Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

In order to bleed the American Horseshoe Crab Limulus PolyPhen, one must hold the crab in a flexed position and insert a syringe needle into the hinge to puncture its heart. Once its circulatory system is accessed, an adult crab can be drained of nearly all of its blood. Why bleed?

The horseshoe crab Its blood contains a highly motile type called an amoeba site that secretes specialized clotting factors which react violently in the presence of gram-negative bacteria. A fact which is led to the adoption of the Limulus amoeba Cyte lysate assay or gram-negative test by the FDA. Hi, I'm Peter Armstrong from the Department of Molecular and Cellular Biology at the University of California Davis.

Over today I'm reporting from the Marine Biological Laboratory in Woods Hole, Massachusetts, where I conduct research during the summer. Today we will show you how to collect blood from the American horseshoe crab. Limitless polyphemus ar arguably the best source for large volumes of blood for any invertebrate.

So let's get started. Horseshoe crabs can be kept in captivity. They require high quality sea water and can be maintained in Marina Aquar that provide aeration and denitrification of the water.

At Woods Hole, seawater is fed directly into our horseshoe crab. Aquarium animals are typically fed shrimp, fish, squid, or lobster as you see here. To feed an animal, remove it from the aquarium, place it on its back, and present the food to its mouth.

If it's hungry, it will eat. Animals should be fed two or three times per week. The three major divisions of the body from anterior to posterior are the pro soma, the EPIs, the soma, and the telson.

The anterior and lateral free margin of the pro soma is the flange, the posterior most indentation of the epithet. Where the telson articulates is the terminal bay, the joint where the pro soma and the epithet articulate is the hinge directly underneath the gap. In the hinge lies the heart.

The pumps blood through the body from a central position located below the carpus, along the dorsal midline. The blood of the horseshoe crab is known as the hemo limp and moves throughout the body via an open circulatory system. Horseshoe crab hemo limp has only one cell type, the granular amoeba site.

The amoeba site is responsible for secreting the proteins of the blood clotting system and proteins and peptides of the immune system. The study of the cells and proteins of the blood is facilitated because of the ease in obtaining large quantities of blood. 50 to 200 milliliters from a single animal in as little as 10 minutes.

Bleeding is tolerated well by the animal. Animals can be bled repeatedly once or twice a month. Now that we've learned a bit about the horseshoe crab, let's move on to preparing to collect its blood.

Prior to the bleeding procedure, chill a healthy looking crab for about an hour By placing it in the cold room while the crab is chilling, prepare your bleeding station. A low chair should be positioned next to an elevated ice bucket so that one can be comfortable while holding a struggling horseshoe crab above the ice bucket. For a good 10 minutes, insert the required number of pre chilled 50 milliliter screw cap plastic centrifuge tubes upright in the ice of the ice bath.

After about an hour in the cold room, bring a crab over to the bleeding station on a nearby bench. Loosen a 14 gauge needle in its sleeve using sturdy forceps. It is imperative to maintain sterile technique during this procedure, so do not touch the needle with any part of your hand.

Just before starting the bleeding operation, remove the caps from all of the tubes you will need and place the caps on an adjacent table. After moistening a kim wipe with 70%ethanol, you can proceed to bleed the crab. Once you've prepared for the blood draw, sit down at your bleeding station with the animal and hold it firmly for bleeding.

In your non-dominant hand, grasp the animal in your offhand so that four fingers are in contact with the front lips of the carpus, and your thumb is reaching back to the rear lip of the carpus. The ventral surface of the animal should face the palm of your hand. The tail should lie along the base of the thumb and project over the wrist.

You can see that the animal is flexed so that the proma and EPIs sections of the carpus come to a right angle. This exposes the hinge joint access to the heart will be through the hinge. Wipe the hinge clean.

Using the ethanol moistened Kim wipe, rotate the hand holding the crab so your thumb is below your fingers. And with your dominant hand, use the forceps to put the needle sleeve between two fingers of the other hand, which holds the crab. Remove the needle from the sleeve using the forceps to puncture the heart.

First, rotate the crab so that your thumb and index finger are up and the animal is upside down. With its dorsal surface and exposed hinge, easily accessible position the butt of the needle above the first of the 50 milliliter collection tubes and orient the animal so that the needle is pointed at the hinge joint and the needle shaft is parallel to the dorsal midline. Now insert the needle through the hinge joint, one centimeter deep, keeping the needle positioned parallel to the dorsal midline and the butt of the needle is still positioned above the collection tube.

As soon as the heart is punctured, blood will flow rapidly in a continuous stream. Keep the needle positioned carefully above the collection tube to keep the flow smooth and even gently move the needle by twisting and making small adjustments to depth and angle. As each collection tube fills with blood, rotate the ice bath to bring the next empty tube beneath the needle.

When the animal stops giving blood, remove the bleeding needle from the heart, recap the tubes and return the animal to the ocean or to its aquarium as quickly as possible after the crab has been bled. Recap the collection tubes and centrifuge at 1000 RPM for five minutes. This will pellet the blood cells at the bottom of the collection tubes.

The blue fluid above the pellet is the plasma. Now inspect the blood for gelatinous material attached to the walls of the tube indicative of clotting or large amounts of clot with entrapped blood cells in the fluid. The material from these tubes is serum, not plasma, and contains the secreted products of blood cells.

Whereas tubes of plasma are free of clots, discard the serum containing tubes. Transfer the plasma to sterile tubes with sterile pipettes. Leave a few milliliters of plasma above the pellet to avoid disturbing the cell pellet.

If the plasma is to be used for protein purification, add the protease inhibitor. If the plasma is to be kept for more than two days, sterilization of the plasma is required and can be achieved by adding sodium azide to a final concentration of 0.2 milligrams per milliliter. Alternatively, ultra filtration can be performed under vacuum to reduce clogging the filter centrifuge the plasma at 20, 000 RPM for 10 minutes for filtering.

Using medium filtration device fitted with a 0.22 micrometer pore filter. It is now safe to store the plasma at four degrees Celsius for months or even years in order to collect blood. For the study of amoeba site cells, blood is collected as previously, but an 18 gauge needle is used instead to reduce the rate of blood flow, collect the blood in a heat sterilized 35 millimeter Petri dish.

After one to two hours at room temperature, the cells settle out from suspension and aggregate to form a tissue like mass at the bottom of the dish. After the cells aggregate pieces of one millimeter square tissue are cut with a sterile 18 gauge syringe needle. For viewing, drop some plasma on the glass slide and then add the piece of tissue that was cut out.

Then add a cover glass to the slide looking through the scope. After about half an hour, amoeba sites should begin migrating from the periphery of the tissue explan onto the cover glass. These cells can now be viewed with phase contrast or DIC optics.

Initially, the migrating cells are compact with hylan pseudopods that are extended above the migration substratum. Later, the cells flatten initiate degranulation and cease locomotion. So today we have shown you how to collect blood from the American horseshoe crab.

The major enemy of the procedure is the premature clotting of the blood before we are able to separate cells from plasma. If you follow the steps outlined in today's procedure, this minimizes the likelihood that clotting will occur. Remember, some animals are have particularly excitable blood cells and even when following these procedures to the letter, blood clotting still occurs before cells can be centrifuge free from the plasma.

This procedure has a happy ending. We can release this hearty creature back into the wild after collecting as much as 200 millimeters of blood. It will survive and carry out the rest of its life.

Thanks for watching and good luck with your experiments.