Abstract
Background
The aim of this study was to produce a dental test soil, with 2 clinically relevant soil components, to be quantified for cleaning process validation. Another goal was to soil diamond instruments with the 2 soil components and validate the efficacy of cleaning instructions, developed and detailed in this study, using both qualitative and quantitative techniques.
Methods
To simulate worst-case clinical use conditions, the authors used each soiled instrument to prepare a 9-millimeter-deep access cavity on a noncarious extracted molar. Afterward, the authors applied a mixture of pooled human saliva and blood test soil to each instrument and air-dried it for 30 minutes. The authors cleaned each instrument using documented multistep cleaning instructions, which were then validated via both qualitative and quantitative assessment of protein and enamel-dentin residues using spectrophotometric analysis and microscopy images.
Results
After thorough cleaning, neither protein nor enamel-dentin residues were found at quantifiable levels (spectrophotometric analysis) on the soiled and cleaned diamond instruments, which was qualitatively verified (microscopy images).
Conclusions
The results of this study show the successful development of a dental test soil with 2 clinically relevant soil components. Furthermore, using these soil components as test markers, the authors found that when the established cleaning instructions are properly followed, a soiled diamond instrument can be cleaned in a quantifiable manner.
Practical Implications
Thorough cleaning is a critical step in reprocessing multiuse dental instruments. In accordance with US Food and Drug Administration guidance, the described process for quantification of soil components, using 2 clinically relevant soil markers, on cleaned diamond instruments can be helpful to dental instrument manufacturers in the development and validation of cleaning instructions for their reusable instruments.
Key Words
Abbreviation Key:
ADA (American Dental Association), ARS (Alizarin red S), B (Negative sample control), BSA (Bovine serum albumin), FDA (US Food and Drug Administration), JWG (Joint Working Group), NC (Negative instrument control), OPA (O-phthaldialdehyde), S (Positive instrument control), SC (Test instrument), SEM (Scanning electron microscopy), TS (Positive sample control)
What are reusable medical devices?.
and the Centers for Disease Control and Prevention.
Single-use (disposable) devices.
In contrast, reusable devices can be reprocessed and reused on multiple occasions, according to a guidance document issued by the FDA
What are reusable medical devices?.
: “These devices are designed and labeled for multiple uses and are reprocessed by thorough cleaning followed by high-level disinfection or sterilization between patients. They are made of materials that can withstand repeated reprocessing, including manual brushing and the use of chemicals.”
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
One of the criteria is that reprocessing instructions should advise users to thoroughly clean the device.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
The FDA classifies several dental instruments, including diamond instruments, as critical reprocessed single-use devices for which manufacturers must submit 510(k)s, which include “validation data regarding cleaning, sterilization, and functional performance.”
Medical devices; reprocessed single-use devices; termination of exemptions from premarket notification; requirement for submission of validation data.
However, dental instrument manufacturers seldom provide detailed cleaning instructions, leading to a lack of consistency
Cleaning and sterilizing SS White Burs, Inc. rotary dental instruments.
,
Brassler USA diamond burs and discs instructions for use.
in cleaning protocols implemented by dental practitioners and an increased risk of introducing cross contamination in the dental setting.
According to the Centers for Disease Control and Prevention, “If a device does not have reprocessing instructions, it should be considered single-use and disposed of after one use, in accordance with local waste management system regulations.”
Single-use (disposable) devices.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
Thus, in the process of proper reprocessing, it is important to recognize that cleaning and sterilization are separate steps in the process, and the terms used in reprocessing have specific, technical meanings. For instance, cleaning is defined as the removal of potential contaminants from an item to the extent necessary for further processing or for intended use.
ASTM F3127-16: standard guide for validating cleaning processes used during the manufacture of medical devices.
