Investigation of calcium disorders and sample preparation.
Dr Stacey A Newton BVSc FRCPath CertEM (Int Med) PhD MRCVS
Stacey graduated from Bristol in 1993. After building up experience in general practice, she became a resident in equine medicine at Leahurst, Liverpool University. She was awarded a certificate in equine medicine (internal) in 1997. Stacey then went on to complete her PhD in equine neuroanatomy and neurology in 2001. She was awarded her FRCPath in 2010. Today Stacey is the Head of Clinical Pathology at NationWide Laboratories.
Parathyroid hormone and ionised calcium measurements are the two most important parameters in calcium investigations and their combined measurement should be considered in both hypocalcaemic and hypercalcaemic situations where the cause is not immediately obvious. Ionised calcium should always be used in the first instance to confirm actual calcium levels. Total calcium measurement may not provide a true reflection of calcium status in all cases. Ionised calcium, which is the biologically active part only makes up 50-55% of the total Calcium.
For example, cases with renal dysfunction may present with high total calcium when in fact Ionised calcium is actually normal or low.
If ionized Calcium is confirmed as high or low, then Parathyroid hormone should be included to determine the cause for the abnormality.
When measuring Parathyroid hormone, an ionised calcium should be measured at the same time since Parathyroid hormone reflects the minute-to-minute changes in calcium levels.
Sample preparation is critical for the accurate analysis of Parathyroid hormone and ionised calcium. Separated EDTA plasma should be shipped frozen for Parathyroid hormone and separated serum for ionised calcium.
The combination of Ionised calcium and Parathyroid hormone measurement usually permits differentiation between the four main categories of calcium regulation disorders:
The first category is Primary hyperparathyroidism or parathyroid dependent hypercalcaemia. Here we talk about functional parathyroid neoplasia (occasionally hyperplasia).
In these cases, the Ionised calcium is HIGH, and the Parathyroid hormone is either HIGH or in the top two thirds of the reference range.
The second category is Parathyroid independent hypercalcaemia. It includes Hypercalcaemia of malignancy (for example: lymphoma, apocrine gland adenocarcinoma), vitamin D toxicosis (calciferol containing rodenticides, calcitriol), Addison’s disease, feline idiopathic hypercalcaemia and granulomatous disease processes.
In these cases, the Ionised calcium is HIGH, and the Parathyroid hormone is LOW or in the lower third of the reference range.
The third category is Primary hypoparathyroidism or parathyroid dependent hypocalcaemia. This is usually idiopathic and relates to parathyroid gland destruction which is thought to involve immune mechanisms or inflammation. Other causes include surgery or spontaneous infarction.
In these cases, the Ionised calcium is LOW, and the Parathyroid hormone is either LOW or in the lower third of the reference interval.
Category four is Secondary hyperparathyroidism or parathyroid independent hypocalcaemia. Here we talk about renal failure, calcium losses for example, eclampsia, pancreatitis, equine diarrhoea/colic, dietary deficiency, rickets, hyperadrenocorticism and hyperthyroidism.
In these cases, the Ionised calcium is NORMAL or LOW with the Parathyroid hormone usually HIGH.
It is important to mention THE ROLE OF VITAMIN D and its metabolites.
Vitamin D3 is intimately involved in calcium regulation. Vitamin D is metabolised in Liver to 25 Hydroxyvitamin D (25(OH)D3) (Calcidiol). Calcidiol is metabolised to 1,25(OH)2 D3 (Calcitriol), in the kidneys.
CALCITRIOL increases serum calcium by:
- Increasing Intestinal absorption of calcium
- Mobilising calcium from Bone
- Calcium reabsorption from the Kidney
Two forms of vitamin D are measurable in veterinary serum samples: 25-hydroxyvitamin D3 (25-OHD3 – Calcidiol) and 1,25-dihydroxyvitamin D3 (Calcitriol).
Calcidiol is produced by the liver and its concentration parallels that of available vitamin D that may be dietary in origin (cholecalciferol (D3), ergocalciferol (D2)) or produced in the skin of certain animals under the influence of UV light.
Please note that dogs and cats have very limited/no synthesis of Vitamin D3 in the skin.
Because the enzymatic hydroxylation of vitamin D3 in the liver depends almost entirely on the availability of the substrate, this test is an excellent marker of overall Vitamin D3 status. It can be used to diagnose both conditions of vitamin excess and deficiency, therefore it is valuable in the investigation of both hyper- and hypocalcaemic disorders.
Cases where abnormal Calcidiol results may be encountered include dietary deficiencies, malabsorption syndromes (PLE, EPI) and calciferol rodenticide toxicities.
Calcitriol is produced by renal tubular cells as a result of enzymatic action (1α-hydroxylase) on Calcidiol substrate. The rate of this process is controlled by Parathyroid hormone concentrations (increasing Parathyroid hormone causes increased 1α-hydroxylase activity).
Phosphorus also has effect on this process as increases in P decrease 1α-hydroxylase activity.
Calcitriol is the most biologically potent form of vitamin D and its main activities are directed at increasing serum calcium concentrations, including increased intestinal uptake of calcium. The failure of renal tubular cells to generate calcitriol in renal disease is one of the contributing mechanisms to renal secondary hyperparathyroidism (and ‘rubber jaw’). The measurement of calcitriol may be of some value in understanding renal tubular function and the effects of Parathyroid hormone on the vitamin D system.
