Understanding the difference between assessing and determining a D-value is important when testing biological indicators.
There is a misconception that U.S. Pharmacopeia (USP) requires the end-users of biological indicators (BIs), except under specific conditions, to perform a D-value determination on incoming lots of BIs prior to acceptance and use. Assessing the D-value is much easier and much less costly than determining it. This article clarifies the difference between assessing and determining the D-value, and discusses what USP requires to perform these evaluations. For either practice, it is essential to be aware of the associated pitfalls.
Certified BIER vessels must document the temperature, time, and pressure during cycle operation.
BIs and the D-Value
Each year, numerous end-users send incoming BIs out to third-party labs for assessment or verification of the D-value or a population assay of the BIs prior to acceptance for use. A D-value is the amount of time (or dose) required to reduce the population of a BI by 90% or one log of the population under specific exposure conditions. For example, when using a steam autoclave, a BI with a D-value of 1.7 minutes would mean that when exposing the BI to a temperature of 121°C for 1.7 minutes, the population of the BI would be reduced by one log or 90%. This is an important BI characteristic for the end-user to be aware of during cycle validation or cycle development work.
To test a sterilization cycle’s effectiveness, BIs may be routinely used to monitor the cycle by being placed into the autoclave chamber along with the items to be sterilized. After the cycle’s completion, the BIs are removed and processed. With a successful delivery of a sterilization cycle, BIs placed into the chamber should also be killed or sterilized. This is a very important way to check cycle lethality delivered. If the BIs show growth after a cycle’s completion, this is an indication that a problem existed with the cycle and that all pertinent cycle parameters were not met. It also means that the sterilized or processed load needs to be held and an investigation conducted before the release of any goods from that particular cycle. As a result, end-users must have accurate D-value information for the BIs that are being used to prevent false positives.
If one is using a certified log 6 BI that has a D-value of 2 minutes at 121°C, then it should die in a sterilization cycle as soon as the temperature has reached 121°C for an exposure time of two minutes for each log of population or approximately 12+ minutes (2-minute D-value × 6 logs of population = 12 minutes). Thus, to check the accuracy of the BI manufacturer’s certified D-value of a given BI, manufacturers may look to the USP for guidance. This is where some misconceptions originate.
Assessing the D-Value
USP 31, “User’s Responsibility,” states:
The user should establish in-house acceptance standards for biological indicator lots and consider rejection in the event the biological indicator lot does not meet the established in-house performance standards. A certificate of performance should be obtained for each lot of indicators, and the user should routinely perform audits of the manufacturer’s facilities and procedures.1
USP is recommending that end-users establish in-house acceptance criteria for BI performance. This may pertain to a user’s acceptable population or D-value range for the cycles they use. In doing so, a company may set a performance standard that states it will not accept a BI for use unless the certificate of analysis shows the D-value to be within a range of 1.5–2.0 minutes, for example, and a population between 1.0 and 3.0 of the desired log. It is further recommended that the end-user obtain a certificate of performance for each lot of BIs obtained, and routinely perform audits of the BI manufacturer’s site and the procedures that are used.
USP 31 also states:
Step 1. A group of BIs ready to be placed into a BIER unit.
Upon initial receipt of the biological indicator from a commercial supplier, the user should verify the purity and morphology of the purchased biological indicator organisms. Verification of at least the proper genus is desirable. Also, a microbial count to determine the mean count per biological indicator unit should be conducted. The manufacturer’s comments relative to D-value range, storage conditions, expiration dating, and stability of the biological indicator should be observed and noted. The user may consider conducting a D-value assessment before acceptance of the lot.
After reading these USP excerpts, many facilities have instituted in-house acceptance criteria for incoming BIs prior to allowing them to be used. As per USP 31, a microbial count should be conducted, and a large number of end-users are performing population counts in-house as a result. If they cannot perform the population assays themselves, they send them out for third-party population verification.
Where D-value is concerned, most facilities do not have the proper equipment to do a D-value assessment, so they send BIs off to a third party for testing. As USP states in the above excerpt, “The user may consider conducting a D-value assessment before acceptance of the lot.” One should note that “may consider” is not a “must” or “should consider.” If one chooses to consider a D-value assessment or the facility protocol requires a D-value assessment prior to use, then a qualified third-party testing lab is usually needed. When the test BIs are sent off to the third-party testing lab, the end-user should request a D-value assessment.
This is not the same as a D-value determination. If requesting such, the same D-value testing methods used initially by the BI manufacturer to determine and certify the D-value for that particular lot of BIs must be used. D-value determination requires that two methods be used. Also, the ISO 11138 series of documents pertain to BI manufacturers for initial resistance testing. ISO allows the use of any two of the following methods:
- Fraction negative method.
