Editorial Type:
Analytic Techniques

Evaluation of changes in haptoglobin and C-reactive protein concentrations caused by freezing of saliva and meat juice samples collected from healthy and diseased pigs

Ana M. GutiérrezDepartment of Animal Medicine and Surgery, School of Veterinary Medicine, University of Murcia, 30100, Espinardo, Murcia, Spain.

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Silvia Martínez-SubielaDepartment of Animal Medicine and Surgery, School of Veterinary Medicine, University of Murcia, 30100, Espinardo, Murcia, Spain.

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Jose J. CerónDepartment of Animal Medicine and Surgery, School of Veterinary Medicine, University of Murcia, 30100, Espinardo, Murcia, Spain.

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DOI:
https://doi.org/10.2460/ajvr.72.1.11
Volume/Issue:
Volume 72: Issue 1
Online Publication Date:
01 Jan 2011
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Abstract

Objective—To evaluate changes in stability of haptoglobin and C-reactive protein (CRP) concentrations caused by freezing of saliva and meat juice samples.

Animals—16 specific-pathogen-free pigs and 16 pigs with clinical signs of disease.

Procedures—Saliva and diaphragmatic muscle were collected immediately before and after slaughter, respectively. Haptoglobin and CRP concentrations of pooled samples were measured before storage (day 0) and after 7, 15, 30, 60, 120, 210, and 365 days of storage at −20°C and after repeated freezing-thawing cycles (up to 7 times). In a second experiment, addition of a protease-inhibitor cocktail to saliva and storage of saliva samples at −80°C for up to 30 days were assessed for effects on CRP concentrations.

Results—Haptoglobin concentrations in saliva did not change for up to 120 days in samples stored at −20°C, but longer storage times and multiple freezing-thawing cycles increased haptoglobin concentrations. Salivary CRP concentrations decreased significantly after 7 days of storage at −20°C, and addition of a protease-inhibitor cocktail did not improve CRP stability. Lower temperatures limited salivary CRP degradation. In meat juice, haptoglobin and CRP concentrations were stable at −20°C up to 210 days.

Conclusions and Clinical Relevance—Acute-phase protein measurements in saliva should be performed as soon as possible after sample collection. When this is not possible, storage temperature of −80°C is recommended. Acute-phase protein concentrations appeared to be more stable in meat juice samples than in saliva samples. Saliva and meat juice could be used as alternatives to serum for haptoglobin and CRP analysis.

Abstract

Objective—To evaluate changes in stability of haptoglobin and C-reactive protein (CRP) concentrations caused by freezing of saliva and meat juice samples.

Animals—16 specific-pathogen-free pigs and 16 pigs with clinical signs of disease.

Procedures—Saliva and diaphragmatic muscle were collected immediately before and after slaughter, respectively. Haptoglobin and CRP concentrations of pooled samples were measured before storage (day 0) and after 7, 15, 30, 60, 120, 210, and 365 days of storage at −20°C and after repeated freezing-thawing cycles (up to 7 times). In a second experiment, addition of a protease-inhibitor cocktail to saliva and storage of saliva samples at −80°C for up to 30 days were assessed for effects on CRP concentrations.

Results—Haptoglobin concentrations in saliva did not change for up to 120 days in samples stored at −20°C, but longer storage times and multiple freezing-thawing cycles increased haptoglobin concentrations. Salivary CRP concentrations decreased significantly after 7 days of storage at −20°C, and addition of a protease-inhibitor cocktail did not improve CRP stability. Lower temperatures limited salivary CRP degradation. In meat juice, haptoglobin and CRP concentrations were stable at −20°C up to 210 days.

Conclusions and Clinical Relevance—Acute-phase protein measurements in saliva should be performed as soon as possible after sample collection. When this is not possible, storage temperature of −80°C is recommended. Acute-phase protein concentrations appeared to be more stable in meat juice samples than in saliva samples. Saliva and meat juice could be used as alternatives to serum for haptoglobin and CRP analysis.

