Histology and cytology

 In the last post we looked at typical tests done from blood and urine. The list was by no means comprehensive, and in fact we only scratched the surface. So continue, let's look at two more sample types which are routinely examined from all kinds of animals (farm, wild and companion).

Histology / histopathology (tissues)

Histology is the study of the processes in, and the structure of, tissues. A typical sample is a piece of tissue removed during a surgery. It could be from the surface, like a lump on a paw, or from the inside of the animal, like a piece of the liver. The tissue is examined under a microscope.

Similar term is pathology or histopathology, which essentially means the same thing. There is a small nuance difference between pathology and histology, though. Histology is the study of tissue, while pathology is the study of disease process in the tissues.  Histological examination can answer questions like:

• is this tissue healthy? If not, what is the likely disease or injury causing the changes?
• describe this tissue and why is it abnormal? (for example on a malformed fetus)
• what is this unknown tissue? (typical question for those removed lumps and bumps)
• for this cancerous mass, what is the expected behavior and malignancy?
• are there signs of damage caused by parasites inside this tissue?

Once a piece of tissue is removed, it starts to change very quickly. It can dry out, change shape, cells can dry or break etc. To stop these changes the tissue is fixated. The fixating agent creates bindings which stabilize the tissue in it's current form. One possibility is to soak the tissue in formalin (formaldehyde mixed with water) for several hours. 

Because the sample must be fixated and prepared more in the laboratory, getting histological results can take several days. Animal owners should also note that the results are often highly detailed and therefore incomprehensible to others than the veterinarian. 

There are plenty of educational videos available. One I like, about the general principles, is available on https://www.youtube.com/watch?v=Xu0QjQ4fbQo. A beautiful look inside a laboratory is provided by IDEXX: https://www.youtube.com/watch?v=wjbmPBQy2F4.

Cytology

Like histology, cytology also looks at cells under a microscope. However, the cytological samples are much smaller. The main difference is then the scale and samples: histology looks at tissues and organs, describing a larges mass and process, while cytology looks at tiny samples, describing the individual cells. Also, cytology can be done on Cytology can answer to questions like

• Are there abnormal cells visible in this sample? (for example abnormal red blood cells)
• Are there parasites, bacteria or other changes visible in the sample?
• Does a fluid show signs of infection, or could it be caused by physical trauma?

A typical sample is a fine needle aspirate (FNA). A thick needle is inserted into the tissue to be analyzed, causing cells to enter the hollow needle. These cells are then ejected on a small slide (object slide). A slide can also be made with blood or any body cavity fluid, such as synovial fluid or fluid from the abdomen. 

Similarly to histology, the report will be very detailed and complicated. A veterinarian is required to understand the implications of the findings.

Comparison

	Histology	Cytology
Sample	Hard and soft tissue	Fluids, cells, smears
Can analyze entire organs?	Yes	No
Invasiveness of sampling	Very invasive	Not invasive
Analysis speed	Slow, due to preparation	Fast
Main tool	Microscope	Microscope
Complexity of preparatoion	Complex	Simple

Veterinary laboratory tests, part 1

Our pets and farm animals get checked by the vet every once in a while. Often the vet needs a sample to test. For us owners the samples are usually classified by the substance needed: there are blood test, tests from urine samples, test from stool samples etc. It's easy to understand and definitely clear enough for everyone involved.

Veterinarians and laboratories, however, use a bit different kind of classification. For their purposes it provides more clarity. Some common types of tests are 

• Hematology
• Chemistry
• Urinalysis
• Cytology and histology

Tests are often run in "panels", meaning a group of tests which are often requested together. Instead of separately requesting for a test for all liver-related values, for example, the vet can just order a "liver panel". The panels available differ per laboratory and testing company.  

Hematology

Hematology tests the different cells in the blood. It studies the life cycle, form and size (morphological features) and the amounts of these cells.  The blood to be sampled is submitted either in a test tube or as a smear on a glass. The sample should be as fresh as possible to avoid lysis (cells breaking down) and clotting. If the sample is shipped to an external laboratory correct packaging and shipping are vital to preserve the sample in good quality. 

