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Sherlock MYCO-LCS Operating Manual

1. 11590 jSummedFeaure 14 1022 228230 jSummedFesur 15 2012 So 30722 j SummedFeaure l 2710 26451 Summed Feature 17 2332 _______ ____ 12933 Summed Feature 18 1140 3266 j SummedFeanme 19 288 ECL Deviation 0 131 Reference ECL Shift 0 034 Number Reference Peaks 2 Total Response 1134562 Total Named 1134562 Percent Named 1 00 0095 Total Amount 1153217 Matches Library Sim Index Entry Name MYCAG1 1 02 0 906 Mycobacterium tuberculo sis complex TB bovis africanum microti To print the profile information and match results directly to the printer use the Print Tool ex in the Toolbar The same options as in Figure 5 12 are available in this dialog box Figure 5 14 with one exception The Print to Current Printer selection Figure 5 14 is not in the Sample Print Preview Options dialog box Figure 5 12 5 14 Figure 5 14 Printing Results for the Current Highlighted Sample wm Sample Print Options Frinter File Options Detail Options General chromat iw Print ta Current Printer iw Save to AIF File Prnt Pre S ample File CASHERLO LESRESLILTSSR T 30523 rtf iw Prnt General Information Iw Prnt Comments Eu cel Print Past 5ample File Select Samples C Pr A Print No Samples
2. E02801738 E02813515 JEO229035684 JEO24314454 JEOZ2B04527A JEO2B065784 E02B156208 EO2Cl 042zA CREATION TIME 2002 05 31 2002 06 03 2002 06 03 2002 06 04 2002 06 07 2002 06 20 2002 06 25 00 08 01 2002 08 15 2002 09 05 2002 10 31 2002 11 04 2002 11 06 2002 11 15 2002 12 10 ALTER TIME To access other Source Volumes select a data volume from the drop down menu click on the down arrow Sherlock data files can be selected for backup in sevaral ways e All files in the Data volume can be selected by clicking on the Select All Tool T in the Toolbar e Individual files may be chosen by clicking the small box to the left of the file name e The nvert Selection Tool LL will reverse the files that have been selected e Allfiles in the data volume can be unselected by clicking the Clear Selections Tool E f Once the data files are selected make sure a backup device is available and click on the Backup Files Tool E Specify the device and folder in the Target Volume field to backup Sherlock data files Browse To choose a Target Volume other than the A click the button A screen like Figure 6 2 will appear and you can then select a new location for the Target Volume After selecting a new location press the OK button and this new target will appear in the Target Volume field Figure 6 2 Browse for Folder Browse for Folder Select Target Volume F E My Documents E 4 My Computer EE
3. Note The Sequence Seq is not alterable Batch Start Batch This function will start the ChemStation automatically and begin sample processing This function is only available when the Sample Processor is not currently running samples A calibration is always the first sample analyzed Batch Stop at end of run Stops the batch after the chromatographic analysis of the current sample is completed and the reports are printed Batch Abort Batch Stops the current analysis immediately Note that the current analysis is lost Note also that since ChemStation takes a few minutes to load completely to prevent potential problems with ChemStation it is prudent to wait until ChemStation has completely loaded before Aborting 3 8 View Reset Resets the sample table showing all the columns Use this option if resizing columns and want to view library search results during a batch The Sample Processor can show results of sample runs in the sample table The right most column of the table can show the similarity index for the library entry with the highest match achieved by that sample If there are multiple high matches the similarity index is preceded by a plus sign You should always review the sample s report for proper interpretation of the results View Reset No Results Resets the sample table hiding results columns Use this option if resizing columns and want to return to a standard state This view is especially usef
4. Review report comments This message may occur when the peaks do not have the correct symmetrical shape i e too broad Possible Solutions e lf there is just one obviously large wide peak re run the sample There may have just been a random hiccup of the system This should not happen on a frequent basis e f this happens on a frequent basis you have chromatography problems Contact MIDI Technical Support Library search results in No matches found in MYCAG1 This indicates the Sherlock parameters have determined that the sample is so different from all of the entries in the database that it will not show a Similarity Index This may be an acceptable answer in that it will tell you that you do not have this species in the database or you may have a mixed culture and the Sherlock software has detected this fault By following the steps below you may be able to find the closest entry in the Library This may help you choose which confirmation test to perform on the culture to make a proper ID of the organism in question To run a NO MATCH sample to find the closest match via Comparison Charts or to reclassify a previously run sample against an updated Library follow the instructions in Chapter 5 Sherlock CommandCenter Sample Preparation Errors Low similarity indices or NO MATCH library results may be caused by mixed cultures or deviations in the sample preparation procedure If low less than 0 5 similarity index
5. Sample ID UN MYCO 1004 4WK GO Profile RT Response Ar Ht ECL Peak Name Percent Comment1 Comment2 2 665 3406 0 088 36 413 lt minrt 2 838 2799 0 091 38 175 min rt 3 017 8922 0 097 40 000 ECL 40 000 standard ECL deviates 0 000 Reference 0 015 3 184 1215 0 093 41 699 Sum In Feature 1 0 11 ECL deviates 0 099 ECL 41 600 5 571 660 0 131 64 427 ECL 64 625 0 06 ECL deviates 0 198 5 930 742 0 190 67 881 Sum In Feature 9 0 07 ECL deviates 0 168 ECL 67 713 6 298 1537 0 177 71 426 Sum In Feature 10 0 14 ECL deviates 0 039 ECL 71 387 6 523 3104 0 110 73 591 Sum In Feature 11 0 27 ECL deviates 0 162 ECL 73 429 index 6 592 3113 0 096 74 252 ECL 74 350 0 27 ECL deviates 0 098 6 816 10131 0 157 76 385 Sum In Feature 12 0 89 ECL deviates 0 107 ECL 76 278 index 7 096 35798 0 130 79 056 Sum In Feature 13 3 16 ECL deviates 0 046 ECL 79 010 index 7 371 115990 0 123 81 688 Sum In Feature 14 10 22 ECL deviates 0 074 ECL 81 614 index 7 648 228230 0 117 84 312 Sum In Feature 15 20 12 ECL deviates 0 028 ECL 84 340 index 7 929 307522 0 112 86 977 Sum In Feature 16 27 10 ECL deviates 0 088 ECL 87 065 index 8 194 264531 0 119 89 450 Sum In Feature 17 23 32 ECL deviates 0 160 ECL 89 610 8 462 129343 0 121 91 951 Sum In Feature 18 11 4
6. E E Methods ToS librar Sim Index Mame ooo worse Speer 0 906 Mycobactenum tuberculosis complex TB bovis aficanur Printing Sample Information Clicking the Print Preview Tool in the Toolbar allows you to preview the results before committing them to paper Figures 5 12a and 5 12b show the dialog box for Sample Print Preview Options This dialog box allows you to select how much information is printed It also allows you to store data as an RTF file which can be opened in Microsoft Word If the Print Chromatogram checkbox is selected the chromatogram plot will be included in the report Note Earlier versions of Sherlock did not save a pointer to the chromatogram plot therefore it is not available for reprinting by Sherlock In this example the general information for Sample Ctr 12 in Figure 5 11 highlighted along with the profile and library match result will be printed Clicking the OK button in the dialog box of Figure 5 12 results in the Print Preview window appearing on the computer screen Figure 5 13 Note Printing all the samples for a large sample list can yield a very large report Consider just printing the match results and turning off Each sample on a new page to minimize paper usage 5 12 Figure 5 12a Sample Print Preview Options Dialog Box General Tab m Sample Print Preview Options Printer File Options Detail Options jw Save to ATF File Print Pre S ample File CASHERLO CESARE SULT S
7. Advanced Sample Selector MOUSE us eoo A dor Mueble iur nre E ant beads 5 5 PRINTING SAMPLE INFORMATION sia tinea tuos nade dies tuo pr eve d dati eoo de CERTI ted eec eda ind 5 12 CONFIGURING THE PRINTER estas tet order a advo cia cans due unas O c Unde ia qeptui o bau bd tes Un ict Vaio 5 15 CORRECTING DATA nidi eM 5 16 VIEWINSINSTAEEEDIIVIETHIODS isisao con cust ha E Uo a 5 16 Displaying Methog Information su ohm qt tue t eue e aee OEA e a x e v tog vide e pea FERRE 5 17 Printing Method Parameters eoe e lateat EN E N E EEE utut at dese eet ula d ERA cuota des ARMS 5 18 VIEWING INSTALLED LIBRARIES coa ica 5 19 PRICING IDEAR FOLIO UO RTT Lm 5 21 COMMANDGENTER TOOLS AND VIEWS ICONS rara A Ee pet diea Deoa R Ue eos TOR 5 22 CHAPTER 6 ROUTINEMAINTENANGCE Siria 6 1 OVERVIEW D 6 1 Data StOF OG Coarse aoa er pu Oh A duris cee A Ue Lotsnt turus ue EAM dc ttr QUE aes 6 1 SHERLOCKU TIMES cc 6 2 A 6 2 DOUG REST OIC and 6 4 SHERLOCK TOOLBOX T 6 6 PS COU Method S esa e Stuart uas pcd our Rdaseni suuni ii 6 8 STON FONS tra 6 9 oodd Methods M 6 9 oodd EIDFOLOS aa 6 10 ROUTINE IVIAINTENANCE OF THE HPUC 5i ictcineuss viaa YU eset eut t euet an Vert a 6 11 Replacing the Purge Valve FINE uotis tide deu tack esu Ortu
8. As you can see in Figure 4 6 in the Mycolic Library a strain with a Similarity Index of 0 660 falls three standard deviations in cumulative variation from the mean The cumulative variance of the mycolic acid composition of an unknown strain can best be visualized by looking at the chart of the mycolic acid composition of that unknown and observing the distance from the mean of each mycolic acid Interpretation Guidelines Use the following guideline when interpreting the Similarity Index SI Strains with an SI of 0 500 or higher with a separation of 0 200 between the first and second choice SI are considered good library comparisons Strains that have an SI of an unknown below 0 500 or with an unacceptably close second choice should be considered indeterminate and a confirmatory test should be used Precaution All interpretations especially those not M tuberculosis should be visually compared using chromatograms Identifications of MOTT regardless of SI should be determined using Sherlock findings along with the results of other laboratory testing and observations Figure 4 6 Population Distribution S l 0 950 S l 0 830 S l 0 660 Interpretation of Library Groups Unknown samples may be identified to the species level or to the species complex group The species complex will typically be organisms having high DNA similarities and or are similar in clinical 4 11 significance Examples are the complexes of Mycoba
9. CL 44 200 44200 0 351 3535 False False Fixed 1000Mo CL 44 800 44 300 0 351 3608 False False Fixed 1 000 Mo CL 45 532 45532 0 282 3675 False False Fixed 1 000 Mo CL 46 069 index 46 069 0256 3731 C Tme False Fixed 1 000 No CL 46 626 46 626 0 175 3789 False False Fixed 1000Mo CL 47 075 47 075 0 275 3837 False False Fixed 1000Mo CL 47 574 4 574 0 225 3889 False False Fixed 1000Mo CL 48 201 48201 0 250 33955 False False Fixed 1000Mo CL 48 650 48 550 0200 4 002 False False Fixed 1000Mo CL 48 175 43175 0 275 4057 False False Fixed 1000 Mo CL 48 630 436530 0 181 4104 False False Fixed 1000 Mo CL 50 095 50 095 Q240 4153 False False Fixed 1 000 No CL 50 693 50533 0 333 4 216 False False Fixed 1000Mo CL 51 275 index 51275 250 4277 C True False Fixed 1000Mo CL 52 035 52 03 4357 False False Fived 1 000 Mo CL 52 600 52 60 4416 False False Fived 1 000 No CL 53 195 53 19 4479 False False Fived 1 000 Mo 53 64 4526 False False Fived 1 000 Mo 1n C 1 En n ids gt EN EM EM EM EM EM EN EN Ce Ce EE Em Em EE EN Em EN EN a iE EMM CL 53 645 I Ba Ine r Utilities Printing Method Para
10. Cleaning MIDI Evaporator and Pierce ReactiTherm Needles 4 1 Daily Wipe the tips with a KimWipe or lint free cloth moistened with isopropanol 4 2 Weekly 4 2 1 Soak needles in warm tap water for 10 minutes 4 2 2 Drain and repeat 2x 4 2 3 Drain and soak in HPLC grade methanol or isopropanol for 10 minutes 4 2 4 Drain and repeat 2x 4 2 5 Soak briefly in chloroform and allow to air dry Caution Perform only in a vented fume hood and wear protective gloves C 2 Appendix D HPLC Performance Qualification Table Neg Contro Pos Control Mobile Phase Calibration second run C albicans 60193 M gordonae 14470 Baseline i Total N d Stans ECL 40 RT ECL 97 RT ii Named Response M Offset Pressure Response Response 2 800 8 000 Initials 20000 1000 FRO Note Key calibration action limits are shown in parentheses in each column Positive and negative control specification values are in parentheses and bolded If either fails the source of the problem should be identified and corrected and sample cultures should be re extracted along with positive and negative controls per Chapter 2 Preparing Extracts and re analyzed FRO For reference only RT Retention Time minutes Consult ChemStation Log or note from on screen display Use values from 2 calibration run D 1 Appendix E Performance Characteristics Overview Table E 1 summarizes an evaluation of the ability of the She
11. M nonchromogenicum terrae and M terrae nonchromogenicum The M bovis BCG not 35737 entry will reliably distinguish BCG strains other than American Type Culture Collection strain 35737 from the M tuberculosis complex The ATCC 35737 strain will give an indeterminate answer between the two library entries and requires orthogonal testing to differentiate A Roman numeral or II following a species name indicates a subgroup of mycolic acid pattern within a species Although there is no taxonomic significance to such subgroups such designation may be useful to infection control personnel Both M haemophilum and M marinum contain the growth condition of 30 C temperature in parentheses following the name field and that M haemophilum should be grown on chocolate agar 4 12 Visual Confirmation An additional feature of the Sherlock Software allows the user to perform a visual comparison of the HPLC pattern to a known reference chromatogram As samples are analyzed by the Sample Processor Sherlock prints visual comparisons like that shown in figure 4 7 The visual comparison is printed for samples that have a top match to a library entry with an SI of 0 400 or higher The chromatogram will be aligned using the internal standard peak times and the reference chromatogram will be scaled to the Same area as the current analysis The reference chromatogram is printed as a mirror image immediately below the current analysis for easy visual comp
12. Summed Feature 5 6 06 3778 Summed Feature 6 3 65 5664 Summed Feature 7 5 47 5181 Summed Feature 8 5 01 6395 Summed Feature 11 6 18 14061 Summed Feature 12 13 58 7612 Summed Feature 13 7 35 7052 Summed Feature 14 6 81 9942 Summed Feature 15 9 60 6890 Summed Feature 16 6 66 4548 Summed Feature 18 4 39 13662 Summed Feature 19 13 20 Total Response 103510 Percent Named 100 00 GOOD PEAK MATCHING PEAK POSITION MATCHING ERROR RMS IS 0 0004 Profile Matches Library MYCAG1 1 02 Sim Index 0 898 Total Named 103510 Total Amount 119184 Entry Name Calibration mix for mycobacteria 4 7 Acceptable Calibration Figure 4 3 represents an acceptable calibration report The key parameters blue italicized on the calibration report that should be recorded on the Performance Qualification Table See Appendix D are the 40 000 and 97 000 ECL Peak Retention Time RT the Similarity Index Sim Index Total Response and Peak Position Matching Error RMS Tracking of these parameters will give an indication of the acceptable performance of the system The expected range for each parameter is given in parenthesis in the corresponding column on the Performance Qualification table Parameters that are out of the expected range give an indication
13. The report will be labeled with the letters RR rerun in the parenthetical portion of the name field with the same Seq Minimum Response If the sample injected results in a Total Response of less than 20 000 the Minimum Response warning message will be printed on the report The system will automatically reanalyze the sample with injection of four times the amount of the initial injection 20ul The report will be labeled with the letters RR rerun in the parenthetical portion of the name field gt max ar ht Indicates that the indicated peak width is wider than the expected value for good mycolic acid peaks The operator may choose to reanalyze the sample and accept the report if no error messages appear If the max ar ht report still occurs it may be necessary to confirm the identification by other techniques or to re extract the sample min rt The peak elutes before the ECL 40 Internal Standard so it is not identified as a mycolic acid of interest The peak is not used in the library search and thus has no negative impact on naming 4 5 gt max rt The peak elutes after the ECL 97 Internal Standard so it is not identified as a mycolic acid of interest The peak is not used in the library search and thus has no negative impact on naming of the organism Reference This peak an internal standard is used as a reference to adjust the ECL values of the other peaks The difference in ECL units betwe
14. is the calculated area divided by the height This is an estimate of the peak width e ECL is the Equivalent Carbon Length of the mycolic acid It standardizes the elution time across instruments and columns e Peak Name a number such as ECL 92 527 is the interpolated carbon length between the two internal standards e Percent the relative amount of each named peak e Comment 1 gives a peak match to the expected position in the naming table e Comment 2 is additional information about peak naming or peak ECL drift Peak Names and Amounts Each peak from the chromatographic analysis is listed by retention time RT Response and area height ratio Ar Ht Also included in the composition report is the ECL which is a linear interpolation of each peak s retention related to the preceding calibration standard and to the two internal standard reference peaks Sherlock software compares the ECL of each peak in the analysis with the expected ECL of the mycolic acids in the Peak Naming Table The mycolic acid name this is the assigned ECL value is printed in the Peak Name column Peaks that do not correspond to ECL values of known mycolic acids are unnamed and are not used in the library search or identification Due to the great diversity of isomers and the difficulty of reproducibly resolving all compounds most peaks will be listed as Summed Feature Summed Features are created by summing adjacent mycolic acid peaks into a single more reli
15. 2 Preparing Extracts the labeled sample vials are inserted into the automatic liquid sampler tray Information about each sample in the tray is entered into the Sherlock Sample Table using the computer keyboard Chapter 3 When the information about each sample has been entered into the Sample Table a batch can be started A batch is a run of one or more sample extracts on the HPLC When a batch is started from the Sample Processor the following occurs e The sample s Method instrument setpoints calibration instructions and sample tray location is downloaded to a ChemStation file ChemStation sets the HPLC parameters and controls the injection by the automatic liquid sampler e The automatic sampler selects the sample vial for the calibration mix The sampler controller causes the injection of a small portion of the extract into the solvent flow stream of the HPLC e The C18 reverse phase column installed in the HPLC column compartment separates the mycolic acids present in the mix as they pass through the column to the detector The mycolic acid sample preparation Chapter 2 converts the mycolics into fluorescent derivatives As the compounds pass through the fluorescence detector the fluorescent tags are excited by 345 nm wavelength light and this causes emission of light at 425 nm that is quantitatively detected by a photo multiplier tube in the detector The amount of light emitted is related to the concentration of the tagged compound
