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LAL-Lab PowerPoint
Biomedical Use of Horseshoe Crabs
and Importance to Human Health
A bit of
history
on the
subject
Long ago people realized that microbes caused certain diseases.
Vaccines were then created to prevent some of these diseases.
However, sometimes the vaccines themselves made people sick.
This was known as “injection fever”.
Some years later, the cause of “Injection Fever” was discovered.
After the invention of the microscope, scientists were
able to distinguish two distinct types of bacteria.
Eventually they found that bacteria could cause illness.
“Injection Fever” was realized to be an immune response
to a component of one of these two types of bacteria.
These two kinds of bacteria came to
be distinguished by a simple procedure:
This procedure known as the: GRAM STAIN test
(after Hans Christian Gram who developed it) works as follows:
Two stains are added in turn to the bacteria sample
If the bacteria stains blue
from the primary stain
GRAM-POSITIVE
If the bacteria does not
take the primary stain,
but counterstains red
GRAM-NEGATIVE
These staining tendencies are determined by the
differences in composition of the bacterial cell wall.
In gram-positive bacteria the cell wall is thin ...
allowing stain to penetrate and turn the cell blue
In gram-negative bacteria, the cell wall has an extra layer
keeping the stain from penetrating, so it won’t turn blue
Comparing the two-types of bacteria:
> thin cell wall [made of protein peptideoglycan]
Grampositive
> stains blue during Gram-stain procedure
> features mainly terrestrial bacteria
> includes bacteria that cause strep throat
& staph infections
> thick cell wall [made of lipopolysaccharide (LPS)]
Gramnegative:
> stains pink during Gram-stain procedure
> features mainly aquatic bacteria
> includes bacteria that cause salmonella, septi-
cemia, dysentery, meningitis & other diseases
Within the outer
Lipopolysaccharide
(LPS) layer of the
double-layered
cell wall of
gram-negative
bacteria are
pyrogens, or
fever-inducing
agents, known as:
ENDOTOXINS
So, what’s the
big deal about
ENDOTOXINS?
ENDOTOXINS are a bacterial toxin found only in gram-negative bacteria.
Endotoxins cause fever in mammals.
Fever is the immune response to combat foreign invaders.
Prolonged intense fever can lead to tissue breakdown, shock, and death.
Once endotoxins enter the bloodstream, there are no effective treatments.
Antibiotics can kill bacteria, but are ineffective against endotoxins.
Bacteria do not have to be viable for endotoxins to produce immune response.
ENDOTOXINS are prevalent in the environment and don’t
always constitute a human health threat. In fact, endotoxins
can be found inside the digestive system of our own bodies.
Dr. Ronald Berzofsky, Technical Director
Cambrex Bio Science, Endotoxin Detection Section
Because endotoxins in our bodies
(outside of the digestive tract)
can cause serious health problems:
IV bags &
solutions
Pharmaceutical companies must
perform intensive screening of all
medical products to avoid introducing
endotoxins into a patient’s bloodstream
during surgery or a routine procedure.
Pacemakers, heart
valves & other
surgical implants
This includes:
Implanted
pins & plates
Numerous other
pharmaceutical
products
Vaccines, allergy or
insulin shots & other
injectable medicines
What does
this guy
have to do
with any
of this?
In the past, rabbits provided the only way of testing products for endotoxins.
A sample of the product to be tested was injected into a live rabbit.
The rabbit’s temperature was monitored for 3 hours.
If the rabbit developed a fever, the sample was considered contaminated and
thus rejected.
In the 1960’s, scientists Frederik Bang and Jack
Levin discovered a more effective endotoxin test:
Dr. Bang, while researching horseshoe crabs, noticed massive
blood clotting in response to injected gram-negative bacteria.
Upon further research,
Dr.’s
Bang and Levin
determined that white
cells (amebocytes)
in
horseshoe crab blood
formed a clot in the
presence of endotoxin.
HSC Amebocytes
Bang & Levin sought to isolate and refine this clotting reaction.
Later, the potential bio-medical applications were recognized.
The procedure they developed is much like the one now in use
by pharmaceuticals worldwide to test for endotoxins.
Horseshoe crabs possess a simple, yet effective,
system to guard against bacterial infections
How horseshoe crab blood responds to endotoxins
 Blood clots at the
site of the wound in
response to endotoxin.
