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J. Dairy Sci. 2007. 90:2253-2259. doi:10.3168/jds.2006-622
© 2007 American Dairy Science Association ®
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Application of a New Portable Microscopic Somatic Cell Counter with Disposable Plastic Chip for Milk Analysis

J. S. Moon*,1, H. C. Koo{dagger},{ddagger},1, Y. S. Joo*, S. H. Jeon§, D. S. Hur§, C. I. Chung§, H. S. Jo§ and Y. H. Park{ddagger},2

* Department of Bacteriology, National Veterinary Research and Quarantine Service, Anyang, Gyeonggi-do, Republic of Korea
{dagger} KRF Zoonotic Disease Priority Research Institute, and
{ddagger} Department of Microbiology, College of Veterinary Medicine and the BK21 program for Veterinary Science, Seoul National University, Seoul, Republic of Korea
§ Digital Bio Technology Co. Ltd., Seoul, Republic of Korea

2 Corresponding author: yhp{at}snu.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The somatic cell count (SCC) is one of the international standards for monitoring milk quality, and it is a useful indicator of mastitis. The current reference method for determining the SCC in raw milk is direct microscopic analysis, but this method requires well-trained staff to maintain its accuracy and reproducibility. To overcome these inconveniences, we developed a portable system (the C-reader system) that utilizes the capillary flow of a microfluidic chamber by surface modification of the hydrophilicity. The microfluidic technology of disposable microchips allows for low consumption of reagents, and a combination of ready-to-use reagents makes the daily work easier. The repeatability test of the C-reader using 10 composite bovine milk samples satisfied the recommended values for SCC equipment. In addition, an acceptable accuracy level of the natural logarithmic-transformed SCC [ln(SCC/1,000): ± 0.059 to 0.112] was achieved using composite raw milk samples and various somatic cell standard solutions from the American Eastern Laboratory and the Korean National Veterinary Research and Quarantine Service. After testing 875 composite milk samples, the C-reader showed a high correlation coefficient (R2 = 0.935 to 0.964) and a low mean difference value in log-transformed SCC (–0.088 to 0.004) compared with 3 automatic commercialized somatic cell counters (Fossomatic 4000, Somacount 150, and Somascope). In conclusion, the C-reader system is a new, easy-to-use automatic on-farm method with acceptable repeatability and accuracy for measuring SCC in large dairies and smaller laboratories.

Key Words: bovine raw milk • somatic cell count • C-reader • disposable plastic microchip


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The measurement of SCC has become one of the most reliable indicators for determining milk quality and the price of raw milk within the dairy industry (Schukken et al., 1992; Ott and Novak, 2001; Jayarao et al., 2004). Several methods such as the direct microscopic somatic cell counting (DMSCC) method, electronic particle counting method, and fluoro-optic electronic cell counting method by disk cytometry or flow cytometry have been used to determine the SCC. Among them, DMSCC with methylene blue staining is the reference method for making a direct estimation of the cells, but this method is slow and requires trained staff (Forest and Small, 1959). In addition, the lack of specificity between the cells and cytoplasmic particles, as well as the time and labor requirements are negative aspects of DMSCC (International Dairy Federation, 1995a).

The Coulter counter (Coulter Electronics Ltd., Luton, Bedfordshire, UK), which is based on electronic particle counting, is a high-speed device for particle size analysis that involves adding a formaldehyde solution to the milk to be examined to fix the somatic cells, and eliminating fat particles by treatment with a lysing solution with an overlapping size range of the cells (Miller et al., 1986; International Dairy Federation 1995a). The other automatic instruments to measure SCC indirectly are the Fossomatic 90, based on disk cytometry, and the Fossomatic 5000 (Foss Electric, Hillerød, Denmark), Somacount 150 (Bentley Instruments Inc., Chaska, MN), and Somascope (Delta Instruments, Drachten, the Netherlands) based on the flow cytometry method (FCM). The FCM-based instruments have been used more frequently than the Coulter counter at DHI organizations because somatic cells can be counted in raw or preserved milk after heating without the need for additional treatment.