Acute-phase proteins (eg, CRP and haptoglobin) are proteins found in serum, and the concentration of APPs changes after tissue injury, infection, or trauma.1 Analysis of APPs in pig production has become an area of increasing interest to evaluate the stress of pigs and to assess the health status and well-being of a herd.2,3 Determination of concentrations of various combinations of APPs is recommended because the response of a particular APP can vary with inflammation and tissue damage.4

The use of saliva and meat juice samples as an alternative to the use of serum has been reported5–8 and has several advantages. First, simple and minimally stressful sample methods, such as the collection of saliva, are in increasing demand for use in current production systems.5 Moreover, repeated collection of saliva samples for serial measurements over the course of minutes, hours, days, or even longer is possible and, in most circumstances, can be performed by personnel that have received minimal training in the collection of saliva samples.6 In addition, meat juice samples have been investigated in pigs to avoid the difficulties associated with obtaining serum at the time of postmortem inspection,7 which allows for the collection of samples from a greater number of pigs without slowing the progress of the slaughter line.8

Because immediate sample analysis is not feasible in many research and clinical settings, the stability of analytes during sample storage should be evaluated. Studies in human medicine have shown APPs, such as CRP in serum and plasma9,10 or whole blood samples,11 to be stable for extended periods or for as little as 3 days when frozen at 4°C. A wide range of variations in the stability of analytes, such as lysozyme and IgA12 or corticosteroids,13 in samples of saliva has been reported. In veterinary medicine, data reported for the stability of APPs pertain mainly to serum or whole blood samples, such as those obtained for CRP stability in pigs14 and dogs15; stabilities were similar to that reported9 in samples obtained for humans. Despite the advantages of the use of saliva and meat juice over serum, no data exist regarding the stability of CRP and haptoglobin in saliva or meat juice.

The purpose of the study reported here was to evaluate changes in the stability of haptoglobin and CRP concentrations caused by freezing of saliva and meat juice samples of pigs and to evaluate the effect of repeated freezing-thawing cycles on the concentrations of CRP and haptoglobin. The results of these evaluations will have important implications with regard to validity of the quantification of CRP and haptoglobin in circumstances in which quantifications are performed on frozen samples.

Materials and Methods

Animals—Samples were obtained from 32 (16 healthy and 16 diseased) pigs to guarantee a sufficient volume of saliva and meat juice to provide the necessary pools for 2 experiments. Healthy pigs came from a specific-pathogen-free farm. Diseased pigs were from a farm on which pigs were affected with several diseases, such as porcine reproductive and respiratory syndrome and postweaning multisystemic wasting syndrome. The diseased pigs had clinical signs of disease, such as cough, nasal discharge, multiple abscesses, and poor body condition. All pigs included in the study were crossbred Duroc X Landrace-Large White males, with a mean age of 190 days. Procedures involving animals were approved by the Murcia University Ethical Committee.

Sample collection—Samples were obtained from pigs during 2 periods. First, samples of saliva and meat juice were obtained from 10 healthy and 10 diseased pigs at the beginning of the study (experiment 1). After 1 year, samples of fresh saliva were obtained from another 6 healthy and 6 diseased pigs of the same age and from the same farms to enable another experiment (experiment 2) to be conducted.

Samples of saliva were collected at the farm of origin by use of specific tubesa containing a sponge, instead of a cotton swab, because a sponge is less absorbent and provides good liberation of saliva after centrifugation. Pigs were allowed to chew on a sponge clipped to a flexible thin metal rod for approximately 1 minute, until the sponge was thoroughly moistened. Sponges then were placed in test tubes and centrifuged for 10 minutes at 3,000 × g.

Samples of meat juice were obtained from pigs at an abbatoir at the end of the slaughter line, after pig carcasses were processed, or at necropsy. For procurement of meat juice samples, pieces of diaphragmatic muscle (3 × 3 cm) were placed in meat extract collectorsb and frozen at −20°C overnight. Afterward, the collectors were thawed at room temperature (21°C) for 2 hours; meat juice samples then were collected from the bottom of each tube.

Samples from diseased pigs were used to provide pools with high concentrations (n = 5 samples/pool; 750 μL from each sample) of APP, and samples from healthy pigs were combined to provide pools with low concentrations (5 samples/pool; 750 μL from each sample) of APP Pooled samples from a combination of both diseased (n = 6 samples/pool; 600 μL from each sample) and healthy (6 samples/pool; 600 μL from each sample) pigs were used to provide pools with intermediate concentrations of APP.