- Survivor curve method.
- Survive/kill method.
In a fraction negative method such as the Spearman/Karber method, various partial or sublethal exposure times are run in a BI evaluator resistometer (BIER) vessel with several groups of BIs from the same lot. This ensures that one exposure results in killing all of the BIs exposed and another exposure keeps alive all of the BIs exposed. Several exposures that run in between these two exposure times will result in exposures where a fraction of the BIs are positive for growth and a fraction are negative for growth, and is thus called a fraction negative method.
|Step 2. A group of BIs are placed into the exposure chamber of a BIER unit.
In a survivor curve method, various groups of BIs are run in a BIER vessel to sublethal exposure times such as 1.5- or 2-minute intervals between exposure times. The BIs from each exposure are processed and quantified for remaining viable organisms. This is done as per USP, and the serial dilutions are plated out on trypticase soy agar and incubated.2 Upon incubation, the colony-forming units on each plate are counted, and the resulting surviving population can be enumerated. As a result, the amount of exposure time to reduce the population by one log can be determined.
In the survive/kill method as described in USP, the USP-calculated survive exposure time and kill time are used.3 Two groups of 20 BIs each are exposed to the survive and kill times in a BIER vessel. All BIs in the survive time must survive and show growth, and all BIs within the kill time group must show no growth for the requirements of the test to be met and thus verify reliability of the D-value being tested.
The methods initially used by the BI manufacturer should be stated on the certificate of analysis provided by the manufacturer with the BI lot. It is important to remember that in a D-value assessment or verification test, you are getting an assessment from the third-party lab, which isn’t the same as a determination. To obtain a D-value assessment, one could use the USP survive/kill resistance performance test for D-value verification. The third-party testing lab would only need to run the two USP-calculated cycles (one for survive time and one for kill time) based on the certified D-value of the BI in question. If the requirements of the test are met, the D-value has been assessed and verified, and the BI can be used.
Even if a manufacturer is requesting verification with use of a BIER vessel from a third-party lab, it is strongly recommended to audit the third-party facility first to ensure that it is following ISO and USP testing procedures. A very important question to ask during the audit would be whether the third-party facility that is using a BIER vessel is ISO/AAMI compliant. In preparation for the audit, the auditor needs to become familiar with ANSI/AAMI/ISO requirements for BIER vessel performance.4 The survive/kill test seems like a very straightforward method to perform, but the use of a BIER vessel is still critical.
To accomplish successful and accurate testing of a BI with regard to resistance, exposure intervals must be as accurate in duration as possible. A 15-minute exposure time needs to be as close to 15 minutes (plus or minus 6–10 seconds) as possible. It cannot be the usual 2–5-minute come-up time, along with a typical 2-minute comedown time at cycle end. If a BIER vessel is used, a 15-minute cycle will typically involve a total time of around 15 minutes and 12 seconds. A BIER vessel’s chamber is very small and responds very quickly to temperature increases. It can maintain a target temperature accurately, within ±0.5°C of the target temperature throughout the entire exposure phase. As with the Spearman/Karber method, a 4.5-minute exposure must be 4.5 minutes. Thus, when auditing a third-party testing lab prior to contracting work, the auditor should review a few BIER vessel run printouts and note whether the test equipment performance requirements are being met. Does the chamber reach target temperature within 10 seconds or less? Is the target temperature stable, and does it remain within ±0.5°C for the entire exposure cycle? Are the BIs removed quickly from the unit following a fast comedown phase? Is the vessel itself capable of recording time, temperature, and pressure as specified within such regulatory standards as ANSI/AAMI/ISO 18472:2006?
To be a certified or compliant BIER vessel, the unit must independently document the temperature, time, and pressure occurring during the cycle operation. Some units claim to be ISO and AAMI compliant, yet they need to use several outside measuring devices to record and document the events that are occurring within the BIER vessel chamber. This does not meet regulatory requirements. Needing a separate instrument other than the actual BIER vessel to record the temperature or pressure will add variables to an already delicate and precise testing process. Knowing what D-value test methods are allowed by ISO or USP, the method’s specific steps, and having the equipment performance documentation should all be part of prequalification process in selecting a lab or facility to conduct third-party testing.
|Step 3. Following the incubation of test BIs, six groups of 10 ampoules each are exposed to different exposure times. The yellow color indicates growth.
If a testing facility is following ISO or USP procedure, obtaining the protocols to review prior to testing should not be an issue, because the procedures are not proprietary. If one is paying for a standard service, it should be privy to the procedure to be used prior to contracting the service.
