It's in the Blood

Blood tests aren't magic, but they do have broad capability that ranges all the way from helping determine pregnancy to diagnosing rare diseases. So helpful are blood tests in making diagnoses that in most equine clinics and hospitals, they are routine. It isn't that the blood test will necessarily pinpoint the malady from which the horse is suffering, but, when added to other circumstantial evidence, it makes a highly important contribution to the diagnosis.

It is somewhat like a detective attempting to solve a crime by using circumstantial evidence. Normally, one piece of evidence isn't sufficient to pinpoint the culprit, but the accumulation of many pieces of evidence, all pointing in the same direction, can serve to build a strong case.

A blood test, for example, might indicate problems with a horse's kidneys, but, generally speaking, it won't pinpoint that problem as to origin. Other pieces of evidence must be used in order to determine whether the problem has arisen as the result of an infection, toxic insult, or whether it stems from the horse's genetic makeup.

Blood tests also help establish baselines early in the diagnosis process. Once the baseline is established, the animal can be monitored for changes which, in many cases, are strong indicators of what's occurring within the body.

Diagnosticians look for changes in red blood cells (erythrocytes) and white blood cells (leukocytes) and also study a variety of enzyme levels in the blood serum. A number of diseases cause measurable changes in the blood chemistry.

In addition, as discussed in an earlier article ("Blood Tests," The Horse of December 1995, page 49), blood tests can tell the practitioner a good deal about a mare's reproductive processes.

Two basic components of blood are red blood cells, which carry oxygen, and white blood cells, which are the body's disease fighters.

The most basic of blood tests is a simple counting of these cells. If the white blood cell count is higher than normal, it is a sure indicator that infection is present and that the animal's body is mounting an attack against the intruder by expanding its fighting forces. If the count is lower than the normal range, it is an indicator that the body does not have the capability to marshall its disease-fighting warriors for one reason or another.

As science and technology have progressed, so has the equipment used to test blood. Many tests are conducted by veterinarians in their own clinics or equine hospitals, with more sophisticated tests sent to commercial or state laboratories or the national diagnostic laboratory.

One of the manufacturers which produces biochemistry and hematology analyzers specifically for veterinary medicine is CDC Technologies of Oxford, Conn. An automated blood testing analyzer which they now have on the market can very quickly perform an analytical assay of both red and white blood cells. Only a small quantity of blood is needed to perform the assay--about 20 microliters.

White Blood Cells

First, a look at the white blood cells, the role they play, and what changes in number and structure might mean.

White blood cells--or leukocytes--are formed in the bone marrow. White blood cells actually spend most of their time outside the circulatory system, patrolling through interstitial fluid (in the vicinity of the body's organs) where most of the battles against invading pathogens are waged.

Listed as the normal range for a white blood cell count in a horse is 5.4 to 14.3 thousand per microliter (a microliter is one thousandth part of a milliliter, or one millionth part of a liter). Normal range, says Paula Perkins, DVM, a veterinarian at CDC Technologies, means that 95% of the horses tested will fall within that range. A white blood cell count lower than 5.4 means the horse has either used up an abnormal amount of white blood cells as the result of an ongoing, debilitating illness, or that the horse simply does not have the capability to produce the needed number of disease fighters. A count that is higher than 14.3 is a strong indicator that the horse's body is under infectious assault and is producing white blood cells to combat the invasion.

There are five major types of white blood cells. They are monocytes, neutrophils, basophils, eosinophils, and lymphocytes. It is their job to fight infections, each in its own, individual way.

For example, monocytes and neutrophils are phagocytes which eat bacteria and debris from our own dead cells. An undue increase in the number of these leukocytes would indicate a bacterial infection.

Listed as normal range combined for monocytes, neutrophils, and basophils by CDC Technologies is 2.3 to 11.6 thousand per microliter. (The listings for normal ranges for both white and red blood cells that follow are all from CDC Technologies, as numerical values to describe ranges vary from laboratory to laboratory.)

Eosinophils react to parasitic invasions of the tissues as well as to allergic attacks. Listed as their normal range for equines is 0 on the bottom side to 1.0 thousand per microliter on top.