Effect of long-term storage at −20°C on CRP and haptoglobin concentrations in saliva and meat juice samples (experiment 1)—The aforementioned saliva and meat juice samples were used to provide 4 pools of saliva (2 with high and 2 with low APP concentrations) and 6 pools of meat juice (2 with high, 2 with intermediate, and 2 with low concentrations of APP) for the haptoglobin and CRP stability evaluations. Eight 100-μL aliquots were prepared from each pool. One aliquot of each pool of saliva and meat juice was immediately analyzed to determine a baseline (day 0) haptoglobin and CRP concentration; the remaining 7 aliquots were analyzed after storage at −20°C for 7, 15, 30, 60, 120, 210, and 365 days.

In addition, a larger aliquot (1 mL) from each of the pools of saliva and meat juice was stored separately at −20°C for evaluation of the effects of freezing-thawing on CRP and haptoglobin concentrations. These aliquots were measured in parallel with the other aliquots but were subjected to repeated freezing-thawing cycles at each of the 8 measurement time points.

Effects of a protease-inhibitor cocktail and storage at −80°C on CRP stability in saliva samples, compared with storage at −20°C (experiment 2)—To determine whether a protease-inhibitor cocktail could efficiently prevent degradation of proteins in saliva samples, 6 pools of saliva (2 with high, 2 with intermediate, and 2 with low APP concentrations) were prepared by use of the aforementioned samples. Each pool was divided into 100-μL aliquots and stored in duplicates at −20°C. A protease-inhibitor cocktailc (1 μL/mL) was added, as previously described,16 to one of the duplicate aliquots before storage to prevent protein degradation by enzymes, whereas the other duplicate aliquot was stored without the addition of the protease-inhibitor cocktail. Concentrations of CRP in the 6 pools, with and without protease-inhibitor cocktail, were determined weekly for 30 days after storage.

For the study of CRP stability in saliva at −80°C, additional aliquots (100 μL) of the 6 pools were stored at −80° or −20°C in the absence of a protease-inhibitor cocktail. Concentrations of CRP were measured in 1 aliquot of each condition after storage for 1, 7, 15, and 30 days.

Quantification of APPs—Haptoglobin and CRP concentrations in saliva and meat juice were measured via validated time-resolved immunofluorometric assays.17–19 Both assays had good precision (coefficient of variation was < 7% and 14% for intra-assay and interas-say precision, respectively) and low limits of detection (0.52 and 0.47 ng/mL for haptoglobin and CRP concentrations, respectively).

Statistical analysis—Results for measurement of haptoglobin and CRP concentrations were evaluated for approximate normality of distribution via the Kolmogorov-Smirnov test. Because the results met the criteria for a normal distribution, a repeated-measures ANOVA and a Dunnett post hoc test were used to evaluate changes in mean haptoglobin and CRP values in saliva and meat juice pools with different concentrations of APPs after long- and short-term storage and to assess the effects of repeated freezing-thawing cycles. Changes that exceeded ± 2 intra-assay coefficients of variation of the haptoglobin and CRP assays and had significant (P < 0.05) differences over time were considered to indicate unacceptable stability for given storage conditions.13 A value of P < 0.05 was used to indicate significance in all analyses.

Results

Effect of long-term storage at −20°C on CRP and haptoglobin concentrations in saliva samples (experiment 1)—Results for haptoglobin and CRP stability in saliva during long-term storage (for up to 1 year) were summarized (Figures 1 and 2). Mean haptoglobin concentration in the pool with high APP concentrations was 3.5 μg/mL at the beginning of the experiment (day 0) and 3.6 μg/mL after storage at −20°C for 120 days. However, concentrations increased, but not significantly, in the pools with high haptoglobin concentrations after storage for 210 and 365 days and after 5 freezing-thawing cycles. In contrast, no variations were observed during the entire experimental period in the pools with low concentrations of haptoglobin.

Figure 1—
Figure 1—

Mean haptoglobin concentrations in pooled saliva samples with high and low APP concentrations collected from 10 specific-pathogen-free and 10 diseased pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; circles] and low [5 samples/pool; squares] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; triangles] and low [5 samples/pool; diamonds] concentration pools). Eight 100-μL aliquots were prepared from each pool of saliva for measurement of haptoglobin concentrations after storage. Concentration in an aliquot was immediately measured to determine a baseline (day 0) haptoglobin concentration, and the remaining 7 aliquots were used for measurements on days 7 15, 30, 60, 120, 210, and 365. In addition, a larger aliquot (1 mL) from each pool of saliva was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.11

Figure 2—
Figure 2—

Mean CRP concentrations in pooled saliva samples with high and low APP concentrations collected from 10 specific-pathogen-free and 10 diseased pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; circles] and low [5 samples/pool; squares] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; triangles] and low [5 samples/pool; diamonds] concentration pools). Eight 100-μL aliquots were prepared from each pool of saliva for measurement of CRP concentrations after storage. Concentration in an aliquot was immediately measured to determine a baseline (day 0) CRP concentration, and measurements of the remaining 7 aliquots were performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a larger aliquot (1 mL) from each pool of saliva was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles. *Within a pool, concentration differs significantly (P < 0.05) from the concentration determined at baseline (day 0).