In dogs, cats and many veterinary species, very little passive intestinal absorption of calcium occurs. Intestinal calcium absorption is almost exclusively mediated by vitamin D.
The situation is different in horses, rabbits (and the hippopotamus), in which there is significantly more passive absorption of calcium and calcium status is controlled more by renal excretion. In these species, the effects of renal disease on calcium status will be different than in most common mammalian species.
Parathyroid hormone related peptide (PTHrP) may be used for the differential diagnosis of hypercalcaemia of unknown origin where other diagnostic tests have not identified the aetiology of the hypercalcaemia and where Parathyroid hormone and ionised calcium suggest parathyroid independent hypercalcaemia.
Parathyroid hormone related peptide is a hormone that can be produced by several different types of tumours in dogs, cats and horses and is considered to be the underlying cause of hypercalcaemia of malignancy in many, but not all cases. Over 50% of dogs with apocrine gland adenocarcinoma of the anal sac are hypercalcaemic at the time of diagnosis. The majority of hypercalcaemic lymphomas and smaller percentages of myeloma and carcinomas are Parathyroid hormone related peptide positive.
Circulating levels of Parathyroid hormone related peptide in normal dogs are almost undetectable (<0.5pmol/l). Levels greater than 1.5pmol/l are considered significant in dogs. Cats appear to show similar values.
It is important to note, that a negative or low Parathyroid hormone related peptide does not exclude a neoplastic cause particularly when the Parathyroid hormone is low normal or LOW.
Feline idiopathic hypercalcaemia is a common cause of hypercalcemia in cats. It is not reported in the dog. It is the most common cause reported in the USA. The pathogenesis is unknown.
Parathyroid hormone is low, and it is not associated with excess Vitamin D or calcitiriol.
In some studies, there is a suggested association with urinary acidifying diets. This is not proven.
Some authors believe the condition to be relatively benign, while others believe that the condition promotes the onset of renal dysfunction. Low dose glucocorticoids have been suggested as a therapy if there is concern about progressive disease.
Other drugs are also currently used including bisphosphonates.
This is important to avoid incorrect or spurious results.
For Parathyroid hormone the Sample must be frozen EDTA plasma, or frozen aprotinin EDTA plasma.
For Parathyroid hormone related peptide, the Sample must be frozen aprotinin EDTA plasma.
The addition of aprotinin is essential. The aprotinin EDTA sample tube supplied with the freezer pack MUST be used to collect the sample.
Follow the collection technique:
Firstly, Take the blood sample into the aprotinin EDTA tube supplied with the freezer pack. Mix well.
Then decant into a cooled plastic EDTA tube, or tubes, kept on ice. A high concentration of EDTA is desirable if you have documented marked hypercalcaemia – it is possible to half fill several EDTA tubes.
Mix well but gently, and centrifuge as quickly as possible (ideally in a refrigerated centrifuge).
Then Transfer the plasma into a cooled plastic (not glass) PLAIN tube kept on ice.
Immediately freeze the plasma sample at <-10°C and keep frozen until dispatch in the transport pack.
The initial sample for Ionised calcium is whole clotted blood in a plain tube filled to the brim to exclude air, but the submitted sample MUST be separated serum and NOT whole clotted blood as this can damage the sample in transit.
EDTA plasma is not acceptable as the EDTA would chelate all the available calcium making it unavailable for analysis.
The collection technique is the following:
Obtain a blood sample in a plain, non-gel tube and fill to the brim with blood to exclude air.
Leave the tube to sit for half an hour to separate out or alternatively (and ideally) centrifuge whole blood, aspirate the serum and decant into a further plain, non-gel tube (this will prevent in-vitro haemolysis which can lead to an erroneously low estimation of the ionised calcium concentration)
Then Close the lid firmly and Mail the sample to the laboratory as soon as possible.
The laboratory will apply a correction formula to take account of the change in pH that will have occurred due to exposure to air. This will result in an estimated Ionised calcium at a standardised pH (7.4).
Coming soon: rapid PTH analysis service from NationWide Specialist Laboratories
NationWide Specialist Laboratories will soon be offering the SAME DAY (<24 hours after sample’s arrival) service for the analysis of Canine, Feline and Equine parathyroid hormone (PTH).
At present, the samples for parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) are assayed once a week on a Wednesday with the results ready on a Thursday.
Going forward clients will be asked to submit 2 separate frozen EDTA plasma samples if they want the rapid PTH analysis service as well as PTHrP. PTHrP will continue to be analysed once a week on a Wednesday.
NationWide Specialist Laboratories, a part of NationWide Laboratories, are experts in veterinary endocrinology. Our diagnostic services are supported by access to world-renowned veterinary clinical and laboratory endocrinologists. We work according to the principles of GLP. All assay procedures are fully controlled using the relevant animal sera (Animal QC) and all assays are fully validated for clinical use in every species if appropriate. NationWide Specialist Laboratories currently organises and runs the European Society of Veterinary Endocrinology (ESVE) External Quality Assessment Scheme. For more information, please call 01223 493400.
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