The principle of FCM methods is that a suspension of cells is stained and forced through a capillary tube that is illuminated in front of a microscope objective. Every passing cell is then registered by photo-electronics attached to the microscope. Therefore, the FCM is an accurate and reliable instrumental method that can be used to quantify SCC in milk very quickly and at low cost. A good correlation between the FCM method and the reference method, DMSCC, was reported in raw milk samples (R = 0.88) at concentrations ranging from 103 to 106 cells/mL (Gunasekera et al., 2003). In addition, the cell count measured by FCM showed good agreement (R = 0.98) with the SCC data obtained using the standard Fossomatic method based on disk cytometry (Gunasekera et al., 2003). The fluoro-optic electronic cell counting methods, however, should be calibrated regularly using standard solutions that have been confirmed by DMSCC for quality control (Schmidt-Madsen, 1975; International Dairy Federation, 1995a).

The California Mastitis Test is a very simple and widely used cow-side test of the milk quality in mastitis control efforts at a dairy farm, but it cannot be used for counting the correct SCC, which is the absolute factor for determining the price of milk, because of its low sensitivity and specificity (Schalm and Noorlander, 1957; Schalm et al., 1971; Sargeant et al., 2001). The DeLaval cell counter (DeLaval, Tumba, Sweden) is a new analytic portable instrument for counting somatic cells optically and automatically within 1 min, but this method has a high coefficient of variation (CV); that is, low repeatability (Nelson, 2004). In this study, an automatic system (C-reader) that is fully compatible with the DMSCC was developed. In this study, the repeatability and accuracy of the portable C-reader system was evaluated by comparing its SCC data in raw milk and somatic cell standard solutions with those from DMSCC. In addition, the SCC data in raw milk samples were compared between the C-reader and 3 major conventional instruments (Fossomatic 4000, Somacount 150, and Somascope) because these 3 machines are popular in large dairies and small laboratories worldwide, including in Korea.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Setup of C-Reader System
The C-reader system comprises disposable plastic microchips, staining solutions, a fluorescence microscopic instrument, an image analysis program operated on a PC, and an LCD monitor with a touch screen and function key, which are built in a single machine weighing only 17 kg (Figure 1Go). A charge-coupled device (CCD) camera captures the fluorescence images of the somatic cells, and the image software analyzes and counts the number of cells automatically.


Figure 1
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  		Figure 1. Portable C-reader with a cell analysis system (A). The system weighs 17 kg; dimensions are 260 (width) x 385 (depth) x 330 (height) mm. Disposable plastic chips: single (B) and 2-channel (C) chips with capacity for loading 1 or 2 samples, respectively.

 
Optical System of the C-Reader.
The disposable plastic chip is mounted on an inverted microscope-type optical system of the C-reader (Digital Bio Technology Co., Seoul, Korea). A green laser at 532 nm and a UV or white light-emitting diode (LED) is used as light source. The image of the sample within the chip is scanned by a CCD camera with the fast stage moving the chip in the X–Y direction. The fluorescence is collected by a 4x objective and passed through a filter block (600 ± 25 nm) and to the CCD camera (Mintron, Taiwan). For accurate counting of the cell number, 90 frames of fluorescence images homogeneously distributed through the count area are processed in a single sample. In addition, images of the cell morphology can be observed using the white LED light. The C-reader system is interfaced with a PC by a serial communication cable and a frame-grabber board. Visual C++-based software is used to operate the cell number counting result display, cell size histogram, and live images.

Disposable Plastic Microchips.
The microfluidic technique was used to design the disposable plastic microchip, which evenly distributes the cell density throughout the channel and generates a natural capillary flow for sample loading. The microfluidic channel is located in the center of these chips, which was developed for a more accurate counting of the cell number as follows: 4 mm wide, 50 mm long (52 mm for the total channel including the sample injection part), and 0.1 mm high. Accordingly, the loading volume of a disposable plastic chip is 20 µL (4 x 50 x 0.1 mm3) and the total analysis volume of the channel area is 8 µL, considering the capture of 90 frames of the cell image (an analysis volume of approximately 0.089 µL per frame), which is 80 times larger than the animal cell counting area of a manual hemocytometer (Freshney, 1993).

