
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72473
10.1038/s41598-024-72473-1
Article
Design and validation of a method for evaluating medical device cleanliness by recovering and quantifying residual proteins on stainless plates
Uematsu Miyuki uematsu@nihs.go.jp

1
Miyamoto Yuko 1
Shimizu Masatake 2
Kajiura Tsuyoshi 2
Saito Atsushi 3
Takashina Masaki 3
Fujita Satoshi 4
Nakano Yuko 4
Shimizu Toshiaki 5
Nagahara Yuki 5
Kosaka Hayato 5
Muramatsu Hiroki 6
Mori Masafumi 6
Suzuki Takamasa 6
Nakamura Takayoshi 7
Tanemura Atsushi 7
Hosaka Junki 8
Mori Takahide 8
Kato Seiichi 9
Itagaki Ayaka 9
Inoue Toshiki 9
Matsumoto Shinichi 10
Naito Tomoko 11
Fujii Shinji 11
Nakaoka Ryusuke 1
Yamamoto Eiichi 1
1 https://ror.org/04s629c33 grid.410797.c 0000 0001 2227 8773 Division of Medical Devices, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-9501 Japan
2 Development Laboratory, Inui Medics Co., Ltd., 2-1-4 Honjo, Higashiosaka-shi, Osaka, 578-0953 Japan
3 https://ror.org/05rnn8t74 grid.412398.5 0000 0004 0403 4283 Central Sterile Supply Department, Osaka University Hospital, 2-15 Yamadaoka, Suita-shi, Osaka, 565-0871 Japan
4 Engineering Department, Clean Chemical Co., Ltd., 4-28-9 Shima, Ibaraki-shi, Osaka, 567-0854 Japan
5 Infection Control Business Unit, Sakura Seiki Co., Ltd., 1122-8 Yawata, Chikuma-shi, Nagano, 387-0023 Japan
6 SUD Re-manufacturing Business Preparation Office, Suzuyo & Co., Ltd., 1-2-12 Shibakoen, Minato-ku, Tokyo, 105-0011 Japan
7 Institute of Surface Science and Technology, Nicca Chemical Co., Ltd., 4-23-1 Bunkyo, Fukui-shi, Fukui, 910-8670 Japan
8 grid.410862.9 0000 0004 1770 2279 Laboratory and Speciality Chemicals Division, FUJIFILM Wako Pure Chemical Corp., 6-1 Takata-cho, Amagasaki-shi, Hyogo, 661-0963 Japan
9 Research and Development Division, Hogy Medical Co., Ltd., 1873-1 Fusa, Miho-mura, Inashiki-gun, Ibaraki, 300-0427 Japan
10 System Sales Department, Muranaka Medical Instruments Co., Ltd., Funakoshi-cho, Chuo-ku, Osaka, 540-0036 Japan
11 R&D Division, Miura Co., Ltd., 7 Horie-cho, Matsuyama-shi, Ehime, 799-2696 Japan
20 9 2024
20 9 2024
2024
14 2198228 12 2023
9 9 2024
© The Author(s) 2024
2024
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We recently reported a method for recovering and quantifying residual proteins bound to surfaces of various medical instruments via thermal coagulation under neutral pH and room temperature. The method effectively recovered and solubilised coagulated proteins at high temperatures in dry and humid conditions, with a protein recovery rate of > 90%. This study validated the previous method by comparing residual protein recovery from test samples using a conventional extraction solution (1% SDS, [pH 11.0]) and proposed solution (1% SDS, 10 mM TCEP, and 10 mM HEPES [pH 7.0]). To mimic soiled medical equipment, pseudo-blood-contaminated stainless steel plates were prepared. Residual protein was recovered using conventional and proposed solutions under varying temperature and humidity conditions. Quantitative protein recovery limits were determined at nine facilities. Compared with the conventional solution, the proposed solution recovered proteins more effectively from samples processed at temperatures > 60 °C. However, low recovery rates were observed for samples processed at 95 °C, possibly owing to differences in protein adhesion due to sample and plate-surface properties. Our findings present a method for quantifying residual proteins on medical instruments exposed to high temperatures during use or disinfection. Further studies should standardise test soiling conditions, materials, and solutions to evaluate cleaning methods.

Subject terms

Biochemical assays
Spectrophotometry
Health policy
Biomaterials - proteins
http://dx.doi.org/10.13039/100009619 Japan Agency for Medical Research and Development 22mk0102174 issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

To ensure the safety of reusable medical devices and reprocessed single-use medical devices, manufacturers must evaluate their cleanliness after washing1–7. The acceptance criteria for residual protein have been determined according to established standards and guidelines (Table 1). For evaluating the performance of washer-disinfectors, ISO 15883-5:2021 defines the alert and action levels for residual protein on all device parts as ≥ 3 and ≥ 6.4 µg/cm2, respectively8. The criteria are consensus-developed expert opinions on the state-of-the-art. The values are a combination of the results obtained using different types of devices, protein recovery solutions, and quantification methods. Table 1 Comparison of currently allowed residual protein levels.

Acceptance criteria in standards or guidelines	
 Standards or guidelines	ISO 15883-5: 20218	ANSI/AAMI ST98: 20229	DGKH, DGSV and AKI guideline: 201712	JSMI guideline: 202114	
 Acceptance criteria

(residual protein per object as BSA)

	Alert level: ≥ 3 µg/cm2 9

Action level: ≥ 6.4 µg/cm2 12

	Limit value: 6.4 µg/cm2	Limit value: 3 µg/cm2

Warning value: > 80 to ≤ 150 μg/device

	Limit value: 200 µg/device	
Evaluation tests for establishing criteria of residual proteins	
 Data collection	Not conducted	Single hospital	Several companies’ database	Seven hospitals	
 Subject instruments	Not conducted	10 bronchoscopes

(Surface area: 45.6 cm2)

	4,122 Crile clamps

(Surface area: 20 cm2)

	91 forceps, needle holder	
 Recovery solutions	Not conducted	10 mL of sterile reverse osmosis water	2 mL of 1% SDS solution	10 mL of 0.2 M NaOH solution	
 Recovery methods	Not conducted	Brushing tubes and rinsing with water

(room temperature)

	Elution of hinged instruments with SDS solution

(room temperature)

	Sonication in an ultrasonic water bath

(50 °C, 30 min)

	
 Quantification methods	Not conducted	Bradford method	BCA method	Bradford method	
Cleaning efficacy testing methods for all reusable medical devices	
 Recovery solutions	Unspecified	Unspecified	1% SDS	1% SDS (pH 11.0)	
 Quantification methods	Unspecified	Unspecified	Modified OPA method, Biuret/BCA method	BCA method, Bradford method, Modified OPA method	
 Considerations for the protein coagulation by thermal effect	The disinfection cycle shall be verified that this does not interfere with analyte detection	Hot water can coagulate protein, making it more difficult to remove during extraction.	When evaluating residual proteins, the wash program must be interrupted and the test objects removed before the beginning of the thermal disinfection phase to ensure that the recoverability of protein is not adversely affected by the disinfection step	

The American National Standards Institute (ANSI)/Association for the Advancement of Medical Instrumentation (AAMI) ST98:20229,10 set the acceptable level of residual protein after washing at 6.4 µg/cm2, based on protein recovered from a brushed endoscope using reverse osmosis water10. In contrast, the Guideline of the German Society for Hospital Hygiene, German Society of Sterile Supply, and a working group for instrument reprocessing set the limit at 3 µg/cm2, based on reports pertaining to 4122 Crile clamps, where 1% sodium dodecyl sulfate (SDS) was used as the recovery solution11,12. As no extensive survey has been conducted since those mentioned above, these values have not been updated.