 The clot encapsulates
any entering bacteria.
This prevents bacteria from infecting the entire
body cavity.
A note about Blue Blood …
Human blood appears red due to
hemoglobin, an oxygen-carrying
molecule containing iron.
When exposed to air it turns
red, just like iron rusting.
Horseshoe crab blood inside it’s
body is actually straw-colored,
but it’s oxygen-carrying molecule,
hemocyanin, is copper-based.
So it turns blue when oxidized.
Please note:
The color of HSC blood
has nothing to do with its
endotoxin-detecting
properties!
Bleeding Horseshoe Crabs for Biomedical Use
HSC’s are bled in
the lab under
sterile conditions
A needle is injected
through the hinge
muscle and into the
heart to start the
blood flowing
The blood flow will
stop due to clotting
after about 30% of
the blood is collected
Effects of bleeding
on horseshoe crabs
HSC’s are returned to the water after
the bleeding procedure.
Spawning continues without ill effects.
Blood cell count returns to normal in
about 2-3 months.
There is about a 10% mortality rate
due to handling.
Research continues on how to
minimize harm to the crabs.
Processing horseshoe crab blood for biomedical use
This clip shows the first step in processing HSC blood for biomedical use
centrifuging to separate amebocytes (white blood cells) from the plasma.
Final steps in
processing of
the blood
product for
biomedical use
Distilled, endotoxin-free water is added to the isolated blood cells.
As water enters the cells, the cells expand and eventually rupture, or lyse.
As the cell bursts, the clotting granules or coagulogens contained within
the blood cell are released into the solution.
Coagulogens are then isolated from the solution and freeze-dried to an
easily-packaged powder. This powdered product is called ‘LAL’.
How
‘LAL’
gets
its
name
LIMULUS (generic name for our Horseshoe Crab)
AMEBOCYTE (blood cells that it’s made from)
LYSATE (refers to ‘lyse’ or rupture of cells)
or
LAL
for
short
Packaging and use of LAL
Use of LAL for testing biomedical products gained FDA approval in the 1980’s.
It has replaced the rabbit test as the preferred means of endotoxin testing.
LAL manufacture is a multimillion dollar industry. It is used globally.
Bottling Lysate
Packaged Product
Photo courtesy of Associates of Cape Cod
Performing the LAL Test
> add sample to be tested to LAL powder
> incubate for 60 minutes at 37º Celsius
> invert the test tube
> check for clot reaction
> if sample remains liquid: endotoxin-free
> if sample forms a clot: endotoxin present
> in lab testing, controls of known amounts
of endotoxin for comparison are used to
quantify amounts of endotoxin present
What Are the Advantages of LAL?
Faster results: 1 hour (compared to 3 hours for the rabbit test)
More accurate results: LAL is more sensitive than rabbit test
(can detect lower levels of endotoxin)
Lower maintenance: HSC’s are returned with little ill effects;
Rabbits must be caged, kept and cared for
So what does all this mean to us as humans?
The risk of infection from
all injected medicines and
implanted medical devices
is greatly minimized.
The risk of patients getting
septicemia (blood poisoning
from endotoxins), a disease
that kills tens of thousands of
people a year, is also reduced.
The speed at which bacterial
meningitis can be diagnosed
and treated is much improved,
also saving many human lives.
AS YOU CAN SEE, HORSESHOE
CRABS PLAY A VITAL YET
LITTLE KNOWN ROLE IN
PROTECTING HUMAN HEALTH!
Isn’ t it amazing that an animal that’s been around for millions
of years is so essential to protecting human health today?
Don’t you think we should protect them as well?
HOORAY FOR HORSESHOE CRABS!
LAL PowerPoint Credits
Inspired by: Glenn Gauvry, Ecological Research Development Group
Developed by: Tricia Cosbey, Melissa Pierce and Gary Kreamer
Delaware Aquatic Resources Education (ARE) Center
Video clips and images supplied by: Michael Oates of Anew, Inc.
Technical support from: Trina Cale-Rosario of Delaware ARE and
Susan Raymond, Teacher, Millsboro, DE
Special thanks to: Dr. Ronald Berzofsky of Cambrex Bio Science for
sharing his ideas, images, expertise and wonderful
way of making complex concepts comprehensible!