BacTx® Bacterial Detection Kit for Platelets
Contents
1. Yy IMMUNETICS BacTx Bacterial Detection Kit for Platelets Immunetics Catalog Number DK B502 032 32 Tests INTENDED USE The Immunetics BacTx Bacterial Detection Kit for detection of bacteria in platelets is a rapid qualitative colorimetric quality control test for the detection of aerobic and anaerobic Gram positive and Gram negative bacteria in leukocyte reduced apheresis platelet units LRAP Apheresis Platelets Leukocytes Reduced as a quality control test following testing with a growth based bacterial detection device cleared by the FDA for quality control testing of leukocyte reduced apheresis platelets and pools of up to six 6 units of leukocyte reduced whole blood derived platelets Platelets Leukocytes Reduced that are pooled within four 4 hours of transfusion INTRODUCTION Bacteria are the most common contaminating infectious agents found in platelet units It has been estimated that the rate of bacterial contamination for apheresis platelet donations is 1 in 5000 making platelets the most frequent source of transfusion related infection Since platelets must be stored at 20 24C in order to maintain function small numbers of bacteria that are mostly introduced from a donor s skin flora into platelet units can multiply to very high numbers in a matter of days Skin flora are not the only source of bacteria that contaminate platelet units many strains of Gram positive and Gram negative bacteria have been2. Assay This test detects the presence of peptidoglycan PG which is a component of bacterial cell walls in both Gram positive and Gram negative bacteria These instructions for use contain the necessary protocols for analyzing LRAP or LR WBDP To test platelets for the presence of bacterial PG a volume of platelets is sterilely sampled from the platelet bag and added to a microfuge tube containing Lysis Reagent and mixed The microfuge tube is then briefly centrifuged to pellet insoluble platelet debris and bacterial cell wall fragments if present The peptidoglycan present at the bottom of the tube is then homogenized in Extraction Reagent The alkaline Extraction Reagent effectively releases PG from bacterial cell walls for optimal detection Lastly the suspension is added to a clean microfuge tube containing Neutralization Reagent and the tube is mixed by inversion From the resulting sample an aliquot is added to a tube containing lyophilized detection reagents the tube is vortexed and the tube is then placed in the BacTx Reader The BacTx Reader is a photometer which automatically monitors the detection reaction and interprets the result using software installed on the provided laptop PC If bacteria are detected within the 30 minute reading time a Fail result accompanied by an optional audible alarm is generated otherwise a Pass result will be recorded The BacTx Kit and associated protocols are covered under US Paten
3. 2010 Bethesda MD AABB 2010 6 29 CFR 1929 1030 Occupational Safety and Health Standards Bloodborne Pathogens 7 Tate J and Ward G Interferences in immunoassay Clin Biochem Rev 25 2 105 120 2004 8 Dimeski G Interference testing Clin Biochem Rev 29 Suppl 1 S43 48 2008 CONTACT INFORMATION Immunetics Inc 27 Drydock Avenue Boston MA 02210 2377 USA Tel 800 227 4765 or 617 896 9100 Fax 617 896 9110 2006 2013 Immunetics Inc All rights reserved Email info immunetics com BacT ALERT and bioM rieux are registered trademarks of bioM rieux SA Internet http www immunetics com BacTx and Immunetics are registered trademarks of Immunetics Inc Page 16 of 16 Effective Date August 16 2013 CF B502 706
4. used during the analytical sensitivity testing Bacterial concentrations in LRAPs were estimated by optical density to be between 1 x 10 CFU mL and 1 x 10 CFU mL The actual titer was confirmed by quantitative plate culture The lowest bacterial concentration at which 10 out of 10 replicates of the BacTx Assay were positive for bacterial contamination i e 10 out of 10 FAIL results was recorded and the higher value between the two clinical sites was taken to be the limit of detection see Table 1 LR WBDP Study Description The limit of detection of the BacTx Assay was determined for 10 species of bacteria four Gram positive aerobes four Gram negative aerobes and 2 anaerobes Spiking studies were performed at two external sites Four lots of BacTx Kits were used during the analytical sensitivity testing Bacterial concentrations in the pooled platelets were estimated by optical density to be between 1 x 10 CFU mL and 1 x 10 CFU mL The actual titer was confirmed by quantitative plate culture The lowest bacterial concentration at which 10 out of 10 replicates of the BacTx Assay were positive for bacterial contamination i e 10 out of 10 FAIL results was recorded and the higher value between the two clinical sites was taken to be the limit of detection see Table 1 LRAP and LR WBDP Study Results The limit of detection for each of the 10 bacterial strains is listed in Table 1 Table 1 Analytical Sensitivity of
5. Reaction Tube use a fine tip permanent ink lab marker to write the same ID on each BacTx Reaction Tube as on the 1 5 mL microfuge tube beneath it Example Reaction Tubes 1 5 mL tubes 2 mL tubes Platelet Samples Figure 1 Example illustrating proper labeling of microfuge tubes and Reaction Tubes In this example eight platelet samples labeled with ID s P1 P8 have been collected 2 mL and 1 5 mL microfuge tubes and Reaction Tubes have been labeled with the corresponding ID Preparing BacTx Samples from LRAP units 1 With a single channel 1000uL micropipettor pipet 0 75 mL of Lysis Reagent into each empty 2 mL microfuge tube in the microfuge rack Close the cap on each tube after addition of Lysis Reagent 2 With a single channel 1000uL micropipettor pipet 0 5 mL of Neutralization Reagent into each empty 1 5 mL microfuge tube in the microfuge rack Close the cap on each tube after addition of Neutralization Reagent 3 Starting with the platelet sample in the farthest left position in the rack P1 in the example in Figure 1 use a micropipettor to transfer 1 0 mL of platelets to the 2 mL microfuge tube containing Lysis Reagent directly above it Immediately close the cap on the 2 mL microfuge tube vortex the tube for 5 seconds at maximum speed and return the 2 mL microfuge tube to its original position in the rack Verify that platelets were transferred only from the platelet sample located directly beneath the 2 mL microf
6. agalactiae 12 15 samples tested no false positives were observed Based on these gt results the following substances and platelet conditions do not Serratia marcescens 0 8 1 2 interfere with the BacTx Assay 50 200 normal platelet _Clostridium perfringens 0 5 0 8 concentration low normal and high pH 0 7 hematocrit hemolysis Propionibacterium acnes 1 2 1 3 hyperproteinemia hypoproteinemia lipemia hypercholesterolemia and hypergammaglobinemia IgA IgG and IgM LR WBDP Study Results Out of the 1300 positive samples tested 100 were detected with the BacTx Assay Out of the 427 negative samples tested no false positives were observed Based on these results the following substances and platelet conditions do not interfere with the BacTx Assay 50 200 normal platelet concentration low and high pH 0 7 hematocrit hemolysis hyperproteinemia hypoproteinemia lipemia hypercholesterolemia and hypergammaglobinemia IgA IgG and IgM Page 13 of 16 Effective Date August 16 2013 CF B502 706 Table 7 Summary of Interfering Substances Conditions and Normal Reference Values Condition Test Concentration Test Concentration Normal Reference for LR WBDP Study for LRAP Study Values 3 0x1011 platelets 50 of normal 50 of normala Kowe ilatele Conceatalion 4 2 6 1 x108 platelets mL 5 0 6 0 x108 plateletsimL 250 300 mL for LRAP 200 of normal 200 of normala 5 5x10 platelets Highieisereonco