Also, disinfection is the process to reduce the number of viable microorganisms to a level previously specified as being appropriate for a defined purpose,
Processing of health care products: information to be provided by the medical device manufacturer for the processing of medical devices, part 1—critical and semi-critical medical devices.
with high-level disinfection being an appropriate step in the proper reprocessing of semicritical instruments when they cannot be sterilized. Finally, sterilization describes a process that destroys or eliminates all forms of microbial life, and it is carried out in health care facilities via physical or chemical methods.
Glossary. Guideline for disinfection and sterilization in healthcare facilities.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
,
ASTM F3127-16: standard guide for validating cleaning processes used during the manufacture of medical devices.
It is unknown if the remaining microbes and proteinaceous material on an improperly cleaned instrument can cause inflammation or infection in the body; however, at the very least, organic material that is not removed completely from an instrument may reduce the efficiency of that instrument greatly.
- Emir F.
- Ayyildiz S.
- Sahin C.
Hence, the cleaning process is a vital step in safe and effective instrument reprocessing. Even with the recognized importance of thorough cleaning as a step in instrument reprocessing, there was no dental standard addressing this issue. Therefore, the American Dental Association (ADA) Standards Committee on Dental Products created a Joint Working Group (JWG) on Cleanliness in Reprocessing.
ADA Standards Committee on Dental Products Working Group structure.
It was a goal of this JWG to develop a technical report to give guidance on method development and validation of cleaning processes for dental instruments.
Involvement in this JWG prompted us to use a draft of the technical report to develop a cleaning process for dental rotary diamond instruments and attempt to validate the process.
as well as for the following reasons. In general, dental rotary instruments, including diamond instruments,
Dental rotary instruments: diamond instruments, part 1—dimensions, requirements, marking and packaging.
finishing burs,
Dentistry: rotary bur instruments, part 2—finishing burs.
and steel and carbide burs,
Dental rotary instruments: burs, Part 1—steel and carbide burs.
are among the most commonly used dental instruments in a daily dental practice. According to Spaulding’s classification,
Spaulding E. The role of chemical disinfection in the prevention of nosocomial infections. In: Proceedings of the International Conference on Nosocomial Infections, 1970. American Hospital Association; 1971;247-254.
dental rotary instruments are classified as critical devices. They come in contact with a patient’s blood, saliva, enamel, dentin, and pulp, as well as the microorganisms associated with the oral cavity.
- Gonzaga C.C.
- Falcão Spina D.R.
- de Paiva Bertoli F.M.
- Feres R.L.
- Franco Fernandes A.B.
- da Cunha L.F.
The small size and complex architecture of dental rotary instruments make quantification of their cleanliness difficult. In particular, it has been shown in a previous study on dental rotary instruments that it is more difficult to clean diamond instruments compared with rotary bur instruments.
Iole Caola, M. Coser, F. Leonardi, P. Caciagli. Cleaning efficiency of pre-sterilization protocols for reusable dental burs. Paper presented at: 10th World Congress of Sterilization Conference; October 7-10, 2009; Crete, Greece.
Furthermore, according to a 2019 ADA Clinical Evaluators Panel report,
Reprocessing of rotary cutting instruments.
nearly 71% of respondents reported that they use a dental rotary instrument “until worn out” regardless of labeling. However, a June 2019 newsletter from the chief dental officer of the US Public Health Service noted that the FDA has not cleared for market any diamond instruments with validated instructions for reprocessing.
Ricks TL. Dental diamon burs: single use. US Public Health Service Chief Dental Officer Newsletter. June 7, 2019; 35.
The newsletter went on to state that the FDA recommends that all diamond instruments (and other similar critical and semicritical reprocessed single-use devices) be considered “single-use regardless of the labeling by the manufacturer” and that “evidence-based cleaning validation information” is necessary if a company wants to market their product as multiuse.
- Chung E.M.
- Sung E.C.
- Wu B.
- Caputo A.A.
,
- Van Eldik D.A.
- Zilm P.S.
- Rogers A.H.
