Pak Lx™ Assay - Immucor, Inc.
Contents
1. Allow the reconstituted vial to sit at room temperature for at least 5 minutes b Do not mix or vortex the Lyophilized Beads with the Bead Diluent at this time e Reconstituted vials may be stored at 2 8 C in the dark for up to 15 days CAUTION DO NOT POOL VIALS RECONSTITUTED ON DIFFERENT DAYS INTO ONE CAUTION DO NOT VORTEX THE BEADS AT ANY TIME DURING THE ASSAY TO AVOID FROTHING Prepare samples by vortexing Centrifuge at gt 10 000 x g for at least 60 seconds to settle any insoluble matter before use Record sample information on the Plate Format Sheet The plate format sheet is available for download from the website www immucor com Cover the unassigned wells of the filter plate with a plate sealer Add 250 uL of wash buffer to the assigned wells a Allow the wash buffer to sit in the wells for at least 3 minutes b Remove the wash buffer by gentle aspiration using the vacuum manifold See manufacturer s recommendations for proper use Mix the reconstituted beads by pipetting up and down about 15 to 20 times taking care to avoid any frothing Add 40 uL of Reconstituted Beads to each test well of the filter plate Ensure that the Reconstituted Beads are mixed during bead addition to the filter plate Pipet up and down 2 3 times after delivery to every 2 3 wells CAUTION Itis important to keep the beads resuspended to ensure uniform bead addition to the wells Failure to mix beads intermittently will cause beads to sett2. If you do not agree to all of the terms and conditions set forth below you must promptly return this Kit for a full refund prior to using it in any manner You the customer acquire the right under Luminex Corporation s patent rights if any to use this Kit or any portion of this Kit including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex instrument Pak Lx Assay 1 303285 IFUEN REV H INTENDED USE Pak Lx Assay is a qualitative immunoassay for use on the Luminex instrument The Pak Lx Assay is designed to detect and differentiate between IgG antibodies to HPA 1 HPA 2 HPA 3 HPA 4 HPA 5 GPIV and Class HLA in human serum SUMMARY AND EXPLANATION Platelets express a variety of polymorphic proteins The polymorphic changes in the proteins while not affecting protein function might become the targets for antibodies as a result of pregnancy or transfusion The presence of antibodies that bind to platelet glycoproteins is associated with life threatening bleeding disorders such as refractoriness to platelet transfusions PR post transfusion purpura PTP and fetal and neonatal alloimmune thrombocytopenia FNAITP 1 The glycoproteins GP targeted by antibodies include GPIlb Illa GPlb IX and GPla Ila GPIV and Class Human Leukocyte Antigens HLA The epitopes found on GPIlb Illa GPlb IX and GPla I
3. Metcalfe P et al Nomenclature of human platelet antigens Vox Sang 2003 85 240 16 Santoso S Human platelet alloantigens Transfus Apher Sci 2003 28 227 236 17 Saw CL Szykoluk H Curtis BR et al Two cases of platelet transfusion refractoriness associated with anti CD36 Transfusion 2010 50 2638 2642 18 Klein J Sato A The HLA system N Eng J Med 2000 343 702 19 Wu GG Kaplan C Curtis BR Pearson HA Report on the 14th International Society of Blood Transfusion Platelet Immunology Workshop Vox Sang 2010 99 375 381 20 Smith GA Ranasinghe E Ouwehand WH The importance of using multiple techniques for detection of platelet antibodies Vox Sang 2007 93 306 308 21 Metcalfe P Ensuring quality in platelet immunology Vox Sang 2007 93 287 288 Pak Lx Assay 10 303285 IFUEN REV H ual Immucor GTI Diagnostics Inc Immucor Medizinische Diagnostik GmbH 20925 Crossroads Circle Adam Opel Strasse 26A Waukesha WI 53186 USA Rodermark 63322 Germany US and International Contact Information Technical Support waukeshatechsupport immucor com www immucor com LUMINEX and xMAP are trademarks and or registered trademarks of Luminex Corporation MATCHIT Pak Lx Immucor and associated logos are trademarks and or registered trademarks of Immucor and or its subsidiaries in the United States and or other countries 2012 2015 Immucor GTI Diagnostics Inc 303285 IFUEN Rev H 2015 05 29 Wa
