INSTRUCTIONS FOR USE

PakPlus® assay
REF PAKPLUS IVD

TABLE OF CONTENTS

INTENDED USE ............................................................................................................................................................ 2

SUMMARY AND EXPLANATION ................................................................................................................................. 2
PRINCIPLE OF THE PROCEDURE .............................................................................................................................. 2
REAGENTS ................................................................................................................................................................... 2
PRECAUTIONS ............................................................................................................................................................. 3
CAUTION ....................................................................................................................................................................... 3
SPECIMEN COLLECTION AND STORAGE ................................................................................................................. 3
PROCEDURE ................................................................................................................................................................ 3
Materials Provided .................................................................................................................................................... 3
Additional Materials Required................................................................................................................................... 3
Test Procedure ......................................................................................................................................................... 4
QUALITY CONTROL ..................................................................................................................................................... 6
INTERPRETATION OF RESULTS ................................................................................................................................ 6
LIMITATIONS ................................................................................................................................................................ 7
SPECIFIC PERFORMANCE CHARACTERISTICS ...................................................................................................... 7
REFERENCES............................................................................................................................................................... 9
TRANSLATION OF SYMBOLS ................................................................................................................................... 11

PakPlus assay 1 303469.IFUEN REV H

INTENDED USE

The PakPlus assay is a qualitative solid-phase enzyme-linked immunosorbent assay (ELISA) designed to screen for antibodies to
HLA Class I and platelet glycoprotein (GP) IV antigens, and to polymorphic epitopes on the platelet glycoproteins IIb/IIIa, Ib/IX, and
Ia/IIa.

SUMMARY AND EXPLANATION

1-5
The existence of platelet-specific antigens on various platelet glycoproteins has been described by many investigators. Antibodies to
6-18
platelet-specific or HLA class I antigens due to pregnancy or transfusion can result in immune destruction of transfused platelets.
Confirming the presence of these antibodies in patient specimens can be helpful in the search for potentially compatible blood products.

The PakPlus Solid Phase ELISA microwells provide monoclonal-captured platelet glycoproteins IIb/IIIa and Ia/IIa obtained from group O
donors of known platelet types. HLA Class I and platelet glycoproteins Ib/IX and IV are provided as affinity purified glycoproteins. The
test is designed to detect and differentiate between antibodies to HLA Class I and platelet-specific antigens. The configuration of the
microwells can be found on the Recording Sheet.

PRINCIPLE OF THE PROCEDURE

Patient serum is added to microwells coated with platelet and HLA glycoproteins allowing antibody, if present, to bind. Unbound
antibodies are then washed away. An alkaline phosphatase labeled anti-human globulin reagent (Anti-IgG/A/M) is added to the
microwells and incubated. The unbound Anti-IgG/A/M is washed away and the substrate PNPP (p-nitrophenyl phosphate) is added.
After a 30-minute incubation period, the reaction is stopped with Stopping Solution. The optical density of the color that develops is
measured in a spectrophotometer.

REAGENTS

Maximum number of tests per kit:

 PAKPLUS: 5 tests per kit

All reagents should be stored as directed by the label.

REF

404553 Microwell Strips: Flat-bottom microwell strips to which platelet and HLA glycoproteins have been immobilized.
MS
The microwells are enclosed in a resealable foil pouch. Ready for use.

403622 Concentrated Wash (10X): Tris (hydroxymethyl) aminomethane buffered solution containing sodium chloride
TCW
and Tween 20. 1% sodium azide. Dilute with deionized or distilled water before use. Store Working Wash
solution up to 48 hours at room temperature or up to seven days at 2 to 8°C.

403831 Specimen Diluent: Phosphate buffered saline solution containing bovine albumin and mouse serum. 0.1%
SD
sodium azide. Ready for use.

403613 Substrate Buffer: This solution contains diethanolamine and magnesium chloride. 0.02% sodium azide.
SB
Ready for use. Protect from light.

403603 Stopping Solution: Ready for use.

ESS

404589 Anti-Human IgG/A/M Conjugate: Alkaline phosphatase conjugated goat affinity purified antibody to human
AH
immunoglobulins (IgG/A/M). 0.1% sodium azide. Dilute in Specimen Diluent before use.

403594 PNPP Substrate: (p-nitrophenyl phosphate) Crystalline powder. Reconstitute with deionized or distilled water
PN
and dilute in Substrate Buffer before use. Protect from light.

