The Horseshoe Crab

Featuring Helen Osborne and Allen Burgenson

ALLEN L. BURGENSON is the Global SME for endotoxin detection with Lonza Walkersville. He has over 40 years of experience in industries regulated by the FDA, including Foods, Drugs, Biologics, Medical Devices, and Cosmetics. He has worked in R&D, QC, QA, Regulatory Affairs, and now as Global SME. Allen is involved in several scientific organizations including, the immediate Past Chair of the Horseshoe Crab Advisory Panel for the Atlantic States Marine Fisheries Commission (ASMFC) and as a member of the Horseshoe Crab Working Group of the International Union for the Conservation of Nature (IUCN). Allen is the immediate Past-President of the Capital Area Chapter of the Parenteral Drug Association (PDA), and was Chair of the 2004 PDA Annual Meeting. He is a coauthor for three PDA Technical Reports including, TR-50 and 51 regarding Mycoplasma detection and filtration, and TR-82 regarding Low Endotoxin Recovery (LER). Allen also contributed to USP Informational Chapter <1228.5> on Endotoxin Indicators. Allen has a BA in Microbiology from Rutgers University, and a MS in Biotechnology Management from the University of Maryland.

In this podcast, Allen Burgenson talks with Helen Osborne about:

  • How the horseshoe crab is not a true crab, but is more related to arachnids such as spiders and scorpions.
  • How the Limulus amoebocyte lysate (LAL) is extracted from blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus.
  • How the LAL test is used to screen for the presence of endotoxin in all injectable products in humans.
  • How the horeshoe crab has blue blood.
  • How blood is collected from the crab and how the crab is returned back to the sea.

Music by Skilsel from Pixabay.

Producer and audio editor: Clair Morgan


HELEN: Welcome to Talking About Blood. I’m Helen Osborne, host of this podcast series and a member of the Advisory Board for The Blood Project. I also produce and host my own podcast series about many aspects of health communication, and it’s called Health Literacy Out Loud. Today’s guest is Allen Burgenson, who is recognized as a global subject matter expert on endotoxin detection. Alan has over 40 years of experience in industries that are regulated by the FDA. These industries include foods and drugs, biologics, medical devices, and cosmetics. Alan is active in several scientific organizations, including the Horseshoe Crab Advisory Panel for the United States Marine Fisheries Commission and the Horseshoe Crab Working Group of the International Union for the Conservation of Nature. Among his many accomplishments, Alan has co-authored several research papers and contributed to a USP informational chapter on endotoxin indicators. Alan, welcome to Talking About Blood.

ALLEN: Glad to be here, Helen, it’s nice to meet you.

HELEN: You and I spoke briefly as we were getting ready for this podcast, and you made a statement that especially intrigued me. The statement was, you can thank a horseshoe crab for all injectables. Whoa, I’ve never had a conversation with that in it before. Do tell. What’s the connection between horseshoe crabs and injectables?

ALLEN: Okay, we have to start from the very beginning of injectable medicines back in the late 1800s. At that time, a physician would give you an injection and almost everybody got something called injection fever where you would rise, you would’ve a temperature rise, and then some of those people got very sick and then some of those people actually died. So the physician at the time had to determine is your case bad enough that they had to risk your life to give you an injection? So at that time, injection was a very risky deal. And then around the 1940s, the FDA, the National Institutes of Health and industry came up with the rabbit pyrogen test, which became the standard for testing injectable products. Now that involved live rabbits. And nowadays folks don’t like to use live animals. So then in the 1960s came the advent of the Limulus amoebocyte lysate test, and Limulus Polyphemus, the North American horseshoe crab, is where that blood comes from. So now we test all of our injectable drugs, and now because of LAL, we don’t have to use animals in the test and nobody thinks twice about going to CVS or the Walmart or wherever you get your injections and getting sick or dying because of endotoxin in the product.

