Contamination during cytotoxic drug preparation

teaser

Marjolijn van Essenberg
PharmD
Department of Pharmacy
de Heel Zaans Medical Centre

Eric JF Franssen
PharmD PhD
Department of Pharmacy
VU University Medical Centre

Hans van Doorne
PhD
University Centre for Pharmacy
State University Groningen

Arie van Loenen
PharmD
VU University Medical Centre

Frits A Boom
PharmD
de Heel Zaans Medical Centre
The Netherlands

In Dutch hospital pharmacies, cytotoxic drugs are prepared in safety cabinets placed in a grade D background environment, according to the Good Manufacturing Practice guidelines.(1,2) Since 2002, the preparation of cytotoxic drugs at the de Heel Zaans Medical Centre, a general hospital in the Netherlands, takes place in an isolator with negative pressure. An isolator offers several advantages over a safety cabinet: lower costs, less restrictive gowning and a higher level of protection for the operator.(3-6)

Low costs are mainly related to the fact that a classified background environment is not necessary when dispensing drugs.(2,7,8) The possibility of contamination is minimal when the isolator is constructed with ventilated transfer hatches with door interlocks and the transfer procedure is validated.(9,10) The main advantage, however, is that an isolator separates the operating personnel from the product. Thus, the main source of microbiological contamination of the product is eliminated.(4,8) There are also some disadvantages. For example, ergonomics can be a problem, and an isolator requires a second technician for the supply and discharge of materials.(4)

Several studies have compared the use of isolators with conventional cleanrooms. In all studies, the isolator appears to be the best technique.(3,5,6,11) However, these studies were carried out in industrial settings. The present study was conducted in a Dutch hospital pharmacy setting where 3,000 preparations of cytotoxic drugs are prepared annually. Based on literature data and cost analysis, an isolator was chosen for cytotoxic preparation.

Landry and colleagues studied the factors that affect the sterility of work areas in isolators and safety cabinets. The microbiological contamination was measured by placing settle plates and taking samples with Replicate Organism Detection and Counting (RODAC) plates. The overall contamination in the isolators was significantly lower than in the safety cabinet (2.6% vs 12.9%; p<0.01). The investigators concluded that more research was required to compare isolators and safety cabinets with respect to sterility.(12) This is especially true for hospital settings with specific standard operating procedures for total parenteral nutrition and cytotoxic drugs preparations, as is the case in Dutch hospitals.

Microbiological monitoring can be used to control the process, although it cannot replace tests on product specifications.(13,14) Media fills are used to estimate the true level of contamination of the product.(15,16) The level of contamination during media fills is very low (1:3,000);(17) therefore, these fills are not useful for a comparison between an isolator and a safety cabinet over a short period of time.

In a prospective study, we compared the microbiological contamination measured with settle plates and finger dabs in a safety cabinet with the contamination in an isolator while preparing cytotoxic drugs.

Methods
The study was conducted in the Department of Pharmacy of the de Heel Zaans Medical Centre. From January to August 2002, seven technicians  prepared cytotoxics in the safety cabinet or in the isolator. The number of preparations per technician was comparable for each cabinet.

The work tray and the sleeves of the isolator were disinfected daily with 70% ethanol, before and at the end of a preparation session. The whole cabinet was cleaned and disinfected weekly, this procedure having been validated by surface sampling with RODAC plates. The materials used for the preparation of cytotoxics (eg, infusion bags, needles or syringes) were not disinfected before being transferred into the cabinet. The septa of vials and infusion bags were disinfected with 70% ethanol inside the cabinet. Gloves were changed every 2h, or when drugs were spilled or a leak was formed. All technicians were trained in aseptic work by a qualified person.

During preparation, a settle plate was opened for approximately 30min, with the precise time being recorded. To obtain maximum sensitivity, the settle plate was placed below the hands of the technician.(13) At the end of a preparation session, finger dabs of the gloved hand were made on a second plate. The plates were then incubated at 30ÞC for 4–6 days, after which the number of colony-forming units (CFUs) was counted.