404601 Positive Serum Control: Human Serum. 0.1% sodium azide. Dilute in Specimen Diluent before use.
PC

404577 Negative Serum Control: Human Serum. 0.1% sodium azide. Dilute in Specimen Diluent before use.
NC

PakPlus assay 2 303469.IFUEN REV H

 503019 Plate Sealers.
PS

PRECAUTIONS

 Do not use reagents that are turbid or contaminated.

 Care MUST be taken to avoid contamination of Specimen Diluent and conjugate. Inadvertent contamination of these reagents with
human serum will result in the neutralization of the Conjugate and subsequently to test failure.
 Do not use reagents beyond their expiration date.
 Microwells and 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.
 Discard any unused portions of diluted Conjugate, diluted Positive and Negative Controls, and diluted and reconstituted PNPP
reagent after each run.
 When using a dry incubator for the heated incubation steps, be careful to limit the number of door openings/closings to avoid
variation in internal temperature.
 When making dilutions, follow pipet manufacturer’s instructions for appropriate dispensing and rinsing techniques.
 The enzyme substrate reaction which occurs in the final incubation is temperature sensitive and should be performed in a
controlled area at 22 to 25°C.

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 of the reagents supplied with this kit contain sodium azide as a preservative.
WARNING: Sodium azide reacts with lead and copper plumbing forming highly explosive metal azides. When discarded in a sink,
the sink should be flushed with a large volume of water to prevent azide buildup. Sodium azide is a poison and is toxic if ingested.
 Discard all components when completed according to local regulations.

SPECIMEN COLLECTION AND STORAGE

Blood should be collected without anticoagulant (serum) using aseptic technique and should be tested while still fresh to minimize the
chance of obtaining false positive or false negative reactions due to improper storage or contamination of the specimen. Serum should
be separated from red cells when stored or shipped. 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 –80°C or below remain in good condition for several years (2-3 years). However, in
order to avoid the deleterious effect of repeated freeze/thaw cycles, it is recommended that samples should be distributed in small
volumes and then stored frozen. Avoid frost-free freezers.

Particulates or aggregates in the sample can cause false positive results or poor duplicate values. Clarify samples containing
particulate matter by centrifugation prior to testing.

PROCEDURE

Materials Provided

Vials may contain more reagent than described on the labels. Be sure to measure the reagent with an appropriate device when making
dilutions.

1. 6 – 2 x 8 Microwell Strips
2. 1 x 50 mL Concentrated Wash (10X)
3. 1 x 14 mL Specimen Diluent
4. 1 x 14 mL Substrate Buffer
5. 1 x 14 mL Stopping Solution
6. 1 x 80 µL Anti-Human IgG/A/M Conjugate
7. 3 x 50 mg PNPP Substrate
8. 1 x 0.3 mL Positive Serum Control
9. 1 x 0.7 mL Negative Serum Control
10. 6 Plate Sealers

Additional Materials Required

1. Polypropylene test tubes for patient sample and control dilutions and for reagent dilutions
2. Transfer pipets
3. Adjustable micropipets to deliver 10 – 100 µL and 100 – 1,000 µL and disposable tips
4. Timer

PakPlus assay 3 303469.IFUEN REV H

5. Microplate reader capable of measuring OD at 405 or 410 and 490 nm
6. Deionized or distilled water
7. Absorbent paper towels
8. Microplate washer or device
9. Centrifuge capable of separating serum from patient samples
10. Waterbath or dry incubator

o o
temperature range 37 C + 1 C
11. Lot-specific Recording Sheet, available on website (www.immucor.com)
12. Premoistened laboratory wipes

Test Procedure

1. Bring all reagents to room temperature.

2. Make Working Wash solution by diluting Concentrated Wash. Add 1 volume of Concentrated Wash to 9 volumes of deionized or
distilled water. Mix well.

3. Determine the number of patient samples to be tested. Using the Recording Sheet, assign each sample to a location consisting of
two (duplicate) columns. Record the identity of each sample on the Recording Sheet.

Prepare Samples and Controls

4. Dilute samples and controls in Specimen Diluent as shown in the table below. Mix well:

Volume Specimen Diluent (SD) Volume sample

PC 150 µL 50 µL
NC 600 µL 200 µL
Patient Sample 600 µL 200 µL

5. Remove microwell frame from pouch. Promptly remove and reseal unneeded strips in the protective pouch.

NOTE: Only one frame is provided in the kit. Do not discard until all strips have been used.