16. 538 8 631 40 20 4 A oe o co wo 3 106 3 277 3 538 3 736 4 010 4 151 4 545 4 677 4 877 5 084 5 373 5 730 6 098 16 359 6 472 6 672 ad 6 977 7 261 8 867 Cop 9 152 9 841 10 026 10 min Sherlock Composition Report The peak retention time width and height data from the ChemStation are transmitted to Sherlock data files at the end of each run The data are processed peaks are assigned names based on ECL values not structural names the mycolic acid pattern for the sample is compared to the library and a report is printed An example report is shown on the following page Figure 4 2 General Information As determined by the user in the Sample Processor Configuration tool the computer stores the chromatographic data in files on the selected data volume see Chapter 5 for a suggested protocol for selection of data volume For each batch of samples a file name is automatically assigned by the computer and is printed at the top of the Composition Report The heading identifies the sample with the sample ID number bottle number sample name and the date and time of the analysis If the data have been edited the date and time of the last edit is also printed Volume DATA Type Samp Created 6 19 2002 7 32 29 PM File E026196 94A Bottle 10 Figure 4 2 Sherlock Composition Report Samp Ctr 12 ID Number 2246 Method MYCOLC1
17. CC Queued M Y COLCI HYCO ABSCE ATCC 19977 CC Queued MYCOLCI VCO FORTU ATCC 6841 CC Oueued Done Done Done Done Done Editing the Sample Table To change an existing entry in the Sample Table Unlock the table click on the field to change and select the choice from that drop down menu or type in the change Figure 3 5 Sample Table entries can be edited at any time even while samples are being run on the LC Samples listed with a status of RUNNING are protected from edits It is a good idea to Lock the table when finished editing The pencil icon on the left side of the screen indicates that changes are outstanding next to bottle 6 in Figure 3 5 Changing the Type Click on the Type field to select the choice for priority e CALIB A calibration bottle There must be at least one calibration logged into the Sample Table If necessary to maintain calibration a calibration will run before next sample even before a STAT sample Its Sequence Number field will automatically be set to 1 and cannot be changed e SAMPLE Normal priority e STAT High priority sample STAT samples are processed before ordinary SAMPLES e QC Indicates a positive control sample e BLANK Negative control bottle or a bottle that contains no sample e EMPTY No bottle in this sample tray position 3 11 Changing the Status The status of the selected sample is either QUEUED DONE or RUNNING To ch
18. Library Representative chromatograms for each entry are available upon request from MIDI The chromatograms are intended for visual comparisons as an aid to confirmatory identification of those species of mycobacteria being identified by additional tests Note The chromatograms are not intended to be used as sole criteria for identification of species of mycobacteria Many mycolic acid pattern variants of some species of mycobacteria may occur and these chromatograms are not inclusive of all patterns only of the ones seen in the MIDI laboratory during creation of the database For optimal comparisons align the internal standards of the unknown with those of the reference strains The Sherlock Mycobacteria Identification System has been shown to reliably differentiate the M tuberculosis complex from other mycobacteria Definitive identification of MOTT requires use of additional laboratory testing to identify clinically significant organisms Sherlock may be useful in conjunction with probe hybridization biochemical testing and other laboratory observations The ability of Sherlock to correctly identify MOTT not listed in the database has not been evaluated See Appendix E B 1 Table B 1 Sherlock amp Mycobacteria Library Entries Mycobacterium abscessus chelonae Mycobacterium asiaticum Mycobacterium aurum vaccae Mycobacterium bovis BCG not 35737 Mycobacterium celatum Mycobacterium chelonae abscessus Mycobacterium flavescen
19. MIDI Part 1600 A add 50 ul of the Sample extract 11 Dispense the extract along the walls of the insert to ensure that the dried internal standards are totally solubilized 12 Once dispensed thoroughly mix by slowly pumping the solution up and down in the pipette 13 After capping the vials the samples are ready for HPLC analysis HPLC Solvents e Solvent A Methanol Shelf life 3 years in unopened container 1 yr if opened Danger Flammable toxic if consumed e Solvent B IPA Shelf life 3 years in unopened container 1 yr if opened Danger Flammable toxic if consumed 2 4 Reagents Reagent 1 Saponification Reagent Shelf life 1 year Potassium Hydroxide 250g Deionized Water q s to 500 ml with continuous stirring Danger Reagent is extremely caustic Wear eye protection Observe splash precautions Substantial heat is released during reagent preparation that may cause the mixture to boil bump or steam Prepare reagent ONLY ina BOROSILICATE GLASS flask inside a chemical fume hood Prepare by slowly adding KOH pellets to 200ml dIH20 until totally dissolved Continue adding H20 until the final volume is 500 ml Note Protect from air absorbs CO Reagent 2 Acidification Reagent Shelf life 1 year To prepare slowly add 250 ml concentrated HCL to 250 ml deionized water with continuous stirring Danger Reagent is extremely corrosive Wear eye protection Observe splash precautions Substan
20. Print Detail Sections General Information f Print Current Sample EI Profile Information Match Results Print Man Lalib 5 nnt Pan cLatlb samples Chramatogram t Print All Samples Ww Each Sample on a Mew Page Cancel Configuring the Printer Use the Setup Printer tool on the file menu dropdown to select and configure the printer for hardcopy output Clicking this tool displays the standard Print Setup dialog box shown in Figure 5 15 In this example The LaserJet 1010 is selected as the printer If there is more than one printer installed on the computer designate which printer will be used in this dialog box This dialog box can also be used to choose between a Landscape or Portrait mode of printing Clicking the Properties button displays the printer specific configuration dialog See the documentation and help files supplied by the printer manufacturer for information about the Properties dialog box 5 15 Figure 5 15 The Print Setup Dialog Box Print Setup LaserJet 1010 r Froperties Ready hp LaserJet 1012 DOT4 001 Comment Paper Orientation Size Letter f Portrait Source Auto Select Landscape coros Correcting Data Entry Errors Sherlock allows you to correct entry errors made when samples were logged into the system via the Sherlock sample table You can correct errors in any of the white fields shown in Figure 5 4 bottom right windowpane or Figure 5 9 bottom windowpane If
21. The bottom left windowpane of Figure 5 8 shows the sample ID prefixes associated with the query done in the previous two steps In this example several of the prefixes were chosen checked boxes These prefixes are then displayed in the bottom right windowpane Pressing the Apply button in the bottom windowpane provides a preview of this data set the samples in the query are displayed in the top windowpane Figure 5 8 Advanced Selector Choosing Prefixes K Sherlock CommandCenter Samples FE Index Volume Flename Botte IDNbr Type Method ID Samp Cir Creates 7 leas seme MYCOLLT UNICO ON AVR e seria ATT DATA Eosissa E 22M Samp MYCOLCI UN MYCO 1002 5wK CHDC 30060 10 13 2002 70213 SES Sano MYSUUT LUNA 1003 SWK ATI SELECTIVE Ln Jenae 2171 10 2246 Samp MYCOLEI UNMYCO 1004 AWKGO 2 8 19 200273228 le Methods Prefixes O CAL STD FOR METHOD MYCOLC LOTH104121 2 O GC B HA UN 4 Lyf wYCO 4 Reset Clear Prefixes Utilities You can then view the details of the samples from the query by clicking on the OK button on the bottom ria LE Panne windowpane or by clicking the Detail Tool in the Toolbar Figure 5 9 illustrates what the sample detail screen looks like from the example query The highlighted sample is the same sample as in Figure 5 4 Note Figure 5 4 and 5 9 are similar as both allow you to view sample details The only difference is that in Figure 5 4 the B
22. This chapter continues with the actual HPLC processing of the samples In this chapter we will look at e Loading the Automatic Liquid Sampler e Sample Processor Configuration e Sample Processing Under Computer Control Loading the Automatic Liquid Sampler The Automatic Liquid Sampler 1100 and 1200 Series Agilent HPLC consists of a sample tray a robotic arm for moving the sample vial from the tray into position to be sampled and a sampling syringe mechanism Figure 3 1 Automatic Liquid Sampler Loading Samples into the Tray The first position in the sample tray numbered 1 should hold a vial containing the Calibration Standard Positions with higher numbers should contain sample vials that correspond to their numbers in the sample table There must not be empty spaces in the sample tray unless those positions are designated as Empty in the Sherlock sample table NOTE This manual generally refers to the physical container as a vial The Sherlock software generally refers to the position in the tray as the bottle number Analyzing Samples When the operator clicks on the Start Batch Y in the Sherlock Sample Processor Toolbar the system will begin a conditioning process that includes a warm up period and a blank run or analysis in which no sample is injected As a result the solvent gradient is performed This conditioning is helpful in enabling the system to equilibrate so that the analysis of the calibration i
23. This could result in erratic flow or pressure This should not be a frequent problem but the seals eventually wear out and should be replaced by a person trained in maintenance of the HPLC Instructions are on the Agilent CD ROM covering maintenance e Missing bottles in the sample tray This causes the system to shut down after a time out period Apparent Software Problems Regardless of the cause these are often resolved by exiting from the Sherlock and ChemStation software and shutting down the computer and restarting 7 6 Performance Qualification Table The purpose of the Performance Qualification Table Performance Qualification Table Appendix D is to help the User determine normal operating conditions for their system Tracking certain key parameters will help alert the User to any potential problems before an error condition occurs Expected ranges of key system and QC organism parameters are given in the Performance Qualification Table A value outside the expected range will not necessarily create a system error however it is an indication that the system is not operating in its optimal condition Listed below are out of range conditions and potential troubleshooting steps e 40 000 97 000 ECL Peak Retention Time Out of Range This is an indication of excessive system drift Run the LC Adjust tool see page 7 4 which will make the appropriate adjustments to bring the retention times back into the expected range If this does not
24. View The CommandCenter application remembers the view from the last time you used it If it is not in the Libraries View as shown in Figure 5 19 click the Libraries View il in the Taskbar on the left hand side of the CommandCenter application window If the Libraries View is not visible click the Views button in the Taskbar so that it is visible and click on it Figure 5 19 shows the MYCAG1 library in the Choose Lib Tool and the dropdown arrow can be used to choose another installed library Initially just the general library information is shown in the top windowpane To view the list of library entries click the Entries tab in the top windowpane as shown in Figure 5 20 The list of entries for that library is displayed 5 19 Figure 5 19 Viewing Installed Libraries Sherlock CommandCenter Libraries File View Library Entry Help Train NewEntry Search Bene aH ERE Samples i Version 1 02 ll El Description M ycabacteria library for HPLC Methods dra Method Mco LC tt Entries n Created 7 27 2000 Madified 4 26 2002 9 59 05 AM Utilities The bottom windowpane displays information for the entry selected in the top windowpane The graphic in the bottom windowpane in Figure 5 20 gives the typical percentages and ranges for each feature mycolic acid or summed feature in the selected library entry The boxes show a 2 sigma range for each feature in the data used to build the library entry The vertical line th
25. and the time of elution is related to the chromatographic properties structure of the compound This signal is stored in the ChemStation data file The plotted and integrated signals called the chromatogram are formatted by ChemStation and printed by Sherlock e Whentherun is complete the retention time peak width and response of each peak are transmitted from ChemStation to Sherlock for processing Peaks in the chromatogram are identified by mycolic acid Equivalent Carbon Length ECL value name e When peak naming is complete Sherlock searches the library to identify the unknown organism The library search uses both the peak name and the peak percentage amount to match with known profiles stored in the library Following the library search the computer prints the Composition Report which includes the peak naming chromatogram plot and library classification results Each library entry is a computer generated composite of the reference strains of each species or subspecies group of organisms taking into consideration strain to strain and experimental variability Reference strains were cultured and processed under carefully controlled conditions The computer system automatically sets the operating parameters of the chromatographic unit There is no need to manually enter the HPLC parameters The system will automatically recalibrate within a preset interval You are permitted to enter sample information into the Sample Table while sample
26. bring the retention times back into the expected range try a different calibration standard bottle and rerun the calibration If the 40 and 97 ECL peak retention times are still out of range contact MIDI Technical Support e Calibration Standard SI lt 0 500 The calibration standard SI is for reference only It is desirable that it be 0 500 or greater However as long as the other calibration parameter specifications including the positive control are within range a calibration SI lt 0 500 will not affect system performance e Calibration Standard Total Response lt 20 000 This is an indication of a calibration standard problem Check the level of the calibration standard and replace with fresh calibration standard if necessary If total response is still low contact MIDI Technical Support e Calibration Standard Peak Matching Error RMS gt 0 0060 This is an indication of excessive system drift Run the LC Adjust tool see page 7 4 which will make the appropriate adjustments to bring the retention times back into the expected range If this does not bring the retention times back into the expected range try a different calibration standard bottle and rerun the calibration If the RMS value is still out of range contact MIDI Technical Support e C albicans Total Response gt 1 000 This is an indication of a contamination problem most likely with one or more reagents The C albicans culture plate may also be mixed Check the
27. chart For each acid the horizontal bar gives a 2 standard deviation window around the mean The library entry mean value for a mycolic acid or summed feature is identified with a vertical line A blue oval is placed on the line opposite the name indicating the amount of that acid or summed feature in the sample of interest Examination of the chart may give the user a better understanding of the quality of the match than the Similarity Index however it must be remembered that the Similarity Index has been calculated using all the features and their cross correlation terms The relationship of acids to each other cross correlation terms is not evident in this type of chart The Similarity Index assumes that species of microorganisms have normal Gaussian distribution the classic bell shaped curve and that the mean of the population in any group of traits e g mycolic acid 4 10 percentages characterizes the group Most of the population falls somewhere near the mean but individuals will differ in composition and thus may show considerable variance from the mean In Figure 4 5 the perfect mean percentage for all mycolic acids in a single species entry no variance on any mycolic acid is indicated by the vertical line that crosses the center of each bar The Similarity Index for a strain that falls on this line is 1 000 As the variance increases the strain falls further and further from the line and the Similarity Index drops
28. concentrated 36 38 ACS Grade Fisher A144 500 Chloroform 1L HPLC Grade ACS Certified Fisher C606 1 Potassium Hydroxide KOH 500g ACS Grade Fisher P250 500 Potassium Hydrogen Carbonate KHCOz 500g ACS Grade Fisher P184 500 4 Bromomethyl 6 7 dimethoxycoumarin F W 299 1 Aldrich 30 145 0 18 crown 6 ether F W 264 3 Sigma C5515 Glass Plastic Other 2 0ml Amber Vials 1000 pack Restek 21143 250 ul Glass insert with spring 100 pack Restek 21776 2 0 ml Clear Vials 100 pack Restek 21154 Short Screw Caps with Liners 1000 pack Restek 24498 Purge Valve Frit 5 Agilent 01018 22707 MIDI Certified SB C18 Zorbax Column 4 6mmx75mm 3 5um pore size MIDI COLUMN L N tank regulator or Air tank regulator Note Only required with the Local Vendor Pierce Evaporating System Gas Drying Unit Note Only required with the Pierce Evaporating System VWR 26668 007 Chemical Fume Hood or Ductless Fume Hood Local Vendor Sterile Applicators 1000 for harvesting Thomas 1132H05 Scientific MIDI Calibration Standard MIDI 1500 A MIDI Internal Standard MIDI 1600 A Media Middlebrook and Cohn 7H10 Agar BD 221174 Fisher B21174x Lowenstein Jensen Slants BD 221388 Fisher BB21388 Quality Control Organisms Mycobacterium gordonae ATCC 14470 Candida albicans ATCC 60193 Appendix B Mycobacteria Library Entries Overview The following pages contain a list of the organisms currently in the Sherlock Mycobacteria
29. either of these fields are edited the Modified field will be set to the date and time of the modification The sample will be permanently marked as modified Printed reports created from a modified entry will be marked with the date and time that they were edited Viewing Installed Methods Sherlock contains tools that allow you to view and print method information The system does not allow you to edit the methods supplied by MIDI Inc The ability to view methods is provided mainly for troubleshooting with the help of MIDI s technical support Double click the Sherlock CommandCenter Icon 3 on the desktop to start the Sherlock CommandCenter application Figure 5 16 shows the Methods View CommandCenter reverts to the view from the last time it was used If itis not in the Methods View as shown in Figure 5 16 click the Methods View in the Taskbar on the left hand side of the CommandCenter application window If the Methods View is not visible click the iew button in the Taskbar to make it visible and then click on it Figure 5 16 Viewing Installed Methods A Sherlock CommandCenter Methods SEs DENIED IRL MO MYYEOLE MYCOLCI Mycobactena method for LE Y LAG 171572002 5 17 2002 9 26 54 Ak A T LAS Samples dE General Sample Control Calib Control Catib Sequence Naming Table Name MYCO Lol Foot Method MyCoLc1 Version f Me Libraries Created 145 2002 Modified 51 2002 9 26 54 AM Description Mucobac
30. in the calculation of percentages e g ECL 40 000 and ECL 97 000 in Figure 4 2 e Total Named the sum of the Response column values for all named peaks excluding zero feature peaks see Total Response e Percent Named 100 Total Named Total Response e Total Amount equivalent to Total Named for the MYCOLC1 method It is used as the denominator for calculating the values in the Percent column Composition Report Messages Messages are printed on the Composition Report to help you evaluate the analysis Each composition report has a Name field and two Comment fields that are used to convey specific information about individual peaks The most common messages are those that describe or identify chromatographic features These messages are listed below with a brief description See Chapter 7 Troubleshooting for more information and potential solutions Detector Overload The fluorescence detector reaches saturation at about 500 000 luminosity units LU Note Sherlock uses a multiplier of 1 000 for LU designation Additional signal then is lost and peak height is incorrectly truncated at ca 480 000 LU To avoid this problem the Sherlock method of analysis causes rejection of any analysis containing a peak with a height greater than 450 000 LU thus giving a margin of safety for the analyses The software will print out a warning message on the report and may automatically re inject a smaller volume of the original sample 2ul
