Pak Lx™ Assay - Immucor, Inc.

Pak Lx™ Assay - Immucor, Inc.
INSTRUCTIONS FOR USE
Pak Lx™ Assay
REF
PLX
IVD
Caution! Consult
Accompanying
Documents.
MATCHIT!
Platelet Antibody software v1.0.1 ( REF 888622)
MATCHIT!
Platelet Antibody software User Manual ( REF LC1371)
Light Sensitive (Keep away
from light)
MATCHIT!
Platelet Antibody software Quick Reference Guide ( REF LC1374)
TABLE OF CONTENTS
INTENDED USE............................................................................................................................................................. 2
SUMMARY AND EXPLANATION ................................................................................................................................. 2
PRINCIPLE OF THE PROCEDURE .............................................................................................................................. 2
REAGENTS ................................................................................................................................................................... 2
PRECAUTIONS ............................................................................................................................................................. 3
CAUTION ....................................................................................................................................................................... 3
INSTRUMENTATION..................................................................................................................................................... 3
SPECIMEN COLLECTION AND STORAGE ................................................................................................................. 3
PROCEDURE ................................................................................................................................................................ 4
QUALITY CONTROL ..................................................................................................................................................... 6
INTERPRETATION OF RESULTS ................................................................................................................................ 7
LIMITATIONS ................................................................................................................................................................ 7
TROUBLESHOOTING ................................................................................................................................................... 8
SPECIFIC PERFORMANCE CHARACTERISTICS....................................................................................................... 8
REFERENCES ............................................................................................................................................................. 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. 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 I 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), post1-13
transfusion purpura (PTP), and fetal and neonatal alloimmune thrombocytopenia (FNAITP).
The glycoproteins (GP) targeted by antibodies include GPIIb/IIIa, GPIb/IX and GPIa/IIa, GPIV, and Class I Human Leukocyte
Antigens (HLA). The epitopes found on GPIIb/IIIa, GPIb/IX, and GPIa/IIa 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 GPIIb/IIIa. HPA-2 is located on GPIb/IX
14-16
and HPA-5 is located on GPIa/IIa.
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
17
exposed to GPIV. Class I HLA is encoded by numerous alleles expressing a large number of diverse epitopes. The vast majority of
18
antibodies produced in response to pregnancy or transfusion will be reactive with the highly polymorphic Class I 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:

PLX:
16 tests per kit
All reagents should be stored as directed by the label.
REF
PLXB
PLBD
CCX
CDX
LMWB
PCX
NCX
Pak Lx Assay
403620
Lyophilized Bead Blend: A blend of beads to which HPA, GPIV and Class I 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.
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 phosphatebased storage buffer containing NaCl, Tween-20, <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.
403586
Conjugate Diluent: A phosphate-based buffer containing NaCl, Tween-20, 0.1% sodium azide, bovine
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
serum albumin. Store at 2 to 8°C.
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 I HLA. Store at 2 to 8°C. Contains 0.1% sodium azide.
403592
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.
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 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 both 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 or 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.
1.
2.
3.
4.
5.
6.
7.
3 x Lyophilized Bead Blend
1 x 850 L Bead Diluent
1 x 120 µL Conjugate Concentrate
1 x 1.2 mL Conjugate Diluent
1 x 50 mL Wash Buffer
1 x 80 µL Positive Control Serum
1 x 80 µL Negative Control Serum
Additional Materials Required:
(as listed or equivalent)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
5 µL to 50 µL adjustable pipets with appropriate pipet tips
250 µL multichannel pipet with matching tips and buffer trough
1.5 mL microcentrifuge tubes for conjugate dilutions
Test tubes
Timer
Marking pen
Plate sealers
Luminex Sheath Fluid
REF 628005 (1X); REF 628025 (20X)
Luminex Calibration and control beads
REF 628006 (CAL1); REF 628007 (CAL2); REF 628008 (CON1); REF 628009 (CON2)
Distilled water
Rotator or vortex with plate adapter
Millipore Multiscreen filter plates
REF 888633
Millipore MSBVN1210/MSBVN1250/MABVN1210/MABVN1250
Multiscreen vacuum manifold
REF 888315
Millipore MAVM0960R; Qiagen 19504; Pall 5017
Microcentrifuge
Lot specific Luminex Acquisition Template file; available on website

IDT file for use with IS 2.3

LXT file for use with xPONENT 3.1
Lot specific Electronic Data Sheet (EDS) file; available on website
Luminex instrument with IS 2.3 or xPONENT 3.1 software
PFS
Plate Format Sheet
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
Table 1
Number of samples
1-4
5-10
11-16
Vial(s) of
Lyophilized Bead Blend
1
2
3
a.
Add 275 L of Bead Diluent to each vial of lyophilized beads. 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.