The tests are further classified by what cells are being studied. Red blood cells, white blood cells and platelets all have their own functions in the body. For red blood cells we can for example test for hematocrit (HCT), which is calculated from the amount of red blood cells and the mean cell volume. We can also calculate reticulocyte percentage to see how much of the red blood cells are immature. A high number of immature cells could indicate blood loss by bleeding or cell death. 

For white blood cells we calculate the total number, morphology and percentage of different types of white blood cells. The changes in these cells form a leukogram. 

Platelets help the blood clot, and are the third type of cells used in hematological tests. Similarly to red and white cells, we count the number and percentage of platelets. We can also determine their morphology. Too many platelets is called thrombocytosis (increased clotting risk), and not enough platelets is called thrombocytopenia (blood is not clotting properly). 

Chemistry

Chemistry tests urine or blood, but in a different way than hematology. These tests can be called clinical chemisty, clinical pathology of blood chemistry. Unlike hematology, chemistry is tested most often from serum or plasma. Serum and plasma are acquired by removing the cells from the blood sample. If the blood is left to clot,  serum remains. If the clotting is prevented with anticoagulant, the result is plasma. 

(c) mouseserum.com

Some examples of chemistry tests are electrolytes, minerals and kidney function.

Electrolytes help cells to function properly. They are carefully regulated, and any deviations can point to a health concern. Sodium concentration can be measured to estimate the the amount of fluids in the body (dehydration). Potassium is crucial to the correct working of muscles. Minerals like calcium and magnesium can reveal organ failure (the body will get lacking minerals from internal organs) or muscle weakness due to lack of calcium (milk fever in cows being a well-known example).   

To estimate kidney function a chemistry test measuring creatinine or urea nitrogen can be done. 

Urinalysis

Urinalysis is the analysis of urine. It focuses on describing the urine (color, cloudiness) and looking at the bacteria and biochemical properties (such as protein, blood and pH). All of these values describe the functioning of the kidneys and urinary tract. 

Much of basic urinalysis can be done at a clinic using a simple dipstick. It's a stick with several pads, which is dipped into the urine. Each pad reacts to a specific substance in the urine. Looking at the colors of the pads the nurse or vet can determine the basic properties of urine. 

 

Basics of immunotherapy

Immunotherapy sounds complicated, but it basically comes down to a simple definition: medical treatments aimed at or using the immune system. Instead of targeting the viruses and other pathogens causing illness, immunotherapy targets the immune system. 

Immunotherapy uses immunomodulators. These are the active ingredients used in the therapy. Examples of immunomodulators  are interleukins, cytokines and specific drugs. They can be administered in several ways:

• SLIT - sublingual immunotherapy, fancy way of saying that the patient gets the therapy (e.g. drops of liquid) under their tongue. In allergy treatment the liquid would contain the allergen. 
• SCIT - subcutaneous immunotherapy. The patient gets the therapy injected under their skin.
• OIT - oral immunotherapy. Patient eats the therapy. For example, a patient eats small portions of the allergen that they're desenziting against.
• TDIT - Transdermal immunotherapy. The therapy is applied on the skin (fancy term is "epicutaneous application")

Immunotherapies can be divided in two basic categories: activating and suppressing.

Activating therapies are used to elicit a specific response from the immune system. For example, vaccinations are designed to train our immune system to recognize a pathogen and to destroy it. Many side effects of vaccinations are caused by our immune system activating (which is exactly what the vaccine was designed to do!). Activating therapies can also take the patient's own blood cells (for example), enhance them and give them back to the patient. 

Cancer treatments belong to the group of activating therapies. It uses the body's own capabilities to recognize and target cancer cells. The therapy essentially re-trains this natural system to help the body destroy cancer.

Suppressing therapies aim to dim or shut down the immune system. Sounds weird - why would anyone want to do that? For example, many patients develop problems after organ donations because their body recognizes the organ as foreign, and is trying to reject it. This rejection is driven by the immune system. 

Allergy desensitization is also an suppressing immunotherapy. An allergic reaction occurs when the immune system reacts defensively to a harmless substance.  In allergen immunotherapy a patient is given increasingly larger doses of the allergen, which gradually teaches their body to tolerate the allergen better.