NOTE: Orient the frame with A1 in the top left corner. Be sure that all strips are properly seated and snapped into their frame. Label or
number each strip to avoid errors. Maintain the same plate orientation throughout the assay.

6. Add 300 µL of Working Wash solution to all wells and allow them to stand at room temperature for 5-10 minutes.

7. Aspirate or decant forcefully and invert on absorbent toweling to prevent drying.

8. Add 50 µL of the appropriate diluted control or sample to the wells as designated on the Recording Sheet. Remove any bubbles
from the microwells, taking care not to carry over between samples.

NOTE: Do not add samples or reagents to blank wells.

NOTE: If multiple patient samples are tested at the same time, only one set of controls is required. LABEL EACH STRIP TO AVOID
ERRORS.

9. Seal the microwells with a plate sealer and incubate:

 30 minutes in a 37°C waterbath, or

o
40 minutes in a 37 C dry incubator

10. Dilute the Conjugate 1 to 100 in Specimen Diluent as shown in the table below. Use a polypropylene container.

#Strips Volume Conjugate (AH) Volume Specimen Diluent (SD)

2–2x8 20 µL 2.0 mL
6–2x8 60 µL 6.0 mL

NOTE: Conjugate is viscous. Prime tip 2-3 times in Conjugate before dispensing and rinse after addition to Specimen Diluent. Mix
well.

PakPlus assay 4 303469.IFUEN REV H

11. WASH STEP

a) Aspirate or decant contents of each well and blot on absorbent toweling.

b) Add 300 µL Working Wash solution.
c) Aspirate or decant.
d) Repeat steps b + c for a total of 4 washes.
e) Vigorously decant to remove all residual wash solution. Invert on absorbent toweling to prevent drying.

12. Add 50 µL of diluted Conjugate (made in a previous step) to all wells EXCEPT those designated as BLANKS. Remove any bubbles
from the microwells.

13. Seal the microwells with a plate sealer and incubate:

 30 minutes in a 37°C waterbath, or

o
40 minutes in a 37 C dry incubator.

14. Dissolve PNPP Substrate by adding 0.5 mL deionized or distilled water to the vial. Replace the stopper and mix well. Protect from
light until use.

15. Dilute the PNPP 1 to 100 in the Substrate Buffer as shown in the table below.

# Strips Volume PNPP Substrate (PN) Volume Substrate Buffer (SB)

2–2x8 40 µL 4.0 mL
6–2x8 120 µL 12.0 mL

Mix well. Protect from light until use.

NOTE: Avoid contaminating the working substrate with conjugate from the previous steps.

16. WASH STEP

a) Aspirate or decant contents of each well and blot on absorbent toweling.

b) Add 300 µL Working Wash solution.
c) Aspirate or decant.
d) Repeat steps b + c for a total of 4 washes.
e) Vigorously decant to remove all residual wash solution. Invert on absorbent toweling to prevent drying.

17. Remove any bubbles from the microwells. Clean the well bottoms using a damp lab wipe.

Proceed promptly through next three steps.

18. Add 100 µL of the diluted PNPP solution to all the wells EXCEPT those designated as BLANKS.

19. Allow the microwells to stand in the dark for 30 minutes at ROOM TEMPERATURE (22 to 25°C).

NOTE: Incubation time and temperature after the addition of PNPP is critical. DO NOT vary the established incubation time or
temperature. For consistency, begin timing promptly after addition of the reagent to the first well.

20. Stop the reaction by adding 100 µL of Stopping Solution to each well in the same sequence as the addition of substrate. Add
200 µL of Stopping Solution to the blank wells.

21. Read the absorbance (OD) of each well at 405 or 410 nm using a reference filter of 490 nm. If the results cannot be read
immediately, return the wells to a dark location for up to 30 minutes.

22. Subtract the values obtained in the blank wells from all sample and control wells. Many ELISA readers are programmed to
automatically perform this step.

23. Record the results on the lot-specific Recording Sheet.

NOTE: Donor phenotypes for GPIIb/IIIa (rows A and B) may change between lots, as noted on the lot-specific Recording Sheet.

