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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.36 No.3 pp.114-120
DOI :

Development of stabilized somatic cell standard solutions for calibration of electronic instruments analyzing bovine raw milk

Yong Ho Park1,2,†, Jin San Moon1, Keum-Chan Jang1, Suk-Chan Jung1, Yi-Seok Joo1, Ji-Ho Kim1, Sung-Hwan Wee1, Hye Cheong Koo2
1Animal, Plant and Fisheries Quarantine and Inspection Agency, 2College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University
(Received June 25, 2012; Revised September 10, 2012; Accepted September 18, 2012)

Abstract

The current standard solutions for somatic cells used for calibration of electronic somatic cell counts as referencematerial in raw milk are preserved with bronopol, boric acid, sodium azide, or potassium dichromate, and have ashelf-life of only up to 6 days at 4 ± 2℃. In the present study, a set of somatic cell standard solutions (SCSS) witha stability of 5 months for calibration of electronic instruments was developed. Somatic cells collected from cow’s milkand stored in a bulk tank at a dairy plant were treated with 10% formaldehyde in order to improve stability, and thenseparated by centrifugation. The resulting somatic cell suspension was preserved with glycerin, thimerosal, and dimethylsulfoxide, and diluted in 3% processed skim milk solution ranging from 200,000~250,000 (low level), 350,000~450,000 (medium level), and 550,000~650,000 (high level) cells/㎖. Each SCSS was verified by direct microscopesomatic cell counting (DMSCC), C-reader, and commercial standard samples. The average somatic cell count determinedby DMSCC was 248, 214, 226 × 103 cells/㎖, 436, 382, 420 × 103 cells/㎖, and 612, 595, 609 × 103 cells/㎖. Thecoefficient of variation representing the repeatability of DMSCC decreased as the number of cells increased, and was<10.0% in almost all SCSS samples (range 4.6~7.1%). No statistically significant difference in somatic cellconcentration was observed after storage at refrigeration temperature (2~6℃) over a period of 22 weeks (5 months).The stabilized SCSS may be useful as a reference material for determination of somatic cell count and quality controlin testing of bovine raw milk.

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Introduction

Somatic cell counting (SCC) in raw milk is one of the main indicators of udder health of lactating animals, and has become a globally reliable indicator for determining milk quality and the price of raw milk within the dairy industry [8, 11, 14]. Typical SCC methods are direct microscope somatic cell counting (DMSCC) and fluoro-optoelectronic-counter by a rotating disk technique or flow cytometry [2, 5, 9]. Among them, DMSCC with methylene blue staining is the reference for calibration purposes. It is based on optical or fluorescence microscopy identification of the cell nuclei after staining [1, 6]. However, the method from the possibility of discrepancies in the values generated by different analysts and even by the same analyst. The accuracy, precision, and repeatability of results in the DMSCC method depend on the training and the skill of the analyst regardless of the instruments or protocol [6, 7].

In milk-test laboratories, SCC is commonly determined using an automated electronic machines, such as Fossomatic 90, 300, and 4000TM (Foss Electric, Hillerød, Denmark) based on disk cytometry and, Somacount 300TM (Bentley Instruments, Chaska, MN, USA), SomascopeTM (Delta Instruments, Drachten, Netherlands), and Fossomatic 5000TM (Foss Electric, Hillerød, Denmark) based on flow cytometry. They may be accurate and reliable instruments that can be used to quantify SCC in milk very quickly and at low cost. However, their performance requires regular calibration using references materials that have been confirmed by the traditional DMSCC method for quality control [7, 13]. References materials are vital to check the exactness and stability of references methods used between two consecutive proficiency tests by comparison with nominal values. They are routinely used weekly [3, 5]. The current standard solutions for somatic cells used for calibration of electronic somatic cell counters (ESCC) as references material in raw milk are preserved with bronopol, boric acid, sodium azide, or potassium dichromate. These standards have a limited shelf-life of no longer than 6 days at 4±2℃ [5]. 

DMSCC and the utilized reference materials can produce inaccurate test results because of the limited precision of DMSCC between 100,000~300,000 cells/㎖, and the low stability and homogeneity of the reference materials. In particular, the limited stability of the reference material can detract from the routine calibration of the mechanized and automated cell-counting methods [8]. With the aim of improving this situation, the present study was undertaken to develop a set of new somatic cell standard solutions (SCSS) having extended stability for 5 month for ESCC calibration. 

