Ask the laboratory expert:
biochemistry results in equine practice

Dr Stacey Newton, Lead Clinical Pathologist at NationWide Labs, answers vets’ questions on biochemistry results in equine practice.

Pathology Focus

Ask the laboratory expert:

Biochemistry results in equine practice

Lead Clinical Pathologist at
NationWide Laboratories,
Dr Stacey Newton answers
vets’ questions on
biochemistry results in
equine practice.

How does fasting affect gamma-glutamyl transferase (GGT) tests? What happens if there is a high protein level in a patient’s urine? And are small increases in alkaline phosphatase significant? Questions like these crop up all the time in veterinary practice and there are always moments where we wish we could “ask the expert”.

#1 In which cases should I run protein electrophoresis?
We can run serum protein electrophoresis (PEP) in all cases but unless the globulins are significantly high, then it does not usually add much information.

High globulin levels of around 60g/l or above (there is no set cut-off) could indicate a neoplastic process, such as lymphoma or multiple myeloma. On a PEP trace, these present with a tall monoclonal peak with a narrow base, usually in the beta or gamma globulin fraction. If there is a peak with a broader base, then this indicates inflammation. It is worth noting that monoclonal peaks may appear similar to oligoclonal ones. The latter, also referred to as “restricted oligoclonal” peaks, consist of polyclonal globulins of a similar size and charge. These are usually due to infectious processes and may have a broader base with a tall narrow spike in some cases.

#2 What is the difference between total proteins and total solids?

The basic difference is that total protein (TP) is run on an analyser that uses a colorimetric method and is measured on serum. On the other hand, total solids (TS) are usually measured on plasma via a refractometer.

The TS value is usually lower than TP due to:

The contribution of TS to the refractive index. This reading is not a measurement of TS, and is only an estimate of protein concentration, since variation in other serum component “solids” (sodium, chloride, phosphate, glucose etc) can also affect refractive index, as can colloids such as hetastarch and gelatin.
The contribution of fibrinogen to TP content in plasma compared to serum. Note that if the chemistry panel is performed on heparinised plasma, the difference between the two measurements is much less as fibrinogen is present in both EDTA and heparinised plasma samples

#3 Can fibrous tissue that compresses the gut after abdominal surgery cause any changes in a horse’s biochemistry results?
Fibrous tissue that develops secondary to any gastrointestinal (GI) tract surgery that involves, for example, the removal of some bowel, or even just performing a laparotomy, can result in loops of intestine adhering together.

If no bowel was removed and there is just fibrous tissue in the abdomen, then there could be an involvement of the midline (where the incision was) or parts of the omentum and other parts of the intestine. This may result in interference in normal peristalsis and the passage of ingesta, which then results in an obstruction (gut twists with complete obstruction) or impaction if material builds up behind a partial obstruction. In turn, these can result in colic.

If there is displacement of some part of the GI tract, particularly the colon, and this compresses the liver, then you may see an increased alkaline phosphatase (AP), GGT, glutamate dehydrogenase (GLDH) and aspartate aminotransferase (AST) depending on whether there is compression of liver tissue or obstruction of the bile ducts.

#4 How does fasting increase GGT?

The mechanism for this is not clear, but it is likely similar to the process wherein increased bilirubin results from hepatic dysfunction.

The increase in GGT is greater the longer the duration of fasting. Furthermore, a longer duration may be due to a metabolic effect on lipid metabolism (particularly in ponies and donkeys), with a resulting mobilisation of triglycerides and development of lipid deposition in the liver causing some hepatocellular swelling and cholestasis.

#5 Regarding the level of GGT, could it increase with the consumption of fermented fruit?

I don’t know the exact answer to this. If the fruit, and stones if the fruit contains them, release some toxins then this could affect the liver with resulting hepatocellular swelling, cholestasis and an increase in GGT. If the toxin is severe enough, there may be more significant liver damage and further cholestasis.