4. HPA 1b 3b 4a Neg Pos Neg Pos Pos Neg HPA 1ab 3ab 4a Pos Pos Pos Pos Pos Neg HPA 1a 3ab 4b Pos Neg Pos Pos Neg Pos Interpretation of results Antibodies to HPA 1 Antibodies to HPA 3 Antibodies to HPA 4 detected detected detected This pattern may mask the presence HPA 4b HPA 4b not HPA 4b HPA 4b HPA 1a 1b HPA 1a of an antibody reactive with masked 3a 3b 1b 3a 3b not masked Heterozygous beads may be positive or negative depending on the titer of the antibody in the sample or the level of background on the negative control beads Any pattern not represented in the table above should be considered an indeterminate result and may be due to the presence of auto antibodies or a combination of auto and allo antibodies This may also include reactivity to polymorphic variants not identified in the assay LIMITATIONS This product has not been designed to detect antibodies of the IgA or IgM class of immunoglobulin The results of this assay should not be used as the sole basis for a clinical decision A positive test only indicates the presence of antibodies specific for HLA Class I GPIV or HPA 1 HPA 2 HPA 3 HPA 4 and HPA 5 and is not intended to diagnose a clinical condition The HPA systems occur in different frequencies in different populations The higher frequency HPA systems are deemed to be public while the lower frequency HPA systems are deemed to be private The Pak Lx
5. aliquoting methods to eliminate carryover of additional Antibody reactivity p samples to adjacent wells Repeat the assay not indicated on the QC certificate Repeat assay following wash instructions User to employ good laboratory technique including Contaminated reagents proper aliquoting methods to eliminate carryover of Negative Control Serum shows samples to adjacent wells Repeat the assay Antibody reactivity Repeat the assay following wash instructions For the Negative Control Photobleached conjugate Use new kit Repeat the assay Serum the MFI value on the Positive Control Bead Probe f 77 is lt 10 000 Repeat the assay following wash instructions For test samples the MFI Photobleached conjugate Use new kit Repeat the assay value on the Positive Control Bead Probe 77 is lt 3 500 Retest the sample following wash instructions MFI values on antigen beads are approximately 30 MFI while Positive Control bead MFI is gt 10 000 Sample not added Retest the sample Beads not mixed well or Mix well with pipet to completely resuspend the resuspended beads Retest the sample _ instrument failures lt out of See Instrument Manual Repeat the assay Bead Failure insufficient bead calibration events counted lt 60 events Instrument failures sample flow See Instrument Manual Repeat the assay collected blocked Photobleached beads Use new kit Repeat the assay Vacuum pressure too strong beads stuck to membrane Reduce v
6. negativity 99 3 95 Confidence Interval 97 6 99 8 MAIPA HPA 2 Positive Negative Pak Lx Positive 0 Agreement 99 7 95 Confidence Interval 98 4 99 1 assa HP A 2 Negative 336 Co positivity 88 9 95 Confidence Interval 56 5 98 0 Total 336 Co negativity 100 0 95 Confidence Interval 98 9 100 0 MAIPA HPA 3 Positive Negative Pak Lx Positive 0 Agreement 99 4 95 Confidence Interval 97 9 99 8 HPA Negative 341 Co positivity 66 7 95 Confidence Interval 30 0 90 3 Total 341 Co negativity 100 0 95 Confidence Interval 98 9 100 0 MAIPA HPA 4 Negative Pak Lx Positive 0 Agreement 100 0 95 Confidence Interval 97 7 100 0 UPAS Negative 160 Co positivity 100 0 95 Confidence Interval 51 0 100 0 Total 160 Co negativity 100 0 95 Confidence Interval 97 7 100 0 MAIPA HPA 5 Positive Negative Pak Lx Positive 29 1 Agreement 99 1 95 Confidence Interval 97 4 99 7 HPA Negative 2 297 Co positivity 93 5 95 Confidence Interval 79 3 98 2 Total 31 298 Co negativity 99 7 95 Confidence Interval 98 1 99 9 MAIPA GPIV Positive Negative Pak Lx Positive 0 Agreement 99 4 95 Confidence Interval 96 7 99 9 GPIV Negative 162 Co positivity 83 3 95 Confidence Interval 43 6 97 0 Total 162 Co negativity 100 0 95 Confi