HELEN: Can I ask you a question there, Alan? You talked about how this got started. It was not safe more than a hundred years ago, and then along the way rabbits came along, but that was animal based. You’re talking about horseshoe crabs, isn’t that animal based?

ALLEN: It is, but the issue is we collect the horseshoe crabs and then we release them after we collect some of their blood. Now it’s only about three tablespoons or so of blood and we return them to the sea where we got them. So our estimates, as well as industry estimates are that over 95% of the horseshoe crabs survive this procedure and come back in future years where we can collect their blood. They’re only blood once a year, by the way. And we mark them so we don’t collect blood twice a year from them.

HELEN: Thank you. You talked about the history of injectables. How in the world did this come up with the connection with horseshoe crabs?

ALLEN: It’s a long and winding road, but to make it a straight road for everybody…

HELEN: Okay, that’d be great.

ALLEN: … back in the 1800s, everybody knew that if you broke a horseshoe crab’s leg, it would bleed and then this clot would form at the injury site, but nobody knew why. And then it was in the 1960s where Frederick Bang and Jack Levin working at Woods Hole actually elucidated the enzymatic reaction, the immune response, the immune reaction in the horseshoe crab blood. And then they derived the product. It comes from the blood cells called amoebocytes that are in the horseshoe crab’s blue blood.

HELEN: You say it’s blue?

ALLEN: It’s blue because it’s not based on iron like ours. Their blood is based on copper and oxidized copper is blue. But anyway, so they came up with the LAL test, which supplanted the rabbit test as the preferred test for injectable products. And it’s been the same to this day. We still use the amoebocyte lysate and actually it was incorporated into the United States Pharmacopeia in the early 1980s in USP 20.

HELEN: That’s fascinating. So it doesn’t hurt the crabs? The crabs are okay? I mean a crab, I don’t know how big you live in the Atlantic region of the United States and you’re talking about horseshoe crabs. I’m thinking about all kinds of crabs. Some of them, I can’t imagine that they have enough blood to give out three tablespoons and go back in the sea and be fine. Tell us more about the crabs that are the ones that keep us safe for injectables.

ALLEN: Sure. Well, actually the horseshoe crabs are not true crabs like you would see with the blue claw crab, which is the state crustacean of my home state of Maryland. But they are more related to arachnids such as spiders and scorpions.

HELEN: Really?

ALLEN: Yeah, they’re actually quite large. And the minimum collections in the state of Maryland is eight inches across the shell.

HELEN: Eight inches across. That’s large.

ALLEN: Well, and that’s the smallest. We actually take them much larger. They can be over a foot across. We collect them, we collect their blood and then we mark their shell and then we put them back. But when we take them and we’ve got videos showing the return to the sea where the crabs are actively moving in the boat before we put them back in the water. So their hands are reaching up. And one of the reasons why there’s such low mortality is because just like the Red Cross, we only collect blood from active healthy people. We don’t collect blood from anybody that’s sluggish or sick. So if you’re sick, there’s a chance for a higher mortality rate. So we only collect from healthy vigorous animals and they do fine.

HELEN: I’m very curious about this process. I’m envisioning. Somehow you caught the crab, you’re in a boat and then a scientist right there or technician right there would take that blood while it’s on the boat.

ALLEN: Oh, no, no, no. The crabs are brought back to a blood collection facility just like we have for the Red Cross where you have to go to the Red Cross to get your blood taken. And we again inspect the crabs and any that are injured or now sluggish get put on the side. And then the ones that are healthy are moved to the collection racks and the horseshoe crabs have a hinge. And when you bend that hinge, you can see a membrane. So just like you with donating blood, the membrane is swabbed with alcohol and then swabbed with iodine, and then a needle is injected to collect blood from that sinus. And once the sinus is drained, the blood stops and that horseshoe crab is removed and put back into another rack to go back to the return bins and then they go back to the fishermen who return them to the sea.