If the consumption of the fruit results in anorexia, then this could further increase GGT due to the reasons mentioned in the question on fasting above. Furthermore, if a lot of this fruit is consumed, there could be fermentation in the gut, which could result in gases that may, in turn, result in enlargement of the gut with obstruction and colic. Ethanol may also be produced which could be toxic.

#6 What is the importance of D-dimers in inflammation in horses, both young and adults? Also, can I combine fibrinogen, serum amyloid A and D-dimers?
D-dimers are not specific for inflammation as they are part of the coagulation cascade and then fibrinolysis. They are a product of fibrinolysis, and increases are seen in any age of patient. A D-dimer is a specific degradation fragment of cross-linked fibrin and a high plasma D-dimer is an indicator of intravascular fibrin formation and plasmin-mediated fibrinolysis.

The measurement of plasma D-dimer concentration is useful to aid in the diagnosis of systemic thrombosis, including pulmonary thromboembolism (PTE) and disseminated intravascular coagulation (DIC). D-dimers can be combined with fibrinogen if there is suspicion of DIC. Fibrinogen and serum amyloid A (SAA) are acute phase proteins that are increased with inflammation. However, SAA has a much shorter half-life than fibrinogen, so combining the two can be used to determine the course of inflammation and whether it is ongoing or resolving.

The measurement of plasma D-dimer concentration is useful to aid in the diagnosis of systemic thrombosis, including pulmonary thromboembolism (PTE) and disseminated intravascular coagulation (DIC).

#7 Can you remind us how we test for D-dimers and what equipment to use?

The tube required is a citrate tube (usually with a green top), and separated citrate plasma is preferred. D-dimer appears to be stable for 24 hours if kept at 2^oC to 8^oC, and for several months if frozen at −20^oC to −80^oC . The sample should then be sent with a frozen ice pack to the lab.

#8 What are the most common bloodwork anomalies in horses with a Fasciola hepatica (liver fluke) infestation?

Given the fact that Fasciola hepatica affects the liver, then cases with an infestation are more likely to have elevated liver enzymes. However, studies suggest that the changes in haematology and biochemistry in horses with liver fluke are variable with some cases having no significant changes (Howell et al., 2019).

There appears to be an increased likelihood of testing positive with recently validated F. hepatica excretory-secretory antibody detection ELISA if there are clinical signs consistent with liver disease.

Given the fact that Fasciola hepatica affects the liver, then cases with an infestation are more likely to have elevated liver enzymes.

#9 Are there any new ways to diagnose polysaccharide storage myopathy type 2 from the blood if even post-exercise basic muscle values are normal, or is a biopsy the only way to confirm or exclude it?
Currently, there is no recommended genetic testing for the diagnosis of polysaccharide storage myopathy type 2 (PSSM2) or myofibrillar myopathy (MFM) as there is currently no scientifically validated evidence that the variants for which genetic testing is available are linked to PSSM2.

To date, there appear to be no peer-reviewed studies offered by the company providing the commercial PSSM2 tests. Diagnosis is thus made from a muscle biopsy. Periodic acid–Schiff (PAS) stains for glycogen reveal aggregates of amylase-sensitive PAS-positive material, likely glycogen, clumped in the cytoplasm of the muscle fibre and under the cell membrane. This is a subjective evaluation of the amount and location of the aggregates because glycogen is a normal part of the muscle cell.

#10 What does bilirubin in urine look like?

The gross appearance of bilirubin in urine depends on the amount of the substance. It may not be visible grossly in small amounts; however, it causes a darker yellow to brown colour in increasingly larger concentrations. Bilirubinuria may precede bilirubinaemia due to the low renal threshold for bilirubin.

Usually, bilirubin is conjugated in urine, but unconjugated bilirubin is bound to albumin and may be present in the urine if there is albuminuria or proteinuria. Bilirubin presence in urine should be confirmed with a dipstick test result as it can be confused in some cases with haematuria or haemoglobinuria. Pigmenturia and some drugs can also give false-positive results.