If a lab follows USP or ISO guidance on D-value testing without variation and uses compliant equipment and suitable recovery media, D-value assessment or determination by a third-party testing lab can be successful and repeatable. When selecting a third-party lab to use for testing results, a quality assurance department can work closely with the selected lab to ensure that all testing parameters are followed and that the third-party testing lab’s testing methods and media are the same as those used by the BI manufacturer during initial D-value testing. BIER vessels should be checked for current calibrations and to verify that they are operating properly.
In Table I, 15 different lots of both ampoule and spore strip BIs were sent out to a third-party lab for D-value testing. Listed is the initial certified D-value found for the BI by the BI manufacturer and the third-party lab’s determined D-value for the lot. In most cases, the Spearman/Karber fraction negative method was initially used by the third-party lab. Once a D-value was determined by fraction negative method, the USP survive/kill method was run.
If verification results pass USP requirements, the D-value can be used as certified on the certificate of analysis and should be accepted as a lot of BIs to be used. The certified D-value has been assessed, substantiated, supported, or confirmed. However, it cannot replace the manufacturer’s determined, certified D-value. Unfortunately, this is happening in far too many cases. When this happens, the end-user uses the third-party testing lab’s result for D-value (or population) as the new certified D-value. Or, if the testing doesn’t pass requirements, the BIs are classified as noncompliant with performance requirements.
Unless two methods have been used and are the same as those used by the manufacturer, the testing result is not an ISO-compliant determination result. Such relabeling cannot be done. A D-value testing assessment cannot replace or be used to relabel the certified D-value determined by the manufacturer. To initially determine and certify the manufacturer’s stated D-value, methods allowed by ISO and USP were used. As stated earlier in ISO 11138 series, to obtain a label claim or certify a determined D-value, one must use two of the three methods outlined in ISO and USP.3,5 A third-party assessment is not a determination. In almost all cases, only one method was used and may not in any way be used to relabel a BI resistance characteristic that was certified by the manufacturer.
|Table I. The initial and the third-party D-values are all within an acceptable +/-20% range of difference.
When sending out a BI for a population assay, a similar situation exists. For the BI to pass the population verification set out in USP, the population result needs to be within +300% and –50% of the labeled population. If the population being verified falls within this range, the requirements of the test are met, and the populationhas been verified. However, the verified population is not the new certified population to be used in further studies or validations done with this lot of BIs. In most situations, BI manufacturers are much more familiar, have validated, and have extensive experience in performing population assays on their particular BIs. Having a third-party lab’s assay result replace a manufacturer’s certified population, even if only slightly different than the certificate of analysis listed population, makes little sense.
The bottom line is that third-party verifications are not intended to replace a manufacturer’s certified BI characteristic such as
D-value or population. Some testing labs are performing recertifications and some end-users are using the third-party verification to replace the certificate of analysis-labeled values. If verification criteria are met, the certified characteristics on the manufacturer’s certificate of analysis should be used.
If a manufacturer cannot do a D-value assessment or verification in-house due to the lack of a compliant BIER vessel and wants to obtain a verification, it should have a contract lab run the USP resistance test, the survive/kill method. This will involve two BIER runs and be much less expensive (around $600–$800) than conducting a full determination involving two methods and costing upwards of $6000–$7000.
When dealing with any third-party lab, before putting any money out or contracting work, perform an audit of the equipment and methods used—even if it has to be a paper audit. Qualifying a lab prior to having work done can save money and numerous needless problems that can cause work delays and frustration. Remember, to consider an assessment is not the same as to determine. To determine a BI’s resistance is very costly, is usually done by the BI manufacturer, and involves at least two test methods. FDA or an auditor might not look favorably upon relabeled or possibly adulterated BI usage.
1. USP 31, “Biological Indicators for Sterilization” (Rockville, MD: U.S. Pharmacopeia), 401.
2. USP 28, “Total Viable Spore Count” (Rockville, MD: U.S. Pharmacopeia), 244.
3. USP 31, “Resistance Performance Tests,” (Rockville, MD: U.S. Pharmacopeia), 1532.
4. ANSI/AAMI/ISO 18472:2006, “Sterilization of Health Care Products, Biological and Chemical Indicators—Test Equipment,” (Arlington, VA: AAMI, 2006).
5. ISO 11138-1:2006, “Sterilization of Health Care Products—Biological Indicators—Part 1” (Geneva: International Organization for Standardization, 2006).
Russ Nyberg is director of retail sales and tech support for Raven Labs (Omaha, NE).
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