- Marin P.D.
measurement of residual protein,
- Vassey M.
- Budge C.
- Poolman T.
- et al.
staining,
- Parashos P.
- Linsuwanont P.
- Messer H.H.
and bacterial growth estimation in colony-forming units per milliliter.
- Miller C.H.
- Tan C.M.
- Beiswanger M.A.
- Gaines D.J.
- Setcos J.C.
- Palenik C.J.
However, bacterial growth estimation is considered a measure of sterilization efficacy, not cleaning efficacy. Moreover, FDA
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
guidance states that the efficacy of cleaning should be validated via visual examination at x20 magnification in addition to quantitative assessment of at least 2 residual soils. Hence, our study was focused on developing validated cleaning instructions for multiuse dental rotary diamond instruments, using both qualitative and quantitative assessments.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
It is also essential that the soil include 2 clinically relevant soil components that can be quantified for cleaning-process validation.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
The 2 clinically relevant soil components we chose for our study were protein and enamel-dentin. However, for the critical step of soiling a diamond instrument in a clinically relevant manner, it was necessary for the production of the dental test soil and its application to an instrument to be 2 distinct, but shared, steps. Therefore, in our study we documented the production of a dental test soil, with 2 clinically relevant soil components and assessed the efficacy of a cleaning method at removing the soil components from soiled diamond instruments. The assessment of cleanliness was done both qualitatively, through microscopy and phloxine B staining, and quantitatively by means of measuring the presence of enamel-dentin debris and residual protein using well-defined spectrophotometric techniques.
Methods
Institutional review board approval (ADA 18.002) was obtained for collecting both natural human teeth, extracted for orthodontic or restorative reasons, from the College of Dentistry at the University of Illinois at Chicago and pooled human saliva from healthy volunteers at the ADA. There was no collection of identifiable information or protected health information. After obtaining informed consent from the volunteers, we asked volunteers to spit approximately 5 L of saliva (unstimulated) into a 50-mL tube. We then pooled all of the saliva samples together and centrifuged them. We stored extracted human teeth in a sealed container with 1 to 10 sodium hypochlorite for at least 5 days. After removing them from storage, we cleaned them with a wire brush and then heat sterilized them before using. We used a multiuse dental rotary diamond instrument (coarse, green, round end 125, 1.8-mm diameter and 1.7-mm length; Premier Dental) to validate the cleaning instructions.
The buffers and chemicals used in our study were concentrated bovine serum albumin (BSA) ampules (23209; Thermo Fisher); o-phthaldialdehyde (OPA) reagent, incomplete solution (P7914; Sigma-Aldrich); 2 β-mercaptoethanol (M6250; Sigma-Aldrich); calf serum (SBS150; Hemostat Laboratories); sheep blood in citrate (DSB150; Hemostat Laboratories); 0.09% sodium chloride (746398; Sigma-Aldrich); 1 mM calcium chloride (C8106; Sigma-Aldrich); alizarin red S (ARS) dye (A5533; Sigma-Aldrich); phloxine B dye (P4030; Sigma-Aldrich).
The equipment used in our study were Sorvall Legend Micro 17R microcentrifuge (75002441; ThermoScientific); Biotek Synergy2 microplate reader (360 nm, 40 bandwidth excitation filter, and a 460 nm, 40 nm bandwidth emission filter) for absorbance reading (300-600 nm) of OPA and ARS assays along with 96-well flat-bottom polystyrene black fluorescence microplates (M33089; ThermoFisher); Quantrex 140 Ultrasonic Cleaner with timer, heat, and drain (L&R Ultrasonics); magnetic block (226-9538; Patterson); and bur caddy (233-3953; Patterson).
The cleaning solutions used in our study were Enzol enzymatic detergent (2552; Advanced Sterilization Products) and Patterson super-concentrated ultrasonic cleaning solution (111-6573; Patterson).