7. 92 Negative Control Serum This serum or sera blend is obtained from individual s with no known antibodies to HPA GPIV and HLA Store at 2 to 8 C Contains 0 1 sodium azide Pak Lx Assay 2 303285 IFUEN REV H PRECAUTIONS For In Vitro Diagnostic Use Do not use reagents that are turbid or contaminated Do not use reagents beyond their expiration date Discard all unused diluted Positive and Negative Controls and Conjugate after use Reagents contained in the kit are not to be used in conjunction with any other test system Substitution of components other than those provided in this kit may lead to inconsistent or erroneous results When making dilutions follow pipet manufacturer s instructions for appropriate dispensing and rinsing techniques Care MUST be taken to avoid contamination of Conjugate Diluent or the anti Human IgG reagent Inadvertent contamination of these reagents with human serum will result in the neutralization of anti Human IgG and subsequently result in test failure Care must be taken during pipetting into the filter plate that beads do not stick to the side of the microplate wells being pipetted while being careful not to touch the membrane with the tip Contacting the membrane with the pipet tip can lead to puncture of the membrane and subsequent failure of the assay Care must be taken to ensure during incubation steps that the beads are not splashing and sticking to the sides of the wells The positive
8. INSTRUCTIONS FOR USE Pak Lx Assay PLX CE 2 Caution Consult MATCHIT Platelet Antibody software v1 0 1 888622 Accompanying Documents MATCHIT Platelet Antibody software User Manual LC1371 8c A Q re Keep away aTCHIT Platelet Antibody software Quick Reference Guide LC1374 rom lig TABLE OF CONTENTS INTENDED USE i ivccticcccncicdissieiscnssacsacisuvsstnectunoacadcundaacsacssansceuastansiun dteceasGnpeanthaadssGaddnauvondagin du ddatsuundeg adGadsuansusdcadpcusacadens 2 SUMMARY AND EXPLANATION 0 ccec ccs ccescesseene sees senesenesseesaeenaessaessoesenesseeseeesaessaesaaesaneseneasnesaeesaeesaesenesenusenesaneoas 2 PRINCIPLE OF THE PROCEDURE cccccssssssssesesenesseesseesseeseessnessnesseessessaeesaessnesanesenesseesaeesaessaesenesenesseesaeesnesanevaneas 2 REAGENTS iscsi setisasssursecsssiaatenteiunsascasatte cncvsavnaarvatiaansscesontnusidsusvuadsosaussiuusvauseuassonsutesedssuusastuin dudsvobautendedaaeusoeusucruseuadaeutens 2 PRECAUTIONS ninanta caavarsaruatvacnsdanseasaudaagesdasaucassaduscunasusayuausunanssldenduasiuaghagiwnda gaa saskvaveaddsasntuasudseduadedenn 3 CAUTION iii ec cecinnsictcvissccectucbsatardscnasctasuscteatesedeathaisiacdancdsstarhaeacinia cual sbiateidaushuddandadebavaastacdnadnadvadesabestsc atshaueanbidasadnaduacadeie 3 INSTRUMENTATION cccccessescessnesseesenesseesseesseesaessneuseesseesaesaaesnoesanasanesaeesaeesaessaessaessaesenssaeesaesaaessnesanasaneasnesseenaeuane
9. ON2 CONS producing three ratios for each antigen specific bead Note when the MFI of any CON bead is less than the Minimum Cutoff MC then the MC is used in the calculation From the three calculated ratios a predetermined ratio the Background Adjustment Factor BAF is subtracted from each antigen specific bead con combination to obtain three Adjusted Ratios e A positive value for two or more of the three Adjusted Ratios indicates a positive bead reaction e A negative value for two or more of the three Adjusted Ratios indicates a negative bead reaction Interpretation of results Refer to the MATCHIT Software report for the results The table below lists the possible results that could be obtained when testing any given sample Antigen GPIV HLA Class HPA 1 3 4 HPA 2 HPA 5 GPIlb IIla GPIb IX GPla lla Possible result Pos Pos Reactive Pos Pos Neg Neg Neg Neg Neg Indeterminate Indeterminate e Results for HLA Class GPIV HPA 2 and HPA 5 are directly reported by the MATCHIT software e Any indeterminate results for HPA 2 and HPA 5 should be repeated using the Pak Lx Assay or using an alternate method e To determine HPA 1 3 and 4 reactivity use the following table HPA coated beads Pattern of Bead Reactivity HPA 1a 3a 4a Pos Neg Pos Neg Pos Neg HPA 1a 3b 4a Pos Neg Neg Pos Pos Neg HPA 1b 3a 4a Neg Pos Pos Neg Pos Neg