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.11

The mean basal concentration of CRP at day 0 in the pools with high concentrations was 925.79 ng/mL. After storage for 7 days, a significant reduction of 36.56% was observed, with measured concentrations of 587.63 ng/mL. In addition, a significant reduction of 82.09% was also detected after storage for 120 days (165.8 ng/mL), followed by a reduction of 84.31% (145.3 ng/mL) and 86.60% (123.9 ng/mL) after storage for 210 and 365 days, respectively. No variation was observed for CRP concentrations in the pools with low concentrations of APPs, which had concentrations that ranged between 28.25 ng/mL at day 0 and 27.45 ng/mL after storage for 365 days. Repeated freezing-thawing cycles during long-term storage also caused a decrease in salivary CRP concentrations in the pools with high concentrations (Figure 2).

Effect of long-term storage at −20°C on CRP and haptoglobin concentrations in meat juice samples (experiment 1)—Concentrations of haptoglobin and CRP were used to evaluate change in stability of these APPs in meat juice samples after long-term storage at −20°C and after 7 cycles of repeated freezing-thawing (Figures 3 and 4). Concentrations of haptoglobin significantly increased in the meat juice pool with high haptoglobin concentrations after storage at −20°C for 210 days, with an increase of 33% (284.6 μg/mL); concentrations also increased significantly after 5 cycles of repeated freezing-thawing, with a 29% increase in haptoglobin concentrations (274.7 μg/mL). Pools with medium and low concentrations of haptoglobin did not differ significantly from the initial concentration of haptoglobin at day 0.

Figure 3—
Figure 3—

Mean haptoglobin concentrations of meat juice pools with high, medium, and low APP concentrations collected from pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; black circles], medium [6 samples/pool; black squares], and low [5 samples/pool; black triangles] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; white triangles], medium [6 samples/pool; white diamonds], and low [5 samples/pool; white squares] concentration pools). Eight 100-μL aliquots were prepared from each pool of meat juice for measurement of haptoglobin concentrations after storage. Concentration in an aliquot was measured immediately to determine a baseline (day 0) haptoglobin concentration, and measurements of the remaining 7 aliquots were performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a large aliquot (1 mL) from each pool of meat juice was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.11

Figure 4—
Figure 4—

Mean CRP concentrations of meat juice pools with high, medium, and low APP concentrations collected from pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; black circles], medium [6 samples/pool; black squares], and low [5 samples/pool; black triangles] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; white triangles], medium [6 samples/pool; white diamonds], and low [5 samples/pool; white squares] concentration pools). Eight 100-μL aliquots were prepared from each pool of meat juice for measurement of CRP concentrations after storage. Concentration of an aliquot was measured immediately to determine a baseline (day 0) CRP concentration, and measurement of the remaining 7 aliquots was performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a large aliquot (1 mL) from each pool of meat juice was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.11

Concentrations of CRP in meat juice did not change significantly during the storage conditions until day 365. Furthermore, CRP concentrations in meat juice samples did not change significantly when repeated freezing-thawing cycles were performed during the storage period (Figure 4).

Effects of a protease-inhibitor cocktail and storage at −80°C on CRP stability in saliva samples, compared with storage at −20°C (experiment 2)—When the stability of CRP in saliva was monitored in aliquots stored at −20°C with and without the addition of the protease-inhibitor cocktail, a decrease in CRP concentrations was detected on day 7 and 15 in the pools with intermediate and high CRP concentrations, respectively, for both types of samples (Figure 5).