For example, the average residual protein on reprocessed gastroscopes and colonoscopes determined using the o-phthalaldehyde (OPA) method was 7.1 ± 5.0 µg/cm213, which does not meet the ANSI/AAMI criterion. However, considering the differences in quantification methods, sample preparation, and operational efficiency, these values cannot be compared. Although the criteria do not specify the applicable devices, they apply to all medical devices.

International guidelines require that residual protein from medical instruments be reported in terms of weight per unit of surface area8–12. Therefore, most facilities must use at least 10 mL of a 1% SDS solution. The volume should fill all parts of a medical instrument to sufficiently recover the residual protein.

The value of residual protein is expressed as the protein concentration multiplied by the volume of protein extraction solution from a medical instrument. The value of residual protein per unit area is calculated by dividing the amount of residual protein by the total surface area of the instrument. Using a large volume of solution results in a low protein concentration, even if most residual protein is localised within a small area. Moreover, the measurements of solutions with low protein concentrations may be inaccurate depending on the spectrometry used in analysis. The protein extraction depends on the recovery rate of the protein solution.

Medical devices differ in their physical size, shape, structure, properties, and mechanisms. In addition, the level of contamination can change considerably, depending on the intended use and direction of the devices. As residual protein may be concentrated in certain parts, we should show the value of residual protein per device instead of per unit surface area. In the German guideline, acceptance criteria for real-use devices are shown for five categories and indicate the value per device for each category except instruments without hinges or cavities. The criteria set the limit for contamination at a maximum of 200 µg/device.

In 2012, the Japanese Society of Medical Instrumentation (JSMI) set the limit for contamination at 200 µg/device for a reusable medical device based on a domestic multicentre test14. The value is the same as that in the German guideline, although their tests differ in terms of protein recovery solutions and quantification methods. The test utilised a 0.2 M NaOH solution for residual protein recovery. With the goal of breaking down protein via hydrolysis, the solution was used to extract and dissolve proteins under sonication at 50 °C for 30 min14,15.

In 2021, the JSMI adopted criteria for real-use instrument groups and an SDS solution similar to those applied in Germany16. However, the value of residual protein was maintained. The SDS solution was adjusted to pH 11 in all cases, according to the JSMI guideline:2021. In contrast, the German guideline states that the solution should be adjusted to pH 11 when using disinfectants with cleaning capability or using instruments with the possibility of slightly soluble residues.

The purpose of the cleaning validation is to evaluate whether the contaminant has been removed from the instrument to prepare it for disinfection and/or sterilization. We explain five aspects that affect cleanliness evaluation: (1) temperature of the cleaning cycle, (2) thermal coagulation of protein, (3) protein denaturation and solubilisation, (4) protein quantification, and (5) protein extraction.

With regard to cleaning cycle temperature, ANSI/AAMI ST98:20229 indicates that hot water can coagulate proteins, complicating their removal during extraction. Therefore, it provides an example for designing validation methods, with the final rinse conducted at a lower temperature if the cleaning step can be performed independently from the disinfection process with hot water. In addition, the World Health Organization guideline17 recommends that instruments be cleaned via sonication with water below 60 °C, thereby avoiding the thermal coagulation of residual protein and its adhesion to instruments. Therefore, all above-mentioned residual protein values were evaluated after the cleaning temperature dropped below 60 °C, even if the full cycle of the cleaning process exceeded 60 °C. However, manufacturers often expect that they will evaluate the full cycle of the cleaning process of the washer-disinfector without stopping at below 60 °C. This is because they sometimes intend to achieve high cleanliness with washer-disinfectors after high-temperature procedures (e.g. 90 °C).

With regard to thermal coagulation, according to the JSMI guideline:2021, test devices should be prepared using sheep blood to evaluate the cleaning effect of washer-disinfectors. The test devices are dried at 45 °C for 1 h in a drying oven at constant temperature. Considering the worst-case scenario, this condition is not appropriate. When surgical energy devices are used for tissue dissection, the temperature of the tip of the device reaches over 100 °C18–20. The temperature of the tip of some devices exceeded 255 °C, and that of the shaft of a laparoscopic hook exceeded 120 °C after only one second of energy application18. This means that surgical instruments may potentially be subjected to high temperatures, both before and after cleaning.

We would also like to know how protein denatures at temperatures over 60 °C when washing medical devices. In our previous study, we assumed thermal coagulation would occur under humid conditions without detergent. A comparison of the two conditions showed that in the conventional recovery measurement using a 1% SDS (pH 11.0) recovery solution, the protein thermally coagulated under dry conditions was solubilised and could be measured, whereas the protein thermally coagulated under humid conditions precipitated and was not completely solubilised, resulting in a recovery rate of only 60%21,22.

When SDS–polyacrylamide gel electrophoresis (SDS-PAGE) is performed, sample buffers are used to dissolve several proteins for separation. A microtube containing the sample was heated with the buffer at 95 °C for 5 min with a heat block in a dry bath. The commonly used buffer contains SDS, dithiothreitol (DTT), and tris(hydroxymethyl)aminomethane (Tris), which cleave disulfide protein bonds reductively, destroy higher-order structures, and promote SDS binding. The temperature can be lowered if the time is extended, and heating can accelerate the reaction23. To simplify the protein solubilisation protocol without heating equipment, we used the solution at 23 °C for 16 h overnight. We previously showed that protein coagulated at over 60 °C could be dissolved in the proposed buffer and quantified unless it carbonizes at approximately 240 °C21,22. Considering this, the limitation of cleaning validation below 60 °C should be reconsidered if coagulated proteins are extracted from medical devices.

With regard to protein solution quantification, although the protein in humid conditions is denatured more strongly than in dry conditions at high temperatures, the SDS-PAGE buffer completely dissolves it. However, buffer components impede quantification when basic quantification assay kits are used. Therefore, we modified the buffer components to a 1% SDS, 10 mM tris(2-carboxyethyl) phosphine (TCEP), 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (pH 7.0) solution to irreversibly break disulfide bonds at neutral pH, and alkylated the recovered protein solution to disregard the sulfhydryl group. The TCEP reaction is irreversible, whereas the DTT reaction is reversible. TCEP is a useful reductant with a wide pH range of 1.5–8.5. However, DTT is used over a pH of 7.024. The pH of the buffer was maintained in HEPES buffer. Finally, we quantified via the micro-BCA method at low protein concentrations (2.5–40 µg/mL)21,22.