10. acuum strength Retest the sample Check to see if the lot number in the IDT LXT file Use of incorrect acquisition used for the acquisition of data matches the Pak Lx template IDT LXT file kit used for testing If incorrect repeat the assay and acquire data using the correct IDT LXT file Use of incorrect EDS BAF file Check to see if the lot number in the EDS file used for performing the analysis by for the analysis of data matches the Pak Lx kit used MATCHIT Platelet Antibody for testing If incorrect repeat the data analysis software using the correct EDS file SPECIFIC PERFORMANCE CHARACTERISTICS The following tables list the co positivity sensitivity co negativity specificity and agreement for the numbers of samples tested for antibodies reactive with each HPA or GPIV when compared to results obtained using monoclonal antibody immobilization of platelet antigens MAIPA or antibodies reactive with Class HLA when compared to results obtained with LifeScreen Deluxe Assay LMX Uninterpretable results Pak Lx Assay 8 303285 IFUEN REV H These data were compiled from two clinical studies conducted at Sanquin Diagnostic Services Amsterdam The Netherlands and The BloodCenter of Wisconsin Milwaukee Wisconsin MAIPA HPA 1 Positive Negative Pak Lx Positive 52 2 Agreement 99 4 95 Confidence Interval 97 9 99 8 HPA Negative 0 292 Co positivity 100 0 95 Confidence Interval 93 1 100 0 Total 52 294 Co
11. and negative controls must be included with each test to help determine if technical error or reagent failures have occurred Care must be taken to control vacuum strength Strong vacuum pressure can cause beads to stick to the membrane causing bead count failure CAUTION All human serum used in the Positive and Negative Controls for this product has been tested and found negative for antibody to HIV HCV and HBsAg by FDA approved methods No test method however can offer complete assurance that HIV Hepatitis C virus Hepatitis B virus or other infectious agents are absent Therefore these materials should be handled as potentially infectious Some reagents contain sodium azide as a preservative which may react with lead and copper plumbing to form explosive metal azides Use large amounts of water when discarding materials down a sink Dispose of all materials after use according to local regulations INSTRUMENTATION The Luminex instrument running the Luminex IS 2 3 or XPONENT 3 1 software is to be used for performing the Pak Lx Assay The Luminex instrument system is based on the principles of flow cytometry and includes the Luminex analyzer the Luminex XY plate handling platform and the Luminex SD sheath fluid delivery system software and PC The Luminex System is the combination of three core xMAP Technologies The first is microspheres a family of 100 fluorescently dyed 5 6 micron sized polystyrene microspheres that act as bo
12. assay uses glycoproteins captured from donors that have only been typed for the public HPA systems HPA 1 2 3 4 and 5 Three of these public HPA systems HPA 1 3 4 are displayed on the same glycoprotein GPllb Illa Serum samples may have antibodies to one or more of these public HPA systems and the presence of antibodies to one HPA system may mask the presence of antibodies to other Pak Lx Assay 7 303285 IFUEN REV H systems For example the presence of a high titer antibody reactive with one HPA 4 epitope may mask the presence of lower titer antibodies reactive with one of the HPA 1 epitopes Please see the section of these instructions labeled Interpretation of Results for further detail The Pak Lx assay uses glycoproteins captured from donors that have not been typed for private HPA systems Serum samples may have antibodies to one or more of these private HPA systems The presence of these antibodies to private epitopes may simulate reactivity patterns that would indicate the presence of an antibody reactive with a public HPA epitope and or cause indeterminate results Some low titer low avidity antibodies including antibodies to low incidence HLA Class antigens may not be detected by this product This assay has not been validated for use in detecting autoantibodies TROUBLESHOOTING PROBLEM POSSIBLE CAUSE SOLUTION P i User to employ good laboratory technique including Positive control Serum shows eee proper