Figure 5—
Figure 5—

Stability of CRP concentrations in pools of saliva with high (A; n = 2 pools), medium (B; 2), and low (C; 2) concentrations of CRP when stored at −20°C with (triangles and diamonds; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) or without (squares and circles; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) a protease-inhibitor cocktailc (1 μL/mL). Concentrations were determined at baseline (day 0) and weekly for 30 days after storage. Notice that the scale on the y-axis differs among the 3 panels. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.11

Pools with low, medium, and high CRP concentrations stored at −80°C and pools with low concentrations of CRP stored at −20°C did not have significant differences when stored for 30 days. However, pools with high and medium concentrations of CRP that were stored at −20°C decreased significantly beginning on day 7 of storage (Figure 6).

Figure 6—
Figure 6—

Stability of CRP concentrations in pools of saliva with high (A; n = 2 pools), medium (B; 2), and low (C; 2) concentrations of CRP when stored at −20°C (triangles and diamonds; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) or −80°C (squares and circles; 5 samples/pool for high and low pools and 6 samples/pool for medium pools). Concentrations of CRP were measured at baseline (day 0) and after storage on days 7 15, and 30. Notice that the scale on the y-axis differs among the 3 panels. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.11

Discussion

Stability is an important consideration in the use of specimens for accurate determination of analyte concentrations.20 Because of logistic, financial, practical, or methodological reasons, it is not always possible to analyze samples immediately after collection, and in these cases, storage prior to sample analysis would be necessary12 Moreover, for epidemiological investigations, a widespread bank of biological specimens stored for a long time typically are used,21 so special attention should be paid to evaluating possible changes of analyte concentrations during storage.

The present study was undertaken to investigate the influence of long-term storage at −20°C and repeated freezing-thawing cycles on haptoglobin and CRP concentrations in saliva and meat juice samples. To that end, we used saliva and meat juice pools with different concentrations of each protein, and we evaluated the stability of proteins at different times during a 1-year period and the influence of up to 7 repeated freezing-thawing cycles. In addition, we evaluated whether the addition of a protease-inhibitor cocktail to samples prior to storage or the use of a storage temperature of −80°C would efficiently prevent CRP degradation in saliva samples. Time-resolved immunofluorometric assays validated for both APPs17–19 with a dynamic range that allowed measurement of CRP and haptoglobin in fluids with low concentrations of these proteins were used in the study reported here.

To perform the experiments, samples were obtained from pigs with 2 different health statuses. Healthy specific-pathogen-free pigs were chosen for collection of saliva and meat juice samples with low APP concentrations, as has been reported elsewhere.22 On the other hand, diseased pigs with porcine reproductive and respiratory syndrome or postweaning multisystemic wasting syndrome were selected for collection of samples with high concentrations of APPs on the basis of the systemic inflammatory component of both syndromes that cause increases in APP concentrations.23

Our findings indicated that haptoglobin in saliva was stable for up to 120 days when stored at −20°C, but longer storage times and multiple freezing-thawing cycles influenced haptoglobin concentrations by yielding higher haptoglobin concentrations. This increase could have been attributable to sample evaporation or progressive changes in precipitability of proteins, as has been reported elsewhere.10 On the basis of these results, it is advisable to avoid, if possible, storage at −20°C for longer than 120 days and repeated freezing-thawing cycles of saliva samples when measuring haptoglobin concentrations.

The storage temperature of −20°C was not optimal for salivary CRP concentrations because after storage for 7 days, a significant reduction of 36.56% in CRP concentrations was detected. This result was in agreement with results of another study17 in which CRP concentrations in saliva samples stored at −20°C had good stability for only 7 days of storage, which was followed by a reduction in CRP concentrations of 38.6%. It has been reported that saliva contains higher amounts of enzymes that may degrade the contents of proteins, compared with enzyme amounts in serum.12 Because it has been reported24 that the use of protease inhibitors could efficiently prevent protein reduction during storage, a protease-inhibitor cocktail was used in an attempt to avoid the degradation of CRP by salivary enzymes. However, use of this protease inhibitor did not provide additional improvement in CRP stability during the experiment. This finding might be explained by the action of microbiota from the oral cavity on salivary CRP integrity because decomposition of analytes by microbial exoenzymes, which are potent destroyers of salivary components even in samples in which specific salivary enzymes have been inactivated, has been reported.13

In addition, CRP stability in saliva samples stored at −80°C for up to 30 days was evaluated as another strategy for improving CRP stability. Although stability improvements were achieved, a decrease of 30% in the concentration for the high CRP pool was still found at 30 days of storage. Further studies during long-term storage should be performed to establish a better estimation of the protective effect of a storage temperature of −80°C on CRP in saliva because the use of other analytes has provided good stability of samples stored at −80°C for long periods.25