The disposable plastic chips are fabricated from poly-methyl methacrylate using a microinjection molding process developed by Digital Bio Technology. A backward step structure is used at the outlet of the channel to achieve an even distribution and rapid precipitation of cells. The internal surface of the plates is chemically treated with gas plasma to alter its wetness, resulting in the generation of a natural capillary flow (Anders and Helene, 2002). Two plates are bonded to build the closed channel structure, one being optically clear and smooth, and the other being an open microchanneled. The bonding chemicals have no accompanying reactions with aqueous liquids, cells, or any other materials used in biological tests, which allows them to accomplish passive flow velocity control in the plastic microchannel chips.

Staining Solutions.
The staining solutions (0.1 M PBS solution, pH 5.8) including the detergent Triton-X 100 (0.2%) for cell lysis and fluorescent dye (0.025 mg/mL of ethidium bromide; Sigma, St. Louis, MO) are used for staining the somatic cells. The detergents break the cell membrane so that the fluorescent dye can penetrate the cell membrane and stain the DNA or RNA of the cell. Ethidium bromide, which has excitation and emission wavelengths of 510 nm (green light) and 590 nm (red light), respectively, was used as the fluorescent dye in the staining solution (Crary and Borysenko, 1986).

Analysis of SCC in Milk
Preparation of Raw Milk and Standard Somatic Cell Solutions.
The composite raw milk samples were collected at random from dairy farms located in Gyeonggi province, Korea, from July to September 2004. A pilot study showed that the SCC in the 5 raw milk samples within the range of cell counts of 105 to 106 cells/mL were similar regardless of whether a preservation treatment had been used. Therefore, 961 composite raw milk samples with a density ranging from 6.0 x 103 to 1.0 x 106 cells/mL were treated with the preservation reagent, potassium dichromate (K2Cr2O7, Sigma), to help stabilize the somatic cells (International Dairy Federation, 1995b).

To test the accuracy of the C-reader cell analysis system, various somatic cell standard solutions were prepared using 12 samples with cell counts ranging from 1.7 x 105 to 9.9 x 105 cells/mL from the American Eastern Laboratory (Fairlawn, OH), and 3 types of solutions with different SCC (low, medium, and high) from the Korean National Veterinary Research and Quarantine Service (NVRQS). Each standard somatic cell solution verified by DMSCC was used to evaluate the accuracy of the C-reader system according to the guidelines of a standardizing somatic cell counting system.

Analysis of SCC by C-Reader and DMSCC.
At least 100 µL of the composite bovine milk sample or standard solution was mixed with the same amount of somatic cell staining solution. Twenty microliters of the mixed sample was loaded into the disposable plastic chip. Within 1 min, the total cell number in 8 µL of stained raw milk or standard solution was measured using the C-reader. The reader analyzes the total fluorescent particles automatically according to the principle of DMSCC and ensures the accuracy of the cell counting. The final number of somatic cells was calculated using the following formulation:


Formula

where A is the average number of somatic cells measured in 90 images, B is 0.089 µL, which is the analysis volume per image, and N is the dilution factor in which raw milk samples or standard solutions are mixed with the same amount of a somatic cell staining solution.

For DMSCC, dried milk smears were stained using 1 of 3 Levowitz-Weber stains, a Canadian modification of the modified Newman-Lampert stain for microscopic counting (Levowitz, 1957; Embert, 1986; Packard et al., 1992).

To measure the repeatability of the C-reader, 10 composite bovine milk samples were tested with different ranges of somatic cells (from 1.4 x 104 to 1.0 x 106/mL), which was repeated 10 times. Before testing using the C-reader, the selection of raw milk samples containing various numbers of somatic cells was performed by measuring the approximate concentrations of the cells in raw milk using the Fossomatic 4000 instrument. Seventy-six milk samples (ranging from 1.0 x 105 to 1.0 x 106 cells/mL) and 2 types of somatic cell standard solutions, whose densities ranged from 1.7 x 105 to 9.9 x 105 cells/mL (verified by DMSCC), were also applied to evaluate the accuracy and calculate the standard deviations of the repeatability of the C-reader system. The determination of the SCC by the C-reader and DMSCC was performed 5 times, and the comparison of the SCC data was analyzed by GLM (for mean comparison) and REG (for differences of regression) procedures of SAS (SAS Institute, 1992). In addition, the SCC in the composite raw milk samples (n = 28, 6.0 x 104 to 1.0 x 106) with and without a preheat treatment at 40°C were determined by the C-reader and compared as described above to analyze whether the heating treatment affected the SCC measured by the C-reader.