Immunotherapeutic testing

A range of veterinary tests and procedures regarding immunotherapy are available. These tests are designated to measure the response of the immune system. Diagnostic immunotherapeutic testing is done prior to treatment in order to determine the correct immunotherapy to be used. For example, blood serum can be tested against different allergens. Once the allergy-inducing substances are recognized, an allergen-specific immunotherapy (ASIT) can be planned.

The names of these tests can sound daunting. In 2013 IDEXX was using a test with a monster of a name: "The canine monoclonal antibody cocktail-based ELISA (macELISA)". Let's break that up, shall we?

Canine = related to canines, mainly dogs. This test is meant for dogs.

Monoclonal = The sample is based on a single cell from the patient. This single cell is cloned. 

Antibody = Antibody, also known as immunoglobulin (Ig), is a protein of the adaptive immune system. That's the part of the immune system that changes and learns. Our immune system uses antibodies to identify antigens (foreign objects such as bacteria and viruses). "Monoclonal antibody"  refers thus to a group of cloned cells which can produce the antibody we're testing for.

cocktail-based = No need for a black dress. This cocktail just means it's a "mix of". In this case it's a mix of antibodies, allowing us to run just one test for several antibodies. 

mac = this is just an acronym of the three previous words, "monoclonal antibody cocktail-based"

 ELISA = enzyme-linked immunosorbent assay. In short, a test that detects antibodies in a sample. Combined with tasty attributes like "sandwich" and "cocktail".

ELISA 

There are several variants of ELISA. In the simplest form (direct ELISA):

1. A sample is taken and processed per the requirements of the test.
2. Antigens (viruses, bacteria, other pathogens) are attached to a surface by the manufacturer of the test.
3. A matching, lab-created antibody is poured over the surface. These antibodies are modified to have a specific active substance called an enzyme.
4. Antibodies match to antigens in the sample (if any are present). This proves that the suspected antigens were present, but cannot be detected.
5. Antibodies, which did not get bound, are removed. Now we only have the antibodies bound to antigens left.
6. A substrate, which reacts with the enzyme, is added. The substrate reacts to the enzyme. If there were any antibodies bound to antigens, they will now show up. 

In short, Elisa takes a sample and shows a specific color if the sought-after antigen was found. The term "cocktail" was already covered: in this case a mix of different antigens is tested in one go. In the "sandwich" ELISA the test begins with specific antibodies, which only respond to specific antigens. The antigens are added, and eventually a another layer of antibodies is added. The term "sandwich" refers to the antigens from the sample getting stuck between two layers of antibodies.  The second layer of antibodies binds to a variety of antigenes. It's less picky than the first one.  Instead of creating specific antibodies with their own enzymes, a generic antibody-enzyme -pair is used.  

More information

https://en.wikipedia.org/wiki/Immunotherapy

Anatomical body planes

In the last post we covered the anatomical terms for directions. Now we'll discuss planes - not the flying kind, but planes that divide the body into two. Planes can be important when considering medical imaging, embryology (how an embryo develops) and descriptions of body motion.

Please note that different terminology seems to be used about humans and about non-human animals. Here I focus on the terminology used about animals.

Median plane: Median plane divides the body into the right and left half, exactly from the center. 

Sagittal plane: If one divides the body just off the median plane, that would be called a sagittal plane. It still divides the body into right and left, but not from the center. The halves are not identical anymore.

Dorsal plane: Dorsal plane divides the body to dorsal and ventral areas, cutting lenghtwise through the midsection of the body.

Transverse plane: Transverse plane is a local plane, meaning that it's in the right angle to the axis of the thing we're looking at. If you're looking at a leg, the transverse plane would go from the front of the leg to the back, at the right angle to the leg itself. If you're looking at the body, the transverse plane is from the back to the stomach (at a right angle to the spine).

The planes are used when talking about the entire animal. When talking about a skeleton, two more distinctions can be made. Now we'll have axial skeleton, which comprises of the head, the vertebral column (spine) and the ribcage. Ventrally to the axial skeleton is the appendicular skeleton, comprising of the limbs.

Anatomically correct directions

An old Internet story claims that a 10-year old boy described a cow thus:

"A cow has seven sides: upside, downside, upper side, below side, right side, left side and inside. The head is on the upper side."

He is right, in a way, but the description could be more specific. We could say that the cow has head, body and legs.. but what about the neck? Is that a separate part, or does it belong to the head or the body? 

