Hematology and clinical biochemistry reference intervals for companion pigs using the ADVIA 2120 and Cobas c501

Introduction

Companion pigs are becoming increasingly popular (Curnutte, 2014), and as such, routine blood testing including complete blood count (CBC) and clinical biochemistry testing is performed more frequently. In the clinical pathology laboratory of the University of Tennessee Veterinary Medical Center (UTVMC), for example, the number of plasma biochemistry profiles performed on porcine samples increased over threefold in the decade spanning 2010–2019 compared to 2000–2009. Because the results of these tests depend on factors such as species, breed, age, sex, and husbandry conditions, the recommendation of the American Society for Veterinary Clinical Pathology (ASVCP) is to generate population-based reference intervals from a reference sample group with similar demographics to the animal population in which they will be used (Friedrichs et al., 2012). These ASVCP guidelines also provide specific recommendations regarding methods for determining and reporting reference intervals, including statistical methods, based on the number of individuals in the reference group. However, the majority of published porcine reference intervals are either not compliant with these recommendations or are from sample groups dissimilar to clinical porcine patients, such as commercial production pigs, research pigs, sexually immature pigs, or pregnant or lactating sows (Brockus et al., 2005; Dimitrakakis et al., 2022; Li et al., 2021; Perri et al., 2017; Radin, Weiser & Fettman, 1986; Verheyen et al., 2007; Yeom et al., 2012).

The main objective of this study was to provide CBC and clinical biochemistry reference intervals for sexually mature companion pigs using ASVCP guidelines. A secondary objective was to compare results based on age group, breed, and reproductive status.

Materials and Methods

Results

Partitioning by age

For the CBC, the following values were significantly lower in <1 year old pigs compared to at least one of the remaining age groups: mean cell volume (MCV, P < 0.001), mean cell hemoglobin (MCH, P < 0.0001), and mean platelet volume (MPV, P < 0.001). Additionally, the following were significantly higher in <1 year old pigs: red blood cell concentration (RBC, P = 0.031), platelet concentration (P < 0.001), and lymphocyte concentration by both automated (P < 0.001) and manual (P < 0.001) methods. For a few of these hematology values, the 1–2 year old pigs were also significantly different from the pigs greater than 2 years old, specifically MCV, MCH, platelet concentration and MPV. The youngest age group also had significantly lower results for total protein by refractometry (P < 0.001). Total protein, albumin, and globulins on the chemistry panel also had a trend toward being lower in the youngest group, but differences did not reach statistical significance.

For all of these values, the reference intervals provided in Table 1 include only pigs that are at least 1-year-old. Calculation of reference intervals for subgroups after partitioning is only recommended if each of the subgroups contains at least 40 individuals (Friedrichs et al., 2012). For this reason, partitioned reference intervals were not calculated separately for each of the three age groups. Instead, graphical representation of results from subgroups is provided in Figs. 1 and 2 for values in which a statistically significant difference was noted between subgroups.

Partitioning by sex and breed

When grouped by sex as intact female, intact male, spayed female, or castrated male, the only significant differences were that intact males had a higher neutrophil count by automated (P = 0.010) and manual (P = 0.016) methods (Fig. 3). A few significant differences were also noted between breeds (Fig. 4). The single Meishan pig was excluded from these comparisons. Vietnamese potbellied pigs had a lower manual lymphocyte count (P = 0.001) than the other two breeds. Total protein on the chemistry panel and total protein estimated by refractometry were both significantly different among the breeds (P = 0.013 and P = 0.011, respectively), with American mini pigs having the lowest values.