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.
4.
Prepare samples by vortexing. Centrifuge at > 10,000 x g for at least 60 seconds to settle any insoluble matter before use.
5.
Record sample information on the Plate Format Sheet. The plate format sheet is available for download from the website
(www.immucor.com).
6.
Cover the unassigned wells of the filter plate with a plate sealer.
7.
Add 250 µL 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.)
8.
Mix the reconstituted beads by pipetting up and down about 15 to 20 times taking care to avoid any frothing.
9.
Add 40 µL 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:
It is important to keep the beads resuspended to ensure uniform bead addition to the wells. Failure to mix beads
intermittently will cause beads to settle 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.
10. Add 10 µL of patient serum or control serum referring to the plate layout sheet.
11. Return unused portions of control sera and beads to storage at 2 to 8°C in the dark for future use.
12. Cover the plate with a plate sealer.
13. 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.
Set up 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 capture 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
Samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Volume of CCX
(µL)
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
65.0
70.0
75.0
80.0
85.0
90.0
95.0
Volume of CDX
(µL)
180.0
225.0
270.0
315.0
360.0
405.0
450.0
495.0
540.0
585.0
630.0
675.0
720.0
765.0
810.0
855.0
15. After the 60 minute incubation, remove the plate sealer and add 200 µL 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 µL of Wash Buffer to each 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 µL 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 µL 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 I bead. If these requirements are not met, the affected samples are
to be considered invalid.
Quality control of the Pak Lx assay is built into 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 >10,000 with the
Negative Control Sera. If values of <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 >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, CON2, CON3), 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.
 A positive value for two or more of the three Adjusted Ratios indicates a positive bead reaction.
 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 I
Possible result
Pos
Neg
Pos
Neg



HPA-1, -3, -4
(GPIIb-IIIa)
Reactive
Neg
HPA-2
(GPIb-IX)
Pos
Neg
Indeterminate
HPA-5
(GPIa-IIa)
Pos
Neg
Indeterminate
Results for HLA Class I, GPIV, HPA-2 and HPA-5 are directly reported by the MATCHIT! software
Any indeterminate results for HPA-2 and HPA-5 should be repeated using the Pak Lx Assay, or using an alternate
method.
To determine HPA-1, -3 and -4 reactivity, use the following table:
HPA coated beads
HPA – 1a-3a-4a
HPA – 1a-3b-4a
HPA – 1b-3a-4a
HPA – 1b-3b-4a
HPA – 1ab-3ab-4a
HPA - 1a-3ab-4b
Interpretation of results
This pattern may mask the presence
of an antibody reactive with:
Pos
Neg
Pos
Neg
Neg
Pos
Neg
Pos
Pos*
Pos*
Pos
Neg
Antibodies to HPA-1
detected
HPA-4b
HPA-4b not
masked
Pattern of Bead Reactivity**
Pos
Neg
Neg
Pos
Pos
Neg
Neg
Pos
Pos*
Pos*
Pos*
Pos*
Antibodies to HPA-3
detected
HPA-4b
HPA-4b
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Neg
Pos*
Neg
Neg*
Pos
Antibodies to HPA-4
detected
HPA-1a, -1b, HPA-1a,
-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
19-21
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 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
1,5,11,14
(HPA-1, -3, -4) are displayed on the same glycoprotein, GPIIb/IIIa.
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 I antigens may not be detected by this
product.