Also, a cow is easy since we all know how they look like. It's relatively easy to say where the head is (in the front, attached to the neck) or where the hind legs are (under the pelvic area, attached to the body). Alright, but where is the liver in relation to the spleen?  Where's the lump you felt, the one kinda close to the ribs but more to the top, if you look directly at the cow and you're about the same height as the cow? There's a wound in the intestines - where is it? 1".5 meters from the start"?

We need anatomical directions. Words that are unambiguous and describe things in relation to the animal itself. With these words we can describe precisely where something is.

Let's start with the main regions. We can divide the body to regions and cavities (hollow areas). A vertebrate has usually

• A head, with three cavities: 
• nasal cavity or rhinarium (the hollow space in the nose)
• oral cavity (mouth)
• the orbit (which contains the eyeballs)
• The neck, attached to the head
• The trunk, with three cavities: 
• the thorax or thoracic cavity (lung area)
• abdomen (stomach(s) and intestines)
• pelvis (urinary tract and reproductive organs)
• Forelegs
• Hind legs

To get more into detail several specific words are used to describe location. Note that they are not related to the animal's position. Think of the phrase "above the dog's hind legs". If the dog is standing, above the legs is the pelvis. If the dog is sitting, above the legs is his stomach!   

Caudal - Cranial

Caudal means to the direction of the tail (or rump in animals without a tail), and cranial means to the direction of the head. For example, an important blood vessel called caudal vena cava , which goes from the heart to the direction of the tail. They can also be used in the meaning "related to or located in", like cranial nerve are nerves emerging from the brain (cranial region). 

Dorsal - Ventral

Dorsal is to the direction of the back, and ventral to the direction of the stomach. Easy to remember when you think about people who "speak from their stomach" - ventriloquists! 

Distal - Proximal

Distal means moving away from the trunk. This could be to any direction, as long as it's away from the trunk. Proximal is to the direction of the trunk. The hooves of an animal are distal to their body, and the shoulders are proximal to the hooves. 

These words have no meaning on some organisms, such as starfish. For these we could use "peripheral" instead of distal - something away from the center.

Medial - Lateral

Let's again use a hind leg as the example. How do we describe something which is on one side of the leg? Right/left is difficult. Are you facing the animal, is it the animal's right/left, and and what if the animal is on it's side? 

Medial refers to "closer to the midline", i.e. inside of the leg. Lateral is away from the midline, i.e. the outside of the leg. Note that the when you face the animal, the right side of the right foreleg is lateral, but the right side of the left foreleg is medial... Hence, medial and lateral rather than right and left.

With these terms we can now say that the lump is 5 cm caudal and 10 cm proximal to the last rib. Or that the horse has soreness on the left medial forelimb, distal to the knee. Much better than "let me show you, if you squeeze here then OOW it kicked me!"

Sources:

Wikipedia https://en.wikipedia.org/wiki/Anatomical_terms_of_location

Coursera, becoming a veterinarian

Tinbergen's four questions

Animals, just like humans, show behaviors. If you just see a picture without a context it's hard to say what is happening - or why. For example you might see an image of a horse running. 

If you can monitor the animal for a while and see it's surroundings, you might be able to deduce what it is doing. A film could show the horse running, and a dog running after it. Looking more closely at the body language of the horse and the dog we can deduce if they're playing or there's a real chase taking place. 

(c) Picography

Now we know what is happening, but we still don't know why.

A Nobel-winning researcher Nikolaas Tinberger has formulated four questions to ask to find out why an animal is showing a specific behavior. These four requirements and their corresponding questions are

1. Cause: What controls the behavior?
2. Development: How and when is the behavior acquired in the animal's lifetime?
3. Evolution: Why the species carry on doing this behavior?
4. Function: How does the behavior contribute to the animal's survival and reproduction? 

A cause is something that motivates the animal to spend time and energy to perform a behavior. It could be an internal or external stimulus. In our example the horse was running. For example,it might have seen a predator (visual stimulus) which caused a fight-or-flight -response.  

Development explains how the behavior is acquired and how it changes. Newborn calves will seek an angle similar to the cow's hind legs and flank to find the udder. When they grow older, this behavior is lost. A behavior could also be learned, like a dog who learns that they get petted when they jump on the owner's lap.

