MALDI Sepsityper Kit

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1 Instructions for Use MALDI Sepsityper Kit Kit for identification of microorganisms from positive blood cultures using the MALDI Biotyper system CARE products are designed to support our worldwide customers with highquality consumables, accessories and dedicated kits. The CARE product range is specifically optimized and certified for use with all Bruker Daltonics systems. Language: en Revision D (December 2015)

2 1 Product description 2 2 Inspection, storage, and stability Inspection on arrival Storage on arrival Storage before and after initial opening 3 3 Risk and safety information Biological risks Limitations of the procedure 4 4 Required chemicals and materials Chemicals Equipment and consumables Recommended materials for harvesting blood culture fluid 5 5 MALDI Sepsityper sample preparation procedure MALDI Sepsityper application training 7 6 Manufacturer 8 1 Product description In clinical practice, blood (approximately 10 ml) from patients suspected of having, for example, a bloodstream infection is collected into blood culture cultivation bottles. The bottles are incubated in an automatic detection system that signals the presence of bacteria, yeasts, or fungi in a sample. The MALDI Sepsityper Kit selectively destroys blood cells and enriches microorganisms from a positively flagged blood culture. The resulting extracts are suitable for analysis using the MALDI Biotyper system. The MALDI Sepsityper Kit enables a highly probable identification of the relevant microorganism species within 30 minutes using the MALDI Biotyper system. In many cases, the susceptibility of the microorganism to a range of antibiotics can be estimated when the species is identified. Kit components The MALDI Sepsityper Kit contains the following components: Lysis Buffer Washing Buffer 1.5 ml microcentrifuge tubes (50 pack) Labels Quick Guide MALDI Sepsityper Kit MALDI Sepsityper Kit Instructions for Use Ordering Information Product Part Number MALDI Sepsityper Kit for 50 samples # Page 2 of 8 MALDI Sepsityper Kit Instructions for Use Revision D

3 2 Inspection, storage, and stability 2.1 Inspection on arrival Check the MALDI Sepsityper Kit on arrival. If the packaging is damaged, check the tubes/bottles inside. If the tubes/bottles are damaged, the MALDI Sepsityper Kit must not be used. Dispose of the MALDI Sepsityper Kit (tubes/bottles and packaging) following the guidelines outlined in the Material Safety Data Sheet for the product and contact Bruker for a replacement. 2.2 Storage on arrival +20 C +25 C The MALDI Sepsityper Kit is shipped at ambient temperature, but must be stored at room temperature (20 25 C). Do not use the MALDI Sepsityper Kit beyond the expiration date printed on the package labeling. 2.3 Storage before and after initial opening +20 C +25 C Before initial opening, check if the MALDI Sepsityper Kit contains precipitates. If precipitates are present, store the Lysis Buffer at room temperature (20 25 C) for at least one hour and shake content before initial opening. After initial opening, the MALDI Sepsityper Kit is stable for up to three months at room temperature (20 25 C). 3 Risk and safety information The MALDI Sepsityper Kit is not classified according to Regulation (EC) No. 1272/2008 and is therefore not classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS). In the manufacturer's experience, the product has no harmful effect when used and handled according to specifications. However, all materials may present unknown hazards and should be handled with caution. Lysis Buffer is classified as a hazardous chemical: it causes skin irritation and serious eye damage. DANGER (H: 315, 318; P: 264, 280, , , 310, 321, , ) For more information, read the Material Safety Data Sheet available for download at Additional chemicals and materials may be required for procedures described in these Instructions for Use. Carefully read any warnings, instructions, or Material Safety Data Sheets provided by the supplier and follow general safety regulations when handling chemicals, biohazards, or other materials. Revision D MALDI Sepsityper Kit Instructions for Use Page 3 of 8

4 3.1 Biological risks The MALDI Sepsityper Kit and MALDI Biotyper system deal with potentially dangerous biological material. Every person working with these systems is responsible for reading and following all necessary health and safety precautions. All patient samples and cultures must be considered potentially infective. Only qualified laboratory staff are allowed to work with the MALDI Sepsityper Kit and MALDI Biotyper system, and they are responsible for taking and following all necessary safety precautions for handling potentially infective material. It is very important to wear appropriate personal protective equipment at all times. This means a lab coat, protective gloves and safety goggles. Make sure that personal protective equipment is in good condition. During the MALDI Sepsityper workflow, some reagents are mixed with microorganisms and consequently with potentially dangerous biological material. Moreover, some of the accessories and consumables used will come into contact with microorganisms. It is the operator s responsibility to carefully handle, correctly dispose of, and decontaminate the relevant substances, accessories, and consumables according to national or local safety regulations. 3.2 Limitations of the procedure The identification of multi-microbial infections or contaminations (mixed cultures) by using the MALDI Sepsityper system is generally difficult and outside of the intended use. In such cases, typically only one microorganism is identified or no identification is obtained at all. To check if mixed cultures are present in positive blood cultures, it is common microbiological practice to subcultivate each blood culture sample on an agar plate (purity plate). The following day, the colonies on the purity plate are examined. The presence of heterogeneous colonies indicates that more than one species of microorganism was present in the sample. Each blood culture identification using the MALDI Sepsityper must be confirmed by checking that the colonies on the purity plates are homogeneous. Due to the nature of positively flagged blood cultures (a huge number of blood cells present in different ratios to bacterial cells) there is no guarantee that every sample preparation will work. Comprehensive surveys show an average success rate of up to 90% 1. Alternative result interpretation which deviates from standard MALDI Biotyper log(score) threshold interpretation can significantly increase the success rate. Such alternative interpretations must be performed by experienced microbiologists familiar with the interpretation of MALDI Biotyper results. 1 Moussaoui, W., B. Jaulhac, A. M. Hoffmann, B. Ludes, M. Kostrzewa, P. Riegel, and G. Prévost Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clinical Microbiology and Infection 16: Page 4 of 8 MALDI Sepsityper Kit Instructions for Use Revision D