With regard to protein extraction from medical devices, as shown in ANSI/AAMI ST98:2022, the spike-recovery test method is commonly used to validate extraction methods. The recovery efficiency is defined as the ratio of the amount of recovered residue to the initial contaminant amount. We used this method to compare the efficiency of protein extraction solutions. This standard requires a recovery efficiency greater than 70%9. Therefore, the target recovery rate was set to 70%.

We reported this newly developed recovery method with 1% SDS, 10 mM TCEP, and 10 mM HEPES (pH 7.0) solutions, which demonstrated high residual protein recovery rates even after thermal coagulation at temperatures above 60 °C in both dry and humid conditions. Owing to the recovery solution, we can perform a recovery test at neutral pH and room temperature (20–25 °C), with the potential for use on pH-sensitive materials in normal working environments21,22. In addition, the proposed solution can quantify small amounts of residual protein (2.5–40 µg/mL) on stainless steel (SUS) plates as test devices22 and scissors as clinically used devices25 after cleaning at up to 90 °C with a washer-disinfector.

Herein, we sought to validate the efficacy of 1% SDS, 10 mM TCEP, and 10 mM HEPES (pH 7.0) solutions and to determine why protein recovery evaluation should be avoided at temperatures > 60 °C. To prioritize quality control, we used a considerably thick layer of proteins on SUS plates, in accordance with our previous reports. Following the ANSI/AAMI ST98:2022 extraction method, we validated the recovery efficiency and confirmed that it was capable of accurately quantifying the defined test soil analyte across a range of values, including the acceptance level.

Results

Residual protein extraction and quantification from SUS plates

The effects of heat treatment on protein recovery using conventional and proposed solutions were investigated. Protein recovery solutions were quantified using the modified OPA (Fig. 1). For samples treated under dry conditions as per the current JSMI guideline, similar protein recoveries were obtained using the conventional (94.2 ± 2.9%) and proposed solutions (97.5 ± 3.4%). However, samples treated under wet conditions showed contrasting results. The protein recovery rates with the conventional solution were significantly low according to the intensity of the wet heat treatment. Meanwhile, the recovery rates using our proposed solution remained high for the sample incubated for 30 min at 60 °C, decreasing to 58.8 ± 10.7% in samples incubated for 15 min at 95 °C in an autoclave with agar medium dissolution mode.Fig. 1 Protein recovery rates from pseudo-blood-contaminated samples treated under various conditions and evaluated using the modified o-phthalaldehyde method and two solutions. (A) Conventional solution: 1% sodium dodecyl sulfate (SDS, pH 11.0) and (B) proposed solution: 1% SDS, 10 mM tris(2-carboxyethyl) phosphine (TCEP), and 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (pH 7.0). In the bars, (A) is represented as the black area, and (B) is represented as the grey area (n = 9. *p < 0.05, **p < 0.01). The total protein concentration of citrated sheep blood using the conventional and proposed solutions was 168.9 ± 1.5 and 178.7 ± 2.3 mg/mL, respectively. Other treatment conditions did not show any significant difference.

After extraction, the test samples were stained with Amido Black 10B (Fig. 2), and the precipitation of each extraction tube was observed (Fig. 3). Visual inspection of the sample plates revealed the residual protein after recovery. Although the initial amount of contaminant was approximately 3.75 mg/cm2, the proposed solution completely removed the residual protein from the SUS plates and clarified the protein-containing solutions in samples incubated at up to 60 °C. Protein-containing membranes on SUS plates and precipitation of protein recovery tubes were observed in samples incubated at 95 °C. In contrast, the conventional solution removed residual protein from the plates and clarified the protein-containing solutions only at 45 °C under dry conditions. The solution could remove almost all contaminants, but could not clarify the protein-containing solution at 95 °C under wet conditions.Fig. 2 Amido Black 10B staining of pseudo-blood-contaminated sample plates after protein extraction using each solution. The upper three rows were extracted using (A) the conventional solution: 1% SDS (pH 11.0), and the lower three rows were extracted using (B) the proposed solution: 1% SDS, 10 mM TCEP, and 10 mM HEPES (pH 7.0). Residual proteins are stained in deep blue on each plate. Each condition was tested using three samples.

Fig. 3 Visual inspection of sample plates showed residual proteins after recovery. Each condition was tested using three samples. Each tube corresponding to the recovery solution after the plate, shown in Fig. 2, was collected. The dark area at the bottom indicates precipitation. The precipitation decreased protein concentration. The conventional solution (A, 1% SDS, pH 11.0) could neither remove residual proteins from plates nor clarify the protein-containing solutions due to precipitation. The proposed solution (B, 1% SDS, 10 mM TCEP, 10 mM HEPES, pH 7.0) removed residual proteins completely and clarified the protein-containing solutions at up to 60 °C. However, solution B retained membranous protein-containing materials on the plates as well as precipitates in the protein-recovered tubes at 95 °C.

Although the conventional solution removed the protein from the SUS plates in samples incubated at 95 °C, the protein-containing solution had a low recovery rate owing to precipitation. Figure 4 shows the blood-contaminated samples. After the samples were dried in a room at 24 °C and 40%, the contaminant exhibited a small crack in the test samples, not observed in previous test samples. Furthermore, the layer of contaminants was partially removed from the surface of some wet samples incubated at 95 °C before solution recovery, although we excluded these samples and re-prepared them for use in the test.Fig. 4 Comparison of pseudo-blood-contaminated sample plates between the previous test and the present test. In previous tests, no obvious cracks were observed in the dried samples (Aa). However, small cracks were observed in all samples before heat treatment in this test (Ba). When heat treatment was performed at 95 °C under humid conditions, certain samples exhibited soil exfoliation in the present test (Bb). However, no exfoliation was observed in the previous test (Bb). Although the difference in treatment time may have impacted the recovery rates owing to increased protein thermal coagulation, the main factor seemed to be the sample drying process at room temperature before incubation in the autoclave.

Equipment performance and estimation of quantitative limits

A calibration curve constructed from bovine serum albumin (BSA) measurements using the proposed solution showed good linearity for both the microplate reader (slope = 0.001884–0.002054, R2 = 0.9984–0.9995) and spectrophotometer (slope = 0.002775–0.003329, R2 = 0.9886–0.9996) when applying the modified OPA method in the range of 25–800 µg/mL. However, at low protein concentrations (2.5–40 µg/mL), the calibration curve linearity was unstable when the absorbance was measured using a spectrophotometer (slope = 0.002971–0.003398, R2 = 0.6646–0.9932). The calibration curve constructed based on the micro-BCA method showed good linearity in the 2.5–40 µg/mL range, regardless of the instrument used (microplate reader: slope = 0.008903–0.01040, R2 = 0.9952–0.9981, spectrophotometer: slope = 0.01425–0.01576, R2 = 0.9606–0.9998).

The measurement efficiencies were evaluated on the basis of the trueness and precision of the quantified BSA concentrations in the respective solutions. While quantification limits varied depending on the instruments used at each facility, the volume reduction of protein solutions had no impact on the lower quantification limit in the spectrophotometer using cell micro-cuvettes.

Discussion

It is currently believed that residual protein evaluation for instrument cleaning should not be conducted at temperatures > 60 °C, owing to thermal coagulation. However, few studies have shown that denatured proteins can be sufficiently recovery from a conventional solution when the cleaning cycle stops below 60 °C. Herein, we aimed to explore the reasons for this limitation and possible resolutions.