Validation of diamond instrument cleaning
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
:
- ▪
Negative instrument control (NC): a new diamond instrument just out of the package and cleaned by means of the defined method (that is, unsoiled and cleaned).
- ▪
Positive instrument control (S): a new diamond instrument just out of the package that has test soil applied to it (that is, soiled and not cleaned).
- ▪
Negative sample control (B): distilled water without soil or contact with a diamond instrument, as a test of the extraction solution (that is, test blank).
- ▪Positive sample control (TS): known amount of test soil (0.5 mg of enamel-dentin powder added to a 5-μL mixture containing both pooled human saliva and American Society of Testing and Materials [ASTM] blood test soil27ASTM International
ASTM F3208-20: Standard guide for selecting test soils for validation of cleaning methods for reusable medical devices.) (that is, quantification of test soil with no instrument).
- ▪
Test instrument (SC): diamond instrument soiled with test soil components, allowed to dry for a defined length of time, and then cleaned via the defined method (that is, soiled and cleaned).
ASTM F2847-17: Standard practice for reporting and assessment of residues on single-use implants and single-use sterile instruments.
,
ASTM F3321-19: Standard guide for methods of extraction of test soils for the validation of cleaning methods for reusable medical devices.
The residual level of the marker equals the SC group minus the NC group, and the percentage removal of the marker equals the following: [((S – NC) – (SC – NC)) x 100]/(S – NC). As detailed below, the dental test soil in our study contains 2 markers, protein and enamel-dentin, that can be quantified for validation testing. Therefore, we made calculations with respect to each marker. We performed descriptive statistics using Excel (Microsoft Office 2013).
ASTM F2847-17: Standard practice for reporting and assessment of residues on single-use implants and single-use sterile instruments.
,
ASTM F3321-19: Standard guide for methods of extraction of test soils for the validation of cleaning methods for reusable medical devices.
The amount of residue that sticks to the diamond instrument can be estimated by means of comparison between the NC and S groups, and the effectiveness of the extraction technique can be estimated by means of comparison between the B and TS groups. Again, because the dental test soil contains 2 markers, which can be quantified, the effectiveness of the extraction method can be evaluated for both markers.
We used 27 multiuse dental rotary diamond instruments in our study, with 9 diamond instruments in each of the following instrument groups: NC, S, and SC. On 3 different days, we performed testing in triplicate for each of the instrument groups (3 instruments per group per day), as well as for both the TS and B groups.
Development of dental test soil with 2 clinically relevant soil components
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
it is recommended that the test soil contain at least 2 clinically relevant components to be quantified. Hence, we developed a dental test soil comprising blood (as defined by ASTM F3208-20),
ASTM F3208-20: Standard guide for selecting test soils for validation of cleaning methods for reusable medical devices.
pooled human saliva, and enamel-dentin powder to represent common contaminants on dental rotary diamond instruments. To generate mixtures of enamel-dentin powder, we first collected a tooth slurry on a 20-μm sieve mesh while cutting access cavity preparations on human teeth with diamond instruments (the diamond instruments used for generation of the slurry were not part of the S and SC groups). We dried the slurry overnight and then crushed and ground it in a mortar and pestle to a uniform texture. We then used the resulting mixtures of enamel-dentin powder (Figure 1) to develop standard curves for the ARS assay, as detailed in the appendix, available online at the end of this article. We ground multiple natural human teeth, extracted for orthodontic or restorative reasons, from the University of Illinois Chicago College of Dentistry to obtain these standardized mixtures, resulting in powder mixtures comprising a wide distribution of enamel-dentin content.
ASTM F3208-20: Standard guide for selecting test soils for validation of cleaning methods for reusable medical devices.
for the TS group was not used to soil the diamond instruments in the S and SC groups. Applying enamel-dentin powder to diamond instruments would not be consistent with FDA recommendations
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
of soiling using worst-case simulated use conditions as detailed in the next section.
