Counting of Somatic Cells in Raw Milk
NucleoCounter®SCC-100TM
www.chemometec.com
NucleoCounter SCC-100
Stand-alone fluorescence microscope
with built in computer for image
analysis
Disposable sampling and measuring device,
preloaded with nuclei staining dye
www.chemometec.com
Easy operation
1. Lyse
2. Load
www.chemometec.com
3. Count
Fast Result
+
= 30 seconds
= 30 seconds
-----------------max 1 minute
www.chemometec.com
Base technology of ChemoMetec
Fluorescence Microscopy
www.chemometec.com
Integrated Fluorescence Microscope
CC D
Emission
F ilter
Lenses
fixture
LED ’s
Excitation
F ilter
Lenses: magnification <1X.
Excitation filter: sorts out green light.
Emission filter: sorts out long wave length including red light.
www.chemometec.com
NucleoCassette
www.chemometec.com
Propidium iodide (PI)
•Immobilized in the interior of the NucleoCassette, 2.8 ug in each
•A fluorescence dye, which intercalates with DNA of cell nuclei
•Cell membrane permeable
www.chemometec.com
Propidium Iodide
1 dye molecule per 4-5 bp
Genome between 2.5 x 109 – 4.0 x 109 bp.
C27H34N42+.2I- Mr 668.4
DNA base pair (bp):
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
www.chemometec.com
Reagent C-100
• Disrupts the plasma membranes of the mammalian cells, rendering the nuclei
susceptible to staining with PI
www.chemometec.com
Total cell count
Viable
Non-viable
Lysis buffer
(Reagents C)
Free nucleus
PI
PI-stained
nucleus
www.chemometec.com
The NucleoCassette
Sensitive parts,
moving parts and
“wear and tear” parts
have been taken out of the instrument
and integrated in the disposable NucleoCassette
-
www.chemometec.com
Pump
Tubing’s
Mixer
Precision optics
Chemicals
Calibration
Waste container
T (code)
Calibrated SCC-cassettes
L
W
Dots for thickness code (T)
Volume LW T  AreaT  0.01 AFT
www.chemometec.com
Highly reliable LEDs
Thermal drift in optical intensity is approximately 0.2 %/°C
Optical intesity versus number of Number of analysis for the
NucleoCounter SCC-100
v
1.10
Optical intensity (a.u.)
1.05
1.00
0.95
0.90
0
250000
500000
Number of analysis
Test conditions:
t = 60°C, If(LED) = 100mA
SCC-100 Operating conditions max:
t = 40°C, If(LED) = 30mA
www.chemometec.com
750000
1000000
Key Advantages
•Simple Operation
–Ready in 5 seconds
–5 minutes operator training
–No cleaning
•Calibration Free
–LED's, CCD and fixed optics assure stable factory calibration
–Individually calibrated cassettes assure long term stability
•Maintenance Free
–Robust and reliable parts used in design
–2 years product guarantee
•Operator Independent
–Objective cell counting
•Safe sample handling
–PI immobilized inside NucleoCassette
www.chemometec.com
Good reproducibility
•Results from different NucleoCounters can be directly compared:
–All NucleoCounters are calibrated to respond as the same Master NucleoCounter at
ChemoMetec.
–The initial calibration will maintain throughout the lifetime of a NucleoCounter.
www.chemometec.com
Repeatability
•Repeatability error dominated by count statistics (Poisson distribution)
•CV = 1/
x 100%
CV% - NucleoCounter SCC-100
NucleoCounter SCC-100 CV%
18.0%
16.0%
14.0%
12.0%
10.0%
8.0%
6.0%
4.0%
2.0%
0.0%
0
200
400
600
800
SCC
NucleoCounter SCC-100
www.chemometec.com
Poisson
1000
1200
Repeatability
•Repeatability*:
• - CV < 8% at 150,000 cells/ml
• - CV < 6% at 300,000 cells/ml
• - CV < 4% at 500,000 cells/ml
• - CV < 3% at 1,000,000 cells/ml
•CV = Repeatability error Coefficient of Variance
www.chemometec.com
Reproducibility
•Comparison of two instruments from different production series - Factory Settings!
NucleoCounter SCC-100 B vs A
NucleoCounter SCC-100 Inst. B
1200
y = 1.0047x - 0.0285
1000
800
600
400
200
0
0
200
400
600
800
NucleoCounter SCC-100 Inst. A
www.chemometec.com
1000
1200
Reproducibility
•Only significant method contribution is repeatability
–In test more than 95% of the intra-laboratory reproducibility explained by repeatability
www.chemometec.com
Routine Milk Samples
www.chemometec.com
Milk Application
• SCC Stability
– The inherent stability of the NucleoCounter SCC-100 offers tools to monitor and assure
stability of SCC results obtained by routine instruments
• Pilot Samples
– The NucleoCounter SCC-100 allows fast and reliable determination of SCC pilot samples
www.chemometec.com
Somatic Cell Counting
• NucleoCounter SCC-100
– Stand-alone fluorescence microscope
– Maintenance free
– Calibration free
• NucleoCassette
– Pre-loaded with nuclei staining dye
– Analysed volume 1µl (working factor 1,000)
– Calibrated measurement chamber (Dot code)
• Cell Counting
– Operator independent
– Reproducibility - Appr. Equal to Repeatability!
– Repeatability Error - Appr. Poisson statistics
• (≈5% @ 500,000/ml)
– Sample throughput – About 100 samples/hour
– SCC - 10,000/ml to 1,000,000/ml
www.chemometec.com