5 4 Required chemicals and materials 4.1 Chemicals In addition to the MALDI Sepsityper Kit components Lysis Buffer and Washing Buffer, the following chemicals and materials are required to perform the MALDI Sepsityper sample preparation workflow. For best results, use freshly prepared solutions of the highest purity available (for example, HPLCgrade or CHROMASOLV LC-MS solvents). Ethanol, absolute Formic acid Acetonitrile HPLC-grade water Bruker Bacterial Test Standard (BTS, # , Bruker Daltonik) HCCA portioned (HCCA, # , Bruker Daltonik GmbH) Standard solvent (acetonitrile 50%, water 47.5% and trifluoroacetic acid 2.5%) from Sigma- Aldrich (# 19182), which has been tested by Bruker Daltonik GmbH and is recommended for solubilization of BTS and HCCA. 4.2 Equipment and consumables MALDI target plate of the type selected for your workflow Vortex mixer Benchtop centrifuge Rack for tubes Pipettes Pipette tips 4.3 Recommended materials for harvesting blood culture fluid BD 1 Vacutainer Blood Collection Tube (8.5 ml), BD # BD Vacutainer Eclipse Blood Collection Needle, BD # BD Vacutainer Needle Holder, BD # Note Carefully read any warnings, instructions, or Material Safety Data Sheets provided by the supplier and follow general safety regulations when handling chemicals, biohazards, or other materials. 1 Becton, Dickinson and Company Revision D MALDI Sepsityper Kit Instructions for Use Page 5 of 8

6 5 MALDI Sepsityper sample preparation procedure Harvesting blood culture fluid 1. Disinfect the septum of the blood culture bottle with 70% ethanol or similar. 2. Collect 7 8 ml blood culture fluid using the BD Vacutainer blood collection tube, blood collection needle, and needle holder. Sample preparation using MALDI Sepsityper Kit Note Additional procedure steps that must be performed when using biomérieux blood culture bottles containing activated charcoal are printed in italics. 3. Transfer 1 ml blood culture fluid from the BD Vacutainer blood collection tube to an Eppendorf tube. 4. Add 200 µl Lysis Buffer and mix by vortexing for 10 (± 5) seconds. a. Apply 800 µl of this mixture to a SigmaPrep spin column (Sigma-Aldrich SC1000-1KT). b. Centrifuge for 2 minutes at 2000 rpm and discard the filter. c. Proceed to step Centrifuge the tube for 2 minutes at 13,000 15,000 rpm. 6. Remove the supernatant by pipetting and discard. 7. Add 1 ml Washing Buffer and resuspend the pellet by pipetting up and down. 8. Centrifuge the tube for 1 minute at 13,000 15,000 rpm. 9. Remove the supernatant by pipetting and discard. Extraction method and preparation of samples onto a MALDI target plate 10. Add 300 µl HPLC-grade water and resuspend the pellet by pipetting up and down. 11. Add 900 µl ethanol and mix the suspension. 12. Centrifuge the tube for 2 minutes at 13,000 15,000 rpm. 13. Remove the supernatant by pipetting and discard. 14. Centrifuge the tube for 2 minutes at 13,000 15,000 rpm. 15. Remove the residual ethanol by pipetting and discard. 16. Allow the pellet to dry for 5 (±1) minutes at room temperature. 17. Add 2 50 μl 1 of 70% formic acid and thoroughly resuspend the pellet by pipetting up and down. 18. Add an equal volume of acetonitrile and mix the suspension by pipetting up and down two to three times. 19. Centrifuge the tube for 2 minutes at 13,000 15, The volumes of formic acid and acetonitrile added to the pellet should be proportional to the size of the pellet. For very small pellets, the volume can be reduced down to 2 μl each. Page 6 of 8 MALDI Sepsityper Kit Instructions for Use Revision D

7 20. Deposit 1 µl of the supernatant onto a MALDI target plate position. 21. For validation of the system, deposit 1 µl reconstituted BTS solution 1 onto two MALDI target plate positions. 22. Allow the samples to dry at room temperature. 23. Overlay each sample with 1 µl reconstituted HCCA matrix solution 2 immediately after samples have dried. 24. Allow the matrix-overlaid samples to dry at room temperature. 25. The sample of the test microorganism is now ready to run through the identification process. Insert the MALDI target plate into the MALDI- TOF mass spectrometer and follow the workflow as described in the software user manual. 5.1 MALDI Sepsityper application training A dedicated MALDI Sepsityper application training is available to enhance the reliability and success rate of sample preparation with the MALDI Sepsityper Kit. 1 Prepared as described in the Bruker Bacterial Test Standard Instructions for Use 2 Prepared as described in the HCCA portioned Instructions for Use Revision D MALDI Sepsityper Kit Instructions for Use Page 7 of 8

8 6 Manufacturer Bruker Daltonik GmbH Fahrenheitstraße Bremen Germany Support Phone: +49 (421) Fax: +49 (421) Sales Information Phone: +49 (421) Web: For research use only. Not for use in diagnostic procedures. # Descriptions and specifications supersede all previous information and are subject to change without notice. Copyright 2015 Bruker Daltonik GmbH Page 8 of 8 MALDI Sepsityper Kit Instructions for Use Revision D

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