Materials and Methods

1. Preparation of SCSS

Somatic cells were collected at random from raw milk contained in a bulk tank of a dairy plant located in Gyeonggi province, Korea. The milk was treated for 24 h at 4℃ with a 10% formaldehyde solution to improve stability by fixing somatic cells. Somatic cells were then separated from other milk components such as fat, protein, and lactose by centrifugation for 30 min at 330×g. The resulting somatic cell suspension was washed three times with phosphate buffered saline solution (PBS, pH 7.2). The final somatic cell suspension was preserved with glycerin (30㎖) and 1% thimerosal (16㎖), dimethyl sulfoxide (30㎖) in 2,000㎖ of 3% processed skim milk solution, counted using a Fossomatic 4000 and then diluted in ranges of 200~250, 350~450, and 550~650×103 cells/㎖ on the basis of raw milk hygiene grades of SCC in Korea. The dilutions represented the low, medium, and high standard, respectively. The final determination of SCC was performed by DMSCC. The SCSS were distributed in 200 ㎖ sample tubes and stored at refrigeration temperature (2~6℃) until analysis. 

2. Verification of the SCC in SCSS

Each SCSS was verified by DMSCC according to the International Dairy Federation (IDF) regulation 148-1 [6] of a standardizing somatic cell counting system. For DMSCC, dried milk smears were stained with Newman-Lampert method. In addition, measurement of SCC with a C-readerTM (Digital Bio Technology, Seoul, Korea) was carried out according to the instruction manual as previously described [10]. The C-reader and DMSCC determinations were performed five and 50 different counts of individual smear with the same samples, respectively. In addition, for the determination of SCC in SCSS, the Fossomatic 4000 and C-reader systems were periodically checked at monthly intervals in our laboratory by a quality control inter-comparative test of known SCC with 12 bovine milk standards containing 1.7~9.9×105 cells/㎖ (American Eastern Laboratory Services, Fairlawn, OH). Measurement of SCC in SCSS was also carried out 10 times for each SCSS sample by inter-laboratory testing with Fossomatic 4000, Somacount 300, and Somascope used at Korean laboratories.

3. Evaluation of the shelf-life stability of scss by automatic ESCC

To determine the shelf-life stability of SCSS, samples were periodically tested at weekly intervals using a Fossomatic 4000 apparatus. In addition, monthly DMSCC examinations were done to observe morphology. Samples were stored at refrigeration temperature (2~6℃), and taken out and tested up to 30 weeks from initial point according to the regular rules for somatic cell counting in our laboratories. Before the experiment, the Fossomatic 4000 system was calibrated with the presently developed SCSS according to the instrument’s instruction manual after checking instrumental fitting by the carry-over and linearity, and overall daily stability by zero-setting and repeatability test. The calibration suitability was checked with results of bios and slope of Fossomatic 4000 based on guidelines for quality assurance on DHI analyses [3]. After calibration, a total of 30 composite bovine raw milk samples containing 6.0×103~1.0×106 cells/㎖ were each tested five times tested in a pre-heating treatment (15 min at 40℃) according to the protocols provided by the manufacturers of Fossomatic 4000 and C-reader. The same 30 bovine raw milk samples were also tested by C-reader without the pre-treatment to analyze the influence of preheating on the SCC values. The coefficient of variation (CV) for the results obtained from each sample was calculated for each SCC method. 

4. Statistical analyses

Repeatability values of each SCSS in three batches with each set of low, medium and high solution were evaluated by the CV calculated from results of SCC with DMSCC. The accuracy and repeatability of the C-reader system, and the mean comparison and regression analysis of the SCC data between the C-reader and the three automatic ESCC instruments have previously been established [10], but they were also evaluated by measuring SCC of SCSS developed in this study, using the 12 bovine milk standards and 30 bovine raw milk samples containing 6.0×103~1.0×106 somatic cells/㎖. Statistically significant differences in SCC of SCSS measured by the C-reader before and after pre-treatment at 40℃ for 15 min were analyzed by a paired-samples t-test, and the weekly changes in SCC of SCSS were analyzed by t-test by comparing SCC data measured 50 times by DMSCC at 0 weeks and SCCs measured 10 times by Fossomatic 4000 after storage at 4℃. T-test analyses were performed using the Analyse-it program [12]. 

Results and Discussion

1. Repeatability of the each standard somatic cell solution

The overall repeatability data of the each SCC in three batch samples of SCSS developed in this study using DMSCC and the C-reader are summarized in Table 1. The average value of the somatic cells measured by DMSCC method were 248×103, 214×103, and 226×103 cells/㎖ (low level), 436×103, 382×103, 420×103 cells/㎖ (medium level), and 612×103, 595×103, 609×103 cells/㎖ (high level). Respective CV values representing the repeatability of the DMSCC were 5.5~7.1%, 4.8~6.5%, and 4.6~4.8%; the decreasing CV with increased cells was <10% in almost all the somatic cell samples (range 4.6~7.1% in the three batches, respectively). The ranges were within those recommended by the IDF for somatic counters (6~10% at 1.5×105~4.0×105 cells/㎖, and 5% at >4.0×105 cells/㎖ [6, 13]). Similar results (CV <4.4%) were evident using the C-reader based on the microscopic somatic cell counter with disposable plastic chip for milk analysis. The C-reader SCC values were less variable than DMSCC; however, the resulting ratios of SCC in the developed SCSS measured by the C-reader system to DMSCC ranged from 0.95~1.03. Therefore, the SCC values of SCCS were acceptably repeatable.