13. dence Interval 97 7 100 0 LMX HLA Cl l Positive Negative Pak Lx Positive 67 1 Agreement 97 7 95 Confidence Interval 94 3 99 1 HLA Cll Negative 3 104 Co positivity 95 7 95 Confidence Interval 88 1 98 5 Total 70 105 Co negativity 99 0 95 Confidence Interval 94 8 99 8 Pak Lx Assay 9 303285 IFUEN REV H Precision The Pak Lx assay showed 100 agreement in reported results for eight samples when tested with one lot in duplicate over 20 separate testing events by two operators The selected samples included samples with antibody reactivity to HPA 1a 1b 3a 4a 5b GPIV and Class HLA Interfering substances Interfering substances studies were conducted using CLSI EP07 A2 Interference testing in clinical Chemistry Approved Guideline The following substances showed no interference in the Pak Lx assay at the concentrations indicated Hemoglobin lt 500 mg dl Triglycerides lt 500 mg dl Bilirubin lt 20 mg dl REFERENCES 1 Mueller Eckhardt C Mueller Eckhardt C Kiefel V Grubert A et al 348 cases of suspected neonatal alloimmune thrombocytopenia Lancet 1989 1 363 366 2 Mueller Eckhardt C Platelet allo and autoantigens and their clinical implications In Nance SJ eds Transfusion Medicine in the 1990s Arlington Va American Association of Blood Banks 1990 63 93 3 Brand A Immunological aspects of blood transfusions Transpl lmmunol 2002 10 183 190 4 McFarland JG Detection and iden
14. hich HPA GPIV and Class HLA glycoproteins have been immobilized along with four control beads The beads are lyophilized in a phosphate based buffer containing bovine serum albumin and 0 02 methylisothiazolone and 0 02 bromonitrodioxane as preservatives LIGHT SENSITIVE Keep out of direct light for extended periods of time 3 hours or less Store at 2 to 8 C in the dark in kit box PLXB PLBD 405160 Bead Diluent A phosphate based buffer containing NaCl Tween 20 ProClin 300 bovine serum albumin and mouse serum Store at 2 to 8 C 405159 Conjugate Concentrate Goat anti Human IgG conjugated to phycoerythrin provided in a phosphate based storage buffer containing NaCl Tween 20 lt 0 1 sodium azide and bovine serum albumin LIGHT SENSITIVE Keep out of direct light for extended periods of time 2 hours or less Store at 2 to 8 C in the dark Dilute 1 10 in Conjugate Diluent prior to use Q gt x lt 0 403586 Conjugate Diluent A phosphate based buffer containing NaCl Tween 20 0 1 sodium azide bovine CDX serum albumin and mouse serum Store at 2 to 8 C 405314 Wash Buffer A phosphate based buffer containing NaCl Tween 20 0 1 sodium azide and bovine euve serum albumin Store at 2 to 8 C PCX 403595 Positive Control Serum This serum or sera blend is obtained from individual s with known antibodies to HPA 1a with or without antibodies to Class HLA Store at 2 to 8 C Contains 0 1 sodium azide NCX 4035
15. la have been characterized into Human Platelet Antigen HPA systems of two alleles each alleles a and b HPA 1 HPA 3 and HPA 4 are all located on GPIlb Illa HPA 2 is located on GPIb IX and HPA 5 is located on GPla lla The epitopes of GPIV are presented only as isoantigens In other words the development of antibodies to GPIV occurs in individuals who do not have or have significantly reduced levels of GPIV expression but have become exposed to GPIV Class HLA is encoded by numerous alleles expressing a large number of diverse epitopes The vast majority of antibodies produced in response to pregnancy or transfusion will be reactive with the highly polymorphic Class HLA PRINCIPLE OF THE PROCEDURE The Pak Lx Beads are reconstituted and incubated with serum sample at room temperature The beads are then washed to remove unbound antibody An anti Human IgG antibody conjugated to phycoerythrin PE is then added After further incubation at room temperature the reaction mixture is diluted and analyzed on the Luminex instrument The signal intensity from each bead is compared to the signal intensity of three negative control beads included in the bead preparation to determine if the bead is positive or negative for bound antibody REAGENTS Maximum number of tests per kit PL 7 16 tests per kit All reagents should be stored as directed by the label REF 403620 Lyophilized Bead Blend A blend of beads to w