The recovery rate of residual protein from test samples treated with 1% SDS (pH 11.0) gradually decreased depending on temperature and moisture (Fig. 1A). The recovery rate using 1% SDS was 70.6 ± 4.5% for the sample incubated at 60 °C for 30 min in the autoclave, which is acceptable. However, the recovery rate of the sample incubated at 95 °C for 15 min was 35.0 ± 1.1%. Our results confirmed that evaluation with 1% SDS (pH 11.0) should not be performed when washing with hot water at temperatures exceeding 60 °C.

The proposed solution was expected to recover > 90% of the total residual protein on a plate incubated for 15 min at 95 °C in the autoclave. However, a recovery rate of 58.8 ± 10.7% was achieved (Fig. 1B). Although the recovery rate with the proposed solution could not reach 70%, it was highly likely to recover total residual protein from the SUS plates more strongly than with the conventional solution. Based on our results, we reconsidered the five aspects that affect cleanliness evaluation.Temperature of the cleaning cycle: We aimed to produce samples heated in humid conditions to denature proteins without detergent in a cleaning cycle of washer-disinfectors and to avoid loss from liquid water during soaking. To do so, we used agar medium dissolution mode in the autoclave. We wanted to know why we should not raise the temperature above 60 °C. A series of treatment temperatures were determined in accordance with cleaning cycle time. Assuming the worst-case scenario, the incubation time at each temperature was doubled as the cleaning time based on a protocol of an adopted washer-disinfector. In a previous test, the heat treatment was performed using the same method as in this study (see Supplementary Figs. S1–S4online). However, for treatment at 95 °C, the time was increased from 10 min in the previous study to 15 min because of a mistake in autoclave settings. The difference in treatment time may have impacted the recovery rates owing to increased protein thermal coagulation.

Thermal coagulation of protein: Fig. 2 shows the remaining proteins on SUS plates. For conventional solution recovery from the SUS plate incubated under wet conditions, the centre proteins remained but the peripheral ones were removed when the plates were heated at 45 °C for 1 h. The opposite was observed after heating at 60 °C for 30 min. Further, almost all protein was removed when the plates were heated at 95 °C for 15 min.

We assume that the unevenly distributed proteins and small cracks on the contaminant surface caused these variations. While we evenly applied a homogeneous blood solution with a pipette, the distribution of the solute might become inhomogeneous with evaporation. The blood gradually grew drier from the peripheral area, and the liquid remained in the centre of plates. In polymer solutions, the solute localises at the interface between the gas and liquid phases, forming a thin solid film, called the skin layer, when the liquid is dried. It is empirically known that the skin layer reduces drying speed, causing bubbles and wrinkles on the coating film surface. Wrinkles are formed when the skin layer is stressed by the non-uniformity of the drying rate around the droplet27. Therefore, protein clusters seemed to be generated in at least two areas: the centre and periphery. When we confirmed heat transfer on the SUS surface with irreversible temperature recording labels, the temperature was higher in the centre than in the periphery at 95 °C under humid conditions, while other conditions resulted in the same heat distribution. Proteins on the plates must have coagulated under the slightly different heat conditions, that is, each protein cluster was individually coagulated on a plate.

In previous tests, no obvious cracks were observed in the dried samples (Fig. 4A a). However, small cracks were observed in all samples before the heat treatment in this test (Fig. 4B a). The vapor entered the gaps between the blood and the SUS surface easily because of the cracks and lifted dirt from the material surfaces, similar to the effect of the steam cleaner. The vapor affected each protein cluster differently in terms of thermal coagulation.

When pseudo-blood contaminated SUS plates were incubated for 15 min at 95 °C under humid conditions in the autoclave, some samples exhibited soil exfoliation (Fig. 4B b). ASTM F3293-18 claims not to use the contaminated samples when contaminants flake during drying28. Therefore, we excluded samples in which contaminants were broken and peeled off. The samples were not exfoliated for use in this test. However, contaminants could have already been removed from the surface prior to recovery testing.3. Protein denaturation and solubilisation: We investigated protein solubility in the solutions. SDS (1%) extraction with a reducing agent is highly efficient as the reductive cleavage of disulfide bonds disintegrates the higher-order protein structure, thus promoting SDS binding29. In contrast, protein recovery was low with 1% SDS alone (pH 11.0) (Fig. 1). Residual protein could not be measured accurately, even though we followed the German guideline, because neither disulfide bonds nor β-cleavages were disrupted at pH below 11. Shimizu et al.15 reported that extraction with 0.2 M NaOH solution at 50 °C for 30 min with sonication followed by Coomassie Brilliant Blue staining is highly effective for quantifying residual proteins on SUS instruments. This method is referenced in the JSMI guidelines and has been widely accepted by Japanese medical device manufacturers. Tanino et al.30 investigated the recovery rate of BSA adsorbed on instruments using various solutions including 2% SDS (pH 11.0) and sonication with 0.4 M NaOH for 30 min at 50 °C. Although BSA extracted using SDS polyacrylamide gel electrophoresis (SDS-PAGE) was significantly hydrolysed to peptide fragments after alkaline treatment, this was not observed with 1% SDS (pH 11.0)30. Therefore, the recovery rate with 1% SDS (pH 11.0) appears unsuitable for evaluating medical instrument cleanliness at temperatures above 60 °C.

4. Protein quantification: We explored the lower limit of quantification using the modified micro-BCA and modified OPA methods (See Supplementary Table S1 online). Accurately measuring residual protein on medical instruments may be impossible without considering the quantification limit for the solution, that is, the point at which little or no residual protein is extracted. The modified OPA method was less precise (see Supplementary Table S1 (b) online) than the modified micro-BCA method (see Supplementary Table S1 (c) online), although trueness was equivalent. The slope of the modified OPA method was small, although it can be applied over a wide concentration range (see Supplementary Table S1 (a) online). Meanwhile, the modified micro-BCA method was applicable in a small concentration range despite its large slope (see Supplementary Table S1 (c) online). The lower limit of quantification depends on the method used. Generally, the values are acceptable when both trueness and precision are within 10%. The accuracy of the modified OPA method was acceptable only at protein concentrations exceeding 25 µg/mL, whereas the modified micro-BCA method accurately determined protein concentrations below 25 µg/mL.

The recovery rates did not vary significantly among assays (see Supplementary Figs. S1 and S2 online). Reducing the solution volume may be necessary to obtain the highest possible protein concentration and ensure accuracy. For spectrophotometric quantification, 1 mL of the solution was used. However, the current findings showed that this volume can be reduced to 100–200 μL with some ingenuity, such as using disposable cell micro-cuvettes with a cell holder or using a mask to adjust the optical axis.5. Protein extraction: Additional visual inspection of the stained residual protein using Amido Black 10B was performed for SUS304 plates from Nippon Steel Stainless Steel Corp. used in this study and Nippon Yakin Kogyo Co., Ltd. in the previous study. Furthermore, we confirmed that previous test results were reproducible for the same spike-recovery test with SUS304 plates from Nippon Steel Stainless Steel Corp. (see Supplementary Fig. S5 online). The initial protein volume was 470 μg/cm2, and the sample was dried at 24 °C and 70%RH using a saturated salt solution in a desiccator because the room humidity was 40%. Owing to the low volume of blood, an uneven distribution was observed on the contaminated blood surface. The results differed for the material surface modification with buff polishing. Proteins on SUS304 2B and the hairline were stained with Amido Black 10B, whereas those on #400 were not stained after protein extraction with the proposed solution. Small cracks were observed on the #400 plate. We need to clarify the plate characteristics that may have affected protein removal to standardise the testing materials for cleaning evaluation.