31. plate for contamination Re streak C albicans for purity if necessary re extract and re run If the source of contamination is not found contact MIDI Technical Support e WM gordonae SI lt 0 600 This is an indication of a potential sample preparation problem or a mixed culture Check the culture plate for contamination and or sample preparation errors Re streak M gordonae for purity if necessary re extract and re run If the source of the low SI is not found contact MIDI Technical Support 7 7 Appendix A Equipment and Consumables Overview Some additional equipment is needed for proper sample preparation for the Sherlock Mycobacteria Identification System These items are listed in Table A 1 to aid in setting up and operating the laboratory Most items should be readily available from your standard laboratory supplies providers Substitutions may be made for the recommended items but you should assure that the materials are equivalent Other items are nonessential but may be useful to increase the ease of the procedure or to improve reproducibility Consult MIDI if you have any questions Table A 1 Sherlock Additional Equipment US Sources ITEM SOURCE PART NUMBER Extraction Sample Preparation 0 5 5ml BrandTech Dispensette bottle top dispensers Brand Tech Fisher 13 688 227 Scientific 4701131 for Reagents 1 and 2 0 5 5ml BrandTech Dispensette organic bottle top dispenser Brand Fisher 13 688 270 Tech Scientific 473113
32. r Disk C ft DVD RW Drive D E a My Network Places As the files are copied into the backup volume the selection box s will clear If the backup volume becomes full Sherlock will ask the user how to proceed The program will prompt the user with an overwrite confirmation box if the selected files already exist in the volume Any files that were not copied will remain checked The user can rename data files in the Backup View by selecting the files of interest one at a time and then pressing the Rename Files Tool AB To delete any data file s choose the files s of interest and press the Delete Files Tool M 6 3 Also under the File menu you can select Exit to exit the Sherlock software To go to another CommandCenter view click the Ies button and select the view of interest Data Restore Double click the Sherlock CommandCenter Icon f Soon the desktop to start CommandCenter application CommandCenter reverts to the view from the last time it was used If it is not in the Restore view as shown in Figure 6 3 click Utilities button in the Taskbar then click the Restore View in the Taskbar so that Restore appears in the Current View The Source Volume label is displayed in the middle windowpane the default is A To change this click the Browse the Source Volume field and choose data files as was done in Figure 6 2 button or anywhere in Figure 6 3 Data Restore Screen Sherlock CommandCenter Rest
33. samples are running will leave the system in an unstable state 3 7 Table Clear Options Each of the table clear options removes samples from the Sample Table by erasing the sample identification number and name and changing the bottle type to EMPTY When the system is running samples the editor will not allow the removal of CALIB or RUNNING bottles from the table Table Clear All Samples Removes all Queued or Done samples but not calibration entries Table Clear If Done Removes samples that have already been analyzed bottles with a status of DONE Table Reset Table Removes all samples and calibration entries and establishes a new calibration entry Table Clear Current Bottle Removes information about the currently selected bottle Note Table Clear functions must be used with caution as there is no Undo command in Sherlock and there is no way to save a sample table Table Set Auto ID Number The Auto ID Number feature should be used to assign and increment the Sequence Number Seq field automatically for each sample run Each sample will then have a unique identifying number Sequence numbers determine the order that samples will be run in but see Stat below for a way to override this behavior By default this number will also be used as the ID number The user may change the ID numbers for samples as needed The numbers don t have to be unique The ID numbers are saved with the sample in the Sherlock data file
34. significant organisms Sherlock may be useful in conjunction with probe hybridization biochemical testing and other laboratory observations The ability of Sherlock to correctly identify MOTT not listed in the database has not been evaluated E 1 Table E 1 Comparison of Sherlock Mycobacteria Identification System with phenotypic biochemical and molecular tests for identification of Mycobacterium species Reference Methods Isolates Definitive Interval Indeter Discrep Low High M abscessus chelonae group 17 17 100 89 100 0 0 M avium complex 50 49 98 92 100 1 2 0 M bovis BCG 2 2 100 E 0 0 M flavescens 3 2 67 1 33 0 M fortuitum group 18 15 83 62 99 3 17 0 M gordonae 23 21 91 76 100 2 9 0 M haemophilum 4 3 75 i 1 25 0 M kansasii 15 13 87 64 100 2 13 0 M marinum 2 2 100 ii m 0 0 M mucogenicum 2 1 50 T z 1 50 0 M neoaurum 1 1 100 l 7 0 0 M simiae complex 4 4 100 i 0 0 M nonchromogenicum terre 10 10 100 81 100 0 0 M thermoresistible 1 1 100 T 0 0 M tuberculosis complex 81 80 99 95 100 1 1 0 M xenopi 2 2 100 0 0 Totals 235 223 95 92 98 12 5 0 Definitive Identification Similarity Index SI 20 500 and second choice SI gt 0 200 from first choice Indeterminate Identification SI 0 500 or second choice SI within 0 200 of first choice SI Discrepancies False Positive or False Negative result by SMIS i Too few samples to calculate co
35. the Mycobacterium avium intracellulare and M tuberculosis bovis africanum complexes Warning The SMIS should be considered a dedicated mycobacterial identification system Any use of the HPLC component for other applications will invalidate the system for its intended purpose Hardware and Software Installation This manual is intended to contain all information necessary for successful operation of the Sherlock system It is assumed that a qualified MIDI technician has properly installed and connected all the components of your chromatographic system Specific hardware installation procedures are not included in this manual There is a hardware installation checklist in the Installation Qualification that should be verified by the MIDI representative and by the customer Consult the Agilent manuals accompanying the individual components for more information involving operation of the hardware without use of Sherlock The MIDI Sherlock software package includes an Installation Qualification that will guide the MIDI service person or the user through the installation of the Sherlock software The installation instructions are specific to your hardware and computer operating system configuration and must be followed carefully The signed and completed Installation Qualification also serves as proof that proper installation procedures were followed for GLP purposes Your software is shipped with a Security Module that must be connected to the co
36. under a dissecting microscope may aid in detection of potentially mixed cultures of the slow growing organisms 2 1 Warning Observe Biosafety Level IIl practices while working with viable cultures An approved biological safety cabinet must be used Sample Preparation There are three steps required to prepare samples to be run on the HPLC and these are outlined in the following sections Figure 2 2 gives a detailed graphical version of these sample preparation steps Harvesting Cultures and Saponification 1 po E Ds ccu Obtain a sufficient number of 13 x 100 mm screw cap tubes for the sample batch Each batch should contain a known strain as a positive control and a negative control For a positive control use M gordonae ATCC 14470 For a negative control use Candida albicans ATCC 60193 Carefully inspect each tube for defects and proper cap fit Discard any defective tubes caps Label tubes with appropriate identifier using a laboratory marking pen that will withstand autoclave conditions Add 1 0ml of Saponification Reagent Reagent 1 to each tube using a repeat dispenser Harvest growth from the agar plate into the tube containing the saponification reagent Only a very small amount of cells is needed therefore an amount of cells barely visible on the end of a sterile applicator stick or disposable plastic bacteriological loop is sufficient It is better to work with isolated colonies Securely tighten a teflon lined cap onto
37. values are frequent e Verify that the media preparation and culture conditions are as specified in Chapter 2 e Use Positive and Negative Controls The easiest way to catch problems with reagent or sample extract preparation is using a positive and a negative sample control with each batch of samples e f sufficient acidification reagent is not used the mycolic acids may not be effectively derivatized e The amount of cells in the extraction tube will affect the total height Too few cells may not yield enough mycolic acids for a reliable comparison to the database In this case a warning will be printed on the profile concerning the TOTAL RESPONSE being less than 20 000 e Contact MIDI Technical Support for additional assistance 7 5 Hardware Problems All hardware problems should be reported to MIDI Technical Support see page 7 1 MIDI will coordinate with the Customer onsite service if necessary through Agilent Technologies The following conditions are examples of potential hardware problems relating to the HPLC e Elevated backpressure This most commonly results from the plugging of the frit in the purge valve Another source may be in plugging of the small diameter tubes between the injector and the column e Solvent leaks The system is designed to minimize need to touch the HPLC plumbing but if it is necessary to do so trained personnel should do this Each module has sensors to detect gross leaks e Pump seals wear out
38. view both windowpanes of library information Allows the user to view the bottom windowpane of library information Chapter 6 Routine Maintenance Overview Routine maintenance of your Sherlock HPLC Mycobacteria identification system comprises the following e Data backup removal and storage e Changing the purge valve frit To prevent loss of data in the event of a computer hard drive crash it is important to regularly back up data to a more permanent storage system Sherlock provides a Windows based program to move batch data out of the Sherlock DATA directories The only user performed routine maintenance procedure is the changing of the purge valve frit A technician trained by MIDI should perform all other maintenance tasks Data Storage When a Sherlock batch is run data is stored in two directories The raw ChemStation chromatogram and integration results are stored in the C SHERLOCK RAW directory while the Sherlock data used to create profiles is stored in selected C SHERLOCK DATA directories The information needed to manipulate Sherlock files in the future is in the C SHERLOCK DATA directory The raw chromatographic data including the chromatographic plot image is stored in the C SHERLOCK RAW directory Without the contents of the RAW directory the Sherlock CommandCenter cannot reprint chromatograms on reports For this reason the C SHERLOCK RAW directory should be included in a backup strategy It is possibl
39. 0 ECL deviates 0 306 ECL 92 257 index 8 697 32646 0 140 94 214 Sum In Feature 19 2 88 ECL deviates 0 109 ECL 94 323 8 988 9733 0 151 97 000 ECL 97 000 standard ECL deviates 0 000 Reference 0 046 9 463 535 0 163 101 535 gt maxrt 9 681 700 0 138 103 617 gt maxrt 9 859 575 0 202 105 324 gt maxrt 1215 Summed Feature 1 0 11 742 Summed Feature 9 0 07 1537 Summed Feature 10 0 14 3104 Summed Feature 11 0 27 10131 Summed Feature 12 0 89 35798 Summed Feature 13 3 16 115990 Summed Feature 14 10 22 228230 Summed Feature 15 20 12 307522 Summed Feature 16 27 10 264531 Summed Feature 17 23 32 129343 Summed Feature 18 11 40 32646 Summed Feature 19 2 88 ECL Deviation 0 131 Reference ECL Shift 0 034 Number Reference Peaks 2 Total Response 1134562 Total Named 1134562 Percent Named 100 00 Total Amount 1134562 Matches Library Sim Index Entry Name MYCAG1 1 02 0 906 Mycobacterium tuberculosis complex TB bovis africanum microti 4 3 A table containing the mycolic acid profile for the sample follows the general information The columns of this table are e RT the retention time is used to calculate the ECL value e Response for HPLC peak height is used rather than peak area as it is more reliable e Ar Ht
40. 1 for Reagent 3 MicroZippette 100 250 uL Cat 268 005N for Reagent 4 and 5 VWR 53410 004 72 place tube holder 13mmx100 mm tube rack Fisher 14 793 14 Pasteur Pipette Bulb 24 Fisher 03 448 26 Pasteur Pipettes 5 Fisher 13 678 20B Borosilicate Glass Tubes Threaded End 13x100mm 1000 count Fisher 14 959 35C Phenolic Caps Fisher 14 930 15D Borosilicate Glass Tubes 13x100mm 1000 count Fisher 14 961 27 Brinkman Eppendorf Series Pipetters 10ul 10011 Fisher 05 402 48 epitips Reloads 2 uL to 200 LL refill 10 x 96 Fisher 05 403 41 Brinkman Eppendorf Series Pipetters 100ul 1ml Fisher 05 402 50 epitips Reloads 50 uL to 1000 pL refill 10 x 96 Fisher 05 403 43 Vial rack 50 Spaces Fisher 03 375 9 Multi tube Vortexer 120 V VWR 58816 115 MIDI N2 Evaporator MIDI MIDI VAP O R Pierce Reacti therm Heating Module Pierce Contact Vendor Pierce Reacti vap Evaporator Pierce Contact Vendor Pierce Reacti Block S 1 Pierce Contact Vendor Pierce Reacti Block A 1 Optional Pierce Contact Vendor Consult Vendor for Additional Size configurations HORE Labconco Rapid Vap Vacuum vortex heat evaporator with lid heater Labconco Contact Vendor and corrosion proof vacuum pump Labconco Sample Block Labconco Contact Vendor Table A 2 Sherlock Consumables Ordering Information ITEM SOURCE PART NUMBER Chemicals Methanol case HPLC Grade ACS Certified Fisher A452 4 2 Propanol case HPLC Grade ACS Certified Fisher A451 4 HCL 5L
41. 19 2002 517 2239 Samp MYCOLCI QC MYCO GORDO ATCC14470 2240 Samp MYCOLCI QC MYCO GORDO ATCC14470 2241 Samp MYCOLC1 QC CAND ALBIC ATCC60193 2 2242 Samp MYCOLCT OC BLANK 6 7 02 GO 2242 Samp MYCOLCI QC BLANK 6 7 02 GORR 2243 Samp MYCOLCI UN MYCO 1001 3wK CHOC 301 2244 Samp MYCOLCI UN MYCO 1002 SwK CHOC 301 6 19 2002 7 027 DO E028017 38A 2245 Samp MYCOLCI UN MYCO 1003 5wK 7H11 SEL 6 19 2002 717 O E028135 154 2246 Samp MYCOLCI UN MYCO 1004 4wk GO 6 19 2002 7 32 Y E029035 68A H O E024314 45A O EU28045 27A Sample 026196 944 12 2246 UN MYCO 1004 4wK GO O E02BD65 78A DO E02B156 20A Taskbar gt O E02C104 22A R DATA1 Sample ID i Bottle 10 DATA2 DATA3 Created 6 19 2002 7 32 29 Modified NEN DATA4 J ID Number 2246 Seq Number 2246 g DATA6 E Sample Type s amp y ethod Name MYCO LC1 x Sequence E025316 314 selected SAMPLE E026033 554 O E026035 904 O E026043 744 HIA ft 6 19 2002 5 32 6 19 2002 5 47 6 19 2002 5 02 6 19 2002 5 17 6 19 2002 5 32 6 19 2002 5 47 E026196 944 olo a eoa colo O E026257 33A e a EF E Ee EC E Data Volume Clear Selections Utilities AAA AAA Windowpane Message Line no Samples View has 3 Panes message showing Sample Selection The Sherlock Command Center contains tools that allo
42. 3 The Print Preview Window w Print Preview Print Preview Volume DATA File E026196 04 A seq Counter 12 ID Number 2246 Type Samp Bottle 10 Method MYCOLCI Created 6 19 2002 7 32 29 PM sample ID UN MY CO 1004 4WK GO Profile LE Regem Art ES PedMeme Percent Commen Coma c c LL s S FZLLSS ass 259 om SIS lema 2017 __ 8922 0097 40 000 ECL 40000 semel ECH devisies 000 _ Refirenes DOS ssn es0 0131 64427 ECL64625 OM6 ECLdeviate 0 198 5930 742 0190 67881 SumInFeaure 9 O07 ECL deviates 0 168 ECL 67 713 6523 3104 0110 73591 SumInFeaure 027 ECLdevistes 0 162 ECL 73 429 index 6592 3113 0096 74252 ECL74350 027 ECLdevies 0 098 8194 264531 0119 89 450 SumInFeamrel 23 32 ECL deviates 0 160 ECL 89610 8462 129 43 0121 91 951 SumIn Feature 18 11 40 ECL deviates 0 306 ECL 92 257 index 8988 9733 0151 97 000 ECL 97000 standard ECL deviates 0 000 Reference 0 046 9463 535 0163 101 535 gt mat Pr 9 681 OO 0138 108617 at 8 0 _________ AAA AAA ep 7 Q lt A A e pp mas e Surmed Featare 1 ee ccs e EE pwn 1987 Summed Feature 10 014 R pp 314 SummedFeaturell O27 1081 jSummedFesi 12 ogo 0 S PO 3598 SummedFeaturel3 316 8 0 00 0 0
43. ATA eoeracosa 2 2241 Samp MYCOLCT QCCAND ALBIC ATCCEUIS 6 8 13 2002 6 02 27 O EO26076 524 E 2242 Samp MYCOLCI C BLANK E 02GU 7 6MB 20026 1722 Y ED26196 04A E 2242 Samp MYCOLCI C BLANK E zU2 GORRA 8 6 19 2002 6 32 10 E026203 914 7 2243 Samp MYCOLCT UN MYCO 1001 3WK CHOC 8 6 19 2002 6 47 13 EO26257 334 8 2244 Samp MYCOLCT UN MYCO I002 EWK CHOC 10 6 19 2002 7 02 13 ED28017 38A 8 2245 Samp MYCOLCT UN MYCO 1003 5wK 7HT1 Tl 6 19 2002 7 17 18 E028135 15 H 2246 Samp MYCOLCT UN MYCO 1004 4wKGO 12 6 19 2002 7 32 28 E029035 654 4 gt E024514 454 ASA SR Sample ED26196 944 12 2246 UN MYCO 1004 4wK GO EO28065 784 E02656156 204 General Raw Data Profie Matches Chromatogram Comments Mgr Comments EF02C 104 224 SamplelD UN MYCO 1004 4wk GO Bottle 10 Created 6 19 2002 7 32 28 P Modified ID Number 2246 Seg Number 2246 Sample Type Samp Method Name wvcaLci H Methods Pr Fo Libraries LI EIETETET ET EI ET ET ET El B E B B E O O n Fla n Ej n B B B B m Clear Selections Utilities Advanced Sample Selector Mode Sherlock CommandCenter also allows you to search for sample data using the Advanced Sample Selector mode In this mode you can search for data files by any combination of the following e Data Volume e Method e Sherlock Data Files e ID Field Prefixes To better understand the operation of the advance
44. At End y This tool is identical to the Batch Stop at end of run menu item Abort amp This tool is identical to the Batch Abort Batch menu item Adding Samples and Calibrations Every Sample Table must contain a calibration entry Sherlock will automatically calibrate twice prior to sample analysis and will recalibrate the system after every 10 samples Samples may be added before or during the processing of samples by Sherlock When the Add Samples tool is selected the Sample Table editor automatically advances to the first empty bottle position The next sequence number will be incremented automatically by the system The 3 10 status of the new sample is set to QUEUED Enter 1 to 42 characters for the sample identification in the NAME field There must be at least one character entered as a sample name Strike the Enter key to advance to the next empty bottle position To stop adding samples press the Done Adding tool the table can also be locked by clicking the Lock Table tool to ensure no inadvertent changes are made Figure 3 5 Editing the Sample Table Sample Processor LC S N 160011 Sample Table e A gt Ye Print Table Add Samples LockTable Start Batch mm smms 7 DATA VOL Status Bottle ID Number Data File s COLA AVAILABLE MYCOLCI Calibration Mix for MYCOLC method Queued IMYCOLCI QC BLANK 5 29 13 CC Queued IMYCOLCI QC MYCO AVIUM ATCC 13956 CC Queued MYCOLCI MYCO TUBER ATCC 27294
45. Print Preview Tool can also be used to view the same library information on the computer screen 5 21 CommandCenter Tools and Views Icons The icons associated with the CommandCenter are described in Tables 5 1 through 5 4 All of the Tools in the Toolbar Tools can be accessed through the Menu Bar but the Tools provide a shortcut to these features Table 5 1 Samples View Basic Selector Name _ ton Description Sherlock Samples Clicking this icon will open up the Sherlock Samples View view where the user can view and edit sample data Print Sample Allows the user to print sample information for the Information number of samples desired to the printer or to a Rich Text Format RTF file Allows the user to preview sample information directly on the computer screen The user can sort the sample table by any of the headers in the sample table Any changes made to the sample table changes are made in the bottom right window pane can be saved Table 5 2 Samples View Advanced Selector Name Ilcon Description Select Samples In the Advanced Selector View allows the user to toggle back to the sample selection mode from the detail AA In the Advanced Selector View allows the user to view the details for the samples in the current query Table 5 3 Methods View ECCE e a Sherlock Methods Clicking this icon will open up the Sherlock Methods View view where the user ca