Table 1. Repeatability of the each somatic cell standard solution (SCSS) examined by the direct microscope somatic cell counting (DMSCC) and Creader system

2. Comparison of ESCC instruments used for analyzing SCSS stability

The linear correlation between the SCC values in the 12 commercially acquired standard solutions (R2, 0.995; 95% CI, 0.990~0.999; p<0.01) measured by DMSCC and the C-reader, as well as the SCC data in bovine raw milk (R2, 0.994; 95% CI, 0.993~0.999; p<0.01) measured by the Fossomatic 4000 and C-reader is shown in Fig. 1. When compared with the SCC data of the 12 standard solutions measured by DMSCC, the C-reader system displayed acceptable and similar repeatability and accuracy to the conventional devices for SCC by Pearson correlation analysis [10]. In addition, after linear regression analysis, a statistically significant correlation (p<0.01) with a high linear relationship (R2, 0.994) with the SCC data was evident between the C-reader and Fossomatic 4000 systems in raw milk samples containing 104~106 cells/㎖. Therefore, both the Fossomatic 4000 and C-reader instruments were acceptable. When SCCs in SCSS were compared after calibration of the Fossomatic 4000, Somacount 300, and Somascope instruments used for determining milk price based on SCC in Korea, the SCC data of the each SCSS obtained from DMSCC were statistically significantly correlated with those from other ESCCs with acceptable repeatability (<7% variation in CV) and high linear relationship (Table 2).

Fig. 1 Pearson correlation and linear regression analysis of somatic cell counting (SCC) data measured by different counting methods. (A) Twelve commercially acquired somatic cell solutions were measured by DMSCC and C-reader. (B) Results from analysis of 30 composite bovine raw milk samples containing 6.0×103~1.0×106 somatic cells/㎖ using the C-reader and Fossomatic 4000 instruments.

Table 2. Comparison of repeatability of three electric somatic cell counting instruments by determining somatic cell counting (SCC) in each somatic cell standard solution (SCSS)

3. Analysis of shelf-life stability of SCSS

The shelf-life stability of each SCSS in the three batches was evaluated by DMSCC and using the Fossomatic 4000 system. No statistically significant differences in cell concentration of SCSS were evident after storage at refrigeration temperature (2~6℃) over 22 weeks (Table 3). In addition, morphology was consistent for up to 6 months after sample production. However, cell lysis was noted at the time when the somatic cell concentration of the standard solutions decreased. The results indicated that the shelf-life of SCSS developed with formalin treatment including glycerin and thimerosal as preserves was superior to compare with the current use of bronopol, boric acid, sodium azide, or potassium dichromate preservatives for calibration of automated ESCC in raw milk as reference material. When the SCC in the raw milk samples measured by the C-reader with and without pre- treatment at 40℃ was compared, no significant difference in SCCs was observed between the two different treatment groups (p>0.05).

Table 3. Weekly change of SCC in SCSS stored at refrigeration temperature

The IDF defines and evaluates the overall accuracy of indirect methods of milk analysis when compared with the reference method [4, 5]. Based on this standard, standardization of SCC counters for raw milk with reference materials is essential in SCC laboratories and equipment to guarantee accuracy and reproducibility of results. Up to now, there has not been a well-defined certified SCC reference material, with a lack of standardized traditional reference materials, and it is questionable whether such a material could be developed and analytically used as outlined by the proposed IDF reference system for somatic cell counting. But, several secondary reference materials from various sources and laboratories exist for calibration purposes. Before these materials can be useful, higher stability over a very long shelf life is needed, and optimization for homogeneity is necessary to enable the construction of a worldwide routine grid of reference points [7]. Since 1999 in Korea, commercial standard solutions have been used at monthly intervals for calibration of approximately 95 automatic instruments including the Fossomatic 300, 4000, and 5000; Somacount 300; and Somascope to establish equivalence in laboratories of the Dairy Herd Improvement Association and dairy companies performing payment testing. The program has improved the accuracy and repeatability of ESCCs by calibration of the routine methods. 

Presently, the overall repeatability of each developed SCSS was analyzed by DMSCC and ESCC; CV values satisfied IDF recommendations for somatic counters. In addition, the novel SCSS had greater stability, as reflected by an extended shelf-life, compared with the current standards solution for somatic cells preserved with bronopol, boric acid, sodium azide, or potassium for calibration of ESCCs in raw milk. Therefore, the stabilize SCSS developed in this study may be useful as reference material for SCC determination and in the quality control in bovine raw milk-testing laboratories. 

Acknowledgements

This study was funded by the budget for veterinary research and development of the Animal, Plant and Fisheries Quarantine and Inspection Agency. 

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