cells did not pellet or settle out and clear the rest of the urine. So now we know she has hemoglobinuria or myoglobinuria.

We spun down her microhematocrit tube and she had pink plasma. And so that supports hemoglobinemia. That free hemoglobin, that excess hemoglobin we can see in the blood, has to come from within red blood cells.

That’s the only place it comes from. So then we know she must have red cell lysis. So we have to figure out what’s causing hemolysis. So we looked at her blood film, we saw those noses on those spheroid looking cells.

We added new methyl and blue stain so that we could see them more clearly. We also were able to better visualize that regenerative response in her reticulocytes. And ultimately that led to the radiographs and the case closed.

Yep.

Jessica: So those are kind of our take home points for this,

the stepwise fashion to determine what pigmented urine is and then also to use that new methylene blue stain when you think that you have HEIMS bodies on your cells.

Holly: Thanks for listening.

Tales from the Lab is a production of Antech diagnostics. The intent of this podcast is to provide education and guidance with the understanding that any diagnostic testing and treatment decisions are ultimately at the discretion of the attending veterinarian within the established veterinarian patient client relationship.