PakPlus assay 5 303469.IFUEN REV H

 QUALITY CONTROL

Quality control of PakPlus is built into the test system by the assessment of the Positive and Negative Serum Controls. These
assessments should be included with each test run to help determine if technical errors or reagent failures have occurred.

Criteria for a valid test:

Negative Control (IIb/IIIa rows) Mean OD  0.175

Negative Control (all rows) Within + 35% of mean OD for the row
Positive Control (HLA) Mean OD > 1.000

OD readings obtained for duplicate Positive test results (for both samples and the Positive Control) should fall within 20% of the
mean of the two values. Samples whose results are outside of this limit should be re-tested. A Positive Control whose results are
outside this limit should be considered invalid and the assay repeated.

OD readings for duplicate results in the Negative range for test samples need not fall within 20% of the mean of the two values.
However, the OD readings should not fall on either side of the cutoff determined for that antigen. Such results are considered invalid
and should be re-tested. Duplicate OD readings for the Negative Control must fall within 35% of the mean OD of the two values.
Readings with greater than 35% variation from the mean OD for the two values should be considered invalid and the assay repeated.

NOTE: Poor duplicates can be the result of reagent or sample omission, uneven addition of reagents, uneven temperature during
incubations, stray light during the final incubation, failure to clean microwell bottoms before reading, or cross-well
contamination. Failure to test in duplicate may lead to acceptance of erroneous results.

NOTE: The Positive Serum Control contains antibodies to HLA Class I antigens. This Control is a check on reagent and technical
and operator performance. The Control is not intended as a check of any particular antigen/antibody reaction.

INTERPRETATION OF RESULTS

Determine the Cutoff for each antigen (row) as follows:

1. Calculate the Mean OD of the Negative Control duplicate results for each antigen (row).

2. Multiply the mean NC OD by the Cutoff Multiplier (listed on the Recording Sheet), and record this value in the appropriate
cell in the Cutoff column.

For each sample, determine the results for each antigen (row) by performing the following steps:

1. Calculate the mean OD for each pair of duplicate results.

2. Compare the mean OD to the NC cutoff for that row. There are 2 possible results for reactivity with each antigen (row):
Negative or Positive, which are determined as follows:

a. If the mean OD is < the cutoff the Result is Negative.

b. If the mean OD > the cutoff the Result is Positive.

3. Determine the final result for each glycoprotein as follows:

a. Uninterpretable result for a sample: a sample that is Positive for rows A – D plus E, or F, or both is considered
Uninterpretable for GPIIb/IIIa, GPIa/IIa, GPIb/IX, and GPIV. Such pan-reactive results do not fit a pattern of allo-
antibody specificity, and may indicate the presence of autoantibodies, non-specific binding or other unknown cause.

Note that this does not apply to HLA Class I.

b. For GPIb/IX, GPIV, and HLA Class I (Rows E, F, G), the final result is the same as the result for that antigen (row).

c. For GPIIb/IIIa:

 If A and B are both Negative, the result for GPIIb/IIIa is Negative.

 If A or B is Positive and the alternate antigen (row) is Negative, the result for GPIIb/IIIa is Positive.

PakPlus assay 6 303469.IFUEN REV H

  If both A and B are Positive, the result for GPIIb/IIIa is Positive.

d. For GPIa/IIa, refer to table below.

Interpretation for GPIa/IIa (Rows C and D)

Row C Result Row D Result Interpretation
Negative Negative Negative for antibodies to GP Ia/IIa

Negative Positive
Positive for antibodies to GP Ia/IIa
Positive Negative

Positive or Uninterpretable for antibodies to GPIa/IIa.

Positive Positive
See ** below.

** For results where both C and D are Positive divide the higher mean OD by the lower mean OD. A ratio > 3.0
should be interpreted as Positive for an alloantibody to GPIa/IIa. Samples with ratios < 3.0 should be assigned as
Uninterpretable for GPIa/IIa.