Discussion

The objectives of this study were to provide hematologic and biochemical reference intervals for companion pigs that are compliant with ASVCP guidelines and to evaluate for differences based on age, sex, and breed. Reference intervals derived from healthy individuals of a species are used to identify potentially pathologic abnormalities on CBC and biochemistry panels from clinical patients. These reference intervals are ideally determined de novo in each laboratory to account for bias between instruments and methods. This can be time-consuming and expensive to perform for all species seen in veterinary hospitals, though. As such, for some species, clinicians must rely on published reference intervals to interpret laboratory data. However, many published reference intervals for pigs are derived from sample groups that do not mimic our clinical patient population of companion pigs, but rather are derived from research colonies, commercial swine, or juvenile pigs (Dimitrakakis et al., 2022; Li et al., 2021; Perri et al., 2017; Radin, Weiser & Fettman, 1986; Verheyen et al., 2007).

Comparison of the methods and results of our study to those of six other porcine reference interval publications is provided in Tables S3–S5. For most values, our reference intervals are similar to previously published values, but some differences are noted. However, the sample groups for these studies varied in breed, age, and number of individuals, and there were also differences in analytical and statistical methods, all of which may contribute to differences in results. For example, the lower reference limit of our lymphocyte reference interval is lower than those published for juvenile pigs (Dimitrakakis et al., 2022; Li et al., 2021), but it is similar to that published for adult potbellied pigs (Brockus et al., 2005), suggesting an effect of animal age. However, compared to the same study of adult potbellied pigs, our chloride reference interval was lower, which might indicate differences in analytical methodology.

Benefits of the current study include that the reference intervals were derived from client-owned healthy companion pigs seen at the UTVMC, so reflect companion pig patients, and were reported using ASVCP recommendations for a sample group of 40–120 individuals. Additionally, the reference sample group included a mix of ages, sexes, and breeds, allowing comparison of results based on these factors. Finally, some of the values reported here are not included in most prior studies, such as WBC differentials by both automated and manual methods, SDH, magnesium, and iron.

The majority of differences noted between pigs less than or greater than 1 year of age have also been identified in other species. The 5 to 11-month-old pigs had lower MCV and MCH compared to pigs at least 1 year of age, which was also reported in 6 to 12-month-old specific pathogen free minipigs compared to those at least 1 year of age (Yeom et al., 2012). Red cells have been reported to be smaller in younger animals of many species compared to adults, with those smaller red cells contributing to decreases in both MCV and MCH (Harper et al., 2003; Monke et al., 1998; Muñoz et al., 2012; Stockham & Scott, 2008a). One proposed mechanism for production of smaller red cells in young rapidly-growing animals is that they may have low iron stores due to an all-milk diet during the neonatal period (Brockus, 2011). Iron deficiency is a common cause of production of microcytic erythrocytes (Li et al., 2021; Stockham & Scott, 2008a). It is possible that the youngest age group in our study either still had low iron stores contributing to decreases in red cell size, or had gained adequate iron stores by the time of sample collection but had not yet replaced all neonatal red cells with normocytic cells. Notably, the serum iron was not significantly lower in the youngest pigs of our study nor of the study of specific pathogen free minipigs (Yeom et al., 2012), supporting adequate iron stores by the time of sample collection. However, iron was only measured in 42 out of the 94 pigs in our study, including 11 pigs <1 year of age, 12 pigs between 1 and 2 years, and 19 pigs >2-years-old, which may have impacted the ability to detect age-related differences. Interestingly, the RBC concentration in 5 to 11-month-old pigs was higher than that of older pigs, suggesting that decreased iron stores, if present, were not measurably impairing erythropoiesis. The cause for the higher RBC count in these young pigs cannot be confirmed but may indicate either increased erythropoiesis or a relative increase due to excitement-mediated splenic contraction. Higher RBC concentration is not a commonly reported feature of young animals.This trend was noted in at least one publication in horses, but not in specific pathogen free minipigs (Muñoz et al., 2012; Yeom et al., 2012). Pigs less than 1 year of age also had higher lymphocyte concentrations than the older pig groups, which has been reported to occur commonly in other species, possibly either due to increased immune stimulation or physiologic leukocytosis (Barton & Hart, 2020; Harper et al., 2003; Monke et al., 1998; Panousis et al., 2018; Stockham & Scott, 2008b).