Soiling the diamond instruments using the worst-case scenario
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
To simulate the clinical use of a diamond instrument, we prepared a 9-mm deep access cavity on a noncarious extracted molar. During the entire soiling process, we used water as a coolant and kept the settings on the dental handpiece consistent. After we used a diamond instrument to prepare the access cavity, we placed it upright in a magnetic block and applied a 5 μL mixture containing both pooled human saliva and ASTM blood test
ASTM F3208-20: Standard guide for selecting test soils for validation of cleaning methods for reusable medical devices.
soil to it via a micropipette. We then left the soiled diamond instrument to dry for 30 minutes (Figure 2). We subjected all of the soiled diamond instruments to the same soiling process, in a consistent manner, and divided them into 2 groups; we subjected the SC group to the cleaning method after soiling and left the S group soiled.
Cleaning protocol
- 1.
presoak in the enzymatic detergent (5 minutes)
- 2.
rinse in distilled water (2 minutes)
- 3.
sonicate in the ultrasonic cleaner filled with ultrasonic cleaning solution (30 minutes)
- 4.
rinse in distilled water (2 minutes)
- 5.
air-dry in a clean container.
Extraction of residue
From groups NC, S, and SC, we placed each diamond instrument in a 2-mL microcentrifuge tube with its working part immersed in 200 μL of distilled water and sonicated it for 30 minutes (pilot testing was performed in our laboratory on 72 diamond instruments to establish the 30-minute extraction time and optimize the quantification techniques).
After sonication, we removed the diamond instrument from its tube and used 30 μL of the extraction solution for protein estimation using the OPA assay described below. We subjected the remaining solution to centrifugation at 14,000 revolutions per minute for 5 minutes. If enamel-dentin debris was present in the solution, this caused the debris to form a pellet that we used to quantify enamel-dentin residues via the ARS assay. We also assessed each instrument qualitatively through both SEM and optical microscopy, as described in the following sections.
SEM assessment of diamond instruments
- Wadhwani C.
- Schonnenbaum T.R.
- Audia F.
- Chung K.H.
as detailed in the next section. We then acquired SEM images at x50 magnification, 10 kV. We used the SEM images to qualitatively assess the extent of soil coverage on the 2 instrument control groups (NC, S) and the test instrument group (SC), as shown in Figure 3. We acquired the SEM and optical microscopy images before the performance of the extraction of residue procedure.
Optical microscopy assessment of diamond instruments using phloxine B staining
- Wadhwani C.
- Schonnenbaum T.R.
- Audia F.
- Chung K.H.
We prepared the staining solution by dissolving the dye in distilled water at a concentration of 400 µg/mL, as described by Wadhwani and colleagues.
- Wadhwani C.
- Schonnenbaum T.R.
- Audia F.
- Chung K.H.
The phloxine B staining solution has been used in dental implant abutment studies to assess the ability of different cleaning protocols to effectively clean these devices.
- Stacchi C.
- Berton F.
- Porrelli D.
- Lombardi T.
,
- Chew M.
- Tompkins G.
- Tawse-Smith A.
- Waddell J.N.
- Ma S.
In our study, we stained the diamond instruments in both the instrument control groups (NC, S) and the test instrument group (SC). We accomplished this by means of placing each diamond instrument in a 2 mL microcentrifuge tube with 800 μL of staining solution. We then sonicated the tubes for 10 minutes to ensure complete interaction of the dye. We then washed the diamond instruments in distilled water to remove any excess dye, followed by air-drying and observation under an optical microscope at ×20 magnification (Figure 4), which is in accordance with FDA
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
guidance on visual inspection to assess cleaning efficacy.
Quantification of residual protein on diamond instruments via OPA assay
- Miller C.H.
- Tan C.M.
- Beiswanger M.A.
- Gaines D.J.
- Setcos J.C.
- Palenik C.J.
,
- Smith A.
