Characterization and Validation of the BCA Protein Quantization (Pro1Micro)
The efficacy of automated cleaning procedures must be validated and monitored on acontinuous base. Tissue and blood proteins are common compounds on contaminated instruments. After cleaning, clinical instruments must be completely free of any residue.Methods for the detection of protein are ubiquitous and often used in food or pharmaceutical industries. Recently protein residues have caught much attention since residual protein on surgical instruments surfaces is important because of the continuing risks of transmission of prions (the causative agent of transmissible spongiform encephalopathies such as variant Creutzfeldt-Jakob disease (vCJD))1,2. It is necessary to use protein detection methods to check for the efficient removal of protein from surgical instruments after processing. Protein levels are used as an indication of the amount of prion protein contamination. The Terragene® Chemdye® Pro1Micro is an easy way to monitor the cleanliness of re-usable medical devices in the context of medical and dental practices.
Guidelines for Protein Measurement
The U.K. Department of Health published the Health Technical Memorandum 01-01 in March 2016. The established guidelines state that there should be no more than 5μg of protein in-situ on the side of any instrument. Prions are hydrophobic proteins and are easier to remove if they have not dried on the surface of an instrument. The attachment of hydrophobic proteins to surfaces becomes less reversible if they are allowed to dry fully. To enable efficient prion removal the hospital staff should ensure that instruments are transported to the sterile service department (SSD) immediately after the close of the procedure, for cleaning and reprocessing as soon as practically possible.
The HTM 01-01 guidance for decontamination management of surgical instruments establishes that the environment around soiled instruments must be kept at or near saturation humidity3. This prevents full attachment of the hydrophobic proteins such that they are more efficiently removed by cleaning, making the cleaning process more effective and reducing the risks to the patients and staff handling the devices. The guidance also indicates that the upper limit of acceptable protein contamination after processing of a surgical instrument is 5 μg of Bovine Serum Albumin (BSA) equivalent per instrument side.
The Bicinchoninic Acid Method for Protein Detection
The bicinchoninic acid (BCA) assay, first described by Smith et al.4 is based on the Biuret Assay, and it depends on the conversion of Cu(II) to Cu(I) under alkaline conditions. The Cu(I) is then detected by reaction with BCA, developing a deep purple color to the solution. Since the production of Cu(I) in this assay is a function of protein concentration and incubation time, the protein content of unknown samples can be determined spectrophotometrically by comparison with known protein standards. A further advantage of the BCA assay is that it is generally more tolerant to the presence of compounds that interfere with the Lowry assay. In particular this assay is not affected by rests of detergent. Depending on the proportion between the reactants two assays are usually described:
• a standard assay (0.1-1.0mg/mL) with minimum or no incubation time and
• a microassay (0.5-20μg/mL) with 1 hour incubation time.
The standard assay is usually used in the food and pharmaceutical industries while the microassay can be adapted for clinical instruments. The microassay uses concentrated reagents and a protocol that utilizes an extended incubation time at an elevated temperature (55-60°C). The result is a sensitive colorimetric protein assay, able to detect proteins in the μg/mL range as required in recent standard revisions.
We had optimized the microassay protocol to give reliable results in the 0.50 y 25.00 μg range (Figures 1 and Figure 2). The assay gives both qualitative and quantitative results. It is compatible with detergents, it is practical since this assay has the advantage that it can be carried out as a one-step process compared to other two steps assays, the reagents are stable indefinitely at room temperature and it exhibits less degree of varying response toward different proteins than other protein assay methods.
我们优化了微量测试法，在0.50 y 25.00μg范围内，该测试法可提供可靠的结果（图1和图2）。该测试法可给出定性和定量结果。它不会受到清洗剂干扰，可与清洗剂兼容，具有可操作性，相比其他需2步反应完成的测试法，该测试法可1步直接反应，与其他蛋白测试法相比，其在室温下稳定性佳，且对不同蛋白质的反应变化差异较小。
Pro1Micro System: The Test Pen
The reaction is time and temperature dependant, i.e. the color develops with time and the speed of color development is slower or faster depending on the temperature. We recommend that results be read within 15 minutes of activation, and then discarded. The reaction takes place in minutes depending on the level of contamination. The test chemistry will turn to purple over a long period of time (4-5 hours) even with the absence of protein. Users may interpret the level of contamination on a surface based on the speed and intensity of the color change. The speed of the reaction and the sensitivity reached is enhanced by incubation. Since the reaction is temperature-dependent, it is important to allow the devices to equilibrate to ambient room temperature (15-25°C) if they have been stored at refrigerated temperatures.
Pro1Micro System: Swab Sampling Procedure Validation
The conditions in which surgical instruments are handled during and after surgery may ,affect the level of tissue protein, prion attachment and the efficacy of subsequent decontamination regimes and sampling methods. In order to evaluate this we investigated the swabbing recovery of BSA and brain homogenates from surgical stainless steel with respect to time and “wet” and “dry” storage conditions.
As it is pointed out in the HTM 01-01 guidance the protein measurement is per side of instrument rather than per unit area of an instrument. Nevertheless “side per instrument” varies greatly from instrument to instrument so we decided to base our tests following the ISO 15883-1:2009 standard that requires that about 10 cm² of an instrument is swabbed 6 . Surgical stainless steel tokens were used since it has been shown that tokens represent surgical instrument surfaces more closely. Tokens of 10 cm² were used for the inoculation with drops of the BSA and brain homogenate samples. Samples were left to dry at room temperature, we also did some experiments drying the samples at 60°C. We considered the initial time when all the water from the protein solutions was evaporated. Sample collection was done thoroughly over the token area with pre-moistened swabs with firm swab contact and movement on the surface for maximum sample recovery. Afterwards the swabs were immersed in Pro1Micro solution and the absorbance at 562 nm was measured (Figure 2). A titration of the Pro1Micro solution was done with BSA as reference for total protein concentration.
正如在HTM 01-01标准中指出的那样，蛋白残留监测是针对器械的每一侧面，而不是器械的单位面积。然而，“每把器械的侧面”因而异，因此我们决定根据ISO 15883-1：2009标准进行测试，该标准要求擦拭约10 cm²的器械6。使用外科不锈钢模拟物，因为已经表明不锈钢模拟物更紧密地代表外科器械表面。10cm²的标记用于接种BSA滴和脑匀浆样品。将样品在室温下干燥，我们还进行了一些实验，在60℃下干燥样品。我们考虑了蛋白质溶液中所有水分蒸发的初始时间。样品收集在不锈钢模拟物区域上进行，使用预先润湿的拭子，使拭子牢固接触并在表面上移动，以最大限度地回收样品。然后将拭子浸入Pro1Micro溶液中并测量562nm处的吸光度（图2）。用BSA作为总蛋白质浓度的参考进行Pro1Micro溶液的滴定。
Long drying times have adverse effect on different cleaning methods so we tested how the recovery of protein and brain homogenate to stainless steel varied with time. At initial time we obtained nearly 90% of recovery from both samples (Figure 3 and Figure 4). As the time of drying increases the protein recovery by swabbing decreases. This goes along with the HTM 01-01 guidance that points out to keep moistened the surgical instruments preventing them from drying.
In general the recovery was less than 50 % and in some samples it was less than 30% (data not