Additionally, younger pigs had increased platelet concentration and decreased MPV compared to older pigs. However, plateletcrit was not significantly different based on age, presumptively due to the summative effects of higher platelet numbers but smaller platelet size. Higher platelet numbers have been reported in younger calves and humans, but in these reports the MPV is either similar to or higher than that of adults (Jeon, 2022; Novak, Tschantz & Krill, 1987; Panousis et al., 2018). The mechanism for these higher platelet counts has not been proven, but in pediatric humans is suspected to be promoted by accelerated thrombopoiesis due to increased circulating thrombopoietin concentrations relative to adults (Jeon, 2022). It is possible that this phenomenon may also occur in pigs, but accelerated thrombopoiesis would be expected to promote an increase in MPV rather than a decrease (Stockham & Scott, 2008c). Therefore, a mechanism for the age-related changes in platelet concentration and MPV in these pigs is uncertain. Lastly, the lower protein concentration noted in the younger pigs has been previously reported in other species (Barton & Hart, 2020; Harper et al., 2003; Monke et al., 1998; Muñoz et al., 2012). In very young animals, this can occur due to variable absorption of colostral immunoglobulins, but this would be an unlikely explanation for pigs of the ages included in our study. A more plausible theory for our study population, given that there was a trend toward decreases in both albumin and globulin in the youngest group, is that non-immunoglobulin protein concentrations may increase as animals mature due to increasing hepatic protein synthetic capability (Muñoz et al., 2012).

Notably, there were a few values in which the differences were also significant between 1 to 2-year-old pigs and pigs greater than 2-years-old, specifically MCV, MCH, platelet concentrations, and MPV, suggesting that some values may not reach adult levels until pigs are older than 1 year. These findings highlight the importance of accounting for patient age when interpreting laboratory results.

In contrast to age groups, there were few significant differences when pigs were partitioned based on sex or breed. Intact males had significantly increased neutrophil concentrations, with possible explanations being increased likelihood of glucocorticoid or epinephrine responses during handling or of subclinical inflammatory disease. There were no other laboratory changes supportive of these conditions in the intact male pigs, such as changes in lymphocyte or band neutrophil concentrations, but that does not exclude these possibilities. Intact males were significantly younger than the other sex groups, but neutrophils concentrations did not differ based on age, so a cause for the neutrophil differences is unclear. For breed groups, a mild but significant difference was noted in the lymphocyte concentration by the manual method, with Vietnamese potbellied pigs having slightly lower values. Vietnamese potbellied pigs were also significantly older than other breeds, so it is unclear if this lymphocyte difference is an effect of age or a true breed difference. Mild differences were also noted based on breed for protein measurements, with lower values noted in American mini pigs. This group did not differ from other breeds based on sex or age, so it is possible that this may be a true breed difference, but there were only 13 American mini pigs in the study, so this would need to be evaluated in a larger number of individuals.

There are a few limitations of this study. According to ASVCP guidelines, the ideal group size for determining reference intervals is at least 120 individuals (Friedrichs et al., 2012). However, they acknowledge that in veterinary medicine there are issues that can make this goal difficult to achieve, such as availability of individuals or cost of sample collection and analytical procedures. For this reason, they also provide recommendations for determination of reference intervals in sample sizes between 40 and 120. Indeed, the goal of the present study was to include 120 pigs, but despite the prolonged course of the study, only 94 clinically healthy companion pigs were available. Additionally, some values were not available for all samples for various reasons, including reticulocytes, iron, triglycerides, and cholesterol. However, there were at least forty samples for most values, so the recommended methods for a sample size between 40 and 120 were used. The exception was reticulocytes, for which there were only 20 individuals. Due to the small sample size, a reference interval was not calculated for reticulocytes, rather descriptive statistics were provided.