7. allow the bacteria to proliferate and reach stationary growth phase Aliquots were withdrawn at 3 4 and 5 days after inoculation for dilution plating on 5 sheep blood agar plates to determine if the bacteria in the unit had reached stationary phase On the fifth day after inoculation a platelet pool was made using the inoculated unit and 5 in date sterile as determined by agar plating BacTx unreactive LR WBDP units For each of the three BacTx Test lots ten samples from the platelet pool were tested in the BacTx Test to determine if any falsely negative test results occur Dilution plating on 5 sheep blood agar plates was performed on the platelet pool to determine final bacterial concentration in the pool Since the two anaerobes Clostridium perfringens and Propionibacterium acnes do not grow to high concentrations in platelet concentrates 4 mL volumes of high titer cultures of each strain were centrifuged and the bacteria pellets were resuspended in separate 4 mL volumes of platelets from single in date sterile WBDP units For each strain a six unit platelet pool was made by combining the bacterially contaminated platelets with platelets from 5 in date sterile as determined by agar plating BacTx unreactive LR WBDP units a 1 5 volume ratio For each of the three kit lots ten replicates from the platelet pool were tested in the BacTx Assay to determine if any falsely negative test results occur Dilution plating on anaerobic culture
8. details Page 2 of 16 Effective Date August 16 2013 CF B502 706 REAGENT PRECAUTIONS Reagents were classified according to OSHA 29 CFR 1910 1200 and applicable European Community EC Directives Applicable Classification Risk R and Safety S phrases are listed below Material Safety Data Sheets are available upon request Lysis Reagent contains n butanol which is classified as Harmful Xn Xn R10 Flammable R22 Harmful if swallowed R37 38 Irritating to respiratory system and skin R41 Risk of serious damage to eyes S16 Keep away from sources of ignition No smoking 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 35 This material and its container must be disposed of in a safe way 36 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show this container or label Lysis Reagent and Extraction Reagent contain sodium hydroxide which is classified as Irritant Xi Xi R41 Risk of serious damage to eyes 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 35 This material and its container must be disposed of in a safe way 36 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show this container or label Neutralization Reagent co
9. identified in contaminated platelet units including Staphylococcus Pseudomonas Bacillus and Streptococcus species To increase transfusion safety the AABB issued directive 5 1 5 1 in March 2004 requiring blood banks and transfusion services to implement bacterial testing methods on all platelet units Further safeguards were made effective in January 2011 with the implementation of AABB Interim directive 5 1 5 1 1 which specified that such bacterial testing methods must have prior FDA clearance or must demonstrate equivalent sensitivity to FDA cleared methods The BacTx Kit is a qualitative colorimetric assay which detects bacteria in platelet samples through recognition of peptidoglycan a ubiquitous component of bacterial cell walls The kit consists of lyophilized peptidoglycan detection reagents sample preparation reagents controls and microfuge tubes In the presence of peptidoglycan the assay generates a red colored product indicating the presence of bacteria in the platelet sample The reaction product is detected by a photometer and results interpreted automatically by PC based software PRINCIPLE The BacTx Assay detects bacteria in both Apheresis Platelets Leukocytes Reduced LRAP and pools of up to six 6 units of leukocyte reduced whole blood derived platelets LR WBDP that are pooled within four 4 hours of transfusion Gram positive Gram negative aerobic and anaerobic bacteria are expected to be recognized by the BacTx
10. on the 1 5 mL microfuge tube and invert the tube 3 times to mix Return the 1 5 mL microfuge tube to its original position in the rack Repeat this step for the remaining 2 mL and 1 5 mL microfuge tubes working from left to right Verify that the IDs for the 1 5 mL microfuge tubes match the IDs on the 2 mL microfuge tubes platelet samples and Reaction Tubes in the same column on the tube rack The samples should be tested immediately in the BacTx Assay PERFORMING THE BACTX ASSAY f Starting with the BacTx Reaction Tube in the farthest left position P1 in the example in Figure 1 remove the green cap from the BacTx Reaction Tube by inserting the barbed end of the decapper under the inner lip of the cap Gently twist the decapper to remove the cap Discard the green cap in the appropriate waste container Return the BacTx Reaction Tube to its original position in the rack Repeat this step with the remaining BacTx Reaction Tubes working from left to right Verify that the IDs for the BacTx Reaction Tubes match the IDs on the 1 5 mL microfuge tubes located in the row directly beneath them Starting with the 1 5 mL microfuge tube in the farthest left position P1 in the example in Figure 1 use a clean sterile pipette tip to transfer 0 3 mL from the 1 5 mL microfuge tube into the BacTx Reaction Tube in the position directly above it Repeat this step with the remaining 1 5 mL microfuge and BacTx Reaction Tubes working from
11. perfringens did not grow during the Time to Detection study Propionibacterium acnes was not detected by the BacTx Assay or by Quantitative Plate Culture during the Time to Detection study cOne or more processed samples gave an ABORT result due to high sample turbidity In these instances the results represented are for an 11 fold dilution of the processed sample as described in the section on INTERPRETATION OF RESULTS The first time point at which 10 out of 10 samples were detected by the BacTx Assay is shaded in grey LR WBDP Study Description To determine the time to detection of bacteria growing in LR WBDP units low titers 0 6 5 0 CFU mL of bacteria were spiked into individual LR WBDP units and incubated on a platelet shaker for 7 days The same bacterial strains used in the analytical sensitivity study above See Table 1 were used for the Time to Detection Study Platelet units spiked with sterile PBS were used as a negative control and also incubated for the 7 days At approximately 48 hours after inoculation a small volume of platelets was withdrawn from the contaminated and uncontaminated units These volumes were each combined with volumes from 5 other sterile LR WBDP units in order to create contaminated and uncontaminated platelet pools respectively Ten samples from the contaminated pool and three samples from the uncontaminated pool were blinded and tested with the BacTx Assay If less than 10 of the contaminated samples
12. plates was performed on the platelet pool to determine the final bacterial concentration in the pool Results Table 9 shows the growth of bacteria in LR WBDPs and the results of the Prozone testing with the BacTx Assay The bacteria titers recorded over the 5 day period for the aerobes indicated that the eight aerobes had either attained stationary phase growth or a very high titer gt 1E9 CFU mL by day 5 A greater than expected decrease in the bacterial concentration was observed upon pooling of the bacteria containing unit with the 5 in date LR WBDP units for almost all of the aerobes tested This loss of viability was attributed to the susceptibility of the stationary phase bacteria to the bactericidal properties of in date LR WBDP platelets upon pooling For each of the den strains none of the 30 samples tested with the BacTx Assay were falsely negative These results indicate that the BacTx Assay is not affected by Prozone effects caused by high bacterial concentrations attainable in LR WBDP pools Table 9 Prozone Testing Results for the LR WBDP Study Bacterial Concentration CFU mL Samples Detected Individual Inoculated WBDP Unit DAY 5 by BacTx BACTERIA DAY 0 DAY 3 DAY 4 DAY 5 6 unit pool out of 30 Bacillus cereus 2 1 x 106 n d 26x10 37x10 9 0 x 105 Escherichia coli 1 3x105 47x108 44x108 45x108 1 1x 108 Klebsiella oxytoca 7 3x104 39x108 1 0x109 1 3x 109 1 6 x 108 Pseudomonas aeruginosa 3 0x105 3 0x109 3 0