LIMITATIONS

 Erroneous results can occur from bacterial contamination of test materials, inadequate incubation periods, inadequate washing or
decanting of test wells, exposure of substrate to stray light, omission of test reagents, exposure to higher or lower than prescribed
temperature requirements, or omission of steps.
 This assay is intended for use as a screening assay. The results of this assay should not be used as the sole basis for a clinical
19-21
decision.
19-21
 Some low titer, low avidity antibodies may not be detected using this assay.
 Antibodies to platelet-specific antigens that are not represented on the Recording Sheet may not be detected.
 The presence of other HPA polymorphic variants located on GPIIb-IIIa (HPA-6, 7, 8, 9, 10, 11, 14, 16, 17, 19, 20, 21), GPIb/IX
(HPA-12) and GPIa-IIa (HPA-13, 18) has not been determined for the antigens captured in the PakPlus wells. Antibodies to these
systems may be reactive in this assay.
 Antibodies to low incidence HLA class I antigens may not be detected using this product.
 PakPlus has not been evaluated for the detection of autoantibodies to platelet antigens.

SPECIFIC PERFORMANCE CHARACTERISTICS

Precision
Between-assay precision was assessed according to CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance;
Approved Guideline. A set of 5 serum samples was tested in duplicate in 20 separate assays. Each of the 5 samples contained an
antibody to one of the 5 glycoproteins detected by PakPlus: GPIIb/IIIa, GPIa/IIa, GPIb/IX, GPIV, and HLA Class I. There was 100%
agreement of the qualitative results across the 20 assays for all samples tested. Total variation of OD values ranged from %CV of 4.4
(1.139 mean OD) to 14.9 (0.064 mean OD), depending on the antigen, and the OD range of the sample.

Reproducibility

Reproducibility was conducted as a multi-site study at three locations: Immucor GTI Diagnostics, Inc., the Blood Center of Wisconsin
(Milwaukee, Wisconsin), and Puget Sound Blood Center (Seattle, Washington). At each site, two operators tested a panel of five (5)
samples with the PakPlus assay to assess between-run variability. Each sample has reactivity for a single PakPlus antigen and is
negative for the others. Each sample was tested in 20 separate assays by six operators (two individuals per site). The table below
shows a summary of the results for the 5 samples tested in 20 different assays by 6 users.

Pak Plus Between-User Reproducibility

Sample # Expected Antibodies Testing events % Positive Testing events % Negative

Positive (of 120) Agreement Negative (of 480) Agreement
1 GPIIb/IIIa (HPA-1a) 120 100% 480 100%
2 HLA 120 100% 480 100%
3 GPIV 120 100% 480 100%
4 GPIb/IX 119 99.2% 480 100%
5 GPIa-IIa (HPA-5b) 120 100% 479* 99.8%

*Sample 5 had a single false Positive result for HLA Class I

PakPlus assay 7 303469.IFUEN REV H

 Total variation (Total CV) of OD values, across all operators, ranged from 7.4% (mean OD = 1.379) to 26.6% (mean OD = 0.048)
depending on the sample, antigen, and OD value.

Lot-to-lot Variability
Variation between lots of PakPlus was assessed in a study comparing the performance testing set of 5 serum samples, each
containing an antibody to one of the 5 glycoproteins detected by PakPlus: GPIIb/IIIa, GPIa/IIa, GPIb/IX, GPIV, and HLA Class I. Each
sample was tested with three PakPlus lots. There was 100% agreement between the qualitative results for the samples tested using
all three lots. Total variation of OD values ranged from %CV of 1.4% (mean OD = 0.827) to 20.5% (mean OD = 0.067), depending on
the sample, antigen.

Method Comparison Studies

The performance of PakPlus for detection of antibodies reactive with GPIIb/IIIa, GPIb/IX, GPIV, or GPIa/IIa was compared to
detection by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and to the Lifecodes QuikScreen assay for
the detection of antibodies to Class I HLA antigens. Three clinical studies were conducted. Two of these were conducted as external
studies at The Blood Center of Wisconsin (Milwaukee, Wisconsin) and Puget Sound Blood Center (Seattle, Washington; MAIPA
testing performed at the Australian Red Cross Kelvin Grove, Queensland, Australia). The third, an internal study, was conducted at
Immucor GTI Diagnostics using samples from Sanquin Diagnostic Services (Amsterdam, The Netherlands) and from Immucor GTI
Diagnostics. Data from all studies were combined for analysis. The 2x2 tables below show the analysis for positive % agreement
(sensitivity), negative % agreement (specificity), and overall % agreement for the samples tested. The 95% Confidence Interval
around these values are shown in parentheses.