46. SAO1061 3 tt v Print General Information w Print Commenta Bi iil Saisie ge Print Post S ample File Select Samples f Pri Print Ho Samples Print Detail Sections General Information Comments Profile Information Print Non Calib Samples Bare C Print All S amples f Print Current Sample W Each Sample on a Mew Page OR Cancel Figure 5 12b Sample Print Preview Dialog Box Chromatogram Tab m Sample Print Preview Options Printer File Options r Detail Options rd css Iw Chromatographic Sections CASHERLOCK RESULTS RO1081 3 1 Print Haw Data W Print Profile Information We Print Match Results Comparison Charts 10 Print Chromatogram Print Sample List Select Samples Pn Print No Samples Print Detail Sections General Information Comments Profile Information Print Non Calib Samples a ae C Print All Samples f Print Current Sample W Each Sample on a New Page OF 5 13 The navigation tools on the left hand side of Figure 5 13 are used to navigate the preview pages You can also set the page range to be printed For example if there were multiple pages you could enter any page range s into the Print Page Range field Clicking the Print button will then send the designated pages to the printer If save to RTF was selected in Figure 5 12a the View RTF button will launch an appropriate viewer e g Microsoft Word to display the file Figure 5 1
47. Sample Table e f no adjustments are needed click on Start Batch e The system will prompt the user to warm up the system Select Yes e The system analyzes the Calibration Standard two runs at the beginning of the batch and if the system is working properly will proceed through the samples in the Sample Table e After all samples have been analyzed the software automatically shuts off the pumps detector and heater column modules of the HPLC and closes the ChemStation software While the system is running samples other commands are still active e Click on the Sample Table to make additions or edits to the sample table Note The Running sample may not be edited Prematurely Stopping a Batch Ordinarily once Sherlock has started a sample batch all samples will be analyzed and the system shutdown as described above However if it becomes necessary to stop a batch before all samples are analyzed there are two options Stop at End and Abort The Stop at End y tool replaces the Start Batch tool after a run has been started Click on the Stop at End tool if it is necessary to stop the operation of the system at the end of the current chromatographic run This will complete the analysis in progress print the results and then stop the batch The Abort Doo also appears in the Toolbar after a run has been started Click on the Abort tool to stop the operation of the system any point before completion of the batch This will immedia
48. Sherlock Mycobacteria Identification System Mycobacteria Identification System Operating Manual Version 6 2B MIDI Inc June 2013 Tel 302 737 4297 125 Sandy Drive Fax 302 737 7781 Newark DE 19713 Email support midi inc com USA www midi inc com Trademarks Sherlock is a U S registered trademark of MIDI Inc All other trademarks are the property of their respective owners This manual may not be copied photocopied reproduced translated or converted to any form electronic mechanical or otherwise without the prior written consent of MIDI Inc Copyright 2013 MIDI Inc All rights reserved First Printing February 2002 Second Printing June 2013 Part Number 2082 06 2013 MIDI Inc 125 Sandy Dr Newark DE 19713 DI Sherlock Software and Libraries LICENSE AGREEMENT AND LIMITATIONS OF WARRANTY IMPORTANT Please carefully read this License Agreement before installing the software contained in this package The right to use this MIDI Inc software product is granted only on the condition that the Customer agrees to the terms of this license For installation to continue you must agree to the license terms If you do not accept the license terms installation will be cancelled In return for payment of a one time license fee for this software product the Customer receives from MIDI Inc a license to use this product subject to the following terms and conditions The product may be used wi
49. Sherlock Sample Processor Configuration screen Figure 3 2b or by specifying this option in the Sherlock CommandCenter Print Preview and Print dialog boxes Figure 5 12 amp 5 14 Chromatographic Report The ChemStation is used to accumulate raw data and to develop the chromatogram In routine use of Sherlock the chromatogram with the peak times plotted by the ChemStation normally need not be evaluated However you should be familiar with the contents of the plotted chromatogram to confirm proper operation of the system Using the Sample Table and the Calibration Sequence parameters specified in the method the Sherlock software selects the sample scheduled for injection The chromatogram heading lists the injection time and date bottle number and the sample name as it was entered in the Sample Table name field The sample ID number appears in the Sample Name fields and within the data file name Once the sample is injected the ChemsStation plots the signal from the fluorescence detector of the HPLC creating the chromatogram as shown in Figure 4 1 Mycolic acids in the sample are separated by the column and identified by the retention time of each peak Retention times are measured to a resolution of 0 001 minute Figure 4 1 chromatogram is typical of M tuberculosis complex 4 1 Figure 4 1 HPLC Chromatogram Plot FLD1 A Ex 345 Em 425 C SHERLOCK RAW E00719 433 A01645A3 D 8 101 7 818 100 8 368 80 60 7
50. TI ZO HOM esum cto ER E EO DRE OQ HDI CIERRE RD Febr TR Dur tu Dd TM aetna 2 4 BEG SONENE 2 4 BEGIN ee T 2 5 duse ue m 2 6 ACENOWEDGEMENTS cee c 2 REFERENCE cece ees 2 7 CHAPTER 3 ANALYZING SAMPLES es nsesessssessesessesessesessesessesecsesessesscsesessssessesessesessesessesessesessesessessssesessesessesese 3 1 OVERVIEW A 3 1 Loading the Automatic Liquid Sampler cccccscecccceeeccssnseccsueecccausecssausesssausecsauuscesausecssausessausessausecssaueessaaanes 3 1 Load ma Samples MOTNE OV E sa 3 1 PA ee PATRIMOINE H 3 1 GODOT OT STANGATA iia 3 2 SAMPLE PROCESSOR CONFIGURATION scncusesausnsasduinnannsa es pantbaina diane a R a a Ea aa ET R a E a RR 3 3 Ie EE e eo 3 3 RPP E Fo a a aos 3 5 Instrument Parameters TOD arab 3 6 THE SIP EE PROCE OR ea a E E E E E E E NT EA E EE E IA EETA 3 7 The Sample Processor Menu BOT sorei arsen r n E e NOE E ATE E TA 3 7 The Sample Processor TOO BGP rancia 3 9 Adding Samples and CAliDrations ccssccccssssccccsnscccsauscccsseccsausecssauseccsauscesausesssau
51. TO ERES to YE Iun tva ro vo ETA 6 11 CHAPTER 7 TROUBLESHOOTING irosip uos eno aure yup o epo venue E 7 1 A M rouel 7 1 FOFJTIDE Technica SabDOEE s etc a x ter duties etuer d nb d eed n oe ne tele ite O d ee eM oed re 7 1 INSTALLATION PROBLEM ctc AAA ser 7 2 Cannot install SOR W LB ssoi oia 7 2 Problems Attempting to Run First Set Of SQIMPIOS ccccccssessecccceeeecccscsseccscaeneecessuunsecesaaeeecessausecessaenseeeseaees 7 2 CHROMATOGRAPHIC PROBLEMS a dias 7 2 CalbFatiomAviessa065 5 eus e ets oa te ae cect D E renee E rac E A ct E A E 7 3 Sample MESSAGES 6b eut ar m its 7 4 SAMPLE PREPARATION ERRORS adi 7 5 FIARDWARE PROBEEM Ss cta 7 6 APPARENT SOFTWARE PROBEENIS ETE RTI a das 7 6 PERFORMANCE QUALIFICATION TABlE is 7 7 APPENDIX A EQUIPMENT AND CONSUMABLES cccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccecccscseccs A 1 OVERVIEW a a saco A 1 APPENDIX B MYCOBACTERIA LIBRARY ENTRIES cccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccceccccccccccccseccs B 1 OER VIE ul S B 1 APPENDIX C PROCEDURAL NOTES acousicisaricinndaraconica acond C 1 EVAPORATION OF LIQUIDS IN TEST TUBES AND VIALS ccccecccccecccccccccccccecceccecceceececcenceccencecceaeecesceecessecceacenceaeeseeseesensenseaces C 1 APPENDIX D HPLC PERFORMANCE QUALIFICATION TABLE cccccccccccccccccccccccccccccceccccccccccccccccccc
52. This usually indicates a problem with the Sample Table entries Confirm that samples were added to the Sample Table properly using the ADD function Confirm that all samples and Calibrations have a Sample ID number and are Queued Verify that there is a Calibration Calib for each Method of queued samples Refer to the examples in Chapter 3 Note Calibration samples must be specified as type Calib in the sample table and will have their ID Num set to 1 Cannot Connect to Chemstation This indicates that Sherlock and ChemStation are not communicating Several things may cause this e Confirm that the Installation Qualification was followed e You should have been able to run a sample using only ChemStation using the Checkout method that is supplied on the Sherlock system CD e Confirm that ChemStation is installed on the C drive e Confirm that ChemStation Instrument 1 is closed before starting a Sherlock batch Chromatographic Problems If your Sherlock fails to calibrate or fails to name peaks there are several potential causes but most are predictable In this section we discuss how chromatographic problems manifest themselves in Sherlock reports Sherlock has been designed to accept and search mycolic acid fluorescent extracts only if specific chromatographic requirements are met Since the system relies on the qualitative and quantitative results of a single analysis for identification of mycobacteria several thresholds are set to a
53. able sum The very distinctive patterns of the mycolic acids allows use of summed features while still giving great precision in matching to the stored library of mycolic acid profiles in the library The two comment columns are used to provide additional information about each peak that may help troubleshoot chromatographic issues Note ECL 40 000 and 97 000 peaks are used as internal standards and thus no response is assigned to these peaks Sherlock refers to them as Zero Features Summary Section The Summary Section of the Composition Report is useful to troubleshoot the system Several of the performance measures listed below are checked by the system during operation and warning messages are printed if limits are exceeded e ECL Deviation the error between the actual ECL values and the expected ECL values It is a measure of the chromatographic accuracy for naming peaks e Reference ECL Shift the ECL drift calculated for designated Reference Peaks It is a measure of the chromatographic stability 4 4 e Number Reference Peaks a count of the number of reference peaks found in the sample For the MYCOLC1 method it should be two the ECL 40 000 standard and ECL 97 000 standard e Total Response the sum of the Response column values for all peaks excluding peaks that are outside the RT of interest lt min rt and gt max rt and zero feature peaks Zero feature peaks are assigned peak names but are not included
54. al but not so high that the liquid is splattered in the tube vial There should be a gentle dimpling of the surface of the liquid 2 6 Position the evaporator head until the needle tips are approximately 25 50 mm above the surface of the tube vial contents 2 7 Monitor the liquid volume and remove the tube vial when dry as evidenced by a slight residue Note If the MIDI or Pierce unit is used with house compressed air a gas drying unit packed with anhydrous silica gel or Drierite desiccant should be used See Appendix A for ordering specifications The desiccant contains a color moisture indicator Change desiccant in the unit when the color of the moisture indicator changes from blue to red Regenerate desiccant by drying overnight in a hot air oven at 120 170 C C 1 3 Labconco RapiVap 3 1 13 x 100 tubes 3 1 1 Temperature 85 C 3 1 2 Vortex 35 full speed 3 1 3 Vacuum Full 3 1 4 Time 20 min 3 2 Sample vials 2 0 ml 3 2 1 Temperature 60 C 3 2 2 Vortex 15 full speed 3 2 3 Vacuum Full 3 2 4 Time 10 min Note Use old 2 ml vials in the heating block as spacers for the sample vials This allows easy access for retrieving the sample vials 3 3 KHCO3 Vials 3 3 1 Temperature 60 C 3 3 2 Vortex 12 full speed 3 3 3 Vacuum Full 3 3 4 Time 30 min Note Use old 2 ml vials in the heating block as spacers for the sample vials This allows easy access for retrieving the sample vials 4
55. ange from QUEUED to DONE or vice versa click on the status field and choose from the drop down menu Samples that have a status of RUNNING cannot be changed If the user wants to process samples out of order they can use the STAT function Removing items from the Sample Table If the table is not Locked samples can be removed from the Sample Table by scrolling to the entry and choosing any of the Table Clear options from the menu bar Record Keeping Enter enough data about the sample into the Name field so that anyone can go back a year later and know what was run If the growth conditions were not standard for the Method being used enter the nonstandard conditions in the name field Also enter any unusual observations that may aid in interpreting results Consistency in the name is extremely important for record keeping as well as for cataloging samples It is advantageous to organize name fields so that the user can look at groups of samples of interest To compare groups of entries it will be necessary to create a system for making groups and subgroups very early in the data collection process The name of a sample consists of two sections separated by an open parenthesis sign A total of 42 characters may be entered We suggest using UN in front of unknown samples The second section of the name begins with an open parenthesis sign and allows the user to enter discrete information about a sample such as the patient id
56. are not intended to replace those provided by MIDI through a service contract through MIDI training courses or appearing in Agilent supplied service and maintenance documentation Replacing the Purge Valve Frit When is this required e When the pump piston seals are replaced or when contaminated or blocked e Blockage may be indicated by increased back pressure of the system during routine operation e tis good policy as part of preventive maintenance to change the frit after each series of ca 500 analyses Tools required e Wrench inch e Wrench 14 mm e Forceps tweezers Part required e PTFE frit from Agilent Technologies pack of 5 01018 22707 6 11 Preparation Switch off the pump and remove the front cover of the pump assembly Steps in replacement of the frit For complete details see the Agilent CD ROM on maintenance of the HPLC 1 Ze 3 10 11 12 Disconnect the waste tubing Take care for leaking solvents due to hydrostatic pressure Using the inch wrench disconnect the outlet capillary at the purge valve Using the 14 mm wrench unscrew the purge valve and remove it Carefully remove the plastic cap with the gold seal from the purge valve Using the forceps remove the frit Figure 6 10 Insert the new frit into the purge valve Replace the cap with the gold seal Note Check the gold seal replace if badly deformed Insert the purge valve into the pump head and turn until it is hand tig
57. arison Figure 4 7 Visual Confirmation FLD1 A Ex 345 Em 425 C SHERLOCK RAW E00623 371 A01570A6 D FLD1 A Ex 345 Em 425 C SHERLOCK SYSFILES METHODS REF FLUORTES MYCOTUBM D 4 13 Chapter 5 Sherlock CommandCenter Overview To facilitate regenerating reports and data retention Sherlock stores data from calibration and sample runs into Sherlock data files which are then stored into data volumes labeled DATA DATA1 DATA7 on the hard disk You can add additional data volumes as well eg DATA8 DATAS etc Typically you will collect data from recent batch runs into the DATA volume and later organize it into other volumes described in Chapter 6 Sherlock saves the raw chromatographic data in the Sherlock RAW folder Sherlock shares the hard disk with the Agilent ChemStation and any other applications that are on the computer A minimum of 20 gigabytes of free disk space available for installation and operation is recommended The Sherlock CommandCenter is the heart of the Sherlock software Instructions on how to use the software for viewing stored sample and calibration data regenerating reports and correcting data entry errors are presented in this chapter The chapter first illustrates how to select and view sample data using either the Basic Sample or Advanced Sample Selector modes Then the chapter describes how to view installed methods and libraries Chapter 6 describes how to install and update methods and also cove
58. asic Sample Selector was used to choose the sample and in Figure 5 9 the Advanced Sample Selector was used to choose the sample From this point on the Advanced Sample Selector screens will be used to demonstrate the operation of the Sherlock CommandCenter The Advanced Sample Selector screens are the same except that they don t show the sample windowpane on the left hand side as shown in Figure 5 4 Otherwise the following operations are the same for both selectors Figure 5 9 Advanced Sample Selector Viewing Sample Details A Sherlock CommandCenter Samples File Sample View E Help ldex voume Fisname Bue ID Nbr Teee Metro ue Created Tar 2243 Samp MYCOLCT UM MYCD 1001 3wK CHOC 30C GO 8 6 19 2002 6 47 13 mem zl ee 6 19 2002 7 02 13 38 2245 Samp MYCOLCT UN MYCO 1003 5wkK 7411 SELECTIVE BO 11 6 19 20027 17 18 10 2246 Samp MYCOLCT UN MYCO 1004 4wK GO 2 E 13 2002 7 32 23 le Methods Libraries Sample E026196 944 12 2246 UN MY CO 1004 4k GO General Raw Data Profie Matches Chromatogram Comments Mgr Comments Sample ID UMMvcO 1004 dWKGO Battle fio Created 6 19 2002 7 32 29 P Modified lo ID Number 246 seq Number pae Sample Type Samp Method Marne MYCOLCT Utilities Selecting the Profile tab in the bottom windowpane displays the list of peaks or compounds found in the selected sample Figure 5 10 This is the same inform
59. ation that can be printed as the samples are run with the Sample Processor You can also view the raw ChemStation data at his point by clicking on the Raw Data tab in the bottom windowpane 5 10 Figure 5 10 Displaying the Mycolic Acid Profile for the 12 Sample continuation from Figure 5 4 K Sherlock CommandCenter Samples File Sample View Help lt SampMethod gt Calc Method EE idesivalame Peneme Bote fohe Mwe dea or et Created MYCOLCI T MYCO 1001 3wK CHOC 30C GO pam 6 19 2002 6 47 13 DATA E026196 944 i MEE prie Eae Tel nesponi Peserai fine Petcenl Commertt _ Comment2 lt min rt a 10m 3m cr E Reference 0 015 ECL 41 600 ECL 67 713 E ECL 71 387 31 E ECL 73 423 index 6 59 ECL 76 278 index 3578 ECL 79 010 index 11599 o 12 ECL 81 614 index ECL 84 340 index ECL 87 065 index ECL 89 610 8 46 12534 ECL 392 257 index 8 a ECL 34 323 E AE Reference 0 046 L 349 saso DIES O no f mat Ij 3 0 0001 103617 Omar E esa sso o 3 oee 0 121 T n 0 O00 O00 Summed Feature 1 i 11 ECL 41 600 ECL 42 282 Total Response 1134562 Percent Named 100 00 ERE 7 03 732 8 13 30752 26403 L L ine 55 5330 _ 5298 L 5523 mE L 6816 Hus L L 58 73 _ 8194 _ 8482 LL 863 na na mo na La mica rm alo oloo o l L ra k l L co oo ps Ix Utilities Se
60. avium intracellulare scrofulaceum avium intracellulare i M MAI M avium 25291 M MAC A avium intracellulare M MAC A avium intracellulare gt camper eee AM uM a es ea gt msme wo ume 8 wsme EA CTE TT ECTS CT E 1 Similarity Index Value Indeterminate first choice SI 0 500 or second choice SI within 0 200 of first choice SI E 3
61. been isolated from clinical specimens by traditional culturing techniques Following observation of growth on the solid medium identification of the mycobacterial isolate is done with the MIDI Sherlock Mycobacteria Identification System Results should be interpreted in conjunction with other laboratory observations and procedures Limitations Sherlock can identify only those microorganisms for which mycolic acid composition profiles of a representative number of correctly named reference strains have been determined and entered into the Mycobacteria Library The library entries have been determined by analyzing reference strains grown under controlled culture conditions These culture conditions and sample preparation procedures must be followed The Sherlock Mycobacteria Identification System has been shown to reliably differentiate the M tuberculosis complex from other mycobacteria Definitive identification of Mycobacterium other than tuberculosis MOTT requires use of additional laboratory testing to identify clinically significant organisms Sherlock may be useful in conjunction with probe hybridization biochemical testing and other laboratory observations The ability of Sherlock to correctly identify MOTT not listed in the database has not been evaluated See Appendix E 1 3 Overview of Sherlock Operation The Sherlock system is completely controlled by the computer After sample extracts have been prepared discussed in Chapter
