19 Oct 2015
Andy Durham
Job Title
Figure 1. Normal variation of haemoglobin concentration though the day (Greppi et al, 1996).
Evaluating erythrocytes in peripheral blood in horses is an extremely common component of blood testing and may be helpful for the purposes of investigation of hydration status and anaemia.
However, many of the values generated by the laboratory to evaluate the erythron are subject to many further influences significantly affecting interpretation.
The commonly generated erythrocyte indices may be divided into three major and three minor indices (Tables 1a and 1b). Generally, the fundamental starting assumption with interpretation of the major red blood cell indices is abnormally high values indicate dehydration or hypovolaemia and low values indicate anaemia. The three minor indices may then be used to further inform or refine a diagnosis of anaemia. However, many additional factors may affect these measures and should always be considered before assuming a horse is anaemic or dehydrated.
Tremendous inter-individual variability exists in these values, largely related to breed, age, activity and possible concurrent illness. As horses enter training, red cell parameters are expected to increase for a couple of months before reaching a plateau. A PCV of 45% is normal in a fit Thoroughbred, but abnormally high in an elderly, retired Welsh pony.
Conversely, a PCV of 26% is normal in the latter and low in the former. A PCV of 50% or more is normal in the presence of hypoproteinaemia. A red cell count greater than 10 × 1012/L would be high in most adult horses, but normal in many foals, which tend to have a larger number of smaller cells, resulting in similar PCV to adult counterparts.
Unfortunately, strict application of laboratory reference ranges (especially those supplied with benchtop analysers) frequently leads to misinterpretation. Recent activity also has a profound effect on major red cell indices due to release of stored red cells from the spleen following catecholamine release. For example, collection of blood immediately following a hard gallop of a normal horse will frequently result in PCV values greater than 50%, but does not reflect hypovolaemia.
High major red cell indices are supportive of dehydration/hypovolaemia, but are far from definitive evidence of such. Two interconnected pools of red cells exist in horses – in the systemic circulation and in the spleen. Physiologic adrenergic splenic evacuation during exercise promotes intravascular oxygen carriage when it is required, resulting in very high intravascular red cell content and increased blood viscosity.
Restoration of a baseline “thinner” blood occurs when exercise is concluded. Stress and pain are also associated with adrenergic splenic evacuation, resulting in high values of major red cell indices in many critical care cases whether or not hypovolaemia is present. Thus, major red cell indices are inconsistent indicators of hydration status and other laboratory markers, such as lactate and creatinine, are preferred alongside clinical indicators.
Even in cases where high major red cell indices genuinely reflect hypovolaemia, they cannot always be used effectively to monitor restoration of fluid balance. For example, a colitis case that has suffered diarrhoea and fluid loss along with significant enteric protein losses, is likely to have relatively high major red cell indices alongside hypoproteinaemia due to low plasma volume. However, intravenous isotonic fluids frequently fail to decrease the red cell values as little of the supplied fluid is retained in the intravascular space due to low colloidal pressure associated with hypoproteinaemia.
Occasionally, cases of neoplastic and non-neoplastic hepatic disease are seen with polycythaemia that appears resistant to fluid therapy and does not seem to be caused by hypovolaemia or splenic contraction. It has been hypothesised such cases might relate to failure by the liver to metabolise and degrade circulating erythropoietin with resulting hyperstimulation of bone marrow. Certain neoplasms are also known to be able to secrete erythropoietin-like substances.
Where abnormally low major red cell indices are seen then consideration of anaemia inevitably follows, although, as aforementioned, the effects of increasing age, inactivity and native breeds should always be considered.
True anaemia is remarkably difficult to establish in horses. Anaemia is defined as decreased oxygen-carrying capacity of the blood. Most oxygen is carried in complex with haemoglobin within red blood cells, although a little
(1% or 2%) is also free and dissolved in plasma. Thus, the total oxygen content of blood (CaO2) is the sum of that associated with haemoglobin (within red cells) and that free in the plasma (outside of red cells). This can be calculated by the formula:
CaO2 = [Hb × SaO2 × 1.34] + [PaO2 × 0.003]
Thus, strictly, anaemia can only be defined following arterial blood gas analysis, although, in reality, it is mainly dependent on haemoglobin concentration. Single measurements of haemoglobin, however, cannot be relied on as considerable variation can occur through the day. For example, one study showed, in a group of 12 healthy horses kept in loose-boxes, the mean haemoglobin concentration varied through the day between 16.1g/dL and 19.1g/dL (Figure 1). Thus, given haemoglobin concentration is a highly variable measurement in horses, it also follows that CaO2 may vary considerably both between individuals and in the same individual at different times of day. Typical CaO2 values range between 12mL to 24mL O2/dL in normal horses – performance animals are expected to be in the top half of this range, while cold-blooded, native, elderly and inactive animals generally appear clinically normal with values in the lower half of this range.
Unequivocal and objective definition of anaemia in horses therefore appears hard to achieve and clinical judgement has to be applied, taking all things aforementioned into consideration. If anaemia is suspected on the basis of clinical signs and unexpectedly low major red cell indices then the minor red cell indices might offer some further guidance. Mean corpuscular volume (MCV) might suggest macrocytosis or microcytosis consistent with regenerative and non-regenerative anaemias respectively. Microcytic anaemias also tend to have low mean corpuscular haemoglobin concentration (MCHC), although this will be normal in most macrocytic anaemias.