Visual inspection revealed that our proposed solution could efficiently extract proteins from test samples at temperatures of up to 60 °C (Fig. 2B). Contrary to our expectations, residual protein was observed on the plates after processing at 95 °C. The thermally coagulated protein may be partly dissolved in the solution on the SUS plate, but cannot be completely removed. A membrane formed after dissolution and re-attached to the SUS surface via the residual protein (Fig. 2B d). Meanwhile, the conventional solution removed proteins at 95 °C, although the protein-containing solution yielded a low recovery rate owing to the precipitate (Fig. 3). Hence, the proposed method and recovery solution have a limit for accurately quantifying residual protein unless it is completely solubilised.

For more effective protein solubilisation, it may be necessary to identify reducing agents that disintegrate higher-order protein structures without interfering with the modified micro-BCA or modified OPA methods, thus facilitating effective cleaning at > 60 °C. Furthermore, fluorescence imaging will be helpful for directly assessing stains on medical instruments. This visual inspection approach requires the development of a measurement scale.

The current study compared the quantification performance of the protein extraction solutions used for different equipment in each facility. However, they did not describe the differences in solution production and residual protein recovery between the facilities. The recovery procedure was conducted at the same place in a controlled environment for the following reasons: the distribution of humid samples could not be guaranteed during transportation, or the participants did not have the equipment necessary to recover the protein solution (such as an orbital shaker and a high-speed centrifuge). In addition, the preparation of the solutions took several days. Therefore, time was reduced by simultaneously using the prepared solutions in one place. To do so, we defined the shelf life and storage methods of the solutions, although the participants originally used their own preparation methods. These aspects should be considered in future research.

ISO 15883-5 Annex A8 provides examples of test soils and contains two types of coagulated blood: heparinised and citrated. The guideline indicates that blood should be stored at 4–8 °C and brought to room temperature before coagulation. Coagulation should then be restarted using protamine sulfate or calcium chloride (CaCl2), whereafter coagulated blood should be applied immediately. The JSMI guideline:202116 recommend the use of heparinised sheep blood in accordance with the German guideline12. However, in this study, we used citrated sheep blood as the contaminated blood, keeping it cool until spreading onto SUS plates to uniformly delay restarting coagulation on the plates. In a previous study, we confirmed that coagulated heparinised and citrated sheep blood did not differ significantly (see Supplementary Fig. S3 and S4 online). Coagulation was reinitiated according to the method provided in the guidelines for heparinised sheep blood and the proposed method for citrated sheep blood. Sheep blood samples were collected from the same individual. Citrated sheep blood was used to prepare more than 100 test samples simultaneously while ensuring quality.

ISO 15883-5 Annex B8 specifies annealed SUS316 L foil (50 mm × 50 mm, thickness = 0.127 mm) as the standard test material. However, we changed the size to enable placement of the test samples into 50-mL tubes to shake them properly in the recovery solutions. In addition, the 316 L and 304 SUS materials were different. As medical devices use various materials, SUS plates were selected as an option in this study.

In conclusion, we reported an extraction solution comprising 1% SDS, 10 mM TCEP, and 10 mM HEPES (pH 7.0), which recovered proteins from pseudo-contaminated plates treated at temperatures above 60 °C more effectively than the conventional solution (1% SDS, pH 11.0). We were unable to determine the cause of protein adhesion to substrate materials. Test soils, materials, and solutions for extracting residual proteins should be standardised to further develop effective evaluation methods for medical instrument cleaning.

Methods

Materials and chemicals

The same lots of disposable items and reagents purchased and distributed by National Institute of Health Sciences were used for all the tests. BSA standard (2 mg/mL) and micro-BCA protein assay kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Amido Black 10B staining solutions were purchased from Inui Medics Corp. (Osaka, Japan), and Clean Chemical Co., Ltd. (Osaka, Japan). SDS solution (10%) was purchased from Nippon Gene Co., Ltd. (Tokyo, Japan). Heparinised sheep blood and citrated sheep blood were purchased from Japan Bio Serum Co., Ltd. (Hiroshima, Japan). Tris, HEPES, iodoacetamide (IAM), TCEP, and CaCl2 were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Protamine sulfate was purchased from Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan). A SUS304 plate (50 × 15 mm, thickness = 1 mm; Nippon Steel Stainless Steel Corp., Tokyo, Japan) was used as the representative medical instrument material. Disposable UV cell microcuvettes (70–850 µL; Thermo Fisher Scientific) and 96-well microplates (AGC Techno Glass Co., Ltd., Shizuoka, Japan) were used in the micro-BCA assay. UV-compatible microplates (Greiner Bio-One, Kremsmünster, Austria) were used in the modified OPA method. Protein low-retention tips (200, 1,000 µL) were purchased from Biotix, Inc. (San Diego, CA, USA). Protein low-binding tubes with capacities of 1.5 and 2.0 mL (Nolato Treff AG, Degersheim, Switzerland), 15 mL (Sumitomo Bakelite Co., Ltd., Tokyo, Japan), 5 and 50 mL (Eppendorf Corp., Hamburg, Germany) were used to avoid decreased residual protein recovery due to adhesion.

Study participants, organisation, and instruments

Table 1 shows the details of the study supervisor, data auditor, participant roles, and equipment. Figure 5 shows the flowchart of the evaluation process from sample preparation to visual inspection and quantification in the test. Solutions for protein extraction and test samples were prepared at National Institute of Health Sciences and were used to evaluate the recovery procedures conducted at participating facilities. The participants conducted a visual inspection and quantification using their instruments. The participants were classified into five categories, A to E, according to the combination of tools, and divided into four groups according to the study date when they gathered and performed a series of recovery procedures at National Institute of Health Sciences.Fig. 5 Flowchart of the evaluation process from sample preparation to visual inspection and quantification in the test. Pseudo blood-contaminated samples were prepared at National Institute of Health Sciences. For extraction using two solutions, participants gathered in the institute for two days. Visual inspection was performed after the extraction process, and the protein-extracted solutions were sent to the facilities for quantification.

Preparing contaminated samples

Test samples were prepared via pseudo-contamination of the SUS304 plates with sheep blood. Subsequently, citrated sheep blood (500 µL) was transferred into 1.5-mL protein low-binding tubes, mixed with an equal volume of 0.025 mol/L CaCl2, and stored on ice to delay coagulation. Aliquots (200 µL) were evenly spread within an area of 4.5 cm × 1 cm on SUS304 plates using a low-binding tip. Surface coverage area was 4.5 cm2 (= 4.5 cm × 1 cm). Therefore, the initial protein surface coverage was 3.75 mg/cm2. The heat treatment for preparing contaminated samples was performed as described in Table 2. Table 2 List of heat-treated pseudo-blood-contaminated samples used in this study.