16. le towards the bottom of the vial This will result in differential amount of beads being dispensed into wells which may adversely affect run times and analysis of results Add 10 uL of patient serum or control serum referring to the plate layout sheet Return unused portions of control sera and beads to storage at 2 to 8 C in the dark for future use Cover the plate with a plate sealer Incubate for 60 minutes at room temperature 21 to 24 C in the dark on a rotating platform approximately 200 rotations per minute or a vortex shaker with a plate adaptor set at a speed that will allow proper mixing without splashing of the samples a During this incubation bring the Conjugate Concentrate and Conjugate Diluent to room temperature 21 to 24 C b During this Incubation warm up and prepare the Luminex instrument c Setup the assay on the Luminex instrument by using the lot specific IDT LXT file acquisition template downloaded from the web site www immucor com Refer to the Luminex software manual on how to add import a template file into the software NOTE When using the Automated Batch Setup feature refer to the MATCHIT Platelet Antibody software User Manual which is available on the Platelet Antibody Software Installation CD PACD and on the website www immucor com CAUTION Failure to use the correct IDT LXT file will result in the collection of data that is out of sequence and will incorrectly or incompletely capt
17. plate file available on website e IDT file for use with IS 2 3 e LXT file for use with xXPONENT 3 1 16 Lot specific Electronic Data Sheet EDS file available on website 17 Luminex instrument with IS 2 3 or xPONENT 3 1 software 18 PFS Plate Format Sheet 19 PACD Platelet Antibody Software Installation CD MATCHIT Platelet Antibody Software v1 0 1 MATCHIT Platelet Antibody software User Manual also available on website MATCHIT Platelet Antibody software Quick Reference Guide also available on website Test Procedure 1 Download the lot specific Luminex acquisition template IDT LXT file from the website www immucor com and save it on the computer connected to the Luminex instrument Note the location of the saved file This file will be imported to the Luminex instrument in a later step 2 Bring all reagents except the Conjugate Concentrate and the Conjugate Diluent to room temperature 21 to 24 C prior to use 3 Reconstitute the Lyophilized Bead Blend as noted in Table 1 below The number of vials provided in Table 1 includes enough volume to run one positive and one negative control along with the number of samples listed in column 1 Pak Lx Assay 4 303285 IFUEN REV H 10 11 12 13 Table 1 Number of samples Vial s of Lyophilized Bead Blend 1 4 1 5 10 2 11 16 3 a Add 275 uL of Bead Diluent to each vial of lyophilized beads
18. r below 20 C remain in good condition for up to 2 years Avoid frost free freezers Avoid repeated freezing and thawing of the serum samples Pak Lx Assay 3 303285 IFUEN REV H PROCEDURE Materials Provided See REAGENTS section for more specific information Vials may contain more reagent than stated on the labels Be sure to measure the reagent with an appropriate device when making dilutions 3 x Lyophilized Bead Blend 1 x 850 uL Bead Diluent 1 x 120 uL Conjugate Concentrate 1 x 1 2 mL Conjugate Diluent 1 x 50 mL Wash Buffer 1 x 80 uL Positive Control Serum 1 x 80 uL Negative Control Serum NOARWON Additional Materials Required as listed or equivalent 1 5 uL to 50 uL adjustable pipets with appropriate pipet tips 2 250 uL multichannel pipet with matching tips and buffer trough 3 1 5 mL microcentrifuge tubes for conjugate dilutions 4 Test tubes 5 Timer 6 Marking pen 7 Plate sealers 8 Luminex Sheath Fluid REF 628005 1X REF 628025 20X 9 Luminex Calibration and control beads REF 628006 CAL1 REF 628007 CAL2 IREF 628008 CON1 IREF 628009 CON2 10 Distilled water 11 Rotator or vortex with plate adapter 12 Millipore Multiscreen filter plates REF 888633 Millipore MSBVN1210 MSBVN1250 MABVN1 210 MABVN1250 13 Multiscreen vacuum manifold REF 888315 Millipore MAVMO960R Qiagen 19504 Pall 5017 14 Microcentrifuge 15 Lot specific Luminex Acquisition Tem
19. rning Warning H317 May cause an allergic skin reaction P261 Avoid breathing dust fume gas mist vapours spray P272 Contaminated work clothing should not be allowed out of the workplace P280 Wear protective gloves protective clothing eye protection face protection P302 P352 IF ON SKIN Wash with plenty of soap and water P333 P313 If skin irritation or rash occurs Get medical advice attention P501 Dispose of contents container to an approved waste disposal plant Pak Lx Assay 303285 IFUEN REV H