We detected 4 factors that could interfere with protein stability: concentration of protein, type of protein, storage conditions, and matrix effects. Samples with high concentrations of protein were more affected than were those with small amounts of protein. In addition, better stability was obtained with lower storage temperatures (ie, −80°C). It appeared that CRP was more easily affected than was haptoglobin, which could have been attributable to the different intersubunit bonds of each protein. Haptoglobin subunits are linked by disulfide bonds,26 which are covalent bonds, whereas CRP subunits interact via salt bridges, which are considered noncovalent weak bonds27; therefore, the aforementioned effects could have been more damaging to CRP bonds than those of the haptoglobin molecule. In addition, the matrix of the sample had a significant effect because stability differences between CRP and haptoglobin were more pronounced in saliva than in meat juice samples, which indicated that meat juice was a better specimen choice for measurement of stability of CRP and haptoglobin in stored samples.

Measurement of CRP concentrations in meat juice samples revealed good stability in samples stored at −20°C for up to 365 days, with no compromise in the validity of results, even when repeated freezing-thawing cycles of samples were performed. However, an increase of 33% was obtained after storage for 210 days at −20°C in the pool with high haptoglobin concentrations in meat juice samples. These differences are similar to those obtained for haptoglobin concentrations in saliva samples; thus, further studies are needed to obtain a better understanding of molecular changes that could influence increases in haptoglobin concentrations after long-term storage.

On the basis of previous observations,17–19 measurements of APPs in saliva or meat juice may be a simpler alternative method for assessing animal health at farms and slaughterhouses, compared with use of serum samples. However, because falsely low CRP values and falsely high haptoglobin values could be obtained in saliva samples stored for > 7 days at −20°C, special cautions have to be taken regarding sample storage. Storage at −80°C is recommended because this improves the stability of CRP in saliva samples, although it does not totally prevent its degradation.

ABBREVIATIONS

APP

Acute-phase protein

CRP

C-reactive protein
a.

Salivette tubes, Sarstedt, Aktiengesellschaft & Co, Nümbrecht, Germany.

b.

Meat extract collector, Sarstedt, Aktiengesellschaft & Co, Nümbrecht, Germany.

c.

Protease-inhibitor cocktail for use with mammalian cell and tissue extracts (P8340), Sigma-Aldrich Inc, St Louis, Mo.

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  • 20.↑

    Moore C, Vicent M, Rana S, et al. Stability of Δ9-tetrahydrocannabinol (THC) in oral fluid using the Quantisal collection device. Forensic Sci Int 2006; 164:126–130.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.↑

    Landi MT & Caporaso N. Sample collection, processing and storage. IARC Sci Publ 1997; 142:223–236.

  • 22.↑

    Petersen HH, Ersbøll AK, Jensen CS, et al. Serum-haptoglobin concentration in Danish slaughter pigs of different health status. Prev Vet Med 2002; 54:325–335.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.↑

    Segalés J, Piñeiro C, Lampreave F, et al. Haptoglobin and pig-major acute protein are increased in pigs with post weaning multi-systemic wasting syndrome (PMWS). Vet Res 2004; 35:275–282.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.↑

    Papale M, Pedicillo MC, Di Paolo S, et al. Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS) from sample collection to data analysis. Clin Chem Lab Med 2008; 46:89–99.

    • Search Google Scholar
    • Export Citation
  • 25.↑

    Lewis MR, Callas PW, Jenny NS, et al. Longitudinal stability of coagulation, fibrinolysis, and inflammation factors in stored plasma samples. Thromb Haemost 2001; 86:1495–1500.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.↑

    Petersen HH, Nielsen JP, Heegaard PMH. Application of acute phase protein measurements in veterinary clinical chemistry. Vet Res 2004; 35:163–187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.↑

    Volanakis JE. Human C-reactive protein: expression, structure and function. Mol Immunol 2001; 38:189–197.

Contributor Notes

Supported by a grant from the Spanish Ministry of Education and Science (AGL 2006-05701).