Equipment
The isolator (Envair CDC ‘D’ 2GD) was under negative pressure relative to the ambient air. Negative pressure was used to avoid exposure of the technicians to cytotoxic drugs. The isolator was placed in an unclassified background environment in accordance with the Good Manufacturing Practice hospital pharmacy guidelines.(2) The safety cabinet (Interflow B&E B4) also operated under negative pressure. The background environment met grade D requirements. Finally, tryptone soya agar plates (90mm), prepared and tested for growth-promoting properties by the microbiology department of the hospital, were used.

Analysis
The number of CFUs on the settle plates was converted to the number of CFU/4h. This level is defined in the Good Manufacturing Practice guidelines.(1,2) For the finger dabs, the number of CFUs/90min (the average time during which gloves are used) was calculated from the number of CFUs on the plates.

Before the study took place, a power analysis was carried out to estimate the sample size. A sample size of 70 per study group was calculated for the finger dabs, assuming an historical contamination level of 54% in the safety cabinet (data Zaandam 1997–2001, not published).

The contamination level of the settle plates was 22% in the safety cabinet before the study. From these data, a sample size of 35 per study group was calculated. However, to enlarge the power of the study we decided to use 70 samples in each arm. A statistical analysis was carried out with a X(2) test. p-values smaller than 0.05 were accepted as significant.

Tables 1 and 2 show the results of the contamination level in the safety cabinet and the isolator.

[[HPE13_table1_60]]

[[HPE13_table2_60]]

Settle plates
Results for the settle plates are presented in Table 1. Although the number of contaminated settle plates was higher in the isolator than in the safety cabinet (12% versus 4%), the difference was not significant (p=0.06).

Growth on a settle plate can be considered as a failure in asepsis. This is not really important if one or two CFUs are present on the settle plate. Whyte postulated a relationship between the number of CFUs on a settle plate and the fraction of containers contaminated. In the equation, the area of opening of the container is one of the factors.(18) Since this area approaches zero in cytotoxic infusions, the relationship does not apply in this case. Therefore, we analysed the data as contaminated versus uncontaminated.

Our study revealed a significant difference between the isolator and the safety cabinet. However,  a learning curve of working in the isolator may be an issue in this case and may result in less contamination in the isolator arm, as the technicians had previous experience with safety cabinets. Although they were trained for working on the isolator before the study, training time was short. The results of the first two months in the isolator are compared with the results of the last two months (see Table 3). Although contamination is higher in the first period, the differences are not significant (16% vs 8%; p=0.25). More measurements should confirm whether there is a trend towards lower contamination.

[[HPE13_table3_61]]

Contamination in the safety cabinet during the study was only 4%, whereas it reached 22% during the period 1997–2001 (data not published). What may have caused this decrease in contamination level?

Performance improvement with safety cabinets
Although all technicians (approximately 20) in the pharmacy usually prepare cytotoxic drugs, only seven experienced technicians participated in this study. Inspection of historical data (see Table 4) shows that this small group is representative of the total group for which the study was designed: in the period 1997–2001, 22% of the settle plates in the safety cabinet were contaminated by the total group of technicians. Data produced by the same group of technicians during the present study were filtered. In this group, contamination was 26%. The difference between the two groups is not significant (p=0.45), as shown in Table 4.

[[HPE13_table4_61]]

Before the study, standard operating procedures were updated and technicians were trained. Appropriate aseptic techniques are necessary to reduce the possibility of contamination;(9) therefore, training is essential.(14) Training programmes can be more easily completed with a small number of technicians than with a large group, and training might have caused the improvement in performance in the small group.

Historically, technicians worked without an assistant technician. For each new preparation, the technician had to go out of the safety cabinet to collect the materials, which may have caused more contamination. Preparing in an isolator, however, is practical only with the help of an assistant technician for supply and discharge of all materials needed. To ensure that procedures in both cabinets were comparable, an assistant technician was also used in the safety cabinet. This change in procedure may have caused lower contamination, as shown in a retrospective analysis by comparing safety and crossflow cabinets. From 1997 to 2001, 20% of the settle plates were contaminated using the crossflow cabinet. During this period, there was no assistant technician for the supply and discharge of materials. From January to July 2002 an assistant technician was used, and contamination in the crossflow cabinet was reduced to 4% (p<<0.01).