- Letters S.
- Lange A.
- Perrett D.
- McHugh S.
- Bagg J.
,
- Smith G.W.G.
- Goldie F.
- Long S.
- Lappin D.F.
- Ramage G.
- Smith A.J.
and when compared with other colorimetric and fluorescent assays, it offers greater sensitivity.
,
- Zhu D.
- Saul A.
- Huang S.
- Martin L.B.
- Miller L.H.
- Rausch K.M.
The OPA assay had root mean square error values ranging from 10 through 70 μg/mL and a calculated limit of detection (LoD)
of 11 μg/mL. We performed the OPA assay as described in detail in the Biotek technical article.
For the OPA assay, we generated standard curves through measurement of known concentrations of BSA in distilled water, as shown in Figure 5. The BSA standards ranged from 0 through 2,000 μg/mL.
ARS assay
- Lucas A.D.
- Nagaraja S.
- Gordon E.A.
- Hitchins V.M.
The dye binds with calcium and releases phosphate, causing a shift in the pH, which can be measured spectrophotometrically. As a consequence of its calcium binding ability, ARS even has been used to determine both osteogenic differentiation of mesenchymal cells
- Castrén E.
- Sillat T.
- Oja S.
- et al.
and mineralization potential of scaffolds.
- Madurantakam P.A.
- Rodriguez I.A.
- Cost C.P.
- et al.
We optimized the ARS assay specifically for the enamel-dentin powder mixture by means of using a concentration of 0.01% dye in distilled water. On each day of experiments, we tested multiple samples of enamel-dentin powder and generated a standard curve, which ranged from 0 through 2 mg (Figure 6). The ARS standard curves had root mean square error values ranging from 0.09 through 0.15 mg, and the assay had an LoD of 0.08 mg. A detailed description of the assay is provided in the appendix, available online at the end of this article.
Results
Visual examination of the diamond instruments
The SEM and optical microscopy images of the cleaned diamond instruments did not reveal any visual evidence of either enamel-dentin debris or protein residue via phloxine B stain retention.
Residual protein quantification
That is, within the LoD of protein via the OPA assay, the residual level of protein marker was 0, when taking the difference between the SC and NC groups. Likewise, because the SC and NC groups had no detectable levels of protein residue, the percentage removal of the marker was 100%.
Enamel-dentin debris quantification
As for the protein marker, this means that within the LoD of enamel-dentin via the ARS assay, the residual level of the enamel-dentin marker was 0, when taking the difference between the SC and NC groups. In addition, the SC and NC groups had no detectable levels of enamel-dentin residue, so the percentage removal of the marker was 100%.
ASTM F3208-20: Standard guide for selecting test soils for validation of cleaning methods for reusable medical devices.
and, as we previously noted, this enamel-dentin mixture was used to generate the standardized curves for the ARS assay. Therefore, unlike for the protein marker, the average amount of enamel-dentin residue that stuck to the diamond instruments compared with the mean enamel-dentin value for the TS did not give an indication of the extent of soiling. Instead, qualitative analysis, through viewing of SEM and optical images of the diamond instruments (as shown in Figures 3 and 4), combined with the mean enamel-dentin value of the S group point to extensive coverage of the diamond instruments with enamel-dentin. No detectable level of enamel-dentin was measured in the B group.
Discussion
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
they can be deemed effectively free of soil components at a quantifiable level and safe to undergo subsequent sterilization or high-level disinfection processes. There is no set acceptance criteria for what is clean enough in the validation of cleaning processes for medical devices; therefore, for dental instrument manufacturers, it is up to each manufacturer to establish and justify end points for their cleaning processes.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
To put the term clean in perspective, in a sample list of available cleaning methods and their effectiveness, the FDA references the Association for the Advancement of Medical Instrumentation technical information report 30,
AAMI TIR30:2011 (R2016). A compendium of processes, materials, test methods, and acceptance criteria for cleaning reusable medical devices.