HELEN: How in the world would you know that the horseshoe crab you collected today and gave its blood you’re not going to see again for a year?

ALLEN: Well, there’s a mark on the shell. There’s a little dimple that is drilled with a Dremel and it’s a dimple that doesn’t hurt the crab, but it makes a scar. So on the right side is even years on the left side is odd years. So if you catch a horseshoe crab say this year and it’s got a dimple on the left side that hasn’t scarred over and the scar is very distinct. So if it still looks fresh, that crab is put back. Matter of fact, if they’re seen on the boats, they’re immediately put back in the water. If they’re missed, they’re put on the side back in that return bin to go back.


ALLEN: We never collect twice.

HELEN: That’s fascinating. And just one more question, I want to hear about the science, but I just am fascinated with this process too. How long would a horseshoe crab tend to live? You talked about doing marks on one side for even years and the other side, do they live for a long time?

ALLEN: Horseshoe crabs will live for about 25 years.

HELEN: My goodness. Who ever knew that.

ALLEN: Yeah, they’re very small. Their eggs are about a millimeter across and it takes them about 10 years to mature to breeding size, which is about eight inches across for males. And the females are slightly larger and then they’re marked. So we may see the horseshoe crab and actually we have seen the same horseshoe crab back for several years with a scar on the shell. And that method is actually used across the industry. Everybody has a mark, so we don’t collect blood that same year because we want to preserve the animal.

HELEN: That’s great. When you keep saying “we” and you are based Mid-Atlantic in the US. Are horseshoe crabs worldwide?

ALLEN: No, actually Limulus Polyphemus, the North American horseshoe crab is only found from Maine to Florida. So on the east coast of the United States, nowhere else. And it’s also found a little bit on the Yucatan Peninsula, but those numbers are dwindling due to pressure from development and everything. There are other species of horseshoe crab on the east coast of the Asian continents, and those horseshoe crabs are threatened or endangered because of the lack of regulatory oversight. But the North American horseshoe crab is absolutely doing fine. Matter of fact, in the Delaware Bay alone, there are more than 43 million horseshoe crabs. And that’s the mean, by the way, that’s the average with error bands. And that number keeps increasing due to the conservation efforts of people like ERDG or the Ecological Research and Development Group as well as the companies that are collecting horseshoe crabs for LAL production. We all have our own conservation programs.

HELEN: This is fascinating. You’re opening up a whole new world to me, but many of our listeners are scientists, are physicians, hematologists, very interested in the mechanisms of blood. So talk at that level please about what happens with the science. You collected this precious blood. What happens with the science that goes from the blood to making sure injections are safe?

ALLEN: Sure. Once the blood is collected, it is centrifuged at low speed to collect all the blood cells. And then the blood cells are lysed or popped due to osmotic pressure using a low ionic strength liquid to make them pop and then sonicated to finish the job. And then they’re put away. And then later on, several of those, they’re called day pools, will be blended together to give us the LAL product, which is then formulated with proprietary formulations. And then that product is then tested for sensitivity and then vialed and then lyophilized. Now the product itself, when it’s used, a small sample of about a hundred microliters of the product is put into a glass tube and a hundred microliters of the LAL is put in with it. It’s incubated at 37 degrees for an hour, 37 degrees C I should say, and incubated for an hour. And at that point, the tubes are gently removed from the heat and gently inverted. And if the gel stays at the end of the tube, you have a positive clot and that shows the presence of endotoxin and that was the original Levin and Bang test. We’ve now since moved past that where we actually measured the rate of turbidity, which is related to the rate of clot formation, or we can measure the amount of time it takes for the turbidity to exceed an optical threshold. And the same thing with our chromogenic reagents. We look for the rate of color formation or the time of color formation and relate that to the endotoxin concentration.

HELEN: Thank you for explaining that. At what point in the development of new injectables would you be introducing the crab blood?