This assay has not been validated for use in detecting autoantibodies.
TROUBLESHOOTING
PROBLEM
Positive control Serum shows
additional Antibody reactivity
not indicated on the QC
certificate
POSSIBLE CAUSE
Positive Control contaminated
with another sample
SOLUTION
User to employ good laboratory technique including
proper aliquoting methods to eliminate carryover of
samples to adjacent wells; Repeat the assay
Poor washing
Repeat assay following wash instructions
Contaminated reagents
User to employ good laboratory technique including
proper aliquoting methods to eliminate carryover of
samples to adjacent wells; Repeat the assay
Poor washing
Repeat the assay following wash instructions
For the Negative Control
Serum, the MFI value on the
Positive Control Bead (Probe
77) is <10,000
Photobleached conjugate
Use new kit; Repeat the assay
Poor washing
Repeat the assay following wash instructions
For test samples, the MFI
value on the Positive Control
Bead (Probe 77) is <3,500
Photobleached conjugate
Use new kit; Repeat the assay
Poor washing
Retest the sample following wash instructions
MFI values on antigen beads
are approximately 30 MFI
while Positive Control bead
MFI is > 10,000
Sample not added
Retest the sample
Mix well with pipet to completely resuspend the
beads; Retest the sample
Bead Failure (insufficient bead
events counted) (<60 events
collected)
Beads not mixed well or
resuspended
Instrument failures - out of
calibration
Instrument failures - sample flow
blocked
Photobleached beads
Vacuum pressure too
strong/beads stuck to membrane
Negative Control Serum shows
Antibody reactivity
Use of incorrect acquisition
template (IDT/LXT) file.
Uninterpretable results
Use of incorrect EDS (BAF) file
for performing the analysis by
MATCHIT! Platelet Antibody
software
See Instrument Manual; Repeat the assay
See Instrument Manual; Repeat the assay
Use new kit; Repeat the assay
Reduce vacuum strength; Retest the sample
Check to see if the lot number in the IDT/LXT file
used for the acquisition of data matches the Pak Lx
kit used for testing. If incorrect, repeat the assay and
acquire data using the correct IDT/LXT file.
Check to see if the lot number in the EDS file used
for the analysis of data matches the Pak Lx kit used
for testing. If incorrect, repeat the data analysis
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 I HLA when compared to results obtained with LifeScreen Deluxe Assay (LMX).
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)
Pak Lx
assay
(HPA-1)
Positive
Negative
Total
Positive
52
2
54
Agreement:
99.4%
95% Confidence Interval = 97.9 – 99.8%
Negative
0
292
292
Co-positivity:
100.0%
95% Confidence Interval = 93.1 – 100.0%
Total
52
294
346
Co-negativity:
99.3%
95% Confidence Interval = 97.6 - 99.8%
MAIPA (HPA-2)
Pak Lx
assay
(HPA-2)
Positive
Negative
Total
Positive
8
0
8
Agreement:
99.7%
95% Confidence Interval = 98.4 – 99.1%
Negative
1
336
337
Co-positivity:
88.9%
95% Confidence Interval = 56.5 – 98.0%
Total
9
336
345
Co-negativity:
100.0%
95% Confidence Interval = 98.9 – 100.0%
MAIPA (HPA-3)
Pak Lx
assay
(HPA-3)
Positive
Negative
Total
Positive
4
0
4
Agreement:
99.4%
95% Confidence Interval = 97.9 – 99.8%
Negative
2
341
343
Co-positivity:
66.7%
95% Confidence Interval = 30.0 - 90.3%
Total
6
341
347
Co-negativity:
100.0%
95% Confidence Interval = 98.9 – 100.0%
MAIPA (HPA-4)
Pak Lx
assay
(HPA-4)
Positive
Negative
Total
Positive
4
0
4
Agreement:
100.0%
95% Confidence Interval = 97.7 – 100.0%
Negative
0
160
160
Co-positivity:
100.0%
95% Confidence Interval = 51.0 – 100.0%
Total
4
160
164
Co-negativity:
100.0%
95% Confidence Interval = 97.7 – 100.0%
MAIPA (HPA-5)
Pak Lx
assay
(HPA-5)
Pak Lx
assay
(GPIV)
Pak Lx
assay
(HLA Cl-I)
Pak Lx Assay
Positive
Positive
29
Negative
1
Total
30
Agreement:
99.1%
95% Confidence Interval = 97.4 – 99.7%
Negative
2
297
299
Co-positivity:
93.5%
95% Confidence Interval = 79.3 - 98.2%
Total
31
298
329
Co-negativity:
99.7%