13. to ID on LRAP Unit Transfer at least 1 mL of platelets to the sterile collection tube using one of two methods e To use an approved sterile connecting device refer to the device manufacturer s instructions dis 2 3 4 5 e To sample from a freshly created segment a b c d Use a tube stripping device to express the platelets present in the segment back into the platelet unit Without releasing the stripping device invert the platelet unit several times Release the stripping device and allow platelets to flow into the empty segment Strip the segment back into the platelet unit Repeat the segment stripping and mixing step several times to obtain a well mixed sample Use a tube sealing device to seal off a segment of tubing sufficiently long to contain 1 mL of platelets For tubing with an internal diameter of 3 mm use a 7 9 length follow the manufacturer s instructions on proper use of the tube sealing device Detach the segment To transfer the platelets into the sterile collection tube f1 Clean one end of the segment using a sterile alcohol prep pad f2 Place the clean end of the segment directly over the sample collection container f3 Cut open the end of the segment using a clean cutting edge scissors scalpel or segment sampling device and drain the platelets into the collection tube f4 To increase the flow rate of platelets from the segment either squeeze the segment from the cl
14. 6 100 testing verified sterile by plate culture Page 12 of 16 Effective Date August 16 2013 CF B502 706 LR WBDP Study Results For the inter assay reproducibility of negative assays 209 out of 210 assays gave the expected negative result 99 5 concordance with a lower one sided 95 confidence limit of 97 9 For the inter lot and inter site reproducibility of negative samples as described in the Specificity Study above 431 assays of 432 sterile 6 unit platelet pools gave the expected negative result a specificity of 99 8 with a lower one sided 95 confidence limit of 99 0 No statistically significant difference in reproducibility was observed among the three lots p 1 0 Fisher Freeman Halton test used for specificity testing For the inter lot and inter site reproducibility testing with the 11 member test panel the expected BacTx result was observed with 395 out of 396 samples No statistically significant difference in reproducibility was observed between the three sites or between the three lots p 1 0 Fisher Freeman Halton test All 360 bacterial panel members were successfully detected with the BacTx Assay as shown in Table 5 POTENTIALLY INTERFERING SUBSTANCES STUDY Turbidity Causing Substances LRAP Study Description In the BacTx Assay System a dedicated photometer is used to monitor the change in absorbance of green colored light that passes through the BacTx Reaction Tube during the 30 minute assay Since t
15. DP Study of Immunoassay Interferents Description As described in the Principle section above the BacTx Assay is an enzyme based assay and is technologically different than immunoassays such as ELISA or lateral flow Peptidoglycan in the prepared platelet sample is bound by a peptidoglycan recognition protein which has a peptidoglycan binding domain that is similar in structure to T7 lysozyme In contrast lateral flow immunoassays typically use mono and polyclonal antibodies from multiple sources mouse rabbit goat for both analyte capture and detection Interference of immunoassays caused by endogenous substances is well documented Substances that interfere with antibody binding are heterophilic antibodies autoimmune antibodies such as rheumatoid factor and antinuclear antibodies and human anti animal antibodies To demonstrate that the BacTx Assay is not affected by these common immunoassay interferents pooled platelets were resuspended in patient plasma or serum containing heterophilic antibodies autoimmune antibodies and human anti mouse _antibody HAMA and tested with Table 8 Samples for Immunoassay Interference Testing in LR WBDP Study the BacTx Assay The platelet samples __ Sample Concentration containing the immunoassay interferent were Heterophilic plasma Positive heterophile antibody test tested using the test panel described in Table 6 ANA Positive qualitative test dsDNA 10 4 123 I
16. U mL Three kit lots were used to prepare and test the Autoimmune Antibodies samples 10 replicates of each test panel member were tested for each interfering l RF 67 1075 IU mL substance The immunoassay interferents Human anti mouse antibody HAMA 37 329 ng mL tested are described in Table 8 Page 14 of 16 Effective Date August 16 2013 CF B502 706 LR WBDP Study Results Out of the 370 positive samples tested all 370 were successfully detected with the BacTx Assay Out of the 91 negative samples tested all 91 tested negative in the BacTx Assay Based on these results the Immunoassay interference substances tested in this study do not interfere with the BacTx Assay LR WBDP STUDY OF PROZONE HOOK EFFECT Description The hook effect is a type of assay interference most commonly associated with immunoassays in which the concentrations of antigen are in such excess that the capture antibodies and labeled antibodies do not simultaneously bind the same analyte unit and leads to falsely negative test results While the BacTx Assay is not an immunoassay testing was performed to determine if a hook effect is present at high concentrations of bacteria present in platelet samples Single LR WBDP units 3 days old were inoculated with moderate concentrations 10 10 CFU mL of each of the eight aerobic bacterial strains tested in the analytical sensitivity study The platelet units were incubated for five days to
17. ach empty 2 mL microfuge tube in the microfuge rack Close the cap on each tube after addition of Lysis Reagent With a single channel 1000uL micropipettor pipet 0 5 mL of Neutralization Reagent into each empty 1 5 mL microfuge tube in the microfuge rack Close the cap on each tube after addition of Neutralization Reagent Starting with the platelet sample in the farthest left position in the rack P1 in the example in Figure 1 use a micropipettor to transfer 0 5 mL of platelets to the 2 mL microfuge tube containing Lysis Reagent directly above it Immediately close the cap on the 2 mL microfuge tube invert the tube 3 times to mix and return the 2 mL microfuge tube to its original position in the rack Verify that platelets were transferred only from the platelet sample located directly beneath the 2 mL microfuge tube Do not discard the platelet samples Repeat this step for the remaining platelet samples working from left to right Centrifuge the 2 mL microfuge tubes at 14 000 20 000xg for 3 min Once centrifugation ends carefully return the 2 mL microfuge tubes to the tube rack making sure that the tubes are placed in exactly the same position as before Verify that the IDs for the 2 mL microfuge tubes match the IDs on the platelet samples Uncap the 2 mL tube on the farthest left position in the rack P1 in the example in Figure 1 and decant the supernatant into the waste container by holding the tube upside down for 3 seconds Remove t
18. acterial strain listed in Table 4 and three lots were also used to test 10 negative samples For each set of 10 samples 3 samples were prepared with one lot another 3 samples with a second lot and the remaining 4 samples with a third lot A summary of the sample conditions tested can be found in Table 7 The concentrations of interfering substances were tested at pathological levels compared to normal or reference levels LR WBDP Study Description To test each of the interfering substances or conditions a six unit pool of LR WBDPs containing the interfering substance was tested using the test panel of bacteria described in Table 6 Three kit lots were Table 6 Test Panel for LR WBDP Interfering Substances Study used to prepare and test the samples by three different users 10 Sample Logs Above replicates of each test panel member were tested for each interfering Limit of Detection substance The concentration of the interfering substance was Escherichia coli 1 0 1 3 measured in the platelet pool and if sufficient volume was available Staphylococcus aureus 11 12 prior to pooling A summary of the sample conditions tested can be i found in Table 7 Bacillus cereus 1 4 Staphylococcus epidermidis 0 6 1 0 LRAP Study Results Klebsiella oxytoca 0 9 1 2 Out of the 1300 positive samples tested with potential interferents Pseudomonas aeruginosa 0 8 1 1 100 were detected with the BacTx Assay Out of the 130 negative Streptococcus