ALLEN: Well, we’re not using the crab blood, we’re using the product arrive from the crab blood. But that goes all the way before you’re looking at preclinical trials before your pharmaceutical product is ever tested in mandatory animal testing and also before, and that is mandatory before you go into human clinical trials. So we’re all the way at early stage development because they don’t want other reactions interfering with the endpoint that they’re looking for in their clinical trials. You don’t want to say that drug X caused a fever without knowing why drug X caused a fever. So if you can eliminate endotoxin, that gets you a long way. And it’s actually a big part of the toxicology when you’re looking at effects of various products on the immune system and on the body.

HELEN: So you said we can thank a crab for all our injectables. Is this a standard protocol now for all new drug development?

ALLEN: Yes, it is. It’s all new drug development and every lot of drug that is released for interstate commerce or to the public is tested using LAL. So every lot, every injection you have ever received, that lot has been tested with LAL and everyone in the world who has ever had an injection the products that they were injected with have been tested with LAL. So now there’s over 8 billion people, so over 8 billion people in the world can thank the horseshoe crab for the safety of their injection.

HELEN: Your statement’s going to stay with me and I hope it is with our listeners too. So you said it’s all our injectables. So the COVID update shot I just got, that’s part of it? The flu shot I got this year, that’s all part of it?

ALLEN: Every injectable or implantable medical device is tested with LAL. So yes, your initial COVID shot as well as all your boosters, every lot has been produced and then tested using LAL to detect the presence of endotoxin. So if you get an IV saline, if you’re in the hospital, if you get vitamins, if you get the flu shot, if it gets injected under your skin, every lot of those products has been tested with LAL.

HELEN: I’m wowed. I’m totally wowed by this. Now. In this era of science and medicine and politicalization, sometimes there’s disinformation and misinformation and confusion and rumors and myths. Is there anything going on that would affect the injectables and the blood that we’re talking about this LAL testing? Is that a factor or is that just a behind the scenes something that’s going along? Should I say swimmingly?

ALLEN: Well, actually, the Atlantic States Marine Fisheries Commission in their 2019 stock status peer reviewed report stated that the collection of horseshoe crab blood for the production of Limulus amoebocyte lysate has a negligible effect on the horseshoe crab population. But however, there are other things that do, and they include development along the shore. If you don’t have a place for the horseshoe crabs to lay their eggs, that affects the population. Also, there’s a lot of detritus that washes up on the beaches, and I’ve seen that it can be up to a foot deep and it’s like peat moss and the horseshoe crabs won’t lay their eggs in that. And there are other fisheries like the bait industry. Whereas we have approximately a 5% mortality, the bait industry is a hundred percent mortality. But there are quotas on the amount of bait that can be crabs that can be taken for bait. And so if you look at 43 million horseshoe crabs in Delaware Bay, the small quota for bait is not really affecting anything. Now as far as some of the disinformation, there’s a lot of folks that hear the words that the horseshoe crabs are threatened and they automatically assume that the horseshoe crabs are threatened by numbers. And that’s not what the IUCN or the International Union for the Conservation of Nature who put the horseshoe crab on the red list meant by that. What they meant was that the horseshoe crab is threatened because of lack of genetic localized diversity, meaning they’re too interrelated. And if something happened, it could potentially wipe out a whole section of horseshoe crabs. Not that there’s not enough numbers because like I said, there’s 43 million in the Delaware Bay area alone. We deliberately tried to keep those numbers up.

HELEN: Okay. So this is so helpful to hear about our understanding of words and your understanding is so deep and profound, like the word threatened, that we as people who are not experts in this subject like you are, can learn from. Thank you for making all of that clear. Now, Alan, our podcast listeners, I’m sure they’re fascinated by this. I certainly am. Our podcast listeners could be often are physicians, hematologists, those in practice somehow very interested and knowledgeable about blood. We also have a large group of podcast listeners who are in their early stages of their scientific or medical careers, whether they’re thinking about going into the the sciences or early in their medical careers. And we also have people like me, everyday people, somewhat knowledgeable perhaps, but just curious about the world and especially the world about blood. I’m curious, what can you offer for each of us? What would you like to say to each audience? Let’s start with the most experienced and savvy group of clinicians and scientists who’ve been working with blood for a long time. What would you like them to know about this connection between the horseshoe crabs and the injectables?