Heat treatment conditions	Reason	
Control	Heat treatment	Storage until use	
RT (23 ℃), 16 h	Dry	45 ℃, 1 h	Vacuum until use for 3–4 days	Sample preparation for evaluating cleanliness (guideline)	
RT (23 ℃), 3–4 days + 16 h	Wet	45 ℃, 1 h	Keep moist and use within 5 h after heat treatment	First cleaning cycle using a WD at a low temperature	
60 ℃, 30 min	First cleaning cycle using a WD at a high temperature

Maximum temperature for ultrasonic cleaning recommended by WHO

	
95 ℃, 15 min	Second cleaning cycle using a WD at maximum temperature	
RT, room temperature; WD, washer disinfector. All samples were made to order for the study dates each week. All JIS Stainless Use Steel 304 (SUS304) plates were washed twice using methanol (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), then twice using ultrapure water prepared using a Milli-Q water purification system (MilliporeSigma, Burlington, MA, USA) to remove adhesions as precleaning. To prepare dried samples, the contaminated plates were allowed to dry for 16 h at 23 ℃, placed in a Petri dish, covered, and heat-treated using a dry-heat oven (SI601; Yamato Scientific Co., Ltd., Tokyo, Japan). These were then vacuumed for 3–4 days until use to ensure the complete removal of residual water. To prepare wet samples, the contaminated plates were allowed to dry for 3–4 days and 16 h at 23 ℃, placed in a Petri dish, covered, and heat-treated using a programmable autoclave (LSX-300; TOMY Digital Biology Co., Ltd., Tokyo, Japan). The treated plates were kept in the autoclave under moist conditions until just before use to simulate treatment without a cleanser during normal cleaning procedures.

Residual protein extraction and quantification from plates

We compared the amounts of protein recovered from pseudo-blood-contaminated SUS plates using a conventional solution containing 1% SDS (pH 11.0) with those obtained using our proposed solution containing 1% SDS, 10 mM TCEP, and 10 mM HEPES (pH 7.0).

A series of recovery procedures were performed over two days at National Institute of Health Sciences, according to the study date set per facility. A test coordinator randomized the plates and developed the correspondence tables. The recovered proteins were quantified in the respective laboratories, and the findings were sent to an independent statistical analyst (Table 3). Table 3 Roles of study supervisors, data auditor, and participants as well as the instruments and equipment used for the test.

Research supervisors from National Institute of Health Sciences in Japan (initial organisation)				
	 Principal investigator	Supervising the validation test				
	 Test coordinator	Making a draft SOP, revising it with participants, coordinating schedules, and randomising test samples				
	 Statistical analyst	Collecting data from participants, sorting it based on the correspondence table made by the coordinator, and analysing it				
	 Technical assistant	Preparing test samples and solutions based on SOP when participants conduct the recovery test				
Data auditor from ISO TC198/WG 13 Japanese mirror committee				
	 Data manager	Checking data integrity and confirming the test was conducted appropriately to achieve goals				
Facilities and their instruments	
Group	Incubator for Micro-BCA method	Ultraviolet and visible spectrophotometer	
Category*	Study date	Dry oven	Water bath	Spectrophotometer	Microplate reader	
A1	IV	IN600	–	–	Power Wave XS	
A2	III	HB-100	–	–	Spark	
B1	IV	KM-450 V	–	V-530	–	
B2	I	MOV-112 F	–	UV-2500	–	
C	II	–	Thermal Robo TR-1α	–	Infinite M200	
D1	I	–	CCS-009	BioSpectrometer Basic	–	
D2	III	–	TBS241HA	U-2910	–	
E1	I	–	Thermal Robo TR-1α	V-630	–	
E2	II	–	Thermal Robo TR-2α	UV-1280	–	
Dry oven: IN600 (Yamato Scientific Co., Ltd., Tokyo, Japan), HB-100 (Taitec Corp., Koshigaya, Japan), KM-450V (AS ONE Corp., Osaka, Japan), MOV-112F(U) (SANYO Electric Co., Ltd., Osaka, Japan), Water bath: Thermal Robo TR-1α (AS ONE Corp.), CCS-009 (Clean Chemical Co., Ltd., Osaka, Japan), TBS241HA (Advantec Co., Ltd., Tokyo, Japan), Thermal Robo TR-1α (AS ONE Corp.), Spectrophotometer: V-530 (JASCO Corp., Tokyo, Japan), UV-2500 (Shimadzu Corp., Kyoto, Japan), BioSpectrometer Basic (Eppendorf AG, Hamburg, Germany), U-2910 (Hitachi High-Tech Corp., Tokyo, Japan), V-630 (JASCO Corp.), UV-1280 (Shimadzu Corp.), Microplate reader: Power Wave XS (Agilent Technologies Inc.. Santa Clara, CA, USA), Spark (Tecan Group Ltd., Mannedorf, Switzerland).

Day 1 (from pm): 10 mL of solution was added to the test sample tubes, which were shaken using an orbital shaker (Orbital Mixer Digital, Thermo Fisher Scientific) for 5 min at 23 °C and 60 rpm to roughly immerse the plate in the solution. The tube was placed on a flat table for 16 h at 23 °C, with the contaminated side of the embedded sample facing down to absorb the solution into the coagulated proteins.

Day 2 (from am): The tube was turned with the contaminated surface facing upward and placed on an orbital shaker for 30 min at 23 °C and 60 rpm to scrub the surface with the solution. After protein extraction, the sample plate was rinsed with Milli-Q water by soaking in another tube, the protein-containing solution was centrifuged at 4000 × g for 15 min at 23 °C, and 1 mL of the supernatant was collected in a 1.5-mL tube. The supernatant was then centrifuged at 14,000 × g for 15 min at 23 °C to remove insoluble substances. The resulting solutions were sent to each facility for protein quantification. The plate was removed from the tube and stained with Amido Black 10B to visualise residual protein.

The total protein in the final solution was quantified using the modified OPA method at each facility within one week of sampling. This method measures the absorbance at 340 nm after proteins react with ο-phthalaldehyde in the presence of a reducing agent12,26 and has been referenced in ISO 15883–5:2021 and JSMI guideline:2021.

Definition of recovery rate for spike-recovery test

Recovery rate Rr is expressed as follows:Rr=Cr/Ct,

where Cr is the concentration of the protein recovery solution, and Ct is the total protein solution of the spiked blood volume. Each plate had a total protein load of 100 µL of citrated blood. The citrated blood was quantified as approximately 160 mg/mL, which was determined by the diluted solution 400-fold with each recovery buffer. Each plate was recovered with 10 mL of recovery buffer. Therefore, the concentration of the protein recovery solution could be measured as the total protein load per 10 mL, i.e., 1.6 mg/mL at maximum. To measure the protein recovery solution with a spectrophotometer or a microplate reader with the correct range, each solution was diluted by four times.