For our example, let's assume that the horse saw a dog. The horse then escaped following it's natural instinct. Had it learned that this particular dog is friendly, this behavior would not have occurred.  Development can alter a behavior, or cause differences in how individuals express that behavior.

Evolution looks at how the behavior impacts the species. Wolves live in packs, because hunting together improves their chances of success and ensuring enough food for adults and pups. The ancestors of horses who ran from predators were more successful in raising their descendants to adulthood. This ensured that the behavior stayed with the species.

Function asks about why the behavior keeps occurring in the species. Suckling, play-fighting and mating are behaviors that survive in the species and directly impact the survival of the individual showing the behavior. Note that trained behaviors don't survive from one animal to another: they're learned. When a horse runs from a predator, the function is to escape from a  threat and to survive.

For example, let's look at the behavior of a cow picking up shredded silage. We can assume that the cow is eating. How can we answer the four questions? 

Cause: Hormonal signals create the feeling of hunger, when the intestines are getting empty. The cow sees others eating, which can promote eating behavior also in animals who are not hungry.  

Development: As a calf, the animal suckles their dam for milk. As their metabolism develops and solid food is offered, the animals learns to eat also silage, hay and similar solid feeds. Their rumen and intestines develop a bacterial flora capable of metabolizing the feed.  

Evolution: Animals who ate high-quality grass were healthier and had better chances of raising their offspring into adulthood. 

Function: To provide the animal's body with nutrients it needs to survive and to grow/produce milk.

For more information, enroll to the free Animal Behavior and Welfare -course on Coursera.  

Sensitization and habituation

Sensitization and habituation are two essential terms when it comes to animal research. They describe the way animals' reactions change to a specific stimuli over time. After every time a stimulus is met, learning occurs. The animal is changed through the experience, and these changes lead to a more profound, noticeable change if the stimulus is repeated. 

Sensitization happens when after repeated occurrences, usually with a lot of time in between them, an animal starts to react more strongly to a stimulus. Their reaction may be more pronounced, and the physiological measurements (blood pressure for one) can be more extreme than before. Examples of sensitization:

• Repeating unpleasant handling. Animal learns that the experience is painful, and can start reacting even stronger every time. (for humans this can be the case with dentist's appointments).
• Loud, sudden sounds like fireworks on New Year's Eve. A dog may not react to the first bangs, but as they keep repeating on uneven intervals, the dog starts reacting more and more strongly during the evening and night. 
• Traumatic event, which leads to fear of smells, sights or other items that coincided with the place of the original event. 

Sensitization usually does not last for very long. The stronger the stimulus, the longer the sensitization lasts. Makes sense, doesn't it - after a quiet "pop!" we're jumpy for a while, but after the bang of explosion the jitters are considerably worse!

Sensitization is not very specific to a stimulus. An unpleasant experience at the veterinarian can cause sensitization towards the vet, but also towards people with similar clothes, the physical location or the smells that were present.  

(c) https://www.dreamstime.com/

Habituation is the opposite of sensitization. According to Lumen Learning, "Habituation occurs when we learn not to respond to a stimulus that is presented repeatedly without change, punishment, or reward". Something happens, but nothing follows from it - therefore it's not needed to react to this stimulus. Broom uses an example where a flock of sheep is moved from a quiet pasture to a field next to a road. At first, they will react to every passing car. As the cars keep on driving by, they get used to it and react less and less. You could say they got bored of the cars, which is in a sense correct. The more scientific terms is that they've habituated.

Other examples of habituation are

• Habituation to humans and human touch
• Habituation to light-dark rhythm.

Habituation is very stimulus-specific. An animal might habituate to a specific type of noise, but if it changes in volume, pitch or sound, the animal will react to it again. In our example the sheep might ignore a car, but react strongly to a Harley-Davidson.

Habituation rely heavily on repetition. Factors influencing the result are regularity (how often does the event repeat), pattern (does it repeat in an predictable interval) and time between repetitions. For example, animals habituate quickly to stimulus repeating often in a short time. However, the recovery (when the habituation wears off) is also quick.

 

More on habituation and sensitization

Biological Basis of Learning | Boundless Psychology (lumenlearning.com)

Microsoft PowerPoint - Learning.ppt (ucsd.edu)