#11 What is the artefactual effect of hyperlipaemia on biochemical electrolyte analysis?

Hyperlipaemia causes a number of analytes to be affected. It causes increased red blood cell (RBC) fragility and thus may cause or accentuate current haemolysis, which is then also an added factor that may affect analytes.

As an effect on direct measurement of analytes, hyperlipaemia can decrease AP/ALP and total calcium. It may, however, increase AST, GGT, GLDH, creatine kinase (CK), phosphorus, proteins, electrolytes, bilirubin and lactate dehydrogenase (LDH). Other effects may be due to clinical hyperlipaemia and its main effect on the liver with cholestasis, and thus AP/ALP, GGT, GLDH, AST, bile acids and also triglycerides.

#12 Is there any explanation for increased alkaline phosphatase activity in eosinophilic lower airway inflammation/eosinophilic pneumonia?

Alkaline phosphatase (ALP) activity could be increased if there is a general inflammatory response due to release from white blood cells, particularly monocytes and neutrophils. These may also be present along with eosinophils. If there is anorexia or other concurrent disease, eg haemolytic anaemia, then this can affect ALP through an effect on the liver.

#13 Can laboratory findings help for diagnosis of laminitis or colitis?

Laboratory tests may help with determining the underlying cause in some cases of laminitis and colitis. For example, laminitis may be due to insulin dysregulation caused by equine metabolic syndrome (EMS) or equine pituitary pars intermedia dysfunction (PPID). Also, underlying systemic disease or neoplasia could be a cause of colitis in rare cases and thus an overall blood picture may be helpful.

Laboratory tests may help with determining the underlying cause in some cases of laminitis and colitis.

#14 Is there any evidence that higher levels of SAA indicate an infectious process rather than just an inflammatory process?

We do not have a particular level of SAA that is more likely to indicate an infectious process, but bacterial disease is more likely to be present (Long and Nolen-Walston, 2020). Elevations in SAA are not specific for a certain disease process and can increase with inflammation in the absence of infection. Therefore, elevations in SAA should be evaluated in conjunction with physical examination findings and the results of other diagnostic tests.

#15 What parameters do I look for to know whether a horse is going into shock?

When a horse is in shock, the bottom line is that its tissues are not being perfused adequately. There are multiple types of shock, including distributive, hypovolaemic, obstructive, cardiogenic and metabolic/hypoxic. The overarching clinical signs of shock are:

Rapid breathing
Shaking and shivering
A weak pulse
Pale or blue mucous membranes
Extremities (eg ears) feel cold
Mentation

I would recommend measuring heart rate, body temperature, capillary refill time, respiratory rate and blood pressure. You can also look for a low blood glucose and partial pressure of oxygen (PaO2) or high lactate levels. Furthermore, a high SAA indicating inflammation, and low fibrinogen with high D-dimer may suggest DIC.

#16 Are small increases in ALP or GGT significant?

If levels of ALP or GGT are just above the reference range, then the result may not be significant. Correlation with other liver enzymes and other parameters is therefore advised. Checking for persistence and if there is an upward trend may then be significant. Normal ALP and GGT can be seen with significant liver disease; this is likely due to the decrease in normal hepatic tissue and the number of hepatocytes/biliary epithelial cells. Please note that bile acids and/or bilirubin are expected to increase in cases of severe or end-stage liver disease. Imaging may also show abnormalities.

#17 What happens if there is a high protein level in the blood of a patient?

High blood protein is not a specific disease or condition, but it might indicate that an individual has a disease. Elevated proteins more often indicate either dehydration (water loss) or inflammation (usually increased globulins), but high blood protein rarely causes signs or symptoms on its own. If the proteins become very high due to either of these causes, then the blood has increased viscosity. This increases the resistance to blood flow and thereby increases the work of the heart and impairs organ perfusion. It may also lead to an increased risk of clots or thromboembolic disease.

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