Featured Case Study: Six-year-old horse, Ursula
by Denise Wunn, DVM, MS, DACVP, Clinical Pathologist, IDEXX Laboratories

Patient: Six-year-old horse, Ursula

Signs: Presented to original veterinarian with laceration on hind leg. Was treated initially for 10 days with topical therapy, antibiotics and nonsteroidal anti-inflammatory medications. Wound developed proud flesh and needed surgical remodeling; referred to surgical facility.

Clinical Presentation: Physical exam was unremarkable except for laceration on hind leg that had excessive granulation tissue.

See the "Methods of Determining Fibrinogen" technical tip below.

Interpretation: There is a mild, mature neutrophilia and a mild hyperfibrinogenemia, consistent with an inflammatory condition (wound). The severe thrombocytopenia was an unexpected finding with the following differentials:

• Artifact of sample collection
  • Clotting of sample during the blood draw
  • EDTA-dependent pseudo-thrombocytopenia
• Infectious diseases
• Bone marrow or splenic disorder
• Immune-mediated destruction
  • Idiopathic dysregulation of immune system
  • Idiosyncratic reaction to drugs

In this case, there had been no problem with the initial blood draw with no platelet clumps seen on the blood film. The platelet count was repeated using a sodium-citrate anticoagulant to rule out EDTA-induced platelet clumping; the platelet count was verified. The horse had a recent negative Coggins test (equine infectious anemia can also be associated with thrombocytopenia), and had no known recent exposure to ticks. Because the horse had been healthy prior to the wound on the leg, the most likely differential was an idiosyncratic reaction to the antibiotic therapy.

Plan and Follow-up: Postpone surgery, discontinue antibiotics, treat topically—platelet count of 50,000/µL or above considered "safe" for surgery.

• Recheck platelet count—count was rising at one-week recheck and was 176,000/µL 10 days after discontinuing oral antibiotics

Outcome
Ursula was taken to surgery and had her wound remodeled; the wound healed well post-op.

Note: Preanesthetic laboratory testing was important to identify a problem that was not evident on physical exam.

The recommendations contained in Equine Edge educational materials are intended to provide general guidance only. As with any diagnosis or treatment, you should use clinical discretion with each patient based on a complete evaluation of the patient, including history, physical presentation and complete laboratory data profile. With respect to any drug therapy or monitoring program, you should refer to product inserts for a complete description of dosages, indications, interactions and cautions.

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Methods of Determining Fibrinogen

A fibrinogen estimate can be performed using the difference between serum and plasma. Plasma is the liquid part of blood when the blood has been anticoagulated with EDTA, heparin or citrate (contains clotting factors), while serum is the liquid part of the blood when the blood is allowed to clot (contains no clotting factors). Here are a few methods to help determine fibrinogen.

Heat Precipitation Method—the most commonly used method for determination of fibrinogen in the horse

Heat Precipitation Method A:

• Sample: EDTA whole blood (purple-topped tube)
• Equipment: Refractometer, microhematocrit tubes, centrifuge, heater block or bath set at 56°C
• Procedure:
  1. Fill two hematocrit tubes and centrifuge them for five minutes.
  2. Break one tube and measure the refractometer protein.
  3. Heat the second tube in the heater block for five minutes.
  4. Centrifuge the second tube for five minutes.
  5. Break the second tube and measure the refractometer protein.
  6. Calculate.
• Example of calculations:
  1. First tube (unheated) had protein of 7.5 g/dL.
  2. Second tube (heated) had protein of 7.0 g/dL.
  3. 7.5–7.0 = 0.5 g/dL
  4. Fibrinogen is expressed in mg/dL. The answer in grams must be multiplied by 1000 to obtain milligrams, 0.5 g/dL x1000 = 500 mg/dL

Heat Precipitation Method B:

• Sample: EDTA whole blood (purple-topped tube)
• Equipment: QBC® VetAutoread™ Hematology Analyzer with centrifuge and fibrinogen precipitator accessory
• Procedure:
  1. The QBC® VetTube is spun in the QBC VetAutoread centrifuge for one cycle (five minutes).
  2. The tube is then placed in the fibrinogen precipitator. The heater block first precipitates plasma protein out of the plasma at a specific temperature. After the tube has been in the precipitation device for five minutes, the plasma layer in the tube will appear to be cloudy (almost like egg whites).
  3. The tube is then spun again in the QBC VetAutoread centrifuge for one cycle (five minutes). This compacts the fibrinogen band on top of the float.
  4. Insert the spun tube into the QBC VetAutoread analyzer. Results are available in 60 seconds.
• Explanation of results: Fibrinogen will fluoresce green in the QBC VetTube (DNA). The fibrinogen measurement is derived from the amount of green fluorescence from the top of the float up. The QBC VetAutoread analyzer then integrates the species-specific algorhythims and produces a quantitative total fibrinogen value.