62. cal plots show how the unknown sample profile compares with library search results There can be up to four comparison charts printed See Chapter 4 for a more detailed explanation of Comparison Charts The nclude Chromatogram checkboxes determine if the chromatogram plot will be printed MIDI recommends that these be printed The Print Pre Sample File and Print Post Sample File checkboxes define a custom header and footer for the reports The header might contain the laboratory s name and address The footer might contain a signoff line for report approvals If Generate Summary Report at End of Batch is checked a list of samples with identifications is printed 3 5 Figure 3 2c Sample Processor Configuration Screen Instrument Tab w Sherlock Sample Processor Configuration LC S N 160011 Internal 100 Vial Tray Instrument Parameters Tab This section will not need to be amended if your instrument was configured by a MIDI technician during the Installation Qualification Note Contact MIDI if the parameters don t match exactly as shown in Fig 3 2c For Sherlock configuration settings the 1100 Series HPLC and the 1200 Series are equivalent 3 6 The Sample Processor The Sample Processor is accessed from the Sample Processor Icon Lon the desktop Sherlock uses the Sample Processor as a Sample Table and as a link with the Agilent ChemStation The top menu bar and toolbars allow manipulation of the Sample Table Figu
63. cecccccceccs D 1 APPENDIX E PERFORMANCE CHARACTERISTICS cccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccecccscceccs E 1 azul M AR E 1 Chapter 1 Getting Started Overview The Sherlock Mycobacteria Identification System SMIS developed and marketed by MIDI Inc Newark DE USA analyzes and identifies microorganisms isolated in pure culture on a solid growth medium Sherlock uses a sample preparation procedure and high performance liquid chromatography HPLC to yield qualitatively and quantitatively reproducible mycolic acid composition profiles This sophisticated chromatographic system has been developed to be used by microbiologists thus chromatographic experience is not essential for operation The Sherlock software calibrates and monitors the system to ensure proper functioning Mycolic acids extracted from unknown mycobacteria are automatically quantified and identified by the Sherlock software to determine the mycolic acid composition The mycolic acid profile is then compared to a library of profiles of reference strains of mycobacteria stored in the computer to determine the identity of the unknown Unknown samples may be identified to the species or to the species complex group The species complex will typically be organisms that have high DNA similarities and or are similar in clinical significance Examples are
64. ces and dust Do not use the HPLC column for other applications Warning Column damage may occur if the peltier heater is at 70 C and there is no solvent flow through the column 1 2 Computer and Software Sherlock runs on an IBM compatible personal computer The computer should be considered to be a dedicated instrument controller All Sherlock files are located in the top level directory named C SHERLOCK MIDI tests Sherlock software with Dell computers and Hewlett Packard printers Minimum requirements e Windows 7 is recommended Windows XP SP3 is supported e Acomputer that fully supports the version of Windows Preferred minimum computer 1 5 GHz 2 GB RAM and 20 GB free disk space e Agilent Technologies ChemStation that is compatible with the version of Windows and the HPLC model and revision prefer B 04 03 or higher e CD ROM drive e 1024x768 Minimum Color Display Resolution e AN interface e Dedicated printer Intended Use The MIDI Sherlock Mycobacteria Identification System is intended to aid in the identification of M tuberculosis and differentiation from other mycobacteria species through the analysis of mycolic acids derived from cultured bacterial samples using high performance liquid chromatography HPLC performed on the Agilent 1100 and 1200 Series HPLCs along with Sherlock pattern recognition software The system is used along with other identification methods to identify mycobacteria that have
65. ck Reports Overview Your Sherlock system reports analysis results in two forms e A Chromatographic Report produced by ChemStation e ASherlock Composition Report including a library search The primary component of the Chromatographic Report is the chromatogram a visual plot or trace of the electronic signal generated by the fluorescence detector as mycolic acids of the sample elute from the column The raw data of the chromatogram is stored in a ChemStation file and can be reintegrated on screen and reprinted if desired Sherlock stores a file containing all peak retention times heights peak widths and a pointer to the chromatogram plot The Sherlock CommandCenter can reprint the chromatogram plot Note Earlier versions of Sherlock did not save a pointer to the chromatogram plot therefore it is not available for reprinting by Sherlock The Sherlock Composition Report comprises a Mycolic Acid Composition Report a Library Search Report and optional Comparison Charts and chromatogram plot The Composition Report contains the mycolic acid composition of the organism The Library Search Report lists the results of comparing the mycolic acid composition to the Sherlock Library Comparison Charts may also be requested These are plots comparing the mycolic acid composition of the unknown to the most similar up to four library entries Comparison charts can be printed by one of two ways either with the Sample Processor report using the
66. cterium aurum vaccae M avium complex M lentiflavum triplex and the Mycobacterium tuberculosis complex that includes M tuberculosis M bovis M africanum and M microti With the exception of M bovis BCG the organisms in the TB complex cannot readily be distinguished by mycolic acid analysis by DNA homology or by DNA sequencing The Mycobacterium MAC complex includes the species M avium M intracellulare and M scrofulaceum Subgroups A and B are predominantly M avium subgroup C is predominantly M intracellulare and Mycobacterium MAIS complex is predominantly M scrofulaceum For definitive speciation DNA probes or biochemical tests may be used with the mycolic acid identification aiding in the choice of test Since treatment following identification of any one of the complexes would be the same there is commonly little value in identification to the species level The Mycobacterium abscessus chelonae and Mycobacterium chelonae abscessus groups contain organisms that cannot currently be reliably distinguished at the species level by mycolic acid analysis An identification report listing of Mycobacterium chelonae abscessus would suggest that the organism is more likely M chelonae rather than M abscessus and is intended to aid in confirmation of identification when a secondary test e g biochemical test is used to help determine the species of the unknown Other similar groups are M fortuitum peregrinum and M peregrinum fortuitum
67. d Version Root 6 8 Install Libraries The user will occasionally need to install custom libraries To install libraries double click on the Toolbox 1 ry Icon m the desktop and then click on the nstall Tool e in the Toolbox The Install Libraries screen should look like Figure 6 7 Make sure that the nstall Libraries tab is selected The default directory to install from will be the A directory Click on the Browse button to search for libraries to install from another location These libraries can be on the hard disk removable media or Local Area Network LAN Choose the libraries of interest and click the nstall Tool e again to install the selected libraries Figure 6 7 Install Libraries z SHERLOCK Install Methods and Libraries Select Install Help X E a Select All Clear Selections Install Refresh Directory Install Methods Libraries to Install Directory u Browse Select Libraries to Install Installed Libraries MYCAG Offload Methods The user will occasionally want to offload custom methods make a copy of a method To offload a 1 ry method double click on the Toolbox Icon m the desktop and then click on the Offload Tool amp in the Toolbox The Offload Methods screen should look like Figure 6 8 Make sure that the Offload Methods tab is selected Next click on the Browse button to search for a destination for a copy of the method The destination path can be on the hard disk removable media o
68. d new samples to the Sample Processor The Add Samples tool will change to Done Adding dl tool while the user is adding samples to the table Figure 3 4 Press this tool to complete adding samples See details in the following Adding Samples and Calibrations section 3 9 Figure 3 4 Sample Processor Add Samples Mode Sample Processor LC S N 160011 Sample Table File Table Batch View Help WA gt Print Table Done Adding LockTable Start Batch wm saos DATA VOL gt taus Bottle ID Number DataFile COLA AVAILABLE Loo SE ENDE MET GUN T Status Result KRC DEE ___1 Galibration Mix for HWCOLC method Queued _ Fi samp HYCOLCI tons tona MPOU TUBER HTOG 27294 00 eve 5 Samp MYCOLCI 1004 1004 MYCO ABSCE ATCC 19977 CC Oueued 8 Bamp_ MYCOLCI 1005 1005 MYCO FORTU ATCC 6841 CC amp Queued b Bamp_ MYCOLC 1006 10008 eet L8 Empty ne L8 Empy ne 11M iFrmnt None bd 4 k Lock Table Unlock Table gt m9 This tool toggles the table between locked and unlocked When the Sample Table is locked no changes can be made inadvertently to the table When adding or editing samples the table must be unlocked Print Table e This tool is identical in function to the File Print menu item Start Batch S This tool is identical in function to the Batch Start Batch menu item Stop
69. d sample selector the previous sample 12th sample in Figure 5 4 will be located using this mode To get to the Advanced Sample Selector mode from the Advanced Samples View Figure 5 1 the down arrow on the Select Style Tool leci tle is pressed and the user drops down to Advanced The screen should then look similar to Figure 5 5 Figure 5 5 Advanced Sample Selector Mode Sherlock CommandCenter Samples File Sample View Help Print Print Preview Index Velune Fieneme Bowe br Type Method ID Stamp CtfCreated Medid Crea DELLI LAA E Methods a Tum Fa Libraries m Select Volumes Files and Samples w Include Calibrations e Include Questioned Samples Clear Files Utilities In the Advanced Sample Selector you can first select samples by choosing complete data volumes and or individual data files To select a complete data volume click on the box directly to the left of the data volume name DATA To expand a data volume in order to choose individual data files click on the plus sign to the left of the data volume The plus sign will change to a minus El sign and the data files in that data volume will be displayed In this example one data file from DATA was chosen Figure 5 6 The data file selected E026196 94A is displayed in the Files and Samples List bottom right windowpane Note You can click on the Apply button in the bottom windowpane at any time in the Advanced Sam
70. e liable for errors contained herein or for incidental or consequential damages including lost profit in connection with the furnishing performance or use of this material whether based on warranty contract tort or any other legal theory Table of Contents CHAPTER L GETTING STARTED coimas 1 1 OVERVIEW e 1 1 Hardware and Software Installation usscstunc ausit A RU ERR A ead VER ba t etr 1 1 Agilent E ieeisro oia EGER RE 1 2 Computer and SO IS seus tiam itv teda uat CRIME eU EUREN 1 3 BITE DEO XE 9 9 1 3 iaa lis oa Log C PER 1 3 OVERVIEW OF SHERLOCK OPERATION e n 1 4 CHAPTER 2 PREPARING EXTRACTS scccscsscsscnssecccssscasesienscnssesieescncsusiwniasesesansnncesessenssoesececensiesesnenteoesseassnseseiieseees 2 1 OVERVIEW H 2 1 CULTURE GROW IU c 2 1 SAMPLE PREPARATION MT 2 2 Harvesting Cultures and Saponification cccccssesecccssnessecccnaeseecessauseeesscaunsecessauneeessaauasecsssaunseesssaunsecsssauseessaaees 2 2 s iuge a gio oa 2 4 DOW G
71. e Sherlock software In the bottom right windowpane you can edit the fields in white Basic Sample Selector Mode To better understand the operation of the Sherlock system a demonstration will be run with real world data For this example the sample data of interest is in DATA Clicking on the plus I sign next to DATA expands it to show the list of Sherlock data files the plus sign will change to a minus El sign Figure 5 3 Figure 5 3 Volume DATA Expanded to Show Individual Samples A Sherlock CommandCenter Samples File Sample View Help Sample lt SampMethod gt Z Print Print Preview ine aa Sort Save Sample Index Volume Fiename Jeotte 0 Nbr Type Method D Samptnftrested Een 0000077 EHE A 0O E025316 314 SAMPLES Y 026033 554 O EO026035 904 p ED2507b6 92 4 Y E0256196 944 O E026203 914 0O Bb2625 334 Y EO2S017 364 p E028135 154 E029035 684 XU 0 E024314 454 F E025045 274 General Comments Mgr Comments ps E0ZB065 784A I E026156 204 sample ID ooo E Battle 0O E02C104 224 0 DATA Created Modified p DATAZ i DATA3 ID Number Seg Number p DAT AF 7 DATAS Sample Type Method Mame 0O DATAS 0O BATA Clear Selections Utilities Each Sherlock data file contains the data from all the sample and calibration runs in that batch Figure 5 3 shows the expanded list of files in DATA To find the data fil
72. e failure is due to the reference peaks falling outside expected time windows consult the Troubleshooting Section starting on page 7 2 A second function of the Calibration Standard is to provide reference retention times for the mycolic acids These retention times are used to calculate Equivalent Carbon Length ECL values by which peaks in subsequent analyses are named The system calculates how much the calibration analysis has deviated from the expected retention times and reports the Root Mean Square RMS fit error If a calibration run is invalid due to a high RMS fit error Sherlock will print a message to warn the user and will repeat the calibration analysis If the system fails to calibrate after two consecutive attempts the error message will be repeated and the sample batch aborted Consult the Troubleshooting Chapter starting on page 7 2 The Calibration Standard is shipped dry in glass inserts An order contains instructions for use and storage of the Calibration Standard The expiration date of the Calibration Standard is stated on the package It should be stored dry in the dark and at ca 4 C The Calibration Standard should be solubilized by addition of 80 ul of IPA If the calibration Standard is not completely used in a batch of analyses it may be recapped with a fresh cap and stored in the dark at ca 4 C for up to one month Before reuse the calibration Standard should be brought to ambient temperature to assure com
73. e s of interest click on the box es to the left of the file name scroll down if necessary In this example the data file E026196 94A is selected Figure 5 4 The data file name is constructed from the year month day and time Eyymddt ttA when the batch started The file names sort in order from oldest to newest The upper right hand windowpane then lists the samples in this data file The 12th sample is selected highlighted The details of the 12th sample are shown in the lower right hand windowpane The General tab shows the Sample ID and its ID Number along with other tracking information The Sample ID is the identifying information e g a patient identifier that was entered in the Sherlock Sample Processor screen when the sample was entered into the system The Advanced Sample Selector mode could have been used to select this sample data This is described in the next section Figure 5 4 Data File E026196 94A Selected with the 12th Sample Highlighted Sherlock CommandCenter Samples File Sample View Help Sample A A src pM Created CHE ef DATA m 1 talib MYCOLCI CalStd for Method MYCOLC 1 6 19 2002 4 39 29 LE 1 1 Caib MYCOLC CalStd forMethodMYCOLC 2 6 13 2002 5 02 35 EA A 5 x6 sam M Ja CaLANC e 1902 DEMO E 3 E 18 2002 5 17 30 rib RE seen E 3 2239 Samp MYCOLCI GC MYCO GORDO ATCCI4 4 5 19 2002 5 32 24 O EO26035 904 4 2240 Samp MYCOLCI GC MYCO GORDO ATCCI4 5 6 19 2002 5 47 26 O EU26043 744 D
74. e to backup C SHERLOCK DATA files using Windows Explorer the details of which will not be discussed here Sherlock provides Backup and Restore functions that allow the user to copy files between Sherlock DATA volumes and backup storage Note As an alternative to the backup and restore procedures described below MIDI strongly recommends that the entire C SHERLOCK directory be routinely copied to an external USB disk or to a network share using standard Windows file management procedures In the event of a computer failure MIDI s Technical Support staff can use this backup to assist in the complete recovery of data methods and libraries 6 1 Sherlock Utilities Data Backup Double click the Sherlock CommandCenter Icon f 3 on the desktop to start the CommandCenter application CommandCenter reverts to the view from the last time it was used If it is not in the Backup View as shown in Figure 6 1 click the Pts button in the Taskbar then click the Backup View in the Taskbar so that Backup appears in the Current View The Source Volume label is displayed in the middle windowpane the default is DATA The Sherlock data files in this volume will be displayed in the bottom windowpane Figure 6 1 Data Backup Screen A Sherlock CommandCenter Backup Q Restore Source Volume Target Volume DATA FILE ED2531531A 4 EDO26033554 e EOZ26035904 4 E0250437 44 4 E025076524 mio2619594A E02625733
75. each tube Vortex each tube for 5 10 seconds Place autoclave indicator tape on the rack containing the tubes Autoclave for 30 60 minutes at 121 C with slow exhaust Turn on heat block 10 Prepare a sufficient quantity of potassium bicarbonate coated vials if necessary Note After autoclaving the tubes are safe to work with at the laboratory bench or a chemical fume hood 2 2 Figure 2 2 Sample Preparation Harvesting Sterile stick applicator Saponification Securely cap tube Vortex 10 sec Extraction f Cap and Mix stand 5 min Cool to RT Add 1 8 ml 6N HCI Reagent 2 Remove Vortex 30 sec bottom layer Derivatization l 8 2 C Heat 260 C for 10 min Add 200 uL Fluor Grown Ether Reagent 4 Transfer to KHCO Precoated Vial Reagent 5 eoc 2 3 Transfer to 13x100 mm test tube Evaporate Suspend in 1 0 ml of KOH Reagent 1 Autoclave 121 C for 30 60 min Add 1 5 ml Chloroform y Reagent 3 Evaporate p 60 C ul Coolandadd Transfer 50 uL 500ul IFA to insert with Reagent6 internal standard Cap Extraction 1 Remove tubes from autoclave and allow them to cool to ambient temperature 2 Add 1 8ml of Acidification Reagent Reagent 2 with the repeat dispenser Cap the tubes and invert as necessary 2 3x to obtain thorough mixing Allow the tubes to stand for 5 minutes 3 Uncaptubes and add 1 5ml of chloroform Reagent 3 using the repeat di
76. ects of the system When contacting MIDI for technical support it is helpful if you have the following information ready e MIDI Support Contract number e Hardware model number e g 1100 e Computer Specifications e g Windows 7 e ChemStation version number e g B 04 03 e Sherlock version number e g 6 2B or higher If applicable record of the exact text of all software error messages including the dialog box title this can be printed You can fax a copy of your Calibration Chromatogram and Sample Composition Report to MIDI for evaluation and assistance This typically saves time For MIDI Technical Support Tel 302 737 4297 Monday Friday 8 AM 5 PM EST Fax 302 737 9035 Email support midi inc com 7 1 Installation Problems This section discusses typical problems encountered with the software installation process Cannot Install Software If you are having trouble loading software on your system Make sure you are following the instructions in the Installation Qualification The IQ is very detailed with instructions that are specific for hardware and software configurations The IQ is located on the Sherlock CD and can be printed Problems Attempting to Run First Set of Samples Invalid Security module or module not in place Consult your Installation Qualification The security module which was included with your software purchase must be installed before Sherlock will operate No Samples to Run