19. bing with an internal diameter of 3 mm use a 4 6 length follow the manufacturer s instructions on proper use of the tube sealing device Detach the segment To transfer the platelets into the sterile collection tube f1 Clean one end of the segment using a sterile alcohol prep pad f2 Place the clean end of the segment directly over the sample collection container f3 Cut open the end of the segment using a clean cutting edge scissors scalpel or segment sampling device and drain the platelets into the collection tube f4 To increase the flow rate of platelets from the segment either squeeze the segment from the closed end and push the sample toward the opening or cut open the closed end of the segment f5 Place the lid on the collection tube f6 Discard the emptied segment and segment sampling device if using as biohazardous waste f7 Clean scissors or scalpel with a sterile alcohol prep pad Pooled platelet samples should be processed within 2 hours after removal from the platelet pool Repeat steps 1 4 with up to eight 8 platelet pools Page 4 of 16 Effective Date August 16 2013 CF B502 706 TEST PROCEDURE PERFORM THE FOLLOWING STEPS BEFORE BEGINNING TEST e Remove the BacTx Reaction Tubes Lysis Reagent Extraction Reagent and Neutralization Reagent from the refrigerator Keep these reagents at room temperature 19 26 C for 45 minutes prior to use e Follow instructions provided in the BacT
20. f the labeling procedure is illustrated in Figure 1 1 Place the platelet samples from the Sample Collection section above in a single row in a microfuge tube rack 2 Place 2 mL microfuge tubes in a row above the platelet samples with one 2 mL microfuge tube for each platelet sample Using a fine tip permanent ink lab marker label each 2 mL microfuge tube with the same ID of the platelet sample beneath it Verify that the ID written on the 2 mL microfuge tube is identical to the platelet sample If the ID is incorrect discard the 2 mL microfuge tube and label a new 2 mL microfuge tube Use only 2 mL microfuge tubes that are supplied with the BacTx Kit 3 Place 1 5 mL microfuge tubes in a row above the 2 mL microfuge tubes with one 1 5 mL microfuge tube for each platelet sample Using a fine tip permanent ink lab marker label each 1 5 mL microfuge tube with the same ID on the 2 mL microfuge tube directly beneath it Verify that the ID written on the 1 5 mL microfuge tube is identical to the 2 mL Page 5 of 16 Effective Date August 16 2013 CF B502 706 microfuge tube beneath it If the ID is incorrect discard the 1 5 mL microfuge tube and label a new 1 5 mL microfuge tube Use only 1 5 mL microfuge tubes that are supplied with the BacTx Kit 4 Place BacTx Reaction Tubes in a row above the 1 5 mL microfuge tubes with one Reaction Tube for each platelet sample In the Sample ID space printed on the label on each BacTx
21. h the OSHA Standards on Bloodborne Pathogens 29 CFR 1910 1930 5 Wear proper personal protection including safety glasses while running the assay 6 Cap bottles tightly after use to avoid contamination 7 Room temperature should be between 19 26 C for all steps 8 Keep lid of BacTx Reader on during an assay Do not attempt to remove tubes from BacTx Reader during an assay REMOVAL OF THE LID DURING AN ASSAY WILL ABORT THE ASSAY RUN 9 Do not use reagents past their expiration date Once opened the BacTx Bacterial Detection Kit Reagent Pack can be used for 21 days 10 The BacTx Reaction Tubes contain human serum albumin No known test method can offer complete assurance that products derived from human sources will not transmit infection Therefore all human sourced materials should be considered potentially infectious It is recommended that the BacTx Reaction Tubes be handled in accordance with OSHA Standards on Bloodborne Pathogens using Universal Precautions The human serum albumin contained within the BacTx Reaction Tubes were found non reactive for antibodies to human immunodeficiency virus types 1 and 2 HIV 1 HIV 2 HIV 1 antigen HIV 1 AG hepatitis C virus HCV and hepatitis B surface Antigen HBsAg at FDA approved centers 11 The Positive Control contains components sourced from inactivated microorganisms No known test method can offer complete assurance that products derive from inactivated microorganis
22. he determination of a Fail or Pass result in the BacTx assay is based on whether the absorbance exceeds 0 5 during the assay endogenous substances or specific platelet conditions that contribute to sample turbidity may potentially interfere with the BacTx assay These include hyperproteinemia hypergammaglobulinemia hemolysis hypercholesterolemia and lipemia In addition specific platelet conditions may also interfere with the proper functioning of the Lysis Extraction or Neutralization Reagents used during sample preparation These conditions include high and low pH platelet concentration and red blood cell concentration The concentrations of interfering substance were tested at pathological levels compared to normal or reference levels To test each of the substances or conditions described above 100 positive samples 10 samples for each of the 10 bacterial strains in which the concentration of bacteria was 0 5 1 5 logs above the limit of detection LOD determined during the analytical sensitivity study for each strain and 10 negative samples were prepared using LRAP containing the interfering substance or condition All of the LRAP units used for this study were 5 days old or less The concentrations of the ten bacterial strains used for interference testing are the same as for the reproducibility study They are listed in Table 4 Three lots of BacTx Bacterial Detection Kits were used to prepare and test 10 samples for each b
23. he remaining droplets of supernatant by tapping the upside down tube against the rim of the waste container or by tapping the upside down tube on a clean dry laboratory tissue or paper towel Close the cap on the tube Inspect the tube to verify that the white gelatinous pellet is still present If the pellet is missing a new sample must be prepared using another 0 5 mL of platelets Return the 2 mL tube to its position in the rack Repeat this step for the remaining 2 mL microfuge tubes working from left to right Use a micropipettor and a clean sterile pipet tip to transfer 0 5 mL of Extraction Reagent into the 2 mL microfuge tube in the farthest left position in the rack P1 in the example in Figure 1 Holding the 2 mL microfuge tube in one hand homogenize the pellet by pipetting up and down 10 times with the pipettor set at 0 5 mL allowing the pipettor piston to return to its initial position before fully depressing the piston This technique will ensure that the entire volume will pass through the pipet tip To avoid excessive foaming always keep the opening of the pipette tip close to the bottom of the microfuge tube when pipetting up and down Return the 2 mL microfuge tube to its original position in the rack Without changing the pipette tip transfer 0 5 mL of the suspension in the farthest left 2 mL microfuge tube P1 in the example in Figure 1 to the 1 5 mL microfuge tube containing Neutralization Reagent directly above it Close the cap
24. imit based on 2 mL of packed red blood cells in 500 mL plasma volume as described in b Upper limit based on complete hemolysis of 0 4 hematocrit Based on normal serum total protein level from David C Dugdale Total Protein http Avww nIm nih gov medlineplus medlineplus html David Zieve Rev May 2009 U S National Library of Medicine U S Department of Health and Human Services National Institutes of Health Accessed June 22 2011 lt http www nim nih gov medlineplus ency article 003483 htm gt sBased on serum triglyceride reference range classified as desirable from Triglycerides http www labtestsonline org March 2011 American Association for Clinical Chemistry Accessed June 22 2011 lt http Awww labtestsonline org understanding analytes triglycerides sample html gt hBased on serum cholesterol reference range classified as desirable from Cholesterol htto www labtestsonline org March 2011 American Association for Clinical Chemistry Accessed June 22 2011 lt http Avww labtestsonline org understanding analytes cholesterol test html gt Based on normal serum levels from David C Dugdale Quantitative Nephelometry http Awww nIm nih gov medlineplus medlineplus html David Zieve Rev June 2010 U S National Library of Medicine U S Department of Health and Human Services National Institutes of Health Accessed June 22 2011 lt http www nim nih gov medlineplus ency article 003545 htm gt LR WB