The results of this study are summarised in Table 5. These results show that using an assistant technician may reduce microbiological contamination.

[[HPE13_table5_61]]

Higher contamination in isolators
Microbiological contamination should be minimal when using an isolator, which is a sealed system. Landry and colleagues found 1.7% contamination of settle plates in isolators,(12) whereas in our study 12% of the settle plates are contaminated. This high contamination could be due to several causes:

• The isolator has been validated, which ensures its functioning.(19) Isolators with negative pressure, however, show in-leakage.(9,20) Through the sleeves or seals of the isolator, some air from the background environment may enter the main compartment. Since the background environment is not classified, this could have theoretically led to contamination. However, a glove-integrity test and a monthly pressure decay test performed after each glove change showed no problem with the isolator. This, in combination with the high air-change rate, makes in-leakage very unlikely.(20)
• Contamination could be the result of opening and closing the settle plates. Again, this is unlikely, as work was carried out by trained technicians.
• There is no significant difference between the results of the first and the last period of the study (see Table 3). Although better results were observed in the isolator at the end of the study, data (n=23) collected six months after the study showed no change in contamination levels. Growth of CFUs was observed on three plates (13%). This level of contamination is comparable to that observed during the study (12%), demonstrating that data in the isolator arm of the study are not biased by a learning curve.

Finger dabs
Results for the finger dabs are shown in Table 2. Contamination rates for the isolator and the safety cabinet are similar (34%), and there is no significant difference between the two groups (p=0.96).

This percentage is an improvement compared with historical data (54%), although it is still high. In comparison, Landry and colleagues observed a 25% contamination of the gloves used in isolators.(12) In this case, contamination is probably introduced by materials not disinfected before use. Guidelines prescribe disinfection of materials used in aseptic preparations with 70% ethanol.(7) Although this is widely accepted, little evidence is available on the effectiveness of disinfecting, as shown by our own retrospective data.

From 1995 to 1997, materials used for aseptic preparations were not disinfected before use, with the exception of the septa. This was the case for the safety cabinet, where cytotoxic drugs were prepared, and for the crossflow cabinet, where parental nutrition was prepared. From 1997 to 2001, the procedure for the crossflow cabinet was different: the whole surface of the materials was disinfected, mostly by spraying with 70% ethanol. The procedure in the safety cabinet was unchanged so it could serve as control group.

Contamination of the gloved hands was compared over the two periods. A nonsignificant increase in contamination was observed in the safety cabinet (47% vs 53%; p=0.21). A significant (33% vs 42%; p=0.044) increase in contamination was also observed in the crossflow cabinet. Results are summarised in Table 6.

[[HPE13_table6_62]]

Thus, complete disinfection of materials cannot prevent microbiological contamination. However, the contamination rate during media fills in our pharmacy was very low (<<1%). Therefore, disinfection of the whole surface of the materials was not included in the standard operating procedure for working in the safety cabinet and the isolator. Only the rubber stops of vials and bags and the neck of ampoules were disinfected with 70% ethanol.

Recently, Hiom validated four different disinfection techniques, with wiping being significantly more effective than spraying.(21) The results of this study have not yet been translated into a practical and validated disinfection procedure in our setting.

Conclusion
Our study demonstrates that there is no significant difference between a safety cabinet and an isolator with respect to microbiological contamination during routine preparation in a general hospital setting. Although this study was not designed to define specific determinants, there are strong indications that training, standardisation of aseptic techniques, working with an assistant technician and a validated disinfection procedure can contribute to lower contamination levels. All these factors seem more critical from a microbiological point of view during the preparation of cytotoxic drugs than the type of cabinet used.

This study also shows that collecting and archiving data can be very useful for understanding the effects of changes in standard operating procedures. It also shows the importance of prospective studies when comparing procedures.

Acknowledgements
The authors wish to express their gratitude to the technicians at the de Heel Zaans Medisch Centrum pharmacy for their contributions to this study.

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