Address correspondence to Dr. Cerón (jjceron@um.es).
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
ISSN:
0002-9645
Publication Date:
01 Jan 2011
  • Figure 1—
    View in gallery
    Figure 1—

    Mean haptoglobin concentrations in pooled saliva samples with high and low APP concentrations collected from 10 specific-pathogen-free and 10 diseased pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; circles] and low [5 samples/pool; squares] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; triangles] and low [5 samples/pool; diamonds] concentration pools). Eight 100-μL aliquots were prepared from each pool of saliva for measurement of haptoglobin concentrations after storage. Concentration in an aliquot was immediately measured to determine a baseline (day 0) haptoglobin concentration, and the remaining 7 aliquots were used for measurements on days 7 15, 30, 60, 120, 210, and 365. In addition, a larger aliquot (1 mL) from each pool of saliva was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles.

  • Figure 2—
    View in gallery
    Figure 2—

    Mean CRP concentrations in pooled saliva samples with high and low APP concentrations collected from 10 specific-pathogen-free and 10 diseased pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; circles] and low [5 samples/pool; squares] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; triangles] and low [5 samples/pool; diamonds] concentration pools). Eight 100-μL aliquots were prepared from each pool of saliva for measurement of CRP concentrations after storage. Concentration in an aliquot was immediately measured to determine a baseline (day 0) CRP concentration, and measurements of the remaining 7 aliquots were performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a larger aliquot (1 mL) from each pool of saliva was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles. *Within a pool, concentration differs significantly (P < 0.05) from the concentration determined at baseline (day 0).

  • Figure 3—
    View in gallery
    Figure 3—

    Mean haptoglobin concentrations of meat juice pools with high, medium, and low APP concentrations collected from pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; black circles], medium [6 samples/pool; black squares], and low [5 samples/pool; black triangles] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; white triangles], medium [6 samples/pool; white diamonds], and low [5 samples/pool; white squares] concentration pools). Eight 100-μL aliquots were prepared from each pool of meat juice for measurement of haptoglobin concentrations after storage. Concentration in an aliquot was measured immediately to determine a baseline (day 0) haptoglobin concentration, and measurements of the remaining 7 aliquots were performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a large aliquot (1 mL) from each pool of meat juice was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles. See Figure 2 for remainder of key.

  • Figure 4—
    View in gallery
    Figure 4—

    Mean CRP concentrations of meat juice pools with high, medium, and low APP concentrations collected from pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; black circles], medium [6 samples/pool; black squares], and low [5 samples/pool; black triangles] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; white triangles], medium [6 samples/pool; white diamonds], and low [5 samples/pool; white squares] concentration pools). Eight 100-μL aliquots were prepared from each pool of meat juice for measurement of CRP concentrations after storage. Concentration of an aliquot was measured immediately to determine a baseline (day 0) CRP concentration, and measurement of the remaining 7 aliquots was performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a large aliquot (1 mL) from each pool of meat juice was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles.

  • Figure 5—
    View in gallery
    Figure 5—

    Stability of CRP concentrations in pools of saliva with high (A; n = 2 pools), medium (B; 2), and low (C; 2) concentrations of CRP when stored at −20°C with (triangles and diamonds; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) or without (squares and circles; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) a protease-inhibitor cocktailc (1 μL/mL). Concentrations were determined at baseline (day 0) and weekly for 30 days after storage. Notice that the scale on the y-axis differs among the 3 panels. See Figure 2 for remainder of key.

  • Figure 6—
    View in gallery
    Figure 6—

    Stability of CRP concentrations in pools of saliva with high (A; n = 2 pools), medium (B; 2), and low (C; 2) concentrations of CRP when stored at −20°C (triangles and diamonds; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) or −80°C (squares and circles; 5 samples/pool for high and low pools and 6 samples/pool for medium pools). Concentrations of CRP were measured at baseline (day 0) and after storage on days 7 15, and 30. Notice that the scale on the y-axis differs among the 3 panels. See Figure 2 for remainder of key.

Figure 1—

Mean haptoglobin concentrations in pooled saliva samples with high and low APP concentrations collected from 10 specific-pathogen-free and 10 diseased pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; circles] and low [5 samples/pool; squares] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; triangles] and low [5 samples/pool; diamonds] concentration pools). Eight 100-μL aliquots were prepared from each pool of saliva for measurement of haptoglobin concentrations after storage. Concentration in an aliquot was immediately measured to determine a baseline (day 0) haptoglobin concentration, and the remaining 7 aliquots were used for measurements on days 7 15, 30, 60, 120, 210, and 365. In addition, a larger aliquot (1 mL) from each pool of saliva was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles.
Figure 2—