25. ld Bacteria detected above assay threshold Falk The result should be confirmed by retesting in duplicate The BacTx Assay software will interpret the result for each tube as Pass or Fail automatically For samples which have not generated a FAIL result during the 30 minute test period the result will be interpreted as PASS A FAIL result will be displayed as soon as it is detected accompanied by an audible alarm A result log will be created and saved for each run See User Manual for further information on operation of the BacTx Reader and software A PASS result means that no bacteria were detected in the sample above the assay threshold A PASS result is valid for up to 24 hours post sampling for LRAP For pools of LR WBDP the BacTx Assay is performed within 4 hours prior to transfusion A FAIL result should be confirmed by re testing in duplicate If either of the retests also FAIL ahis means that bacteria were detected at a concentration above the assay threshold A flowchart of the BacTx Assay Testing algorithm is shown in Chart 1 An immediate ABORT displayed by the BacTx Reader within 30 seconds of initiating an assay i e within 30 seconds of pressing the Start All Samples button may indicate a highly contaminated platelet unit that is too turbid to be read by the BacTx Reader In the case of an immediate ABORT dilution and retesting of the sample is recommended as follo
26. left to right Page 7 of 16 Effective Date August 16 2013 CF B502 706 Vortex each of the BacTx Reaction Tubes for 3 seconds at maximum speed to mix and return each BacTx Reaction Tube to its original position in the rack Verify that the IDs on the BacTx Reaction Tubes match the IDs on the 1 5 mL microfuge tubes located in the row directly beneath them Place the BacTx Reaction Tube located in the farthest left position in the tube rack into tube slot 1 of the BacTx Reader Make sure that the bottom of the Reaction Tube contacts the bottom of the tube slot in the BacTx Reader Enter the platelet ID into the Sample ID field 1 in the BacTx software window Verify that the sample ID entered into the Sample ID field matches the ID on the 1 5 mL 2mL and platelet sample tubes in the same column on the tube rack Repeat this step with the remaining Reaction Tubes working from left to right The ID of the technologist 2 40 characters should be entered into the Technician ID field at the top of the BacTx software window Once all the Reaction Tubes have been placed in the BacTx Reader click on the Start All Samples button The test will run for 30 minutes Save the BacTx Assay results by following the instructions in the BacTx Reader User Manual Follow institution guidelines for handling data files INTERPRETATION OF RESULTS 6 Result Interpretation PASS No bacteria detected above assay thresho
27. ms will not transmit infection Biosafety level 2 or other appropriate biosafety practices should be used for materials that contain or are suspected of containing infectious agents 12 Performance characteristics of the BacTx Bacterial Detection Kit were established using LRAPs with ACD A anticoagulant and LR WBDPs with CP2D anticoagulant 13 Users should validate the BacTx Assay System according to a scientifically sound sampling method LIMITATIONS 1 LRAP and LR WBDP platelet samples should be tested within 2 hours after sampling 2 LR WBDPs to be tested should be prepared within 8 hours of collection and suspended in plasma 3 LRAPs to be tested should be suspended in plasma 4 For optimal performance leukocyte reduction of LR WBDP should take place at the time of processing pre storage Pooling with subsequent filtration prior to BacTx sampling may reduce the bacterial load in the leukocyte reduced platelet pool to a level that is lower than the limit of detection of the BacTx Assay 5 For optimal performance LRAPs should be leukoreduced at the time of the apheresis collection process Filtration of apheresis products prior to BacTx sampling may cause false negative results 6 The recommended optimal sampling time of 72 hours was based only on the detection of eight aerobic organisms The anaerobic organisms tested did not grow in the Time to Detection Study Consult the section of Performance Characteristic below for
28. n Tubes by inserting the barbed end of the decapper under the inner lip of the cap Gently twist the decapper to remove the cap Discard the green caps in the appropriate waste container 4 Using a calibrated single channel 1000uL micropipettor transfer 0 3 mL of positive and negative control to the respective BacTx Reaction Tubes 5 Vortex each BacTx Reaction Tube for 3 seconds at maximum speed to mix 6 Immediately place the BacTx Reaction Tubes in the BacTx Reader Make sure that the lt pottoms of the Reaction Tubes contact the bottom of the wells in the BacTx Reader Replace the cover on the BacTx Reader 7 Inthe BacTx Assay window on the laptop PC click on the Start All Samples button The test will run for 30 minutes Save the assay results by following the instructions in the BacTx Reader User Manual 8 At the end of the 30 minute assay the BacTx Assay Software should report a result of PASS for the negative control tube and FAIL for the positive control tube If control results do not meet quality assurance acceptance criteria contact Immunetics Do not initiate testing of platelet pools in the absence of satisfactory performance of controls PREPARATION OF BACTX SAMPLES Labeling Microfuge Tubes and BacTx Reaction Tubes This section describes the proper labeling of both microfuge tubes and Reaction Tubes in order to minimize the risk of sample mix up and misidentification An example o
29. nded and tested with the BacTx Assay If less than 10 of the contaminated samples were detected at the 48 hour time point this testing was repeated at approximately 72 hours after inoculation All LRAPs were also tested at approximately 7 days after inoculation When BacTx testing was performed quantitative plate culture QPC was carried out to determine the bacterial titer in the contaminated LRAP unit at that time point Culture plates made at 24 hours and 7 days after inoculation from the spiked units were submitted for bacterial identification to confirm the strain that proliferated in the LRAP was the same as the strain that was inoculated Page 9 of 16 Effective Date August 16 2013 CF B502 706 BacT ALERT testing was performed at day 0 to confirm sterility of the LRAP unit Testing was conducted at two sites with multiple lots of BacTx Assay Kits LRAP Siudy Results The results of the BacTx testing and quantitative plate culture are shown in Table 2 Of the 8 aerobes tested six species were detected at 48 hours at both sites S agalactiae was detected at 48 hours at one site and at 72 hours at the second site P aeruginosa was detected at 72 hours at both sites As expected Clostridium perfringens did not grow during the Time to Detection study Propionibacterium acnes was not detected by the BacTx Assay or by Quantitative Plate Culture during the Time to Detection study Based on these results the optimal time to detection fo
30. negative and positive spiked assays The analytical sensitivity study above served as an inter assay reproducibility study for spiked pools as 10 10 positive BacTx Assay results were required to be positive for the presence of Table 5 Inter lot and Inter site Reproducibility Test Panel for LR WBDP Study bacteria at a given titer before the assay Logs was validated as positive For the inter Above Epered Detected Detection assay reproducibility of negative BacTx Sample ID Limit of BacTx out of 36 Rate assays 21 unique sterile 6 unit LR Detection Result WBDP pools were tested with 3 kit lots at DETEN 3 10 FAIL e 100 a single site with 10 replicates of the Escherichia coli Cee ee BacTx Assay for a total of 210 assays Staphylococcus aureus 1 2 FAIL 36 100 For the inter lot and __ inter site Bacillus cereus 1 4 FAIL 36 100 reproducibility a test panel was used for Staphylococcus epidermidis 1 0 FAL 36 100 testing at three sites using three lots of Klebsiella oxytoca 12 FAIL 36 100 BacTx Assay Kits on three different days i The test panel consisted of 10 bacterial Pseudomonas aeruginosa 0 8 FAIL 6 100 members and one negative member and Streptococcus agalactiae 1 2 FAIL 36 100 the composition of the positive panel Serratia marcescens 1 2 FAIL 36 100 members is listed in Table 5 Sterile 6 Clostridium perfringens 0 8 FAL 36 1o Unt sewer poole Were USEAN Propionibacterium acnes 1 2 FAIL 3