77. en the expected and measured ECL is given Index Index peaks are those peaks used to calculate ECL values They occur in the Cal standard Sum in Feature The ECL value of the peak corresponds to one of the mycolic acids that cannot be resolved reliably from another mycolic acid by the chromatographic conditions used This acid comprises a portion of a Summed Feature The total percentage of all the mycolic acids that are grouped as one feature are printed at the end of the mycolic acid composition list Summed Feature The total percentage of those peaks identified as a member of a summed feature is given at the end of the Composition listing Calibration Reports Calibration analyses are automatically run according to the Calibration Sequence parameters specified in the method as described in Chapter 3 Analyzing Samples When a calibration analysis is scheduled the computer checks the results against the Peak Naming Table for a specific number of peaks and a pattern of retention times If the analysis results in a report with peak data outside of the tolerance range set for the calibration a warning is printed on the message line of the report and the calibration is repeated A second failure results in the system shutting down and requires corrective measures See Chapter 7 Calibration Standard The calibration standard contains 16 compounds prepared by MIDI for use in the calibration standard The first and last peaks
78. entification number type of media age etc It is helpful to use the initials or some personal identifier for the person logging the sample into the sample table That person is verifying that the samples are placed in the correct positions on the auto sampler tray It is not necessary to close the parenthesis or use capital letters or hyphens after the open parenthesis Once it sees the left parenthesis the software looks no further for cataloging functions While the user will be able to view this information in any file listing it is for personal use only and the software does not use it Starting a Batch Sherlock is ready to analyze samples when the following are achieved e Verified that the proper printer parameters are in use and that the printer has paper Note See Page 3 3 if immediate printing of results is not desired e Confirmed that each sample has a unique sequence number and logged the sample information into the Sample Table e Loaded the sample tray with the samples and the Calibration Standards corresponding to the Sample Table entries e Confirmed that ChemStation is not open or running e Verify that all instrument modules are on and in a ready state e Verify that there is sufficient solvent in the solvent bottles 3 12 When the above steps have been taken the system is ready to start To start a batch press on the Start Batch S tool in the Toolbar e f prompted make any necessary adjustments to the
79. erlock Sample Processor Icon Aro restart the analysis Precaution Never Abort Sherlock while ChemStation is loading There will be time after ChemStation is loaded and before the first injection to halt the Sample Processor If the ChemStation exits abnormally and or Sherlock locks up you should reboot your computer to clear any remaining errors in Windows Failure to reboot will most likely result in another lockup 3 14 Sample Processor Tools Icons The icons associated with the Sample Processor are described in Table 3 1 Most of these Tools in the Toolbar can be accessed through the Menu Bar but the Tools provide a shortcut to these features Table 3 1 Sherlock Sample Processor Tools Tool Description Icon Print Table E Used to print the sample table in the Sherlock Sample Processor Add Sample EN Allows the user to add samples to the sample table Done Adding v Press this button when done adding samples to the sample table Lock Table e Allows the user to lock the sample table from editing Unlock Table EN Enables the user to unlock the sample table Start Batch Allows the user to automatically load the Agilent ChemStation and begin sample processing Stop at End Stops the batch at the end of the current chromatographic run Stops the current chromatographic run immediately End Warmup Yi Terminate the instrument warmup period at the start of a batch and begin processing samples Chapter 4 Interpreting Sherlo
80. fy that the proper Calibration Standard bottle was in the correct tray position Repeated use of a Calibration bottle may have allowed evaporation of the solvent Cannot match peaks to expected relative positions in calibration mix Compare with past calibration analyses for excessive retention time drift The LC ADJUST program Figure 7 2 in the Sherlock Toolbox folder can be used for a semi automatic adjustment of the solvent gradient Contact MIDI Technical Support 7 3 Bad peak matching peak position matching error RMS is 0 XXXX The system will reject a calibration if the RMS is above 0 0150 The LC ADJUST program in the Sherlock Toolbox folder can be used for a semi automatic adjustment of the solvent gradient This calculates the changes in the solvent gradient necessary to move the reference peaks to acceptable positions and then offers a help box that will automatically implement the changes by clicking on the OKAY button If the changes in solvent concentrations fall outside logical ranges the HPLC Adjust algorithm will issue a warning and offer to change the pump flow rate and the solvent gradient Contact MIDI Technical Support Precaution The requirement to change the pump flow rate suggests chromatography problems that may soon require maintenance Figure 7 2 LC Adjust Screen TES Adjust Version 6 2 S N 160011 Method Mco LET Data Volume DATA Expected Current Expected Current HT HT HT LC Ad
81. h the suggested identities are listed in order of descending similarity index Figure 4 4 Figure 4 4 The Library Search Results Matches Library Sim Index Entry Name MYCAG1 1 02 0 725 Mycobacterium simiae 0 437 Mycobacterium lentiflavum triplex Similarity Index Some microbiology identification systems present results as a probability percentage Thus the system may report a 98 probability for the identification of an isolate The basic assumption behind a probability assignment is that species are well defined groups of organisms with little variation in how they perform certain biochemical tests Since comparisons have traditionally been made between two or more biochemical test systems the comparisons are simply how well the systems perform similar enzyme assays Even when the naming is incorrect the probability of the identification may be quite high and may be confirmed using a similar enzyme assay system The technique used by the Sherlock system is based on a Similarity Index The Similarity Index is a numerical value that expresses how closely the mycolic acid composition of an unknown compares with the mean mycolic acid composition of the strains used to create the library entry listed as its match The database search presents the best matches and associated similarity indices This value is a software generated calculation of the distance in multi dimensional space between the profile of the unk
82. herlock Reports are stored while a batch is running Checking the RESULTS TO PRINTER checkbox will print Sherlock Reports after each sample and calibration is run MIDI suggests this option so results are immediately available The Margin menu controls the margins for the text when printing during a batch run or from CommandCenter The options are and inch If you need to put your reports in a 3 ring binder choose the inch option to avoid loss of text from a hole punch Checking the SAVE RESULTS TO FILE checkbox will send Sherlock reports to a formatted text file in Rich Text Format RTF The file name is chosen automatically when the option is chosen The user can print the files later using MS Word 3 3 To print the files in MS Word e Open MS Word e Open file in C Sherlock Results xxxxxxxxx rtf e You can print the entire file or choose the data you want to print Note Each report will be in a separate file Data Storage This controls the storage location of data in the Sherlock DATA directory There are eight storage locations DATA and DATA1 through DATAZ This is where data are accessed for further manipulation by CommandCenter functions Data can be transferred to other data volumes within this directory or transferred from this directory to the archival system of the customer s choice Data File Suffix Sets the letter used as the suffix on Sherlock data file names Typically this is set to A For site
83. ht Position the valve with the waste outlet pointed down Tighten the purge valve using the 14 mm wrench so the waste outlet is pointed down Replace the outlet capillary and the waste tubing Open the purge valve turn the knob counterclockwise two turns and operate the solvent pump for 1 minute to flush the new frit and then close the purge valve Figure 6 10 Replacing Purge Valve Frit 6 12 Chapter 7 Troubleshooting Overview Sherlock is an easy to use system Except for the time required to carry out routine maintenance procedures Chapter 6 Sherlock should operate with little downtime The software usually prints messages to alert you when the system is unable to function properly These messages may appear ina message box on the computer screen or on the final composition report The easiest way to ensure dependable system functioning is to be certain to follow the sample preparation instructions in Chapter 2 very carefully If reagent preparation sample extraction procedures and routine maintenance protocols are followed instrument downtime can be kept to a minimum and reproducible sample identifications will be obtained This chapter discusses the most common sources of problems Reading this chapter before problems arise will result in prevention and easy trouble shooting of problems should a complication occur If you encounter difficulty the MIDI Technical Support staff is available to assist you with all asp
84. just Using Sequence File First IS TD 3 095 3 07 LastISTD 9 076 9 029 Ede rB 734 Sample Counter 2 Stat 2 Current Value 23 8 Hew Getting Retention Times Value 235 Calculating Mew Values Updating Method Sample Messages Total response less than 20 000 Concentrate and re run Extracts that are too dilute will not result in valid searches since the smaller mycolic acid peaks will not be detected Usually this occurs when the isolate is a very slow grower and too few cells were harvested The system may automatically reanalyze the sample injecting four times the original injected amount 20ul If many samples give this error there may be a problem with the HPLC or the extraction derivatization protocol Column overload A peak s response is greater than 450 000 Dilute and re run Peaks with heights greater than 450 000 Luminosity Units LU cause saturation of the fluorescence detector and thus the height assigned to them will be inaccurate This is detected by the Sherlock software and the sample may be automatically reanalyzed by injecting only 2ul If this occurs commonly you are harvesting too many cells and should reduce the amount put into the tube PERCENT NAMED IS LESS THAN 85 Percent named is less than 85 This error usually occurs when there are many unnamed peaks This may suggest retention time drift or other failure of the chromatographic method Re run the sample or re extract
85. lecting the Matches tab displays the sample name as identified from a search of the library or libraries specified in the method see Figure 5 11 In this example the MYCAG1 library was searched for an organism match As a result this sample is matched to the Mycobacterium tuberculosis complex library entry with extremely high confidence Similarity Index 0 906 Selecting the Chromatogram tab displays the chromatogram plot for the selected sample It appears as shown in figure 4 1 Note that the plot will not be displayed for samples analyzed with older versions of Sherlock that did not save the plot image Selecting the Comments tab displays user comments that are associated with the sample The Set Comment button available in this tab provides a mechanism for adding and editing the comment The comment can be printed in the report header see Figure 5 12a The Mgr Comments tab will be empty unless the optional Sherlock ERS module is installed This function is described in the separate ERS User s Manual 5 11 Figure 5 11 Matches against the MYCAG1 Library for the 12 Sample A Sherlock CommandCenter Samples Adex vokme Filename Bote MD Nbc Type Method 10 SO 2 Created EE ES lee IMYCOLET UN MYCO 1081 3wWK CHOC 30C GU 3S El8 2002 47 13 Em DATA Enega E 2244 Samp MYCOLCI UN MYCO 1002 5wKk CHOC 30C GO 5 13 2002 7 02 13 aas Some Wo UN MYCO i 003 SwK 7HT1 SELECTIVE GB Lut E si 3 2002 7 17 18 EN A E a U ee ie iT
86. meters To print the method parameters click the Print Tool ex in the Toolbar The Method Print Options dialog box will then appear Figure 5 18 You can then choose the portions of the method to print by checking the appropriate boxes and clicking the OK button The method information can also be saved to an RTF file which can be opened in Microsoft Word The Print Preview Tool in the Toolbar can also be used to view the same method information on the computer screen 5 18 Figure 5 18 Method Print Options Dialog Box m Method Print Options Frinter File Options iw Print to Current Printer iw Print Method List Print Details for MYCOLC1 c SSHERL LCESRESLLTS sRMESUT 27 rt Detail Options r Print Calib We Print General Information Seguence We Print Sample Control We Print Naming T able Iw Print Calib Control OF Cancel Viewing Installed Libraries Sherlock contains tools that allow you to view and print library information The system does not allow you to edit the libraries supplied by MIDI Inc The ability to view libraries allows you to determine the list of Species a particular library can recognize Deviations from standard growth conditions are also described If the library does not contain a particular species then the system will not be able to name that species Double click the Sherlock CommandCenter Icon 3 on the desktop to start the Sherlock CommandCenter application Figure 5 19 is the Libraries
87. mputer s USB port The Sherlock software and libraries are locked to the serial number of the module and will not operate without it 1 1 Note If your computer requires service or is replaced be sure to retain the Security Module and connect it to any replacement system Loss of the security module may require the purchase of new Sherlock software Agilent Technologies HPLC The Sherlock Mycobacteria Identification Software can be used only with the Agilent Technologies 1100 and 1200 series HPLCs A computer with compatible version of the Agilent Technologies ChemStation software installed is also required For specific information and requirements regarding any of these hardware components refer to the accompanying Installation Qualification included with your system Figure 1 1 Agilent HPLC 1200 Series m ua TEE TED This manual assumes that a qualified MIDI representative has properly installed and connected all the components of your chromatographic unit during a standard installation Environmental Considerations The chromatographic unit will operate within temperatures of 4 40 C 39 104 F and 95 relative humidity See Chapter 1 of each Agilent HPLC component manual for detailed environmental information however an environment comfortable for human habitation reasonably constant temperature and humidity conditions is recommended for optimum performance and instrument lifetime Avoid exposure to corrosive substan
88. n view the entry information for each method Print Method Allows the user to print information pertaining to each Information L method to the designated printer or to a Rich Text Format RTF file Any changes made to a custom method can be saved changes can only be made with the Library Generation Software upgrade installed Create Method Allows the user to create a custom method based ona previously installed method Only available with the library generation software installed Delete Method The user can delete an active method Table 5 4 Libraries View NEM a AA e Sherlock Libraries Clicking this icon will open up the Sherlock Libraries View view where the user can view the entry information from each library Print Library Allows the user to print information pertaining to each Information library to the designated printer or to a Rich Text Format RTF file Train New Entry Search Library The user can locate specific entries based on up to three features specific mycolic acid or summed feature and the values of those features Not active without Library Generation Software license Save changes to a custom library Allows the user to view general library information Not active without Library Generation Software license Tools to explore the quality of library entries Enables the user to view the top windowpane of library information Save Both View Bottom View Allows the user to
89. nfidence interval Precaution Since these results were compiled 9 02 2 strains of the rare isolate M kubicae which currently is not a library database entry were incorrectly identified as M tuberculosis One additional strain provided the correct designation of indeterminate This species can be readily differentiated from M tuberculosis by its bright yellow color and comparison to the reference chromatogram E 2 Table E 2 INTERLABORATORY REPRODUCIBILITY DATA Sample Identification Lab 1 Lab 2 Lab 3 ATCC Name Name Name M malmoense 28571 M malmoense M malmoense M malmoense M xenopi 19250 M xenopi 662 M xenopi 881 M xenopi 907 M fortuitum peregrinum M fortuitum peregrinum M peregrinum II fortuitum M fortuitum 6841 M peregrinum I fortuitum M peregrinum II fortuitum M fortuitum peregrinum a Dime ja sies m Pi vaccinarea anun S36 M MAC mvacsluae avum 40 M MAC nacaluae amumy S4 d CATIA TT 85 pireeememeserie m wremehonemegmen re M nonchromogenicum M nonchromogenicum terrae M nonchromogenicum terrae M nonchromogenicum terrae 19530 M terrae nonchromogenicum M MAIS complex M MAIS complex M MAIS complex M ful 19981 scrofulaceum avium intracellulare FN scrofulaceum avium intracellulare scrofulaceum avium intracellulare M MAI M MAI E MN 841 SCORE zs 885 M MAC A avium intracellulare 868 scrofulaceum
90. ngs of the Sherlock system These tools as well as all of the icons associated with these tools are described in Table 6 2 The following tools are described in more detail Install and Offload a a ie To access the Toolbox double click on the Toolbox Icon m the desktop A window will appear similar to Figure 6 4 Some tools are not available for this application and can be ignored Figure 6 5 Figure 6 4 Figure 6 5 Sherlock Toolbox Unavailable Tools E Sherlock Toolbox AE Ea Sherlock Toolbox AE Hie Heb Tools Unavailable Tools SI Set Pressures MI Configure Methods Jh LC Adjust eL Logon ERS Configuration s Install ERS Extract eh Offload Unavailable Tools 6 6 Table 6 2 Toolbox Associated Tools mem ee A el Sample Allows for adjustment of the instrument configuration Processor ila and Sample Processor output parameters Configuration Activate el Installs MIDI Inc s factory copies of the methods and Methods and lk libraries ordered by the customer Libraries Install Allows the user to install methods and or libraries from any location including network stores Offload Allows the user to copy current methods and or libraries to any location including network stores Select All In the Install or Offload tool allows the user to select all the libraries or methods in the bottom left windowpane Clear In the Install or Offload tool allows the user to deselect Selecti
91. nown and the mean profile of the closest library entry Thus it is not a probability or percentage but an expression of the relative distance from the population mean An exact match of the mycolic acid makeup of the unknown and the mean of the library entry would result in a Similarity Index of 1 000 As each mycolic acid varies from the mean percentage the Similarity Index will decrease in proportion to the cumulative variance between the composition of the unknown and the library entry 4 9 Figure 4 5 Comparison Chart IMYCAG1 Mycobecterium tuberculosis complex TB bovis africanum microti Distance 1 459 Summed Feature 1 Summed Feature Summed Feature 10 Summed Feature 11 Summed Feature 12 Summed Feature 13 Summed Feature 1 Summed Feature 15 Summed Feature 18 Summed Feature 17 Summed Feature 1 Summed Feature 19 Sherlock Comparison Charts A visual representation of the results of the library search is optionally given after the listings of the best possible matches and corresponding Similarity Indices You can select O 1 2 3 or 4 charts as an option when searching libraries An example of the comparison chart of an unknown extract with a library is shown in Figure 4 5 All mycolic acids and summed features a grouping of mycolic acids found in the extract and the library entry are listed in elution order on the left side of the chart A scale of percentages is printed on the bottom of the