Lymphocytes are white blood cells that respond to viral diseases by producing antibodies, so an undue increase in their number would indicate the body is combating a virus. Their normal range for a horse is listed as 1.5 thousand per microliter on the bottom to 7.7 on the top.

Red Blood Cells

Now, a look at red blood cells or erythrocytes. Like the white blood cells, erythrocytes are formed in bone marrow. Unlike their white counterparts, they are much more numerous. For example, each cubic millimeter of human blood contains five to six million red cells. There are about 25 trillion of these tiny cells in the human body's five liters of blood. A horse, obviously, has a good deal more blood than a human, with volume depending on size of the animal.

The major function of the red blood cell is to carry oxygen. The red blood cell is tiny and this suits its task of carrying oxygen very well. For oxygen to be transported, it must diffuse across the plasma membranes of the red blood cells. The smaller the cells, the greater the total area of plasma membrane in a given volume of blood.

While the red blood cell is extremely small, it still contains about 250 molecules of hemoglobin, a protein containing iron. As the red blood cells pass through the lungs, they load up on oxygen. Hemoglobin binds oxygen to the cell. This process is reversed in the capillaries of the systemic circuit as the hemoglobin unloads its cargo of oxygen.

The number of red blood cells formed in the red bone marrow is determined by a negative feedback mechanism that is sensitive to the amount of oxygen reaching the tissues via the blood. When the tissues receive an inadequate supply of oxygen, the kidney secretes a hormone--erythro-
poietin--which stimulates increased production in the bone marrow.

Conversely, if there are too many red blood cells being produced, the kidney produces a restricted amount of erythropoietin and red blood cell production is cut back.

Red blood cells are not long-lived. On the average they will circulate through the system for three to four months, then are destroyed by phagocytic cells. Much of the iron of the hemoglobin is cycled back to bone marrow, where it is reused in red blood cell production.

Listed by CDC Technologies as the normal range in the horse for red blood cells is 5.50 to 12.90 million per microliter. The normal range for hemoglobin content for horses is 8.0 to 19.0 grams per deciliter.

Also measured is the hematocrit or the percentage of blood volume that is comprised of red cells. The normal range for horses is listed at 8.0% to 19.0%. A reading on the low end of the range would indicate anemia.

In addition, the mean corpuscular volume (MCV) is measured. This measurement involves determining the average size of the red blood cells. Most of the red blood cells are about the same size in a normal horse. When they vary greatly in size, it is an indicator of potential problems. The normal range for horses is listed at 37.0 to 58.5 femtoliters. (A femtoliter is one-quadrillionth of a liter.)

The measurement of mean corpuscular hemoglobin concentration (MCHC) and MCV gives the diagnostician a reading on hemoglobin content in individual cells. The normal range in horses, using the CDC Technologies parameters, for MCHC is from 31.0 grams per deciliter to 38.6; and the normal range for MCV is 12.3 to 19.7 picograms. Readings below the normal range are an indication that the red blood cells do not have sufficient hemoglobin to do their job adequately.

A measurement for red blood cell distribution width (RDW) is an indicator of the variation in sizes of the red blood cells. The normal range is listed as between 12% and 27%.

A final measurement of red blood cells involves thrombocytes, which gives an indication of the presence of platelets in the blood. It is the job of platelets to plug any breach in a blood vessel. The normal range for equines is 100 to 350 thousand per microliter.

More to Blood Tests Than Blood

Analyzing red and white blood cells is only the tip of the iceberg in blood testing. Tests that analyze the chemical makeup of the blood can screen for metabolites and electrolytes as well as do an albumin measurement of the liver and detect phosphorus content of the kidneys...the list goes on.

The blood tests can be broad in scale or, in the case where a particular disease is the chief suspect, highly specific.

One of a number of diagnostic laboratories that serves the equine industry is the Livestock Disease Diagnostic Laboratory in Lexington, Ky.

"We don't try to second guess the expertise of the veterinarians," says Lenn Harrison, DVM, director of the laboratory, in discussing the approach taken by the lab when a blood sample arrives. "The veterinarian often will ask for specific tests based on his or her observation of the animal. We haven't seen the animal. It's up to the vet to make the diagnosis."