Equipment performance and lower quantification limit

To examine the differences caused by instruments and operators, participants performed both the modified OPA and the modified micro-BCA methods, which can quantify proteins in the presence of thiol-containing chemicals21,22,24,26,29,31. The total protein of the citrated sheep blood was in the final solution and BSA was used as the reference standard for protein quantification.

Calibration curves were constructed using BSA solutions to validate the proposed method. A series of dilution operations were repeated six times to analyse variance. For the modified micro-BCA, the test solution was pre-incubated for 20 min at 37 °C after mixing with a quarter volume of 16% IAM aqueous solution and mixed with the same volume of reagent. For the modified OPA method, the test solution was immediately mixed with the same volume of the reagent. The absorbance values of the final mixed solutions were measured to quantify the extracted proteins, according to each protocol.

Volume reduction for absorbance measurement

Absorbance was measured using either an ultraviolet–visible (UV–VIS) spectrophotometer or a microplate reader with UV disposable cell microcuvettes (70–850 µL) and 96-well microplates. Small volumes of the protein solutions were measured using an ultra-micro cell holder for the UV-2500 spectrophotometer (Shimadzu, Kyoto, Japan) (B1) and a mask for a cuvette for the V-530 spectrophotometer (JASCO Corporation, Tokyo, Japan) (B2), which narrows the optical pathway. A BioSpectrometer (Eppendorf Corp. (Hamburg, Germany) (D1) did not require an attachment to measure small volumes. The spectrophotometers used could analyse only 100 µL of the solution, whereas the others required 500–600 µL (D2, E1, and E2).

Statistical analyses

The recovery process and quantification were conducted by nine facilities. Test samples for all facilities were prepared by one institution. One facility tested 24 samples (3 samples × 4 conditions × 2 recovery solutions). The mean of three samples was determined for each condition. The recovery rates were presented as the mean the mean ± standard deviation for nine facilities. The calibration curve equations were obtained from six replicates in each facility. The definition of measurement accuracy (trueness and precision) follows that outlined by ISO 5725-1:202332. One-way analysis of variance was used to determine significant differences in the recovery rates among the groups. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA).

Supplementary Information

Supplementary Figures.

Supplementary Table S1.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72473-1.

Acknowledgements

This study was supported by grants from the Japan Agency for Medical Research and Development for Research on Regulatory Science of Pharmaceuticals and Medical Devices (22mk0102174, 24mk0121296). The funders confirmed project progress semi-annually and had no further involvement in the publication. The authors are grateful to Mr. T. Hiramatsu from Shimadzu Corp. and officials from JASCO Corp. for their technical assistance in the use of spectrophotometers with minute protein solution volumes. We also thank Mr T. Ikenoya from the Japanese Society of Medical Instrumentation; Mr K. Matsumoto and Dr F. Hasegawa from the Japan R-SUD Association; and Dr Y. Haishima, the former director of the Division of Medical Devices at the National Institute of Health Sciences for inviting public participation in the test. We would like to thank Editage (www.editage.jp) for English language editing.

Author contributions

Miyuki Uematsu: Conceptualisation, Writing – original draft, Data curation, Formal analysis, Supervision, Validation. Yuko Miyamoto: Conceptualisation, Data curation, Formal analysis, Supervision, Validation. Ryusuke Nakaoka: Writing – original draft, Data curation, Formal analysis, Supervision, Validation. Eichii Yamamoto: Writing – original draft, Funding acquisition, Project administration. Masatake Shimizu, Tsuyoshi Kajiura, Atsushi Saito, Satoshi Fujita, Yuko Nakano, Toshiaki Shimizu, Yuki Nagahara, Hayato Kosaka, Hiroki Muramatsu, Masafumi Mori, Takamasa Suzuki, Takayoshi Nakamura, Atsushi Tanemura, Junki Hosaka, Takahide Mori, Seiichi Kato, Ayaka Itagaki, Toshiki Inoue, Tomoko Naito, and Shinji Fujii – Data acquisition, Writing – review and editing. Shinichi Matsumoto: Data curation. All authors had full access to all the study data and approved the submission for publication.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethical approval for human and animal experimentation