92. o completely dry the contents of the container without splattering onto the sides of the container Consult the vendor operation manual for specific usage information The settings should be adjusted to dry the samples without causing boil over and within the time frame indicated in the procedure Slight adjustments may be needed depending on the number of samples processed Consult MIDI for advice with your particular model if necessary 1 MIDI Evaporator 1 1 Use a clean dedicated needle for each sample 1 2 Set Temperature to 60 C A right angle thermometer is provided with the unit 1 3 A heating block with 10 slots for 13x100 tubes and 10 slots for 2mL GC vials is provided 1 4 Turn on air N2 to obtain a flow high enough to rapidly evaporate the liquid in the tube vial but not so high that the liquid is splattered in the tube vial There should be a gentle dimpling of the surface of the liquid 1 5 Position the evaporator head until the needle tips are approximately 25 50 mm above the surface of the tube vial contents 1 6 Monitor the liquid volume and remove the tube vial when dry as evidenced by a slight residue 2 Pierce Reacti Therm Reacti Vap 2 1 Use a clean dedicated needle for each sample 2 2 Set Temperature to 60 C 2 3 For 13x100 tubes use the S 1 heating block 2 4 For 2 ml sampler vials use the A 1 heating block 2 5 Turn on air N2 to obtain a flow high enough to rapidly evaporate the liquid in the tube vi
93. of the chromatographic profile of the calibration standard are the same as the internal standards The calibration standard is used to 1 assign the ECL values of peaks in the unknown samples 2 check the chromatographic performance of the system and 3 allow the LC Adjust algorithm to make all systems using the MIDI HPLC Mycobacteria Identification System software achieve highly similar results The ECL values of the calibration standard are those by which peaks in subsequent analyses are named The system calculates how much the calibration analysis has deviated from the expected relative retention times and reports the Root Mean Square RMS fit error If a calibration run is invalid due to a high RMS fit error Sherlock will print a message to warn the user and then will repeat the calibration analysis If the system fails to calibrate after two consecutive attempts the error message will be repeated and the sample batch aborted The user should consult Troubleshooting Chapter 7 4 6 Figure 4 3 Sherlock Calibration Report of Acceptable Analysis Volume DATA File E026196 94A ID Number 1 Type Calib Bottle 1 Created 6 19 2002 5 02 35 PM Sample ID Cal Std for Method MYCOLC lot 104121 Samp Ctr 2 Method MYCOLC1 Profile GOOD PEAK MATCHING PEAK POSITION MATCHING ERROR RMS IS 0 0004 RT Response Ar H
94. ons i all the libraries or methods in the bottom left windowpane Refresh T mm In the Install or Offload tool this feature is used when Directory the user changes media and needs to refresh the directory Empty This feature is used in the Offload View to delete the Directory libraries and or methods from the destination directory in the bottom right windowpane LC Adjust A feature for use with High Performance Liquid y Chromatography HPLC that aids in moving the reference peaks to acceptable time windows 6 7 Install Methods The user will occasionally need to install custom methods To install methods double click on the 1 ry Toolbox Icon m the desktop and then click on the nstall Tool e in the Toolbox The Install Methods screen should look like Figure 6 6 Make sure that the nstall Methods tab is selected The default directory to install from will be the A X directory Click on the Browse button to search for methods to install from another location These methods can be on the hard disk removable media or Local Area Network LAN Choose the methods of interest and click the nstall Tool e again to install the selected methods Figure 6 6 Install Methods SHERLOCK Install Methods and Libraries File Select Install Help S 3 Select All Clear Selections I Refresh Directory Methods to Install Directory A Browse Select Methods to Install Installed Methods Selected Method Version Root Metho
95. ore _He Help A E o h i e Refresh Select All Clear Selections InvertSelections Restore Files EN Source Volume CNTUBERCULOSISATB DATA Browse Mp Target Volume DATA Backup DATA FILE CREATION TIME ALTER TIME My p ME003093824 2000 03 09 09 11 05 ME003106444 2000 03 10 15 27 33 wjEO00313596A 2000 03 13 14 19 04 w E00313712A 2000 03 13 17 05 29 E003144054 2000 03 14 09 44 06 LJE003145754 2000 03 14 13 49 22 EO0315345A 2000 03 15 08 17 09 EO0315510A 2000 03 15 12 14 57 E D On J be E 4 2 2 The Sherlock data files contained on the disk or directory will appear in the bottom windowpane To access other Target Volumes select a data volume from the drop down menu click on the down arrow The default Target Volume location is DATA but you can select the down arrow to select other target volumes to restore the data to Data files can be selected in several ways e All data files in the Data volume can be selected by clicking on the Select Al Tool T in the Toolbar e Individual data files may be chosen by clicking the small box to the left of the file name e The Invert Selection Tool IL will reverse the data files that have been selected e All data files in the data volume can be unselected by clicking the Clear Selections Tool cor Once the data files are selected click on the Restore Files Tool 8 As the files are copied onto the hard drive the selection box s will clear The program
96. ple Selector to view all the samples in the current query These samples will appear in the top windowpane see Figure 5 8 5 6 Figure 5 6 Advanced Sample Selector Choosing Data Files Sherlock CommandCenter Samples AE i LIBE DELIS T T ELA Da e e a E A pmo AA ee a ee la Methods Ta ri Libraries m Select Volumes Files and Samples Iw Include Calibrations e Include Questioned Samples DATAIEU25135 344 O EUZ2801 7 384 Reset Clear Files Utilities You can then select samples by choosing the method under which the sample was run To choose a method click on the Methods tab in the bottom windowpane In this example Figure 5 7 the Methods tab was pressed and the MYCOLC1 method was chosen This allows for a narrower search of Sherlock data files chosen by only including samples in the data files that were run under the MYCOLC1 method Figure 5 7 Advanced Selector Choosing Methods K Sherlock CommandCenter Samples File Sample View Help e Print Preview Index volume Filename Botle IDNbr Type Method D Samp t Cieated SS Medfed Ceat a Se a SSS eS SS as a A NNI td 44 15 Methods r WU Fa Libraries Eu Prefixes Select All Clear Selections Clear Methods Utilities You can finally narrow the sample search by selecting specific sample ID field prefixes To choose a prefix click on the Prefixes tab in the bottom windowpane
97. plete solution of all mycolic acids Each bottle placed into the sample tray must be logged entered into the computer s Sample Table This procedure is discussed in the Sample Processor section see page 3 7 Before starting a sample batch be certain that the descriptions logged into the Sample Table match the bottles in the sample tray 3 2 Sample Processor Configuration 1 ry Sample Processor Configuration is accessed from the Sherlock Toolbox Icon on the desktop or through the Programs Sherlock group under the Windows Start button other Toolbox items will be described in Chapter 7 Routine Maintenance This screen has three tabs and controls where Sherlock results are sent and stored important instrument control parameters and the level of detail that report parameters are recorded The Update and Exit button will save all changes made to the window while Cancel and Exit button will not save any changes Figures 3 2a 3 2b and 3 2c show the three configuration tabs Figure 3 2a Sample Processor Configuration Screen Output Tab w Sherlock Sample Processor Configuration LC S N 160011 iw RESULTS TO PRINTER MARGIN 172 inch SAVE RESULTS TO FILE Browse In Falder CASHERLOCKNRESULTS DATA STORAGE D ata Volume DATA Data File Suffix Front Column 5uffis A Back Column 5 uffis B Update and Exit Cancel and Exit Output Tab Results to Printer Results to File These sections determine how S
98. r Local Area Network LAN In this example the MYCOLC1 method is offloaded to the A directory The LC settings will be remembered with this technique 6 9 Figure 6 8 Offload Method z SHERLOCK Offload Methods and Libraries Select Offload Export Help amp gt A S Clear Selections Offload Refresh Directory Empty Directory Offload Methods Offload Libraries Offloaded Methods Directory ETT to Uftload Offloaded Methods Selected Meter enn Root Method Version Root Offload Libraries The user will occasionally want to offload custom libraries make a copy of a library To offload a 1 ry library double click on the Toolbox Icon on the desktop and then click on the Offload Tool in the Toolbox The Offload Libraries screen should look like Figure 6 9 Make sure that the Offload Libraries tab is selected Next click on the Browse button to search for a destination for the copy of the library The destination path can be on the hard disk removable media or Local Area Network LAN In this example the MYCAG1 Library is offloaded to the A directory 6 10 Figure 6 9 Offload Libraries z SHERLOCK Offload Methods and Libraries File Select Offload Export Help E Refresh Directory Empty Directory Ofiloaded Libranes Directory pas Browse Selected Libray Version Diray Version T IMYCAG Routine Maintenance of the HPLC Note The instructions below are for your convenience and
99. re 3 3 The middle windowpane gives Sherlock and ChemStation status information currently running sample status and the data storage location The bottom section is the Sample Table where the Calibration Standard and sample identification information is logged Figure 3 3 Sherlock Sample Processor Screen Current State of Menu Bar Tool Toolbar Sample Table Sample Processor LC S N 16001 Sample Table MEE e phe meh view mo Vd amp HA es Status PrintTable Add Samples LockTable idis mmsmms DATA VOL Status Bottle lID Number Datafile mE COLA AVAILABLE M Y COLCI Calibration Hix for HVCOLC method Queued MYCOLCI QC BLANK 5 29 13 CC Queued IMYCOLC1 1002 QC HYCO RUIUM ATCC 13958 CC Queued MYCOLCI 1003 HVCO TUBER ATCC 27294 CC Queued MYCOLCI 1004 MYCO ABSCE ATCC 19977 CC Queued IMYCOLCI 1005 MYCO FORTU ATCC 6841 CC Queued Done Done Done Done Anna Sample Table The Sample Processor Menu Bar File Print Allows printing the current sample table optionally including result information The Sample Table can be printed at any time even when running samples The table is printed as soon as the printer is available File Exit Allows exiting the Sample Processor If the Sample Processor is running samples it is strongly recommended that the user abort or stop the batch before exiting Exiting the Sample Processor while
100. reused if they are the screw on type and the septum is replaced for each reuse All work with alkali acids and solvents must be performed in an approved explosion resistant chemical fume hood Eye protection and other personal protective equipment must be worn during all work with alkali acids and solvents 2 6 Acknowledgements The MIDI extraction protocol was adapted from the methodology established at the Texas Department of Health Bureau of Laboratories Mycobacteriology Mycology Branch References Richard J Wallace Jr Barbara A Brown Elliott Leslie Hall Glenn Roberts Rebecca W Wilson Linda B Mann Christopher J Crist Sher H Chiu Robbie Dunlap Maria J Garcia J Todd Bagwell and Kenneth C Jost Jr Clinical and Laboratory Features of Mycobacterium mageritense 2002 J Clin Microbiol 40 2930 2935 Brown B A B Springer V Steingrube R W Wilson G E Pfyffer M J Garcia M C Menendez K C Jost Jr S H Chiu G O Onyi E C Bottger and R J Wallace Jr 1999 Description of Mycobacterium wolinskyi and Mycobacterium goodii two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections a cooperative study from the International Working Group on Mycobacterial Taxonomy Int J Syst Bacteriol 49 1493 1511 2 7 Chapter 3 Analyzing Samples Overview The previous chapter discussed the preparation of HPLC ready extracts from cell cultures
101. rlock Mycobacteria Identification System to identify routine clinical isolates of Mycobacterium spp Each of 3 participating laboratory sites was asked to analyze all suspect mycobacterial isolates submitted to them during the evaluation period 3 02 7 02 Thus the distribution of species tested is reflective of the frequency of occurrence during that period Some additional isolates included CAP proficiency test samples and archived infrequently encountered species Table E 2 provides a measure of the interlaboratory reproducibility for a diverse sampling of mycobacterial species Each of the 3 participating laboratory sites was sent a set of blind coded specimens consisting of well characterized ATCC strains M tuberculosis was not sent as a result of safety issues Following onsite training by MIDI personnel each laboratory cultured the specimens under the standard conditions described in the MIDI protocol and analyzed on their respective system All sample identifications from the participating laboratories were in agreement with the ATCC strain designation Only 2 out of 36 specimens produced indeterminate results i e first choice SI lt 0 500 or second choice SI within 0 200 of first choice SI The Sherlock Mycobacteria Identification System has been shown to reliably differentiate the M tuberculosis complex from other mycobacteria Definitive identification of MOTT requires use of additional laboratory testing to identify clinically
102. rough the middle is the mean for the library entry data 5 20 Figure 5 20 Selecting and Displaying Library Entries K Sherlock CommandCenter Libraries e a 8 m Print Preview Train Mew Entry Search Views Bn General Entries Parameters ATT f Ent Selection 1454 Library MYCAG1 ere Single Selection Multiple Selection Samples lg EntyMum Name Date Features 0 EN ENT TU cacao tomara 4 26 2002 9 58 05 AM TD Methods Myc abac tem tuber ulozis complex TB bovis aficar 1272572002 9 59 05 AM 10 ma Munnhantetumxennni RMI MY CAG Mycobactenun tuberculosis comples TB bovis aficanum microti Mycobacterium tuberculosis complex TB bowiz a Index 38 Mum Features f Date Time 4 26 2002 9 59 05 AM Mycobacterium tuberculosis complex TH bovis africanum microti Summed Feature 1 LL Summed Feature 11 0 Summed Feature 12 Summed Feature 13 Summed Feature 14 Summed Feature 15 Summed Feature 16 Summed Feature 17 Summed Feature 18 Summed Feature 15 cH Utilities Printing Library Information Selected library information can be sent to the printer or viewed on the screen the same as was done for methods Clicking on the Print Tool will bring up a dialog box similar to Figure 5 21 You can then choose the portions of the library to print by checking the appropriate boxes and clicking the OK button The library information can also be saved to an RTF file which can be opened in Microsoft Word The
103. rs general maintenance functions such as how to backup and restore data Chapter 6 outlines the Sherlock disk structures and how to remove old data to maintain the free disk space needed for reliable operation Note The Sherlock CommandCenter is icon intensive and descriptions of all the icons used in the CommandCenter are given in Tables 5 1 through 5 4 at the end of this chapter Sherlock Terminology The Sherlock software uses some terminology that you should be familiar with to better follow the manual text and understand the operation of the software Figure 5 1 illustrates some of the commonly used terminology with the Sherlock interface All of the Tools in the Toolbar can be accessed through the Menu Bar but the Tools provide a shortcut to these features By clicking on any of the Column Headers you can sort that column in alphabetical or numerical order Also when the mouse cursor is placed over any horizontal or vertical resizing bar within the CommandCenter window the cursor bb ud Ww changes shape to one of the following or Youcanthen resize this field 5 1 Figure 5 1 Sherlock CommandCenter Terminology Current View Tool Column Header Index Volume Filename Botle ID Nbr_ Type Method J D SampCt Created 1 1 1 Cali MTESCLI Cal Std for Method MYCOLC lotH 6 19 2002 4 39 1 Caib MYCOLC1 Cal Std for Method MYCOLC lotH 6 19 2002 5 02 2238 Samp MYCOLCI QC BLANK 6 19 02 DEMO EXTF 6
104. s Mycobacterium flavescens II Mycobacterium fortuitum peregrinum Mycobacterium gordonae I Mycobacterium gordonae ll Mycobacterium haemophilum I 30C chocolate Mycobacterium haemophilum II 30C chocolate Mycobacterium haemophilum III 30C chocolate Mycobacterium interjectum Mycobacterium intermedium Mycobacterium kansasii Mycobacterium lentiflavum triplex Mycobacterium MAC A avium intracellulare Mycobacterium MAC B avium intracellulare Mycobacterium MAC C intracellulare avium Mycobacterium MAIS complex scrofulaceum avium intracellulare Mycobacterium malmoense Mycobacterium marinum 30C Mycobacterium mucogenicum Mycobacterium mucogenicum II Mycobacterium neoaurum Mycobacterium nonchromogenicum terrae Mycobacterium peregrinum l fortuitum Mycobacterium peregrinum ll fortuitum Mycobacterium simiae Mycobacterium szulgai Mycobacterium szulgai II Mycobacterium terrae nonchromogenicum Mycobacterium terrae nonchromogenicum II Mycobacterium thermoresistible Mycobacterium triviale Mycobacterium tuberculosis complex TB bovis africanum microti Mycobacterium xenopi Mycobacterium xenopi II B 2 Appendix C Procedural Notes Evaporation of liquids in test tubes and vials Evaporation of liquids in 13 x 100mm tubes or 2 ml sampler vials is performed with either a heat block air N2 evaporator e g MIDI or Pierce or a Labconco RapidVap evaporator See Appendix A for ordering information The objective of the evaporation is t
105. s are being processed This allows for continuous operation of your system Note The figures in this manual show the method and library versions at the time the screen shots were taken The version installed on your system may be newer Chapter 2 Preparing Extracts Overview The mycolic acids of mycobacteria are part of the structure of the cell wall Figure 2 1 To release the compounds from the cell wall and to make the samples safe to work with the harvested cells are Saponified in an autoclave Figure 2 1 Mycobacteria Cell Wall i Mycolic acids Glycolipids e Proteins CHO Arabinogalactan 2 LAM A A Peptidogl ycan The mycolic acids are converted to free acids by lowering the pH extracted into chloroform to remove materials not soluble in organic solvents and derivatized by a fluorescent derivatizing agent Because the mycobacteria are killed by the saponification procedure the procedure is safe from a microbiology standpoint from the first step forward Culture Growth The mycobacteria cultures typically are grown on solid medium such as Middlebrook 7H10 or 7H11 at 35 37 C until visible growth is noted Some species of mycobacteria may normally be grown under different conditions and the database has been constructed to reflect this Examples would be the culturing of M marinum and M haemophilum at 30 C with the latter organism being grown on chocolate agar rather than on Middlebrook Examination of cultures
106. s not only successful but 3 1 provides optimal times for the peak naming table to use in naming peaks in subsequent samples At the completion of the blank run the robotic arm will pick up the vial in position 1 Calibration Std move it to a space between the injection syringe which has raised to accept the vial and the syringe seat The syringe will then pick up the amount of sample designated by the Sherlock method The sample vial is returned to its original position and the syringe moves down into the syringe seat causing injection of the sample into the flowing stream of carrier solvents The entire sample is put into the analysis and the syringe is continuously flushed by solvent during the entire analysis This virtually eliminates carry over of sample to the next analysis Calibration Standard The calibration standard is used for the first two injections of a batch and is reanalyzed after every 10th Sample injection to ensure system stability When a calibration analysis is completed the computer checks the results against the Sherlock Method s Peak Naming Table for a specific number of peaks and a pattern of retention times The Peak Naming Table contains the expected retention time for each peak in the calibration analysis Deviations from the expected values may result in a failure to calibrate with a warning message to the user If the system fails to calibrate on the first attempt it will try once more to calibrate If th