31. ntains 4 morpholinopropanesulfonate which is classified as Irritant Xi Xi R 36 37 38 Irritating to eyes respiratory system and skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 35 This material and its container must be disposed of in a safe way 837 39 Wear suitable gloves and eye face protection S46 If swallowed seek medical advice immediately and show this container or label STORAGE AND STABILITY e Store BacTx Reaction Tubes Extraction Reagent Controls and Neutralization Reagent at 2 8 C e Store Lysis Reagent and microfuge tubes at 2 26 C e Use BacTx Reaction Tubes Lysis Reagent Extraction Reagent Positive Control Negative Control and Neutralization Reagent prior to expiration date on kit label Once opened the BacTx Bacterial Detection Kit Reagent Pack can be used for 21 days RECOMMENDED TECHNIQUES PROPER CLEANING OF WORK AREA This test can be performed on the bench top as long as careful laboratory technique is used and regular cleaning is performed to prevent the introduction of contaminants into test reagents e Clean surfaces where the test will be performed with disinfectant 70 alcohol spray or similar and clean paper towel on a routine basis as good laboratory practice Ideally disinfection should be performed before performing each test e The micropipettor microfuge tube rack vortexer and centrifuge required for sample p
32. ntalion 1 7 2 4 x10 platelets mL 2 0 2 4 x10 platelets mL 45 65 mL for LR WBDP Low pH 5 5 5 68 5 5 6 26 Normal pH 7 15 7 36 7 16 7 21 i High pH 8 4 8 8 8 5 TASTAR Red Blood Cells 0 7 hematocrit 0 7 hematocrit 0 0 4 Hemolysis 0 25 g dL hemoglobin 0 25 g dL hemoglobin 0 0 13 g dLe Hyperproteinemia 10 1 10 7 g dL 10 4 g dL i Hypoproteinemia 2 3 3 3 g dL 3 2 g dL Domo ae Lipemia 206 484 mg dL 1350 mg dL lt 150 mg dL9 Hypercholesterolemia 212 436 mg dL 234 mg dL lt 200 mg dLh Hypergammaglobulinemia IgG 3600 4000 mg dL 3500 mg dL 560 1800 mg dL Hypergammaglobulinemia IgM 730 mg dL 1000 mg dL 45 250 mg dLi Hypergammaglobulinemia IgA 1675 mg dL 2205 mg dL 100 400 mg dL aDecember 2007 Guidance for Industry and FDA Review Staff Collection of Platelets by Automated Methods Rockville MD City State U S Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research Downloaded on June 22 2011 21 CFR Chap 1 Subpart C section 640 24 4 1 12 Edition Normal range of blood serum from Merck Manual of Diagnosis and Therapy Section Endocrine and Metabolic Disorders Subject Acid Base Regulation and Disorders Topic Introduction http www merckmanuals com James L Lewis Ill MD ed July 2008 Merck Sharp amp Dohme Corp Accessed June 22 2011 lt http www merckmanuals com professional sec12 ch157 ch157b html gt dUpper l
33. on Table 3 Summary of BacTx Testing and Quantitative Plate Culture Results for LR WBDP Time to Detection Study 48 hours after 72 hours after 168 hours after Bacterial Inoculation Inoculation Inoculation Titer in unit Detected Bacterial Detected Bacterial Detected Bacterial at by BacTx Titer in by BacTx Titer in by BacTx Titer in inoculation Assay Pool Assay Pool Assay Pool Species ATCC CFU mL outof10 CFU mL outof10 CFU mL out of 10 1 1 9 2 2x107 0 2 6x107 2 5 0 6 5x107 0 6 0x107 1 2 2 5 2x106 0 4 0x107 2 2 0 1 2x106 0 1 9x108 1 2 0 2 8x107 0 1 5x107 2 3 5 9 9x107 0 2 4x107 1 0 7 3 5x108 0 3 0x108 2 1 5 7 5x107 0 2 6x108 1 2 1 2 EE EA Escherichia coli 25922 Pseudomonas aeruginosa 27853 Klebsiella oxytoca 43863 Serratia marcescens 43862 Aerobe 1 8 3 6x105 5 4x105 3 7 5 4x106 8 9x105 3 4 1 0x107 5 6x107 2 0 8 7x108 7 0x107 Bacillus cereus 11778 Staphylococcus aureus 27217 Staphylococcus epidermidis 49134 Streptococcus agalactiae 12386 2 7 6 6x102 1 5x107 3 4 7 0x104 5 5x107 0 8 8 7x102 6 2x108 2 1 3 6x108 4 9x108 0 6 0 2 3 0 0 47 0 0 2 3 7 0 0 0 0 0 Second of two TTD studies performed at Site 1 with S agalactiae In the first attempt S agalactiae did not readily proliferate in the platelet unit with measured titers of 60 and 660 CFU mL at 48 and 72 hours after inoculation respectively A Time to Detection
34. osed end and push the sample toward the opening or cut open the closed end of the segment f5 Place the lid on the collection tube f6 Discard the emptied segment and segment sampling device if using as biohazardous waste f7 Clean scissors or scalpel with a sterile alcohol prep pad LRAP samples should be processed within 2 hours of removal from the platelet unit Repeat steps 1 3 with up to eight 8 LRAP units For each LR WBDP pool to be tested Write Pool ID with fine tip permanent ink lab marker on a sterile collection tube microfuge or capped centrifuge tube Verify that ID written on collection tube is identical to ID on Pool Transfer at least 0 5 mL of platelets to the sterile collection tube using one of two methods e To use an approved sterile connecting device refer to the device manufacturer s instructions 1 2 3 4 5 e To sample from a freshly created segment a b c d Use a tube stripping device to express the platelets present in the segment back into the platelet unit Without releasing the stripping device invert the platelet unit several times Release the stripping device and allow platelets to flow into the empty segment Strip the segment back into the platelet unit Repeat the segment stripping and mixing step several times to obtain a well mixed sample Use a tube sealing device to seal off a segment of tubing sufficiently long to contain 0 5 mL of platelets For tu
35. ples include a 50 mL conical tube or a sterile specimen cup 7 Sterile containers with lids to receive the initial platelet samples prior to sample preparation microfuge or centrifuge tubes 8 Sterile sampling device or tube stripper tube sealer clean cutting edge scissors scalpel or segment sampling device and single use alcohol prep pads 9 BacTx Reader Immunetics Part BTX 001 10 Laptop PC with BacTx Assay Software Immunetics Part AA C018 000 11 Personal protective equipment including safety glasses 12 Canned compressed air for cleaning tube slots on BacTx Reader when necessary Refer to BacTx Reader User Manual Immunetics Doc GN U0001 for guidance 13 Clean plastic forceps for removing debris from tube slots when necessary Refer to BacTx Reader User Manual Immunetics Doc GN U0001 for guidance WARNINGS 1 For in vitro diagnostic use 2 Use nitrile gloves preferably powder free at all times when handling kit components Latex gloves should not be worn as latex particulates may cause erroneous results 3 For optimal reproducible and accurate performance of the test technicians should be trained on the procedure and must follow all instructions in this package insert Deviations from instructions may lead to false results 4 All samples and materials used in the BacTx Assay should be disposed of in a biohazard waste container Handle all platelet containing solutions in accordance wit
36. r all of the bacterial strains that proliferate in platelet units is 72 hours Table2 Summary of BacTx Testing and Quantitative Plate Culture Results for LRAP Time To Detection Study 48 hours after 72 hours after 168 hours after Bacterial Inoculation Inoculation Inoculation Titer in unit Detected Bacterial Detected Bacterial Detected Bacterial at by BacTx Titer in by BacTx Titer in by BacTx Titer in inoculation Assay LRAP Assay LRAP Assay LRAP Species ATCC CFU mL outof10 CFU mL outof10 CFU mL out of 10 mL 1 8 10 3 0x108 5 2x108 3 8 10 6 4x108 4 6x108 3 1 7 7x10 1 1x109 2 6 9 2x108 5 1x108 2 7 1 9x108 3 9x108 3 0 3 4x108 10 5 4x108 1 3 1 8x108 10 8 1x108 2 1 4 1x108 2 0x109 5 3 8 4x106 2 3x106 3 0 6 2x106 8 3x106 3 9 1 5x107 1 7x108 4 4 TNTCa 6 6x108 i 1 6 5 0x103 1 9x107 Staphylococcus epidermidis 49134 lt 1 9 6 0x104 5 2x108 1 3 7 4 9x106 5 0x108 2 24 0 5 4x102 10 3 1x107 1 3 7 0 0 2 2 5 0 1 0 2 0 Escherichia coli 25922 Pseudomonas aeruginosa 27853 Klebsiella oxytoca 43863 Serratia marcescens 43862 Bacillus cereus 11778 Staphylococcus aureus 27217 Streptococcus agalactiae 12386 Propionibacterium acnes 11827 Anaerobe 3 4 2 1 Clostridium perfringens 3629 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q aTNTC Too numerous to count after dilution plating gt Clostridium