which sets an acceptable level of residual protein on a flexible endoscope instrument after cleaning at less than 6.4 μg/cm2. In our study, the residual protein level extracted from a diamond instrument was undetectable at an LoD below 11 μg/mL. Like residual protein, there is no acceptance criteria for the amount of residual enamel-dentin after cleaning of a diamond instrument. After thorough cleaning using the method used in our study, the soiled and cleaned diamond instruments exhibited no measurable level of residual enamel-dentin, as indicated via the ARS assay with an LoD of 80 μg. The LoD for the enamel-dentin debris was relatively high compared with the assay for residual protein. However, the absence of quantifiable levels of protein and enamel-dentin on the soiled and cleaned diamond instruments was verified qualitatively through examination of SEM and optical images, which also showed no identifiable evidence of either enamel-dentin debris or protein residue. Our research is focused on quantification of enamel-dentin debris through Raman spectroscopy to improve the limit of detection of this clinically relevant marker.
Furthermore, it has been suggested that the interval between the use of an instrument and its reprocessing should be kept as short as possible to reduce the risk of developing corrosion.
Instrument reprocessing: reprocessing of instruments to retain value.
Hence, in our study, we tested a drying time of 30 minutes after soiling and increased the sonication time to 30 minutes, as a result of pilot testing performed in our laboratory. Ultimately, we found all of the soiled and cleaned diamond instruments to be quantifiably clean, as defined by the absence of measurable levels of 2 markers (protein residue and enamel-dentin debris), with no visual evidence of corrosion. The soiling process in our study followed the principle of validation of the cleaning process using worst-case testing, as suggested in FDA guidance for medical devices.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
Therefore, the 9-mm deep access cavity prepared on extracted molars combined with the mixture of pooled human saliva and ASTM blood test soil
ASTM F3208-20: Standard guide for selecting test soils for validation of cleaning methods for reusable medical devices.
was meant to represent a worst-case clinical use of a dental rotary diamond instrument, creating the greatest challenge to the cleaning process. Also, because manual cleaning is difficult to control and achieve consistently and increases the risk of experiencing sharps injuries and disease transmission for the staff members performing it, we only focused on ultrasonic cleaning in our study.
- Cafruny W.A.
- Brunick A.
- Nelson D.M.
- Nelson R.F.
Theoretically, the process described in our study for quantification of soil components, using 2 clinically relevant soil markers, on diamond instruments can be used in the development and validation of cleaning instructions for other types of dental instruments (of higher commercial value) with larger sample sizes and different cleaning parameters, such as alternative ultrasonic cleaning cycles. Furthermore, although the process described was performed on instruments soiled using the principle of validation of the cleaning process using worst-case testing for standardization purposes, it is feasible to follow this process on instruments that have been used clinically.
Conclusions
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
Furthermore, the soiling process used in our study was consistent with worst-case simulated clinical use conditions suggested in FDA guidance for creating the greatest challenge to the cleaning process.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
Using levels of both protein residue and enamel-dentin debris as test markers, we found that when the cleaning instructions established in our study are properly followed, a soiled diamond instrument can be cleaned in a quantifiable manner, as validated through the use of ARS and OPA assays and verified through visual inspection of SEM and optical images. Because there is no universal, standard method
for cleaning dental instruments, we suggest that the process described in our study for quantification of soil components, using 2 clinically relevant soil markers, on diamond instruments, which were soiled using a worst-case scenario and then cleaned, can be helpful to dental instrument manufacturers in the development and validation of cleaning instructions for reusable instruments, in accordance with FDA guidance.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
Appendix. Alizarin red S dye assay.
- 1.
1. The mixtures of enamel-dentin powder were suspended in 200 microliters of distilled water in their individual tubes and sonicated for 30 minutes. Each tube was labeled appropriately for identification.
- 2.