This study focused primarily on comparing the methodologies for protein quantification/residual protein recovery and included human handlers who solely operated the equipment and were not involved in clinical procedures or specimen collection. Given the absence of direct human involvement beyond equipment operation and clinical data or specimens, the nature and scope of the study did not necessitate human ethics clearance. As commercially purchased animal blood was used in the study, approval was not required by the Committee for Proper Experimental Animal Use and Welfare, a peer review panel established at National Institute of Health Sciences. Consequently, there were no ethical parameters, ethics approval, or informed consent issues related to human or animal experimentation.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. United States Food and Drug Administration. Guidance for Industry and for FDA Staff: Enforcement Priorities for Single-Use Devices Reprocessed by Third Parties and Hospitals. Document Issued on 14 August 2000 (2000).
2. United States Food and Drug Administration. Draft Guidance for Industry and FDA Staff: Reprocessing Medical Devices in Health Care Settings: Validation Methods and Labeling, Draft Document Issued on 2 May 2011. Latest Edition Issued on 9 June 2017 (2017).
3. Commission for Hospital Hygiene and Infection Prevention (KRINKO) & Federal Institute for Drugs and Medical Devices (BfArM). Hygiene requirements for the reprocessing of medical devices. Recommendation of the commission for hospital Hygiene and infection Prevention (KRINKO) at the Robert Koch Institute (RKI) and the Federal Institute for Drugs and Medical Devices (BfArM). Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 55, 1244–1310 (2012).
4. Ministry of Health, Labour and Welfare. Points to Consider for the Reprocessing of Single-Use Devices: Co-signed Notification of the Managers of the Evaluation and Licensing Division(0731-No. 8), the Pharmaceutical Safety Division (0731-No. 5), and the Compliance and Narcotics Division and Safety (0731-No. 1). Issued on 31 July 2017 (2017).
5. Ministry of Health, Labour and Welfare. Points to Consider for Writing an Application for Marketing Authorisation for the Reprocessing of Single-Use Devices, Co-signed Notification of the Managers of the Evaluation and Licensing Division(0816-No. 3), Ministry of Health, Labour and Welfare. Issued on 16 August 2017 (2017).
6. Ministry of Health, Labour and Welfare. A Standard for the Reprocessing of Single-Use Devices, Ministerial Notification No. 261 (2017).
7. Ministry of Health, Labour and Welfare. Ministerial Ordinance on Standards for Manufacturing Control and Quality Control for Medical Devices and In-Vitro Diagnostic Reagents, MHLW Ministerial Ordinance No.169, Issued on December 17, 2004. American National Standard Institute Inc. and Association for the Advancement of Medical Instrumentation (ANSI, 2022)/AAMI ST98:2022 Cleaning validation of health care products—Requirements for development and validation of a cleaning process for medical devices (Association for the Advancement of Medical Instrumentation, 2022).
8. ISO 15883-5:2021. Washer-Disinfectors—Part 5: Performance Requirements and Test Method Criteria for Demonstrating Cleaning Efficacy (2021).
9. American National Standard Institute Inc. & Association for the Advancement of Medical Instrumentation. ANSI/AAMI ST98. Cleaning Validation of Health Care Products—Requirements for Development and Validation of a Cleaning Process for Medical Devices (Association for the Advancement of Medical Instrumentation, 2022).
10. Alfa MJ DeGagne P Olson N Worst-case soiling levels for patient-used flexible endoscopes before and after cleaning Am. J. Infect. Control 1999 27 392 401 10.1016/S0196-6553(99)70004-0 10511485
Alfa, M. J., DeGagne, P. & Olson, N. Worst-case soiling levels for patient-used flexible endoscopes before and after cleaning. Am. J. Infect. Control 27, 392–401 (1999).10511485
11. Michels W Roth K Eibi R Assessment of cleaning efficacy based on the protein–surface relationship Central Serv. 2013 21 212 215
Michels, W., Roth, K. & Eibi, R. Assessment of cleaning efficacy based on the protein–surface relationship. Central Serv. 21, 212–215 (2013).
12. German Society for Hospital Hygiene (DGKH), German Society for Sterile Supply (DGSV), Instrument Reprocessing Working Group (AKI). Validation and Routine Monitoring of Automated Cleaning and Thermal Disinfection Processes for Medical Devices. Zent Steril Suppl, 2017 (2017).
13. Guan A Wang Y Phillips KS An extraction free modified o-phthalaldehyde assay for quantifying residual protein and microbial biofilms on surfaces Biofouling. 2018 34 925 934 10.1080/08927014.2018.1521959 30362370
Guan, A., Wang, Y. & Phillips, K. S. An extraction free modified o-phthalaldehyde assay for quantifying residual protein and microbial biofilms on surfaces. Biofouling. 34, 925–934 (2018).30362370
14. Japanese Society of Medical Instrumentation. Guideline for Evaluation and Decision Making in Cleaning Efficacy (2012).
15. Shimizu M Development of the high sensitivity measurement method for checking the cleanliness of medical instruments Jpn. J. Med. Instrum. 2005 75 28 33
Shimizu, M. et al. Development of the high sensitivity measurement method for checking the cleanliness of medical instruments. Jpn. J. Med. Instrum. 75, 28–33 (2005).
16. Japanese Society of Medical Instrumentation. Guideline for Sterility Assurance in Healthcare Setting (2021).
17. World Health Organization & Pan American Health Organization. Decontamination and Reprocessing of Medical Devices for Healthcare Facilities (2016).
18. Brinkmann F Temperature profile and residual heat of monopolar laparoscopic and endoscopic dissection instruments Surg. Endosc. 2022 36 6 4507 4517 10.1007/s00464-021-08804-4 34708296
Brinkmann, F. et al. Temperature profile and residual heat of monopolar laparoscopic and endoscopic dissection instruments. Surg. Endosc. 36(6), 4507–4517 (2022).34708296
19. Matsuura M Study of laparoscopic monopolar devices and its thermal effects Eur. J. Gynaecol. Oncol. 2020 42 1 118 121
Matsuura, M. et al. Study of laparoscopic monopolar devices and its thermal effects. Eur. J. Gynaecol. Oncol. 42(1), 118–121 (2020).
20. Hefermehl LJ Lateral temperature spread of monopolar, bipolar and ultrasonic instruments for robot-assisted laparoscopic surgery BJU Int. 2014 114 2 245 252 10.1111/bju.12498 24127773
Hefermehl, L. J. et al. Lateral temperature spread of monopolar, bipolar and ultrasonic instruments for robot-assisted laparoscopic surgery. BJU Int. 114(2), 245–252 (2014).24127773
21. Uematsu M Novel measurement method of residual proteins in cleanliness evaluation of reusable and reprocessed medical devices Int. J. Cars. 2022 17 S126 S127
Uematsu, M. et al. Novel measurement method of residual proteins in cleanliness evaluation of reusable and reprocessed medical devices. Int. J. Cars. 17, S126–S127 (2022).
22. Uematsu M Novel method to recover and quantify residual proteins for cleanliness evaluation of reusable and reprocessed medical devices Jpn. J. Med. Instrum. 2022 92 400 414
Uematsu, M. et al. Novel method to recover and quantify residual proteins for cleanliness evaluation of reusable and reprocessed medical devices. Jpn. J. Med. Instrum. 92, 400–414 (2022).
23. Temperature of the denaturing process–Notes on sample preparation for SDS-PAGE https://www.cytivalifesciences.co.jp/technologies/protein_preparation/sds_page.html (2022).
24. Getz EB Xiao M Chakrabarty T Cooke R Selvin PR A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry Anal. Biochem. 1999 273 73 80 10.1006/abio.1999.4203 10452801
Getz, E. B., Xiao, M., Chakrabarty, T., Cooke, R. & Selvin, P. R. A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273, 73–80 (1999).10452801
25. Uematsu M Clinical efficacy for a new method of recovering and quantifying residual protein from reusable medical devices Jpn. J. Artif. Organs. 2022 52 2 1 200
Uematsu, M. et al. Clinical efficacy for a new method of recovering and quantifying residual protein from reusable medical devices. Jpn. J. Artif. Organs. 52(2), 1–200 (2022).
26. Frister H Meisel H Schlimme E OPA method modified by use of N, N-dimethyl-2-mercaptoethylammonium chloride as thiol component Z. Anal. Chem. 1988 330 631 633 10.1007/BF00473782
Frister, H., Meisel, H. & Schlimme, E. OPA method modified by use of N, N-dimethyl-2-mercaptoethylammonium chloride as thiol component. Z. Anal. Chem. 330, 631–633 (1988).
27. Arai S Doi M Skin formation and bubble growth during drying process of polymer solution Eur. Phys. J. E. 2012 35 57 10.1140/epje/i2012-12057-2 22772595
Arai, S. & Doi, M. Skin formation and bubble growth during drying process of polymer solution. Eur. Phys. J. E. 35, 57 (2012).22772595
28. American Society for Testing and Materials. ASTM F3293-18. Standard Guide for Application of Test Soils for the Validation of Cleaning Methods for Reusable Medical Devices (2018).
29. Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature. 1970 227 680 685 10.1038/227680a0 5432063
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685 (1970).5432063
30. Tanino M Uematsu M Nomura Y Miyamoto Y Haishima Y Precautions for the cleanliness evaluation of reusable medical devices by alkaline extraction of residual proteins Jpn. J. Med. Instrum. 2020 90 490 500
Tanino, M., Uematsu, M., Nomura, Y., Miyamoto, Y. & Haishima, Y. Precautions for the cleanliness evaluation of reusable medical devices by alkaline extraction of residual proteins. Jpn. J. Med. Instrum. 90, 490–500 (2020).
31. Suttapitugsakul S Xiao H Smeekens J Wu R Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics Mol. Biosyst. 2017 13 2574 2582 10.1039/C7MB00393E 29019370
Suttapitugsakul, S., Xiao, H., Smeekens, J. & Wu, R. Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics. Mol. Biosyst. 13, 2574–2582 (2017).29019370
32. ISO 5725-1:2023. Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 1: General Principles and Definitions (2023).