Automated Instruments for SCC.
To compare the C-reader system with other indirect conventional instruments (Fossomatic 4000, Somacount 150, and Somascope), a total of 875 composite bovine raw milk samples with a different range of somatic cells (6.0 x 103 to 1.0 x 106) were tested in duplicate with (for examination by 3 conventional instruments) or without (for C-reader system) a preheat treatment of 15 min at 40°C according to the protocols provided by the manufacturer of each instrument.

Statistical Analysis
For the analysis of the SCC variable, a natural logarithmic transformation of the SCC [SCCt = ln(SCC/ 1,000)] was performed to approximate the normal distribution as suggested by De Vliegher et al. (2004). To determine the repeatability of the C-reader, which is a measure of the variation between the replicate determination in a single instrument using the same samples, the analysis of SCC in 10 milk samples was performed 10 times, from which the CV were calculated. The accuracy, which is a measure of the bias between the reference method and the C-reader, was determined by analyzing the agreement (means comparison, standard deviations of repeatability, and regression analysis) between the SCC obtained with the reference test (DMSCC) and C-reader using the GLM and REG procedures of SAS (SAS Institute, 1992) according to the International Dairy Federation (IDF) standard 128A (IDF, 1999; Gonzalo et al., 2004). The mean comparison and regression analysis of the SCC between the C-reader and 3 automatic instruments in commercial use (Fossomatic 4000, Somacount 150, and Somascope) was also performed.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Repeatability of the C-Reader System
Table 1Go shows the overall repeatability data obtained from the C-reader system. The CV values, which represent the repeatability of the C-reader, decreased with an increasing number of cells and were <5% in almost all the somatic cell ranges, but 7% at 1.4 x 104 cells/ mL. This satisfies the IDF recommendations for somatic counters, which are 5 to 10% at 1 x 105 to 2 x 105 cells/mL, and 4 to 5% at 4 x 105 to 5 x 105 cells/ mL (IDF, 1995a). The acceptable standard deviation of repeatability (Sr, 0.005 to 0.009) for the SCCt in the raw milk samples and standard somatic cell solution from the American Eastern Laboratory and Korean NVRQS was calculated and the results are shown in Table 2Go. Both low CV and Sr values represent greater repeatability of the C-reader system determined in bovine milk than the DMSCC (Schmidt-Madsen, 1975).


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  		Table 1. Analysis of the repeatability of the C-reader cell counting method using composite raw milk samples with different somatic cell counts
 

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  		Table 2. Evaluation of accuracy and repeatability of the C-reader system in measuring SCC using different samples1
 
A digital camera in the DeLaval cell counter takes 2 images of the nuclei of the somatic cells stained by a DNA-specific fluorescent reagent within the cassette and counts the cell nuclei one by one (Nelson, 2004). It was reported that the DeLaval cell counter makes an incorrect estimation of the SCC at the 2 x 105 cells/ mL level in 4% of cases compared with the Fossomatic instrument, and has CV values of 12, 8, and 7% at 1 x 105, 4 x 105, and 1 x 106 cells/mL, respectively (Nelson, 2004). This is higher than that recommended by the IDF for somatic counters (IDF, 1995a). The C-reader instrument, whose CCD camera processes 90 frames of fluorescent images for a single sample, however, can reduce the high CV value (low repeatability) of the De-Laval cell counter, which might be due to taking only 2 pictures of nuclei per sample.

Comparison of SCC Measured by the C-Reader System and DMSCC
Using the DMSCC method, the nucleus of the somatic cells is shown as dark blue or blue in the microscopic image (Figure 2AGo), whereas the C-reader system analyzes the somatic cells as ethidium bromide-stained fluorescent particles (Figure 2BGo). When the statistical significance of the differences between the DMSCC and C-reader results for those parameters was analyzed by the value of t observed (tobs) under the hypotheses (mean of the difference in SCCt = 0.00; slope of regression line = 1.00; intercept = 0.00), there was no significant difference in the SCCt obtained by the C-reader and DMSCC with the lower tobs values compared with the reference t values in the composite raw milk samples or the standard somatic cell solutions (Table 2Go).


