Investigation about Log Reduction ofBlood Contamination byWasher-Disinfectors

Tomozo Yamamoto, Ryo Fushimi, Yushi Uetera, Kazuhisa Tomozo Yamamoto, Ryo Fushimi, Yushi Uetera, Kazuhisa Matsuda, Yutaka Shimazaki, Toshiaki Matsuyama, Ken Sakai, Matsuda, Yutaka Shimazaki, Toshiaki Matsuyama, Ken Sakai,

Yoji Harada, Masatake Shimizu, Yoji Harada, Masatake Shimizu, Ken Okubo,Ken Okubo, Hiroyoshi Kobayashi Hiroyoshi Kobayashi

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Target of Investigation

• We tried to figure out the log reduction of blood contamination by washer-disinfectors with practical cleaning cycles, using the test pieces contaminated quantitatively by sheep blood.

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Hierarchic Structure of Working Group

• JAPANESE SOCIETY OF MEDICAL INSTRUMENTATION （ JSMI ）

–Accrediting Committee of Reprocessing Technicians

–Working Group of Cleaning Evaluation

The WG was set up in April 2006 for cleanliness evaluation with 11 group members assigned.

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Members of Working GroupRyo Fushimi Osaka Univ. Hosp.

Yushi Uetera Univ. of Tokyo Hosp.

Kazuhisa Matsuda Saiseikai Fukuoka General Hosp.

Yutaka Shimazaki Kainan Hosp. Aichi Prefectural Welfare Federation of Agricultural Cooperatives

Toshiaki Matsuyama Univ. Hosp. of Occupational & Environmental Health

Ken Sakai Kyushu Univ. Hosp.

Yoji Harada Clean Chemical Co., Ltd.

Masatake Shimizu Inui Medics Corporation

Ken Okubo Tokyo Healthcare Univ.

Hiroyoshi Kobayashi Tokyo Healthcare Univ.

Tomozo Yamamoto Muranaka Medical Instruments Co., Ltd.

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Design of Test piece

• Base Plate– Stainless Steel– 220x120mm

• Wire Mesh– stainless Steel– 200x90mm

• Fixing Bars – stainless Steel– 120x8mm

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Test soil• Heparinized Sheep Blood

– The content rate of protein in sheep blood varies a lot according to blood drawing date and also donor sheep.

– The content rate of the individual lot was measured by OPA method.

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Quantitative Control of Sheep Blood on each test piece

Date of Blood Drawing

Protein Content in blood (w/w %)

Applied Amount of Blood (g)

Applied Amount of Protein (g)

2008.11.27 10.4 19.3 2.0

2009.01.14 12.2 16.4

2009.03.23 15.5 12.9

2009.04.07 17.2 11.7

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Application of Sheep Bloodon a test piece

Sheep blood contained 2 g of protein was applied in thin layer on each mesh and base plate and then fixing bars were screwed on the plate.

Each prepared plate was dried at room temperature over a night and then vacuum-packed in a PE bag.

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Loading Configuration of test pieceinto WD

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Extraction of Residual Protein

The processed test piece by WD was put into PE bag with 10 ml of NaOH (0.2 mol/l) and immersed in a water bath at 50 for ℃120 min.

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SO3Na

CH2 N

CH3

C

H3C

N+

H2C

C2H5

NH

OC2H5

C2H5

SO3-

　

Coomassie® Brilliant Blue G-250(Bradford Method)

CBB

400 600500 700 8000.00

0.20

0.40

0.60

0.80

1.00

wave length (nm)

CBB + Casei n 100μｇ

Absorbing Spectrum（ OD）

Chemical Structural Formula

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Measuring Procedure by Bradford Method

• Put 1 ml of residual protein extract and 3 ml of Coomassie® Protein Assay Reagent into test tube.

• Measure the optical density of the reactant at 595 nm after 20 min.

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Calibration Curve

• BSA solution (2 mg/ml) was diluted in NaOH solution (0.2 mol/l)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 1 2 3 4 5 6

BSA (μg)

OD(595nm)

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Recovery Rate of BSAApplied Amount of BSA

on a test piece (μg)25 50 100

measurements 　　　　　　　(μg/test piece, N=５ )

25 48 9924 51 9823 53 9624 48 9926 48 95

Average (μg/test piece) 24.4 49.6 97.4 Standard Deviation 1.0 2.1 1.6

Coefficient of Variation (%) 4.1 4.2 1.6Recovery Rate (%) 97.6 99.2 97.4

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Measurement Result in LaboratorySetting Parameters of Main Wash Residual

ProteinAverage

(μg)℃ min. Detergent (type, v%, pH)

90 5 Alkaline 0.3 11.3 30 30.845

36

23

20

60 10 Alkaline 0.3 11.3 37 31.621

34

29

37

50 10 Enzymatic 0.5 8.2 32 42.458

33

56

33

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Measurement Result in HospitalsSetting Parameters of Main Wash Residual

ProteinAverage

(μg)℃ min. Detergent (type, v%, pH)

93 10 Alkaline 0.3 11.3 97 68.75851

93 10 Alkaline 0.3 11.3 20 29.73732

60 8 Alkaline 0.5 12.0 21 16.01413

40 5 Alkaline 0.5 11.5 28 28.32631

pretreatment: immersing into enzymatic detergent solution (0.8 v%) for 5 min. prior to processing by WD

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Conclusion

• The measurements of the residual protein in the laboratory varied between 20 and 58 μg and the rate of protein decrease showed 1/100,000 down to 1/34,000.

• The measurements of the residual protein in the hospitals varied between 13 and 97 μg and the rate of protein decrease showed 1/153,000 down to 1/20,000.

• Washer-disinfectors have power 5 of the log reduction factor of sheep blood.

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Thank you for your attention.