37. re This study also served as a test of reproducibility of negative assays as described in the reproducibility study section LR WBDP Study Results Out of the 432 BacTx Assays 431 were negative for the presence of bacteria in the BacTx Assay i e a PASS result corresponding to a specificity of 99 8 with a lower one sided 95 confidence limit of 99 0 REPRODUCIBILITY STUDY LRAP Study Description Reproducibility of the BacTx Assay Kit was determined inter assay inter lot and inter site for both negative and positive spiked LRAPs The analytical sensitivity study above served as an inter assay reproducibility study for spiked LRAPs as 10 10 positive BacTx Assay results were required to be positive for the presence of bacteria at a given titer before the assay was validated as positive For the inter assay reproducibility of negative BacTx assays 21 unique sterile LRAP units were tested with 3 kit lots at a single site with 10 replicates of the BacTx Assay for a total of 210 assays For the inter lot and inter site reproducibility a test panel was used for testing at three sites using three lots of BacTx Assay Kits on three different days The test panel consisted of 10 bacterial members and one negative member and the composition of the positive panel members is listed in Table 4 The sterility of LRAPs used in the study was confirmed by either BacT ALERT culture or plate culture LRAP Siudy Results For
38. reparation should be routinely cleaned to avoid contamination of samples e Workspace should be a draft free area with low foot traffic PROPER HANDLING OF SAMPLES e Use nitrile gloves preferably powder free at all times when handling kit components Latex gloves should not be worn as latex particulates may cause erroneous results e Avoid touching tops of tubes and caps as this could potentially introduce contaminants e Gloves should be changed frequently to avoid contamination of samples e Keep all tubes closed opening only briefly when necessary This will prevent contamination from the air or other surfaces e During sample preparation use only 1 5 mL and 2 0 mL microfuge tubes that are supplied with the BacTx Kit Page 3 of 16 Effective Date August 16 2013 CF B502 706 SAMPLE TYPE For optimal performance a Sample LRAPs from 72 hours after collection of the unit through the expiration date b Sample LR WBDP pools 72 hours after collection of the freshest unit in the LR WBDP pool through the expiration date of the oldest unit in the pool k Platelets should be sampled from units stored on a platform shaker at 20 24 C 1 2 SAMPLE COLLECTION Sampling volume is 1 0 mL for LRAP and 0 5 mL for LR WBDP pools For each LRAP unit to be tested Write Unit ID with fine tip permanent ink lab marker on a sterile collection tube microfuge or capped centrifuge tube Verify that ID written on collection tube is identical
39. t 7 598 054 and other pending patents MATERIALS SUPPLIED 1 BacTx Reaction Tubes Part CB B005 032 32 sealed glass tubes each containing lyophilized detection reagent for 1 test 2 Lysis Reagent Part CC L001 060 Aqueous solution containing sodium hydroxide surfactant and n butanol 1 x 60 mL Extraction Reagent Part CC E001 060 Aqueous sodium hydroxide solution 1 x 60 mL Neutralization Reagent Part CC N001 060 4 morpholinopropanesulfonate MOPS solution with gentamycin added as a preservative 1 x 60 mL 5 Positive Control Part CB P012 000 Purified peptidoglycan from Bacillus subtilis suspended in MOPS buffer with gentamycin added as a preservative 2 x 0 9 mL Negative Control Part CB N031 000 MOPS buffer with gentamycin added as a preservative 1 x 1 8 mL Sterile 2 mL Microfuge tubes microcentrifuge tubes Part AA T029 000 qty 50 Sterile 1 5 mL Microfuge tubes microcentrifuge tubes Part AA T032 000 qty 50 Poe rp EQUIPMENT REQUIRED BUT NOT PROVIDED 1 Bench top microcentrifuge with capacity to spin at 14 000 20 000 x g examples include Eppendorf Models 5418 and 5424 Beckman Coulter Microfuge 18 and Thermo Scientific Sorvall Legend Micro 21 2 Rack for 1 5 2 mL microfuge tubes 3 Vortexer 4 A calibrated single channel 1000uL micropipettor 5 Sterile disposable 1000uL pipette tips preferably with filters 6 Clean disposable container for liquid waste exam
40. the BacTx Assay Gram Positive robe Limit of Limit of GP or r ATCC Detection with Detection with Gram An erob Number LR WBDP LRAP Negative CFU mL CFU mL GN Escherichia coli GN Aerobe 25922 Pseudomonas aeruginosa GN Aerobe 27853 Klebsiella oxytoca GN Aerobe 43863 Serratia marcescens GN Aerobe 43862 Bacillus cereus GP Aerobe 11778 Staphylococcus aureus GP Aerobe 27217 Staphylococcus epidermidis GP Aerobe 49134 Streptococcus agalactiae GP Aerobe 12386 Clostridium perfringens GP Anaerobe 3629 Propionibacterium acnes GP Anaerobe 11827 TIME TO DETECTION BACTERIAL GROWTH STUDY LRAP Study Description To determine the time to detection of bacteria growing in LRAP units low titers 1 3 5 8 CFU mL of bacteria were spiked into LRAP units and allowed to proliferate for 7 days The same bacterial strains used in the analytical sensitivity study above See Table 1 were used for the Time to Detection Study In order to ensure growth of bacteria in LRAPs for each strain tested four LRAP units were spiked the LRAP that best supported growth was used for testing and the other units were discarded An LRAP spiked with sterile PBS were used as a negative control and also incubated for the 7 days At approximately 48 hours after inoculation a small volume of platelets was withdrawn from the contaminated and uncontaminated LRAPs Ten samples from the contaminated LRAP and three samples from the uncontaminated LRAP were bli
41. the inter assay reproducibility of negative assays 210 out of 210 assays gave the expected negative result 100 concordance with a lower one sided 95 confidence limit of 98 7 For the inter lot and inter site reproducibility of negative samples as described in the Specificity TET Study above 501 assays of 505 sterile Table 4 Inter lot Inter site Reproducibility Results for the LRAP Study LRAP units gave the expected negative Logs Expected result reproducibility of 99 2 with a Sample ID Above BacTx Detected Detection lower one sided 95 confidence limit of p Limit of Result out of 36 Rate 98 2 Detection esU For the inter lot and inter site escherichia col reproducibility testing with the 11 member Staphylococcus aureus 08 FaL 3 100 test panel the expected BacTx result _ Bacillus cereus was observed with 396 out of 396 Staphylococcus epidermidis samples No statistically significant Klebsiella oxytoca difference in reproducibility was observed Pseudomonas aeruginosa os FAL 3 100 between the three sites or between the 5 three lots p 1 0 Fisher Freeman Halton Streptococcus agalactiae test All 360 bacterial panel members Serratia marcescens were Successfully detected with the Clostridium perfringens all sterile samples were negative in the BacTx Assay LR WBDP Study Description Reproducibility of the BacTx Assay Kit was determined inter assay inter lot and inter site for both
42. uge tube Do not discard the platelet samples Repeat this step for the remaining platelet samples working from left to right 4 Centrifuge the 2 mL microfuge tubes at 14 000 20 000xg for 3 min Once centrifugation ends carefully return the 2 mL microfuge tubes to the tube rack making sure that the tubes are placed in exactly the same position as before Verify that the IDs for the 2 mL microfuge tubes match the IDs on the platelet samples 5 Uncap the 2 mL tube on the farthest left position in the rack P1 in the example in Figure 1 and decant the supernatant into the waste container by holding the tube upside down for 3 seconds Remove the remaining droplets of supernatant by tapping the upside down tube against the rim of the waste container or by tapping the upside down tube on a clean dry laboratory tissue or paper towel Close the cap on the tube The peptidoglycan present will adhere to the bottom of the tube although a pellet may not be visible to the user Even if a pellet is not visible the sample should be processed as per the protocol Return the 2 mL tube to its position in the rack Repeat this step for the remaining 2 mL microfuge tubes working from left to right 6 Use a micropipettor and a clean sterile pipet tip to transfer 0 5 mL of Extraction Reagent into the 2 mL microfuge tube in the farthest left position in the rack P1 in the example in Figure 1 Holding the 2 mL microfuge tube in one hand homogenize the peptidogl