20. s 3 SPECIMEN COLLECTION AND STORAGE cccccscscsenessnesseesseesseessessaessnessneseeesessaesaaesaneseneasneaseesaeesaeseneseneseneeaeeas 3 PROCEDURE o nna atana tarine eanna anae h Arae rA Aaaa raaa a AaS Aaa A AAA aa teach sabiadedeunandSectuanedscauvandnisvadadevnontsuctadeeandanstva 4 QUALITY GONTROL Eea A E E E A E 6 INTERPRETATION OF RESULTS 0 ccsecssstessesseessesseesseesseessesnseseoessnesenesaneuseeseesaessaeseaessneseesaeseaesaaesanasaneasnenseesaeeaass 7 LIMITATIONS io cctietiensctssdccddeantcessunsacatceTatuaseuasavadcaastaueantiadstcelsdn nantes adassusddsnupavaagsstesadedagubanedsdunacedeswadugiatendsededalnisduccbstadeds 7 TROUBLESHOOTING ccccccccssssesseeseeeseesseesenesseesseesaeesaessoessoesenesenesenesaeeeaesaaesaneseneseneseesaeesaeseaesenesenasanesaeeaaesaaesanauaneas 8 SPECIFIC PERFORMANCE CHARACTERISTIGG ccccscssstsssesseesseessessesseesseesseesaesnaessnesenessnesseesaeesaessneseneseneeaneoas 8 REFERENC E S eaa aaaea E aa a aaa aaa aada aa Aaaa A aaa aa aa Eaa aa E Ea Ea aaaea iaaea 10 Restricted Use Label License By opening the packaging containing this Kit which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this Kit in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you
21. th the identifier and the solid surface to build the assay The second is a flow cytometry based instrument the Luminex analyzer which integrates key detection components such as lasers optics fluidics and high speed digital signal processors The third component is the software which is designed for protocol based data acquisition Information on the features of the software and the instrument installation operation maintenance and safety are provided in the Luminex IS Training Manual The user should contact the Luminex Corporation for specific services regarding the Luminex instrument system The assay files required to use the Pak Lx Assay are provided by Immucor on their website for download by the user The user should contact Immucor GTI Diagnostics for any product related concerns for Pak Lx Assay and the MATCHIT Platelet Antibody Software SPECIMEN COLLECTION AND STORAGE Collect blood without anticoagulant using aseptic technique Process blood while still fresh to minimize the chance of obtaining false positive or false negative reactions due to improper storage or contamination of the specimen Only serum is suitable for this assay Serum should be separated from red cells when stored or shipped Samples containing particulate matter should be clarified by centrifugation prior to testing Serum samples that cannot be tested immediately should be stored at 2 to 8 C for no longer than 48 hours or frozen Samples frozen at o
22. tification of platelet antibodies in clinical disorders Transfus Apher Sci 2003 28 297 305 5 Davoren A Curtis BR Aster RH McFarland JG Human platelet antigen specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia Transfusion 2004 44 1220 1225 6 Rebulla P A mini review on platelet refractoriness Haematologica 2005 90 247 253 7 Kanhai HH Porcelijn L Engelfriet CP et al Management of alloimmune thrombocytopenia Vox Sang 2007 93 370 385 8 Stroncek DF Rebulla P Platelet transfusions Lancet 2007 370 427 438 9 Arnold DM Smith JW Kelton JG Diagnosis and management of neonatal alloimmune thrombocytopenia Transfus Med Rev 2008 22 255 267 10 Bussel JB Primiani A Fetal and neonatal alloimmune thrombocytopenia progress and ongoing debates Blood Rev 2008 22 33 52 11 Curtis BR McFarland JG Detection and identification of platelet antibodies and antigens in the clinical laboratory Immunohematology 2009 25 125 135 12 Vassallo RR Recognition and management of antibodies to human platelet antigens in platelet transfusion refractory patients Immunohematology 2009 25 119 124 13 Kaplan C Ni H Freedman J Alloimmune Thrombocytopenia In Michelson A eds Platelets 3rd Edition Academic Press Elsevier 2012 46 953 970 14 Rozman P Platelet antigens The role of human platelet alloantigens HPA in blood transfusion and transplantation Transpl Immunol 2002 10 165 181 15