Results at the extremities of distribution were retained in most cases to maximize sample size; however, a few outlying results were eliminated. These included the platelet measurements from a sample with numerous large platelet clumps, and high AST and CK values from two pigs suspected of having muscle injury during transport or sample collection. Additionally, two high GGT measurements were excluded from the two most lipemic samples in the study. The manufacturer of the GGT assay used in this study states that the degree of lipemia based on the lipemic index in those samples is not expected to interfere with GGT measurement (Roche Diagnostics, Basel, Switzerland). This suggests that analytical interference from lipemia might not explain the high GGT results in these pigs, although the manufacturer’s claim is based on experiments using an artificial lipid additive that may not correspond to endogenous porcine lipids. Both pigs were 9-month-old males that appeared clinically healthy, and neither had high total bilirubin measurements (0.1 mg/dl in both animals), arguing against cholestasis as a cause of increased GGT. A cause for the extreme GGT values in these individuals cannot be confirmed. Because this finding was only noted in the two samples with marked lipemia, an unverified lipemia interference is suspected; however, subclinical liver disease such as biliary hyperplasia or cholestasis cannot be ruled out.

Additional limitations include that these samples were measured on specific analyzers, so the resulting reference intervals may not be completely transferrable to other instruments. These results should be used as guidelines only if internal reference intervals are not available from the laboratory evaluating the samples. Also, the confidence intervals, particularly of upper reference limits, were often wider than ideal, indicating that mildly increased values may not always indicate pathology. Additionally, almost half of the reference group was made up of intact females, likely because many of the samples were collected prior to ovariohysterectomy. However, there were very few significant differences in results based on sex, so this might not have appreciably skewed reference intervals.

Finally, the majority of pigs were sedated or anesthetized prior to sample collection, with the most common drugs being xylazine, midazolam, ketamine, and isoflurane so we cannot exclude that some values may have been impacted. Specific effects of these drugs on hematology and biochemistry in companion pigs is uncertain, but there are reported effects in other species. The drugs used in our study have been associated with lower values for RBC concentration, hemoglobin, hematocrit, or packed cell volume in several species, possibly due to splenic sequestration and fluid shifting (Custer et al., 1977; Dhumeaux et al., 2012; Kullmann et al., 2014; Marini et al., 1994). Mild increases in CK were observed in dogs administered xylazine and ketamine (Franco et al., 2009), so it is possible that this might have affected the CK results from some of the pigs in our study. Hepatotoxicity and increased liver enzymes have been reported after repeated ketamine doses in people (Cohen et al., 2018), but ketamine was only administered once before blood collection in these pigs so this is considered unlikely. Ketamine is reported to cause increased plasma cortisol in calves, horses, and rabbits, so it is plausible that there may be cortisol-related effects on laboratory results, such as increases in neutrophils and glucose, and decreases in lymphocytes (Plumb, 2024). Lastly, isoflurane has been reported to cause mild increases in AST or GGT in some species (Humann-Ziehank & Ganter, 2012).

In contrast, unsedated pigs may become stressed during blood collection, with the resultant epinephrine response also impacting results, such as promoting increased concentrations of blood neutrophils, lymphocytes, and glucose. This highlights that animal handling and medications may impact hematology or biochemical results in various ways. It is therefore ideal that the blood collection procedure for the reference sample group is as similar as possible to that used for blood collection from the patient population in which the reference intervals will be used. At the UTVMC, clinical porcine patients are commonly, but not always, sedated or anesthetized prior to blood collection, similar to the reference sample group in this study. Care should be taken in using these reference intervals in unsedated pigs, as alterations may result from the differences in patient preparation.

Conclusions

This study provides hematology and biochemistry reference intervals for companion porcine patients, including a comparison of results based on age, sex, and breed. Some values were significantly different based on age, all of which have also been described in other species, but few values were significantly different based on sex or breed. To minimize bias introduced by differences in methodology, reference intervals are ideally generated de novo in the same laboratory in which patient samples are measured. However, because reference interval determination is a time-consuming and costly endeavor, these published values can be used cautiously to interpret data from companion pig patients when local reference intervals are not available.

Supplemental Information