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Counting of Somatic Cells in Raw Milk

NucleoCounter®SCC-100TM

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NucleoCounter SCC-100

Stand-alone fluorescence microscope with built in computer for image analysis

Disposable sampling and measuring device, preloaded with nuclei staining dye

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Easy operation

1. Lyse 2. Load 3. Count

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Fast Result

+ = 30 seconds

= 30 seconds

------------------

max 1 minute

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Base technology of ChemoMetec

Fluorescence Microscopy

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Integrated Fluorescence Microscope

Lenses: magnification <1X.

Excitation filter: sorts out green light.

Emission filter: sorts out long wave length including red light.

LensesCCD

Emission Filter

LED’s

Excitation Filter

fixture

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NucleoCassette

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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

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1 dye molecule per 4-5 bp

Genome between 2.5 x 109 – 4.0 x 109 bp. Propidium Iodide

DNA base pair (bp):

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

C27H34N42+.2I- Mr 668.4

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Reagent C-100• Disrupts the plasma membranes of the mammalian cells, rendering the nuclei

susceptible to staining with PI

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Total cell count

Viable Non-viable

Free nucleus

PI

PI-stained nucleus

Lysis buffer

(Reagents C)

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Sensitive parts,

moving parts and

“wear and tear” parts

have been taken out of the instrument

and integrated in the disposable NucleoCassette

- Pump- Tubing’s- Mixer- Precision optics- Chemicals- Calibration- Waste container

The NucleoCassette

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Calibrated SCC-cassettes

W

T (

co

de

)

Dots for thickness code (T)

TAF0.01TAreaTWLVolume

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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

0.90

0.95

1.00

1.05

1.10

0 250000 500000 750000 1000000

Number of analysis

Optica

l inte

nsi

ty (

a.u

.) v

Test conditions:t = 60°C, If(LED) = 100mASCC-100 Operating conditions max:t = 40°C, If(LED) = 30mA

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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

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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.

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Repeatability•Repeatability error dominated by count statistics (Poisson distribution) •CV = 1/ x 100%

CV% - NucleoCounter SCC-100

0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

14.0%

16.0%

18.0%

0 200 400 600 800 1000 1200

SCC

Nucle

oC

ounte

r SC

C-1

00 C

V%

NucleoCounter SCC-100 Poisson

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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

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Reproducibility•Comparison of two instruments from different production series - Factory Settings!

NucleoCounter SCC-100 B vs A

y = 1.0047x - 0.0285

0

200

400

600

800

1000

1200

0 200 400 600 800 1000 1200

NucleoCounter SCC-100 Inst. A

Nucle

oC

ounte

r SC

C-1

00 I

nst.

B

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Reproducibility

•Only significant method contribution is repeatability–In test more than 95% of the intra-laboratory reproducibility explained by repeatability

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Routine Milk Samples

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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

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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