23. to the test system by the inclusion of four control beads one positive control bead and three negative control beads The Positive Control Bead is coated with human IgG and should yield MFI values gt 10 000 with the Negative Control Sera If values of lt 10 000 MFI are obtained with the Negative Control serum the assay may be insufficiently washed or the conjugate may be compromised Samples may show a wide range of reactivity with the Positive Control Bead but should produce a signal of gt 3500 MFI If these requirements are not met the affected samples are to be considered invalid Pak Lx Assay 6 303285 IFUEN REV H The interpretation of sample data is based on having a minimum number of each bead collected during an assay When too few bead events are acquired for a sample a Bead Failure error is noted and all antibody results for the affected sample are to be considered invalid Please see the troubleshooting section of these instructions for further details INTERPRETATION OF RESULTS Interpretation by software The MATCHIT Platelet Antibody software v1 0 1 is required for the interpretation of the results For the analysis a lot specific EDS file is required and is available for download from the website www immucor com Determination of individual bead reactivity To determine if an antigen specific bead is positive or negative the MFI of the antigen specific bead is divided by the MFI for each Negative Control Bead CON1 C
24. ure the assay data from the Luminex instrument Pak Lx Assay 5 303285 IFUEN REV H 14 Prepare diluted Conjugate Concentrate by mixing the volumes of Conjugate Concentrate CCX with Conjugate Diluent CDX as listed in Table 2 below The volumes shown in Table 2 below include enough volume to run one positive and one negative control along with the number of samples listed in column 1 a Cover the diluted conjugate with foil or store in the dark at room temperature until used b Return the unused portion of Conjugate Concentrate and Conjugate Diluent to storage at 2 to 8 C in the dark for future use Table 2 Number of Volume of CCX Volume of CDX Samples uL uL 1 20 0 180 0 2 25 0 225 0 3 30 0 270 0 4 35 0 315 0 5 40 0 360 0 6 45 0 405 0 7 50 0 450 0 8 55 0 495 0 9 60 0 540 0 10 65 0 585 0 11 70 0 630 0 12 75 0 675 0 13 80 0 720 0 14 85 0 765 0 15 90 0 810 0 16 95 0 855 0 15 After the 60 minute incubation remove the plate sealer and add 200 uL of Wash Buffer to each well Remove the wash buffer by gentle aspiration using the vacuum manifold CAUTION Use of excessive vacuum strength will cause beads to stick to the membrane and can result in an assay failure Apply the minimum vacuum pressure required to aspirate samples Refer to the Manufacturer s instructions for specific use of the filter plates and vacuum manifold 16 Add 250 uL of Wash Buffer to each
25. well aspirate Repeat two more times CAUTION Failure to wash completely may reduce the ability of the conjugate to detect IgG bound to sensitized beads 17 Add 50 uL of diluted conjugate to each well Cover with plate sealer Incubate for 30 minutes at room temperature 21 to 24 C in the dark on a rotating platform approximately 200 rotations per minute or a vortex shaker with a plate adaptor set at a speed that will allow proper mixing without splashing of the samples Do not save the diluted conjugate for future use 18 Remove the plate sealer Add 150 uL of Wash Buffer to each well containing the sample Return the unused portion of Wash Buffer to storage at 2 to 8 C for future use 19 Immediately collect data with Luminex instrument using the manufacturer s instructions NOTE A minimum of 60 events must be collected per bead region QUALITY CONTROL The Positive and Negative Control Sera serve as external assay controls and must be included with each test run The Positive Control Serum is prepared from available serum donors and may change with each lot of Pak Lx The Positive Control Serum should have a positive reported result for those antibodies reported on the Certificate of Quality Control The Negative Control Serum should have a negative reported result for any HPA GPIV or HLA Class bead If these requirements are not met the affected samples are to be considered invalid Quality control of the Pak Lx assay is built in
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