Figure 2
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  		Figure 2. Image comparison of the direct microscopic somatic cell counts (DMSCC) and C-reader when SCC is 4.77 x 106 cells/mL: A) Stained somatic cell image with methylene blue (DMSCC, x1,000) and B) fluorescent image of stained somatic cells captured by the C-reader system.

 
An acceptable level of accuracy of ± 0.059 to 0.112 SCCt [ln(SCC/1,000)]/mL was achieved using the C-reader system compared with the reference method (DMSCC; P < 0.01). The high linear correlation coefficient values were obtained between the SCCt in the 2 types of standard solutions (R2 = 0.994 to 0.996; P < 0.01) as well as raw milk (R2 = 0.992; P < 0.01) measured by DMSCC and the C-reader. The C-reader system is based on the principles of the DMSCC method; however, the C-reader system avoids the negative influences of the experimenter through automation of instruments and the use of disposable plastic chips. It requires only a premixing procedure of raw milk and staining solution by the analyst.

Comparison of the C-Reader System with Other Conventional Automatic Instruments
When analyzing the SCC using conventional fluoro-optical electronic cell counting methods (Fossomatic 4000, Somacount 150, and Somascope), a pretreatment step of raw milk (heating for 15 min at 40°C) was needed to allow the staining of DNA with the fluorescent dye (Miller et al., 1986; Hinz et al., 1992; Paape et al., 2001). When the SCC in the raw milk samples measured by the C-reader with and without a preheat treatment at 40°C, however, were compared using the GLM procedure of SAS (SAS Institute, 1992), there was no significant difference in the SCC observed between the 2 groups.

When the results of the C-reader were compared with 3 other commercialized automatic methods based on indirect FCM by linear regression analysis, the C-reader system was found to have a high linear correlation coefficient (R2) of 0.964 [95% confidence interval (CI), 0.980 to 0.984] with the Fossomatic 4000; an R2 of 0.935 (95% CI, 0.962 to 0.971) with the Somacount 150; and an R2 of 0.960 (95% CI, 0.977 to 0.982) with the Somascope. With the high correlation, the SCCt data from each type of conventional equipment showed a linear relationship with those from the C-reader (P < 0.01). In addition, a low mean difference in the SCCt (D, –0.088 to 0.004) between the C-reader and the other conventional automatic instruments was calculated. The high R2 and low D values indicate that the C-reader can be used as a reliable instrument for SCC analysis.

The maintenance of the C-reader instrument requires neither tubing nor washing system as a result of the use of disposable plastic chips and a separate staining solution, which makes the C-reader system more suitable for small laboratories with an inexpensive automated SCC test. In addition, it is suitable for use as an on-farm tool because of its ease of use, low cost of the instrument, low reagent consumption, use of a touch screen and function keys, and portability. The single machine system weighs only 17 kg including the PC and LCD monitor, with a ready-to-use type in under a minute. Although the C-reader instrument is more expensive and complex than the California Mastitis Test for monitoring the SCC level of quarter milk at a dairy farm, it can be used as accurate method for the daily measurements of the SCC in raw milk from cows or bulk tanks at dairy farms. Moreover, the C-reader can provide the farmer with a means of achieving economic profitability through an increase in the premiums paid by milk purchasing cooperatives if the milk from high SCC cows is not sold.

In conclusion, the portable C-reader is an easy-to-use automatic system for measuring SCC, without the need for calibration and heating the milk samples, in large dairies and smaller laboratories. This method has greater or comparable repeatability and accuracy compared with the manual DMSCC method and other expensive automatic instruments.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
This study was funded by Digital Bio Technology Co. Ltd. (Seoul, Korea) as well as the National Veterinary Research and Quarantine Service (Gyeonggi-do, Korea). H. C. Koo and Y. H. Park were supported by the Korean Research Foundation Grant (KRF-2006-005-J02903), Research Institute of Veterinary Science, Department of Veterinary Microbiology, College of Veterinary Medicine, and the BK21 Program for Veterinary Science, Seoul National University. The authors thank G. C. Jang from NVRQS; S. Shin, Y. K. Park, and S. Y. Hwang from College of Veterinary Medicine, Seoul National University; and S. Y. Gang from Dang Jin Dairy Livestock Cooperative Federation for their technical assistance in many ways.


   FOOTNOTES
 
1 These authors contributed equally to this study. Back

Received for publication October 5, 2006. Accepted for publication January 5, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 


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