Fibrinogen Estimation Method

• The difference between plasma and serum is clotting factors.
• Fibrinogen is the most abundant clotting factor in the blood.
• The difference between the protein content of plasma and serum is a reasonable estimate of fibrinogen.

Fibrinogen Estimate Procedure

• Sample: EDTA whole blood and red-topped tube of blood
• Equipment: Refractometer, microhematocrit tubes and centrifuge
• Procedure:
  1. Centrifuge the red-topped tube of blood to obtain serum. Fill the hematocrit tube and centrifuge it to obtain plasma.
  2. Measure EDTA plasma protein from the hematocrit tube. Measure serum protein on the refractometer.
  3. Calculate as before.

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Learn from the ACVIM consensus statement on Streptococcus equi management

Recently, the American College of Veterinary Internal Medicine (ACVIM) issued a consensus statement on Streptococcus equi management. Along with the release of this consensus statement, awareness from the veterinary and horse-owner communities is increasing with respect to this disease. For many years, we have just put out the fires during an outbreak. Treating horses with clinical disease, and vaccination of those at risk, were all we had to offer our clients.

Two of the tests featured in the ACVIM consensus statement are the Strep equi ELISA and the Strep equi PCR (polymerase chain reaction) with culture test. Both tests are available from IDEXX’s Equine Biodiagnostics laboratory in Lexington, Kentucky.

With the Strep equi ELISA, veterinarians now have the ability to evaluate the immune status of an individual prior to vaccination or following an outbreak. With the Strep equi PCR test and culture, we can now effectively screen for asymptomatic carriers of this disease and make the important management decisions for prevention in groups of horses.

The Strep equi PCR from IDEXX/EBI is a very useful tool to manage strangles outbreaks that occur in your practice. It is very important to wait at least 30 days after cessation of clinical signs to begin evaluating the healthy-appearing or chronic shedder of Strep equi. In the natural recovery from the disease, many individuals will shed the DNA from a Strep equi infection for up to three weeks. These horses are recovering and will not become chronic shedders of the organism or pose a threat in that regard. Because PCR testing is three times more sensitive than culture, and will not distinguish between live and dead organisms, false-positive results are possible, and we are premature in testing these herds. It is very important to run a culture along with PCR sampling because the PCR is specific for Strep equi, and other strangles organisms may be causing disease. Therefore, a culture may be necessary to catch those other infections.

Hopefully, with these valuable new diagnostic tools from IDEXX/EBI, the practice of treating strangles and Strep equi cases when they occur, year after year, will become a thing of the past.

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Introducing the IDEXX EquiView® Digital Radiography System

The IDEXX EquiView® Digital Radiography System is a portable, direct-digital radiography system designed specifically for the equine practitioner. Both the portable and in-house systems are user-friendly and provide fast and reliable radiographic images:

• Preview image in four seconds
• DICOM-compatible
• PACS image enhancement software
• Daylight readable monitors
• Archiving and database storage

The IDEXX EquiView® Digital Radiography System is the newest addition to the IDEXX Digital Radiography product line, which also includes two computed radiography (CR) products, the IDEXX Digital Radiography Compact System for equine practitioners and the IDEXX Digital Radiography System for companion animals. With the addition of the IDEXX EquiView® system, IDEXX offers you quality choices in digital imaging solutions based on your needs. For more information about IDEXX digital radiography products, call
1-877-433-9922 or send us an e-mail.

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Can you identify this cell?

Test your hematology knowledge by trying to identify the cell and structures (indicated by arrows) in the picture below.

History: blood film from an adult mare

Send your answer in an e-mail to: diagnosticedge@idexx.com. Please include your name, practice name, address and telephone number.

The first 10 respondents to correctly identify the cell and structures correctly will appear in the next issue of the Equine Edge.

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