2. The tubes were then centrifuged at 14,000 revolutions per minute for 5 minutes, creating enamel-dentin pellets out of the powder mixture in each tube.
- 3.
3. The supernatant was removed from each tube and discarded, leaving an enamel-dentin pellet at the bottom.
- 4.
4. 1 milliliter of 0.01% ARS dye was added to each tube, which was then mixed with a vortex mixer for 10 seconds to dislodge the pellet at the bottom. There was an immediate color change of the solution from orange to pink when the dye was added to the enamel-dentin pellets.
- 5.
5. Each tube, containing the ARS dye, was then placed on a rotating shaker for 30 minutes. The vortexing and shaking was to ensure complete interaction of the dye with the enamel-dentin pellet in each tube.
- 6.
6. The centrifugation detailed in step 2 was then repeated for each tube. When this step was performed, the pellets appeared dark red in color.
- 7.
7. After centrifugation, the supernatant, which contained unbound dye, was extracted and discarded. The pellet in each tube was then washed with 1 mL of distilled water, and each tube was mixed with a vortex mixer for 10 seconds to dislodge its pellet.
- 8.
8. The centrifugation detailed in step 2 was then repeated a third time for each tube with the supernatant extracted and discarded afterwards. When this step was performed, the remaining pellets in the tubes appeared dark red in color.
- 9.
9. The pellets in each tube were then destained via adding 1 mL of 10% acetic acid, and each tube was then mixed with a vortex mixer for 10 seconds to dislodge its pellet.
- 10.
10. The destaining of the pellets in each tube then continued, with each tube being placed on a rotating shaker overnight (18 hours).
- 11.
11. While destaining was going on, a new set of tubes was labeled (corresponding to the labeling on the tubes noted in step 1) and filled with 600 μL of 1 gram equivalent per liter sodium hydroxide.
- 12.
12. The centrifugation detailed in step 2 was then repeated a fourth time for each tube. The enamel-dentin pellet at the bottom of each tube appeared white, and the supernatant, which contained the bound dye, appeared yellow in color.
- 13.
13. After centrifugation, 400 μL of supernatant was withdrawn from each tube and added to the corresponding tubes from Step 11 and vortexed.
- 14.
14. After vortexing, for each tube described in the previous step, 200 μL was pipetted into a well of a 96 clear polystyrene microplate. This procedure was repeated 2 more times (for a total of 3 times) for each tube.
- 15.
15. The absorbance of the solutions in the wells of the microplate were then read with the microplate reader, using a wavelength setting of 495 nanometers.
- 16.
16. From the absorbance readings, a standardized curve was plotted of mass of enamel-dentin powder (x axis) versus absorbance (y axis).
References
What are reusable medical devices?.
Single-use (disposable) devices.
Reprocessing medical devices in health care settings: validation methods and labeling guidance for industry and Food and Drug Administration staff.
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Biography
Dr. Gopal is a senior manager, microbiology and chemistry, American Dental Association Science and Research Institute, 211 E Chicago Ave, Chicago, IL 60611
Ms. Claussen was a research assistant, Microbiology, American Dental Association Science and Research Institute, Chicago, IL, when the work described in this article was conducted. She is now a visiting research specialist, Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL.
Ms. Azzolin is a manager, research and evaluations, American Dental Association Science and Research Institute, Chicago, IL.
Dr. Megremis is the director, dental materials and devices research, American Dental Association Science and Research Institute, Chicago, IL.
Article Info
Publication History
Published online: January 06, 2022
Publication stage
In Press Corrected Proof
Footnotes
Disclosure. None of the authors reported any disclosures.
This research was supported by American Dental Association Science and Research Institute research funding.
The authors acknowledge Dr. Parthasarathy Madurantakam for his useful discussions on the applications of the alizarin red S assay.
Identification
DOI: https://doi.org/10.1016/j.adaj.2021.08.003
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© 2021 American Dental Association.
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