Another prime function of a diagnostic laboratory, such as the one in Lexington, is to run blood tests to determine whether horses are carriers of infectious diseases.

In the Bluegrass country of Kentucky, where Thoroughbred breeding farms abound, one of the key concerns is equine viral arteritis (EVA), which can cause abortion. There are state laws in Kentucky regarding vaccination and negative EVA testing before breeding, and most countries insist on a negative EVA test before permitting horses to cross their borders. The Kentucky diagnostic facility is one of 12 in the country authorized to perform EVA blood tests.

In tests of this type, the presence or absence of antibodies is the key indicator of whether the horse's body has been invaded by a virus.

"When checking for EVA," says Harrison, "we don't want to see any evidence of antibodies--none."

A problem arises when one wants to export a horse which has been vaccinated for EVA. The vaccine causes the animal to produce antibodies, and they will remain in the body once it has been vaccinated, even though the horse is free of the disease.

"Basically," says Harrison, "if an animal's body has been invaded, it will have produced antibodies--specific antibodies for that specific disease. The disease may not be active in the animal, but the horse's body nevertheless would still be producing antibodies."

While there are many state and commercial laboratories that handle blood tests, there is only one National Veterinary Services Laboratory, and it is located in Ames, Iowa.

It is the only laboratory that tests horses being imported for piroplasmosis, a parasitic disease of the red blood cells; dourine, a contagious disease of horses and donkeys characterized by swelling of lymph glands, genital inflammation, and paralysis of hind limbs; and glanders, a contagious disease of horses that is communicable to man. Glanders is characterized by inflammation of the mucous membranes and the eruption of nodules on the skin which turn into deep ulcers that can invade cartilage and bone.

With the exception of imports from Canada, all horses entering the United States must be tested for piroplasmosis, dourine, and glanders.

Blood samples for the tests are delivered from quarantine stations to the National Veterinary Services Laboratory in Ames by courier. Getting the blood to the national laboratory in timely fashion is important, says Jim Pearson, DVM, acting director.

The time range generally runs from eight to 48 hours, he says, with eight hours being on the fast side for delivery to the lab from quarantine centers in California, New York, and Miami. The outside time range from the taking of blood to the testing process is about 48 hours. After 48 hours the quality of the blood is questionable for testing purposes.

The range of time from when the blood test is performed in the laboratory until results are known ranges from four to 72 hours. The shortest time frame is for piroplasmosis, dourine, and glanders--four to six hours--and one of the longest is for EVA--72 hours.

The tests that are performed to detect disease are about as varied as the diseases themselves, and they carry names like Virus Neutralization Test, to detect EVA; Agar Gell Diffusion Test, to detect equine infectious anemia (EIA) and histoplasmosis; Microscopic Agglutination Test, to detect leptospirosis; Indirect Fluorescent Antibody Test, for detecting Potomac horse fever; and Complement Fixation Test, for contagious equine metritis and piroplasmosis.

Each has its own sophisticated protocol to identify a specific disease, with the key element in common being the detection of antibodies that the body has produced to battle a specific disease.

One of the main responsibilities of diagnostic laboratories, such as the one in Lexington, says director Harrison, is the prevention of disease outbreaks.

"Our goal is to prevent a disease from breaking out," he says. "If we can actually stop it from happening--that's what we're here for."

Underlining that approach was the national laboratory's Pearson. He said that about 80% of the tests for encephalitis are done in Ames.

"We have had a concern about Venezuelan Equine Encephalitis (VEE)," he says, "because of an outbreak in Colombia and a scare in Panama. As a result, we have been welcoming submission of blood samples any time there is a suspicion the disease might have cropped up, and we don't charge for them. However, we do charge for routine encephalitis tests for horses which are being exported."

Thus, blood tests play a dual role. They are of invaluable help in making a diagnosis, and they also serve as disease outbreak watchdogs.

About the Author

Les Sellnow

Les Sellnow is a free-lance writer based near Riverton, Wyo. He specializes in articles on equine research, and operates a ranch where he raises horses and livestock. He has authored several fiction and non-fiction books, including Understanding Equine Lameness and Understanding The Young Horse, published by Eclipse Press and available at www.exclusivelyequine.com or by calling 800/582-5604.

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