ALLEN: Well, as I stated earlier, if you’re administering any type of injectable, everyone has a horseshoe crab to thank for that. And it’s the horseshoe crab blood’s unique chemistry that allows such safety in the use of injectable pharmaceuticals and implantable medical devices. And that immune system, we all contain clotting factors in our blood. So the horseshoe crab’s immune system with its clotting system is a precursor to how our own immune clotting system developed. So there is that link there, and it was Jack Levin who’s a hematologist, who is out at the University of California, San Francisco, who discovered all of this as his studies in hematology progressed.

HELEN: Thank you for that. What about people earlier in their career?

ALLEN: Earlier in your career, depending upon what you’d like to do. If you’re like me, I’m a microbiologist by training. I’m not a physician, but I’ve been studying horseshoe crabs. And I mentioned this story to you earlier, Helen, about the three-year-old Alan out on the beach with my dad fishing. And my dad, it was pitch dark and when the sun came up, he said, my job was to find all the horseshoe crabs that were turned over and put them back in the water. And despite the protestations from my father’s friends, my dad just took me by the hand and said, “put ’em back in the water”. And that’s how I did what I did. So I’ve been doing that ever since. And I’ve been involved with horseshoe crab ecology growing up on the Jersey shore ever since. So it was a running joke in my family though, with my dad telling me to put them back. We weren’t sure if he was doing that to do the right thing or whether it was just to keep me out of his hair, but either way, it worked.

HELEN: Okay. I’m just

ALLEN: I’ve been involved ever since.

HELEN: When I’ve asked about people earlier in their career, I wasn’t thinking particularly of three year olds, but I think that there’s a time and place for increasing this awareness and this curiosity and for you as I hope many of our listeners are going to somehow turn that into action, even if it’s a somewhat novel development. So last group, it’s all about me. What do you want the lay public to know?

ALLEN: Well, if you see a horseshoe crab, if you live on the east coast of the United States and you see a horseshoe crab turned over, it won’t hurt you. It won’t bite you. There are pictures of me on the internet with my hands actually in the horseshoe crab’s mouth. It’s not biting me. It won’t hurt. You just flip them over and put them back in the water or near the shoreline so that they can right or get themselves back in the water. One thing that people don’t understand is that due to spawning activity and stranding, 10% of the horseshoe crab population dies every year due to that stranding, and you’ll see them on the beaches. So if after a high tide and you’re walking along the beach, don’t be afraid to pick them up and you’re actually helping the horseshoe crab survive. And these animals have been around for 450 million years now and they’ve survived five extinction events, but now they need our help. So if you see them, just give them a hand.

HELEN: Thank you. Oh boy. You’ve really added something for all of us. You’ve talked about videos of this. I know you have a lot of published papers. We’ll have perhaps some of the links to your work on the talking about blood website, particularly the transcript for this podcast. But if people have questions and want to know more, what’s one way you’d recommend?

ALLEN: Well, you can email me at aburgenson1@aol.com. That’s A-B-U-R-G-E-N-S-O-N, and then the number1@aol.com. And yes, I’m still one of those Luddites that uses AOL, but I’ve had the same email address for decades, so I’m not going to change it.

HELEN: Thank you. Alan, thank you for making yourself so available too, because I’m sure a lot of people will have a lot of questions. And mostly I want to thank you for all your amazing work and your leadership and championing this and sharing this great information with us on talking about blood. Thank you, Alan.

ALLEN: It’s been my pleasure. Thank you very much.