43. were detected at the 48 hour time point this testing was repeated at approximately 72 hours after inoculation All units were also tested at approximately 7 days after inoculation When BacTx testing was performed quantitative plate culture was carried out to determine the bacterial titer in the contaminated pool at that time point Culture plates made at 24 hours and 7 days after inoculation from the spiked units were submitted for bacterial Page 10 of 16 Effective Date August 16 2013 CF B502 706 identification to confirm the strain that proliferated in the unit was the same as the strain that was inoculated Testing was conducted at two sites with multiple lots of BacTx Assay Kits LR WBDP Study Results The results of the BacTx testing and quantitative plate culture are shown in Table 3 All titer values at 48 72 and 168 hours reflect the concentration after pooling Of the eight aerobic strains tested seven of the strains were detected at 48 hours with 159 of the 160 contaminated platelet detected by the BacTx Assay at this time point All 10 samples of the contaminated pool containing S epidermidis were detected at the 72 hour time point Neither of the two anaerobes tested C perfringens and P acnes exhibited any detectable growth in the aerobic environment of the platelet unit over the 7 day study and were not detected by the BacTx Assay Based on these results the time to detect all eight aerobes is 72 hours after collecti
44. within the 5 day shelf life of the platelet unit could not be determined and this study was repeated The first time point at which 10 out of 10 samples were detected by the BacTx Assay is shaded in grey Propionibacterium acnes 11827 Anaerobe Clostridium perfringens 3629 O Q 3 Z SPECIFICITY STUDY LRAP Study Description Specificity of the BacTx Assay was tested at two external sites using six lots of BacTx Assay Kits and 505 unique LRAP units Sterility of the platelet units were confirmed by culture on blood agar plates This study also served as a test of reproducibility of negative assays as described in the reproducibility study section LRAP Study Results 505 LRAP units were tested of which 501 were negative for the presence of bacteria in the BacTx Assay BacTx Assay result PASS Of the 4 LRAP units 0 79 that were positive in initial testing 3 were negative in duplicate retests Thus 1 LRAP unit out of 505 was Repeat Reactive This corresponds to a specificity defined as 1 the frequency of Repeat Reactive samples of 99 8 with a lower one sided 95 confidence limit of 99 1 Page 11 of 16 Effective Date August 16 2013 CF B502 706 LR WBDP Study Description Specificity of the BacTx Assay was tested at two external sites using three lots of BacTx Assay Kits and 432 unique 6 unit platelet pools Sterility of the platelet pools was confirmed sterile by plate cultu
45. ws e Using a clean sterile pipette place 0 5 mL of Extraction Buffer into a 1 5 mL microfuge tube e Add 0 5 mL of Neutralization Buffer to the tube and pipette up and down 5 times to mix e Add 0 1 mL of the remaining platelet extract from the platelet sample that ABORTED e Close the lid of the tube and vortex for 3 seconds e Add 0 3 mL of the diluted sample to a new BacTx Reaction Tube and perform the BacTx Assay following the standard procedure Please report any immediate ABORT of an assay to Immunetics Technical Service Deviations from the procedure may lead to aberrant results Results from assays with protocol deviations should be invalidated and the assay repeated Follow the assay instructions carefully For potentially interfering substances see the Performance Characteristics section below Chart 1 BacTx Assay Testing Algorithm BacTx Result Retest in Assay 1 Fal Duplicate One or Both oma Both BacTx BacTx Retest PASS Retest Results Results FAIL PASS No Bacteria Bacteria Detected Detected Page 8 of 16 Effective Date August 16 2013 CF B502 706 PERFORMANCE CHARACTERISTICS ANALYTICAL SENSITIVITY STUDY LRAP Study Description The limit of detection of the BacTx Assay was determined for 10 species of bacteria four Gram positive aerobes four Gram negative aerobes and 2 anaerobes Spiking studies were performed at two external sites Three lots of BacTx Kits were
46. x Reader User Manual document GN U0001 to turn on the BacTx Reader and open the BacTx Assay software program In the BacTx program window all eight channels should display Ready The ID of the technologist 2 40 characters should be entered into the Technician ID field at the top of the BacTx software window CONTROLS Controls should be run once per shift on each BacTx Reader that is intended for use or in accordance with the laboratory s quality control policies Controls should also be run after each calibration of the BacTx Reader This calibration procedure is described in the BacTx Reader User Manual Document GN U0001 Each laboratory is responsible for using BacTx controls to establish an acceptable quality assurance program to monitor the performance of the test under its specific laboratory environment and conditions of use Control solutions are added directly to BacTx Reaction Tubes The positive control must be vortexed thoroughly before use in order to thoroughly resuspend the peptidoglycan solution 1 To run controls first vortex the positive control tube at maximum speed for 30 seconds The negative control tube requires only pulse vortexing before use 2 Using a fine tip permanent lab marker designate two BacTx Reaction Tubes as positive and negative by writing on the ID blank printed on the BacTx Reaction Tube label 3 Remove the green caps from each of the BacTx Reactio
47. x101 33x 109 2 2 x 108 Streptococcus agalactiae 4 0 x 105 n d 24x10 1 2 x 108 3 9 x 106 Staphylococcus aureus 2 6 x 105 n d 5 6x 108 5 0 x 108 2 0 x 108 Staphylococcus epidermidis 5 0 x 10 17x109 2 6x 10 1 0 x 109 3 7 x 107 Serratia marcescens 3 1x104 8 0x108 46x109 37x 109 4 2 x 108 Clostridium perfringens Propionibacterium acnes n d not determined Aerobe Anaerobe Page 15 of 16 Effective Date August 16 2013 CF B502 706 REFERENCES 1 Eder AF Kennedy JM Dy BA Notari EP Weiss JW Fang CT Wagner S Dodd RY Benjamin RJ American Red Cross Regional Blood Centers Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions the American Red Cross experience 2004 2006 Transfusion 47 1134 1142 2007 2 Brecher ME Holland PV Pineda AA Tegtmeier GE Yomtovian R Growth of bacteria in inoculated platelets implications for bacteria detection and the extension of platelet storage Transfusion 40 1308 1312 2000 3 Jacobs MR Good CE Lazarus HM Yomtovian RA Relationship between bacterial load species virulence and transfusion reaction with transfusion of bacterially contaminated platelets Clin Infect Dis 46 1214 1220 2008 4 Standards for blood banks and transfusion services Bethesda American Association of Blood Banks 2004 5 Interim standard 5 1 5 1 1 Association bulletin 10 02 May 3
48. ycan by pipetting up and down 10 times with the pipettor set at 0 5 mL While pipetting always keep the pipet tip near the bottom of the tube and allow the pipettor piston to return to its initial position before fully depressing the piston This technique will ensure that the entire volume will pass through the pipet tip To avoid excessive foaming always keep the opening of the pipette tip close to the bottom of the microfuge tube when pipetting up and down Return the 2 mL microfuge tube to its original position in the rack Page 6 of 16 Effective Date August 16 2013 CF B502 706 Without changing the pipette tip transfer 0 5 mL of the suspension in the farthest left 2 mL microfuge tube P1 in the example in Figure 1 to the 1 5 mL microfuge tube containing Neutralization Reagent directly above it Close the cap on the 1 5 mL microfuge tube and invert the tube 3 times to mix Return the 1 5 mL microfuge tube to its original position in the rack Repeat this step for the remaining 2 mL and 1 5 mL microfuge tubes working from left to right Verify that the IDs for the 1 5 mL microfuge tubes match the IDs on the 2 mL microfuge tubes platelet samples and Reaction Tubes in the same column on the tube rack The samples should be tested immediately in the BacTx Assay Preparing BacTx Samples from LR WBDP pools 9 10 11 12 13 14 15 16 With a single channel 1000uL micropipettor pipet 1 0 mL of Lysis Reagent into e
Download Pdf Manuals
Related Search