Mean CRP concentrations in pooled saliva samples with high and low APP concentrations collected from 10 specific-pathogen-free and 10 diseased pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; circles] and low [5 samples/pool; squares] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; triangles] and low [5 samples/pool; diamonds] concentration pools). Eight 100-μL aliquots were prepared from each pool of saliva for measurement of CRP concentrations after storage. Concentration in an aliquot was immediately measured to determine a baseline (day 0) CRP concentration, and measurements of the remaining 7 aliquots were performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a larger aliquot (1 mL) from each pool of saliva was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles. *Within a pool, concentration differs significantly (P < 0.05) from the concentration determined at baseline (day 0).
Figure 3—

Mean haptoglobin concentrations of meat juice pools with high, medium, and low APP concentrations collected from pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; black circles], medium [6 samples/pool; black squares], and low [5 samples/pool; black triangles] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; white triangles], medium [6 samples/pool; white diamonds], and low [5 samples/pool; white squares] concentration pools). Eight 100-μL aliquots were prepared from each pool of meat juice for measurement of haptoglobin concentrations after storage. Concentration in an aliquot was measured immediately to determine a baseline (day 0) haptoglobin concentration, and measurements of the remaining 7 aliquots were performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a large aliquot (1 mL) from each pool of meat juice was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles. See Figure 2 for remainder of key.
Figure 4—

Mean CRP concentrations of meat juice pools with high, medium, and low APP concentrations collected from pigs; samples were stored at −20°C for up to 365 days (high [n = 5 samples/pool; black circles], medium [6 samples/pool; black squares], and low [5 samples/pool; black triangles] concentration pools) and underwent up to 7 repeated freezing-thawing cycles (high [5 samples/pool; white triangles], medium [6 samples/pool; white diamonds], and low [5 samples/pool; white squares] concentration pools). Eight 100-μL aliquots were prepared from each pool of meat juice for measurement of CRP concentrations after storage. Concentration of an aliquot was measured immediately to determine a baseline (day 0) CRP concentration, and measurement of the remaining 7 aliquots was performed on days 7, 15, 30, 60, 120, 210, and 365. In addition, a large aliquot (1 mL) from each pool of meat juice was stored separately at −20°C, subjected to 7 repeated freezing-thawing cycles at each of the measurement time points after baseline, and used for measurements in parallel with the aliquots that were not subjected to repeated freezing-thawing cycles.
Figure 5—

Stability of CRP concentrations in pools of saliva with high (A; n = 2 pools), medium (B; 2), and low (C; 2) concentrations of CRP when stored at −20°C with (triangles and diamonds; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) or without (squares and circles; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) a protease-inhibitor cocktailc (1 μL/mL). Concentrations were determined at baseline (day 0) and weekly for 30 days after storage. Notice that the scale on the y-axis differs among the 3 panels. See Figure 2 for remainder of key.
Figure 6—

Stability of CRP concentrations in pools of saliva with high (A; n = 2 pools), medium (B; 2), and low (C; 2) concentrations of CRP when stored at −20°C (triangles and diamonds; 5 samples/pool for high and low pools and 6 samples/pool for medium pools) or −80°C (squares and circles; 5 samples/pool for high and low pools and 6 samples/pool for medium pools). Concentrations of CRP were measured at baseline (day 0) and after storage on days 7 15, and 30. Notice that the scale on the y-axis differs among the 3 panels. See Figure 2 for remainder of key.
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    Segalés J, Piñeiro C, Lampreave F, et al. Haptoglobin and pig-major acute protein are increased in pigs with post weaning multi-systemic wasting syndrome (PMWS). Vet Res 2004; 35:275–282.

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    • Search Google Scholar
    • Export Citation
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    Papale M, Pedicillo MC, Di Paolo S, et al. Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS) from sample collection to data analysis. Clin Chem Lab Med 2008; 46:89–99.

    • Search Google Scholar
    • Export Citation
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    Lewis MR, Callas PW, Jenny NS, et al. Longitudinal stability of coagulation, fibrinolysis, and inflammation factors in stored plasma samples. Thromb Haemost 2001; 86:1495–1500.

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    • Search Google Scholar
    • Export Citation
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    Petersen HH, Nielsen JP, Heegaard PMH. Application of acute phase protein measurements in veterinary clinical chemistry. Vet Res 2004; 35:163–187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Volanakis JE. Human C-reactive protein: expression, structure and function. Mol Immunol 2001; 38:189–197.

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