107. s with multiple Sherlock systems a different suffix is used for each system Figure 3 2b Sample Processor Configuration Screen Report Parameters Tab Sherlock Sample Processor Configuration LC S N 160011 Output Report Parameters instrument Calibration Report Sample Report Report Type Report Type Profile with Classify v Profile with Classify v Number of Charts none Number of Charts none I Include Chromatogram I Include Chromatogram v Print Pre Sample File Edit Edit Generate Summary Report at End of Batch Update and E xit Cancel and Exit Report Parameters Tab This section controls the amount of sample identification detail that will appear on a Sherlock Sample or Calibration Report that is generated from the Sample Processor Reports that are generated from CommandCenter have their own control The menu choices are name and warning message peaks in the chromatogram Brief with The sample name warning Classify messages match to the library Profile with Full page report listing all Classify peaks in the chromatogram and match to the library MIDI strongly suggests using the Profile with Classify option for routine operation Note that all raw data is stored in a separate file in Sherlock Through CommandCenter Sherlock can generate a more or less detailed report at any time desired by the operator The Number of Charts field refers to Comparison Charts These graphi
108. secssuecessusesssauseessauscesauseessans 3 10 Eating he SOO TODE RITTER 3 11 BUILT GO rs c 3 12 Premaluro y SEONG AO os 3 13 sample Processor Tools 00n arta una e ud 3 15 CHAPTER 4 INTERPRETING SHERLOCK REPORT sscssccsscssccsccnscnsscsccnscnsscsccnscnsscsccnscnsscsccnscnssesccnscnssesccnsoess 4 1 OVERVIEW r 4 1 COFOmatoarabhIe REB sad 4 1 Seno Ek COMPOSITION REPOSAN ete pedea FERA AAA AAA AA 4 2 CONDEGEOD REDO S oce A RA EA A A A 4 6 Pe E E A s re Oa sete AE EA a 4 8 SHERLOCK LIBRARY SEARCH ad dais 4 8 Interpreting the LI DIGI SC ak CD AN REP yond sac E AA ced naa det quiis wea IPod 4 9 SHAVING OX et C 4 9 SherocK COmparisoD COMES iia 4 10 INTERPRETATION GUIDELINES oisi tier stica tona a a aa 4 11 Interpretation of Library GFOUDS ict ott e OE t nid b a t A an ea VR P CR RR RR 4 11 Visual Con ALON Eom ETE 4 13 CHAPTER 5 SHERLOCK COMMANDCENTER ssessssessssecsssecsssecsssecssoecsssecsssecsesecsssecsssecsssecsesecsssecsesecsssecsesecssseoe 5 1 OVER VIE Wissinia liado Dine ne Loud 5 1 SIVCPIOGK PEU OO Vit AA AAA AAA AAA AAA AAA AAA AAA 5 1 SAMPLE SELECTION e 5 2 Basic Sample Sele ctor Mode oai o ei ee aL Coen d EE 5 3
109. spenser 4 Recap each tube and vortex vigorously for 30 60 sec Visually inspect each tube to insure full vortex 5 After phase separation transfer the bottom chloroform layer to a clean 13 x 100 mm borosilicate glass tube Note It may be necessary to briefly centrifuge the tube if an emulsion is present The tube must be labeled with the corresponding label number from the original 13 x 100mm tube 6 Evaporate the chloroform from each tube See Procedural Note 1 0 for instructions using the Pierce Reacti Therm or Procedural Note 2 1 for the Labconco system 7 Cool the tubes to ambient temperature Derivatization 1 Add 200ul Derivatizing Reagent Reagent 4 with dispenser 2 Gently swirl the tube to dissolve its contents 3 Transfer the mixture from each tube into a correspondingly labeled amber vial 2 ml size which has had the interior precoated with 100ul of 2 Methanolic Potassium Bicarbonate Reagent 5 4 Swirl the vial gently by hand to assure mixing of the contents 5 Heat the vials uncapped for 10 minutes at 60 2 C 6 Slowly evaporate the contents of each vial See the Appendix C procedural notes for the MIDI Evaporator Pierce Reacti Therm or Labconco System 7 Remove the vials and cool to ambient temperature 8 Add 500uL of isopropyl alcohol IPA Reagent 6 and cap the vial 9 Swirl to totally dissolve the contents and mix the sample extract 10 To a tapered insert vial containing the internal standards
110. ssure the quality of the analyses If results are outside the acceptable tolerance windows error messages are printed in the Composition Report to assist you in troubleshooting the system Usually these problems 7 2 are identified by Sherlock during the initial calibration runs Figure 7 1 shows a chromatogram of a good calibration run Figure 7 1 Good Calibration Run Chromatogram FLD1 A Ex 345 Em 425 C SHERLOCK RAW E00904 479 A01926A2 D LU S D ii a Oo 20 m N s 7 o E M T q 0 109 3 689 3 995 4 503 4 821 5 346 5 695 6 739 7 586 8 686 AAN JW PA 25 T T T T T T T T T T T T T T T T T T T T T T T T T T T T 3 4 5 6 7 8 9 10 min Calibration Messages Sherlock is trained to recognize the Calibration Mix The calibration analyses require a specific number of mycolic acid peaks with a set pattern retention times With each calibration analysis the following messages may be printed Good peak matching peak position matching error RMS is 0 XXXX The normal deviation from the best fit RMS root mean square is less than 0 0060 This message indicates that the values were within allowable tolerance Large deviations from the expected calibration values result in one of the error messages that follow Not enough good peaks to match expected calibration mix One or more peaks were rejected or are missing Veri
111. t ECL Peak Name Percent Comment1 Comment2 3 016 8141 0 098 40 000 ECL 40 000 standard 3 300 6845 0 155 42 884 Sum In Feature 1 6 61 Peak match 0 0006 ECL 42 884 index 3 636 5605 0 123 46 069 Sum In Feature 1 5 41 Peak match 0 0008 ECL 46 069 index 4 179 6274 0 109 51 275 Sum In Feature 5 6 06 Peak match 0 0003 ECL 51 275 index 4 519 3778 0 112 54 537 Sum In Feature 6 3 65 Peak match 0 0002 ECL 54 537 index 5 060 5664 0 103 59 526 Sum In Feature 7 5 47 Peak match 0 0004 ECL 59 526 index 5 423 5181 0 102 63 000 Sum In Feature 8 5 01 Peak match 0 0004 ECL 63 000 index 6 508 6395 0 107 73 429 Sum In Feature 11 6 18 Peak match 0 0006 ECL 73 429 index 6 807 14061 0 105 76 278 Sum In Feature 12 13 58 Peak match 0 0006 ECL 76 278 index 7 094 7612 0 117 79 010 Sum In Feature 13 7 35 Peak match 0 0004 ECL 79 010 index 7 367 7052 0 118 81 614 Sum In Feature 14 6 81 Peak match 0 0001 ECL 81 614 index 7 655 9942 0 110 84 340 Sum In Feature 15 9 60 Peak match 0 0002 ECL 84 340 index 7 942 6890 0 106 87 065 Sum In Feature 16 6 66 Peak match 0 0004 ECL 87 065 index 8 500 4548 0 127 92 257 Sum In Feature 18 4 39 Peak match 0 0003 ECL 92 257 index 8 747 13662 0 118 94 654 Sum In Feature 19 13 20 Peak match 0 0002 ECL 94 654 index 8 993 7534 0 119 97 000 ECL 97 000 standard 12450 Summed Feature 1 12 03 6274
112. tely stop analysis in progress and then stop the batch The Abort tool should be used with caution If the sample injection has occurred any non eluted compounds will remain on the chromatographic column If there is sample left on the column a blank must be run to clear the column of any residual sample This is typically done automatically at the beginning of the next batch see Analyzing Samples page 3 1 3 13 In addition to the Toolbar icons two Sherlock menu options describing the options for correctly terminating a batch are also available in the menu bar e Click on the Batch Stop at end of run menu item to complete the analysis in progress print the results and then stop the batch e Click on the Batch Abort Batch menu item to immediately stop the analysis in progress and stop the batch Caution It is not possible to stop the batch correctly using the ChemStation Abort function from the ChemStation menu bar The LC will stop the run and return to a standby status but Sherlock will not be able to proceed with the batch or communicate with ChemStation and will be difficult to close It is then necessary to completely exit back to the Windows screen and then click on the Sherlock Sample Processor to restart the analysis It may be necessary to use the Windows Task Manager Ctrl Alt Delete Task Manager Applications and end the Sherlock application to completely exit back to the Windows screen and then click on the Sh
113. teria method for LC Libraries De Mm Utilities Displaying Method Information The top windowpane lists the methods found on the system Click on the method row in the top windowpane to select a method In the current example the MYCOLC1 method is selected highlighted Details for this selected method are shown in the bottom windowpane Note None of the bottom windowpane information can be edited In Figure 5 17 the Naming Table tab is selected to show the MYCOLC1 peak naming table Clicking on other tabs such as Sample Control will display other details for the method 5 17 Figure 5 17 Viewing the MYCOLC1 Peak Naming Table Sherlock CommandCenter Methods Seles S542 dci Save Create Delete Se ES ET EE Noo NS MYrEOLE MY COLI Mycobactena method for LC MH CAO 171572002 57172002 9 26 54 AM DELLI LAA Samples General Sample Control Calib Control Cab Sequence Naming Table METHODS Add Peak E dit Peak Delete Peak Use Weights E Mom Mame Mom Cal Indes Ref Quant Hom Check Min Max E jeep name A AAA I 0 350 Libraries ECL 40 000 standard 40 000 0 350 True True Fixed 1 000 Mo CL 41 600 41 600 350 3263 Fale False Fixed 1 000 Na CL 42 282 42282 0 333 3334 False False Fixed 1000Mo CL 42 884 index 42884 0270 3387 C True False Fixed 1 000 Mo CL 43 500 43 500 0 350 3462 False False Fixed 1000Mo
114. that action may need to be taken to continue proper performance The user should consult Troubleshooting Chapter 7 Note The match against the library for the calibration mix as measured by the Similarity Index SI is for reference only It is desired that the SI will be above 0 500 however since each batch of calibration mix will have variance in relative size of peaks the actual SI may vary significantly As long as the other calibration parameter specifications are met and the positive and negative control specifications are met an SI 0 500 will not affect subsequent sample runs Quality Control Each batch of samples should contain both a positive and negative control as described in Chapter 2 Harvesting Cultures and Saponification Use M gordonae ATCC 14470 for the positive control and C albicans ATCC 60193 for the negative control C albicans does not produce mycolic acids and thus is primarily used to measure reagent purity Incubate both on Middlebrook 7H10 11 media at 35 37 C The same plate can be repeatedly harvested for up to 1 2 months for M gordonae and 1 2 weeks for the C albicans Routinely subculture to maintain working cultures M gordonae should be subcultured once every month and C albicans once each week For each batch of runs record critical parameters from the process controls on the Performance Qualification Table see Appendix D The positive control must name as M gordonae at a similarity index SI
115. thout time limit on one personal computer or workstation The customer may not modify this software security ID module or copies of them in any way 3 To use this product on additional computers the user must contact MIDI about additional licenses and security ID modules 4 Purchase of this license does not transfer any right title or interest in the software product to the Customer except as specifically set forth in this license agreement 5 The Customer shall not use this software product to create and distribute competing products including but not limited to identification libraries methods or software The Customer may use this software product to create libraries and methods for use on MIDI supplied systems within the Customer s organization 6 The license may not be transferred to another person or organization without the express written consent of MIDI Inc 7 The Customer is on notice that this software product is protected by copyright laws 8 With prior written approval MIDI may grant other license rights 9 By installing the software the Customer signifies acceptance of this license agreement 10 This agreement is subject to the laws of the United States of America and the jurisdiction of the U S A courts MIDI Inc makes no warranty of any kind with regard to this software product including but not limited to the implied warranties of merchantability and fitness for a particular purpose MIDI Inc shall not b
116. tial heat is released during preparation that may cause the mixture to boil bump or steam Prepare reagent ONLY in a BOROSILICATE GLASS flask inside a chemical fume hood Add acid slowly to water Never add water to acid Reagent 3 Chloroform Shelf life 1 year in unopened container 6 months if opened Danger Toxic Reagent 4 Derivatizing Reagent Shelf life 6 months in amber container and stored in the dark Dissolve 100 mg of 4 bromomethyl 6 7 dimethoxycoumarin F W 299 1 and 100 mg of 18 crown 6 ether F W 264 3 in 100 ml of chloroform Danger toxic lacrimator Reagent 5 2 Methanolic Potassium Bicarbonate Solution Shelf life 1 year Dissolve 2 0 g of potassium bicarbonate KHCO in 100ml of 50 aqueous methanol 50 ml methanol 50 ml deionized water Prepare vial by adding 100ul of Reagent 5 to a clean amber 2 ml autosampler vial Dry the contents of the vial completely See Procedural Note 1 0 for the Pierce Reacti Therm System or Procedural Note 2 3 for the Labconco system The coated vials may be prepared in batches and stored at room temperature in a dried covered condition Shelf Life 6 months Reagent 6 Sample Extract Diluent HPLC grade isopropyl alcohol ACS certified preferred Shelf life 3 years in unopened container 1 yr if opened Warning Flammable 2 5 Precautions Extracts may be stored refrigerated and in the dark for 7 days prior to analysis Mycolic acids of rapid growers tend
117. to deteriorate more quickly than those of slow growers The Reagent 4 the Derivatizing Reagent should be stored in an amber container in the dark for maximum shelf life The extraction derivatization procedure may be stopped for as much as 24 hours following the autoclaving saponification step Do not reuse any glassware Use of plastic autosampler vials or plastic inserts may cause problems with back pressure elevation of the HPLC and should not be used The isopropanol in the sample diluent may dissolve some of the plastic components and carry that into the methanol isopropanol mobile phase where it is less soluble and thus may come out of solution plugging the plumbing and damaging the column Repeating pipetters make dispensing the reagents safer and more reproducible The dispensers should be well primed before use in order to expel air bubbles in the pickup and delivery tubes This is especially important with low volume chloroform solution dispensers Avoid pumping the dispensers too fast since air bubbles can be introduced that lead to inaccurate reagent delivery When pipetting or dispensing tilt tubes or dispenser tip or pipet so that reagent runs smoothly down the side of the tube Samples must be completely dry after evaporation steps for subsequent reactions to be efficient The use of 200 ul sample inserts in the autosampler vials allows easy injection by the system and permits reuse of the vials Vial caps may also be
118. ul for initially setting up a sample table The Sample Processor Tool Bar Note A description of all the Sample Processor icons is given in Table 3 1 at the end of the chapter Add Samples Before putting a sample bottle into the sample tray the proper identification information must be logged into the corresponding bottle number in the Sample Table The sample bottle type can be a calibration sample Calib or a sample extract Samp to be processed for identification In addition the bottle type can be labeled as Stat which indicates a priority sample that will be run next after any required calibrations Blank for the negative control QC for a positive control or Empty This places the system in a mode that simplifies adding new samples to the sample table When the Add Samples tool is pressed the Sample Table editor automatically advances to the first empty bottle position The sample type method and status will be entered automatically by the system based on the previous sample s information while the user will enter sample name and pertinent sample information Figure 3 4 The Seq is automatically incremented and displayed Note The first sample in the analysis should be the QC negative control C albicans Under the Type column select Blank as the type The second should be a positive control with type QC selected All subsequent specimens will automatically default to Samp This is the only correct way to ad
119. value 0 600 A SI value of 0 600 for M gordonae suggests improper processing of samples or possibly a mixed culture Record the Total Named response and the Percent Named result on the Performance Qualification Table These results are for reference only The result for negative control C albicans should not match any library entry and must have the Total Response 1000 If the negative control has a total response 1000 reagent contamination is suspected If the M gordonae SI 0 600 or the C albicans Total Response 1000 the source of the problem should be identified and corrected Subsequently the sample cultures should be re extracted along with positive and negative controls per Chapter 2 Preparing Extracts and re analyzed Sherlock Library Search Once a microorganism has been cultured processed and properly analyzed by Sherlock its mycolic acid composition is matched with those of known organisms that are stored in the Mycobacteria Library The Library profiles have been carefully developed to take into account strain to strain and experimental variation 4 8 The library search is rapid The naming of the unknown is available within seconds of the completion of the HPLC chromatographic analysis The Sherlock Library Search Report lists the most likely matches to the unknown composition and provides a similarity index for each match Interpreting the Library Search If the search results in more than one possible matc
120. w you to view and print sample and calibration data stored on the hard disk Double click the Sherlock CommandCenter Icon ion the desktop to start the CommandCenter application Figure 5 2 shows the CommandCenter main screen The CommandCenter always calls up the view from the last time it was used If it is not in the Samples View as shown in Figure 5 2 click the Samples View Wea in the Taskbar on the left hand side of the CommandCenter application window If the Samples View is not visible click the ews button in the Taskbar to make it visible and then click on it 5 2 Figure 5 2 Sherlock CommandCenter Main Screen Sherlock CommandCenter Samples File Sample View Help e E Print Print Preview Sample Index Volume Filename Botte ID Nbr Type Method D Samp Cr Created pgp O DATA a AR Lati DATA1 O DATA2 DATA3 O DATA4 O DATAS O DATAG O DATA is Methods Libraries Sample General Comments Mgr Comments Sample ID ON Bottle Created a Modified il ID Number Seq Number gt Sample Type o Method Name Clear Selections Utilities In the Toolbar the Select Style dropdown should be in the Sample mode default This mode is the Basic Sample Selector mode and is recommended for customers new to the Sherlock software An Advanced Sample Selector mode discussed later in this chapter is available and is recommended for customers who are more familiar with th
121. will prompt the user with an overwrite confirmation box if the selected files already exist on the data volume Any files that were not copied Da will remain checked In the Data Restore View the Refresh Tool Ws allows the user to update the data directory if new media is inserted Under the File menu you can select Exit to exit the Sherlock software or to go to another CommandCenter view click the ews button and select the view of interest The icons associated with various Tools in Sherlock Utilities are described in Table 6 1 Table 6 1 Utilities Associated Tools Select All f In the Backup or Restore View allows the user to Igi choose all the Sherlock data files Clear Selections i In the Backup or Restore View allows the user to deselect all the Sherlock data files Invert Selections LI In the Backup or Restore View allows the user to invert the current selections Backup Files ER In the Backup View pressing this icon enables the user PE to backup the selected data files to the target volume Rename Files Allows the user to rename a selected file in the Backup 3 View Delete Files In the Backup View this allows the user to delete any of SS the selected Sherlock data files Refresh Directory p In the Restore View this feature is used when the user changes media and wants to refresh the directory 6 5 Sherlock Toolbox The Sherlock Toolbox contains ancillary tools that are used to manage the worki

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