95% Confidence Interval = 98.1 - 99.9%
Total
5
Agreement:
99.4%
95% Confidence Interval = 96.7 – 99.9%
Positive
MAIPA (GPIV)
Positive
Negative
5
0
Negative
1
162
163
Co-positivity:
83.3%
95% Confidence Interval = 43.6 - 97.0%
Total
6
162
168
Co-negativity:
100.0%
95% Confidence Interval = 97.7 - 100.0%
Total
68
Agreement:
97.7%
95% Confidence Interval = 94.3 – 99.1%
Positive
LMX (HLA Cl-I)
Positive
Negative
67
1
Negative
3
104
107
Co-positivity:
95.7%
95% Confidence Interval = 88.1 - 98.5%
Total
70
105
175
Co-negativity:
99.0%
95% Confidence Interval = 94.8 - 99.8%
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 I 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
Triglycerides
Bilirubin
≤ 500 mg/dl
≤ 500 mg/dl
≤ 20 mg/dl
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Mueller-Eckhardt C. Mueller-Eckhardt C, Kiefel V, Grubert A et al. 348 cases of suspected neonatal alloimmune
thrombocytopenia. Lancet 1989;1:363-366.
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.
Brand A. Immunological aspects of blood transfusions. Transpl.Immunol. 2002;10:183-190.
McFarland JG. Detection and identification of platelet antibodies in clinical disorders. Transfus.Apher.Sci. 2003;28:297-305.
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.
Rebulla P. A mini-review on platelet refractoriness. Haematologica 2005;90:247-253.
Kanhai HH, Porcelijn L, Engelfriet CP et al. Management of alloimmune thrombocytopenia. Vox Sang. 2007;93:370-385.
Stroncek DF, Rebulla P. Platelet transfusions. Lancet 2007;370:427-438.
Arnold DM, Smith JW, Kelton JG. Diagnosis and management of neonatal alloimmune thrombocytopenia. Transfus.Med.Rev.
2008;22:255-267.
Bussel JB, Primiani A. Fetal and neonatal alloimmune thrombocytopenia: progress and ongoing debates. Blood Rev.
2008;22:33-52.
Curtis BR, McFarland JG. Detection and identification of platelet antibodies and antigens in the clinical laboratory.
Immunohematology. 2009;25:125-135.
Vassallo RR. Recognition and management of antibodies to human platelet antigens in platelet transfusion-refractory patients.
Immunohematology. 2009;25:119-124.
Kaplan C, Ni H, Freedman J. Alloimmune Thrombocytopenia. In: Michelson A eds. Platelets 3rd Edition. Academic Press –
Elsevier. 2012; 46:953-970.
Rozman P. Platelet antigens. The role of human platelet alloantigens (HPA) in blood transfusion and transplantation.
Transpl.Immunol. 2002;10:165-181.
Metcalfe P et al. Nomenclature of human platelet antigens. Vox Sang. 2003; 85:240.
Santoso S. Human platelet alloantigens. Transfus.Apher.Sci. 2003;28:227-236.
Saw CL, Szykoluk H, Curtis BR et al. Two cases of platelet transfusion refractoriness associated with anti-CD36. Transfusion
2010;50:2638-2642.
Klein J, Sato A. The HLA system. N Eng J Med. 2000; 343:702.
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.
Smith GA, Ranasinghe E, Ouwehand WH. The importance of using multiple techniques for detection of platelet antibodies. Vox
Sang. 2007;93:306-308.
Metcalfe P. Ensuring quality in platelet immunology. Vox Sang. 2007;93:287-288.
Pak Lx Assay
10
303285.IFUEN REV H
EC
Immucor GTI Diagnostics, Inc.
20925 Crossroads Circle
Waukesha, WI 53186 USA
REP
Immucor Medizinische Diagnostik GmbH
Adam-Opel-Strasse 26A
Rodermark 63322
Germany
US and International Contact Information:
Technical Support :
[email protected]
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
Warning
H317
P261
P272
P280
P302 + P352
P333 + P313
P501
Pak Lx Assay
Warning
May cause an allergic skin reaction
Avoid breathing dust/fume/gas/mist/vapours/spray
Contaminated work clothing should not be allowed out of the workplace
Wear protective gloves/protective clothing/eye protection/face protection
IF ON SKIN: Wash with plenty of soap and water.
If skin irritation or rash occurs: Get medical advice/attention.
Dispose of contents/container to an approved waste disposal plant.
11
303285.IFUEN REV H
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