Intravascular haemolysis can be accompanied by high MCHC and mean corpuscular haemoglobin due to free haemoglobin in plasma. Also, bear in mind individual expectations with, for example, normal foals tending to have relatively small red cells (low MCV) and, in contrast, normal donkeys having relatively large red cells (high MCV).
A logical approach to investigation of anaemia should enable significant diagnostic progress and either a specific diagnosis or at least a narrowed differential diagnosis list.
Classification and sub-classification of anaemias are important diagnostic steps that enable a shorter list of differential diagnoses (Figure 2). Anaemias can be initially classified into regenerative – associated with appropriate (increased) bone marrow activity – and non-regenerative – associated with inappropriate (normal to decreased) bone marrow activity. Regenerative anaemias may be acute, subacute, chronic or recurrent, whereas non-regenerative anaemias tend to be subacute to chronic.
Regenerative anaemias are caused by haemorrhage or haemolysis. Haemorrhagic anaemias always require consideration of haemocoagulation as a possible cause and also where blood loss is occurring. Some cases of blood loss may be obvious – for example, chronic epistaxis or haematuria. Others may be far more cryptic such as enteric blood loss into the lumen or into endoparasites.
Blood may be lost from the vasculature, but remain within the body (intracorporeal). This may be into a body cavity (for example, pleural or peritoneal cavity following neoplasia, trauma or phenylephrine therapy) or held within tissues such as broad ligament (foaling complication) or intramuscular sites (for example, bone fracture and haemangiosarcoma).
Bleeding into body cavities such as the pleural or peritoneal space is generally effectively restored back into the circulation and the temptation to drain it should generally be resisted unless clinical circumstances (for example, dyspnoea) dictate otherwise.
Haemolytic anaemias may be intravascular or extravascular, with the latter probably predominating. It is not unusual for concurrent thrombocytopenia to occur.
Acute, immune-mediated haemolysis is associated with complement activation and red cell lysis within the circulation may occur in adults or foals (neonatal isoerythrolysis) and may also be seen with infectious cases, such as piroplasmosis or equine infectious anaemia and in microangiopathic haemolysis where abnormal vasculature leads to haemolysis (for example, disseminated intravascular coagulation, vasculitis and haemangiosarcoma).
Both piroplasmosis and equine infectious anaemia have been seen in the UK in recent years and should definitely be included in differential diagnosis considerations of anaemia. Extravascular haemolysis may occur as subacute to chronic/recurrent anaemia following immunoglobulin G opsonisation of red cells, which are then phagocytosed by screening macrophages in the reticuloendothelial system, most notably in the spleen. Non-regenerative anaemias are often associated with a primary disease process involving chronic inflammation or neoplasia (“anaemia of chronic disease”). This probably arises as a result of iron sequestration as part of a chronic inflammatory response, and also from the general catabolic effects of chronic inflammation.
Alternatively, toxic, drug-induced or viral processes may be associated with suppression of bone marrow cellular activity, although this aetiology often remains speculative.
Specific dyspoiesis of erythrocyte lines may occur, or more widespread suppression of white cell and/or platelet precursors. Occasionally, myelophthisic disease is seen where bone marrow haematopoietic precursor cells are normal, but occupation of bone marrow by non-haematopoietic tissue limits their numbers and productivity (for example, leukaemia). There are many aspects to investigation of anaemia, the detail of which is beyond the scope of this article, but should include basic clinical examination, endoscopy, ultrasonography, peritoneal tap and urinalysis.
Bone marrow examination is a very important, but underused, technique in equine practice. Bone marrow sampling is relatively straightforward in horses and adds significantly useful information to the investigation of cases of anaemia and also horses showing other persistent abnormalities of leukogram or platelets.
Two techniques are possible: aspiration and core biopsy. The preferred site for both techniques in adult horses is the midline sternum level, with the points of the elbows in a horse standing square. In foals, the tuber coxa can be used. Bone marrow aspiration is performed following sedation and sterile preparation.
A 9cm, 18G spinal needle can be slowly “drilled” into the sternum and then the stylet removed and a 5ml syringe attached containing a bead of ethylenediamine tetra-acetic acid (EDTA) solution in the hub (from adding sterile water to an EDTA vacutainer). Very short and gentle vacuum is then applied to the syringe in an attempt to obtain a single drop of bone marrow in the hub of the syringe.
If the sample flows spontaneously or very easily then it is likely significant blood contamination will have occurred and the sample will be of poor diagnostic quality. The best sample is one the operator feels he or she has worked quite hard for – after perhaps three or four gentle suctions. Air-dried smears of this are then prepared and submitted for evaluation. Very occasionally, no sample is obtained, despite several attempts, in which case, again, another site is chosen as the needle may well be sitting in an intersternebral space rather than in a sternebra itself.
Bone marrow core biopsy is performed at the same site as above. After sterile preparation, 5ml of local anaesthetic is infiltrated subcutaneously and in the tissues to the depth of the midline sternum. A small stab incision is made with a number 15 scalpel blade.
An 8G Jamshidi biopsy needle is then inserted and drilled into the bone (Figure 3). This collects a small core of bone marrow, which is preserved in formalin for a better evaluation of cells within bone marrow.
Some operators like to check for sternebral deposits using ultrasound, although this author has never found the need for this and simply ensures midline penetration of the sternum at the level of the points of the elbows with invariable entry into bone marrow deposits.
Anaemia is probably suspected more often than genuinely seen in horses, but, nevertheless, lends itself well to a logical and systematic investigative approach as outlined. In the climate of vigilance versus exotic diseases, the UK practitioner should not forget piroplasmosis and equine infectious anaemia when investigating such cases.