Method Comparison: PakPlus vs. QuikScreen for detection of antibodies to HLA Class I antigens
Lifecodes QuikScreen
Positive Negative Total
Positive 202 12 214 Overall % Agreement: 92.32% (89.95 – 94.28%)
PakPlus Negative 36 375 411 Positive % Agreement: 84.87% (79.68 – 89.18%)
Total 238 387 625 Negative % Agreement: 96.90% (94.65 – 98.39%)

Three samples were excluded from the study because of uninterpretable or invalid QuikScreen results.

Samples with discordant results in the PakPlus comparison to QuikScreen were further assessed by testing with a Luminex-based
assay (Lifematch LMX) for detection of antibodies to Class I HLA antigens. Of the 12 PakPlus positive – QuikScreen negative
samples, all were negative by LMX. Of the 36 PakPlus negative – QuikScreen positive samples, 14 were also negative by LMX.

Method Comparison: Detection of antibodies to GPIIb/IIIa, GPIa/IIa, GPIb/IX, or GPIV

The analysis was performed in two ways. The table below shows the overall comparison of PakPlus to MAIPA for any HPA antibody
detected in the assay. The subsequent tables compare the result of PakPlus to MAIPA by each specific glycoprotein present in the
PakPlus assay. For both types of analysis, samples with uninterpretable MAIPA results (allospecificity could not be determined or
broad reactivity to multiple targets) were excluded from the comparison. The performance of PakPlus on such samples has not been
evaluated.
Analysis based on Antibodies to Any HPA Antigen

MAIPA
Positive Negative Total
Positive 118 39 157 Overall % Agreement: 91.15% (88.27 – 93.52%)
PakPlus
Negative 4 325 329 Positive % Agreement: 96.72% (91.82 – 99.10%)
Total 122 364 486 Negative % Agreement: 89.29% (85.65 – 92.27%)

A total of 17 samples were excluded from analysis due to uninterpretable MAIPA results. Nine samples were excluded from the
analysis due to an Uninterpretable result (pan-reactive) in the PakPlus assay. These samples were excluded from any further analysis
of PakPlus performance compared to MAIPA.

Testing for GPIV was only performed at one clinical study site as described in the Analysis of Antibodies to GPIV section below.

Analysis based on Antibodies to GPIIb/IIIa

MAIPA
Positive Negative Total
Positive 75 28 103 Overall % Agreement: 94.33% (91.91 – 96.20%)
PakPlus
Negative 0 391 391 Positive % Agreement: 100.00% (95.20 – 100.00%)
Total 75 419 494 Negative % Agreement: 93.32% (90.49 – 95.51%)

PakPlus assay 8 303469.IFUEN REV H

 Analysis based on Antibodies to GPIa/IIa

MAIPA
Positive Negative Total
Positive 41 12 53 Overall % Agreement: 96.58% (94.51 – 98.03%)
PakPlus
Negative 4 411 415 Positive % Agreement: 91.11% (78.78 – 97.52%)
Total 45 423 468 Negative % Agreement: 97.16% (95.10 – 98.53%)

Twenty four samples were determined to be Uninterpretable by PakPlus due to Positive results for both GPIa/IIa preparations (rows C
and D) and a ratio of mean ODs < 3.0 (see Interpretation section). These were excluded from the comparison. All were negative by
MAIPA.

Analysis based on Antibodies to GPIb/IX

MAIPA
Positive Negative Total
Positive 4 20 24 Overall % Agreement: 95.52% (93.29 – 97.17%)
PakPlus
Negative 2 465 467 Positive % Agreement: 66.67% (22.28 – 95.67%)
Total 6 485 491 Negative % Agreement: 95.88% (93.70 – 97.46%)

Analysis of Antibodies to GPIV

The performance of PakPlus for detection of antibodies reactive with GPIV was compared to detection by MAIPA in a single
study conducted at The Blood Center of Wisconsin (Milwaukee, Wisconsin).

MAIPA
Positive Negative Total
Positive 3 1 4 Overall % Agreement: 98.68% (95.30 – 99.84%)
PakPlus
Negative 1 146 147 Positive % Agreement: 75.00% (19.41 – 99.37%)
Total 4 147 151 Negative % Agreement: 99.32% (96.27 – 99.98%)

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 PakPlus assay at the concentrations indicated:

Hemoglobin ≤ 500 mg/dl

Triglycerides ≤ 500 mg/dl
Bilirubin ≤ 20 mg/dl

IVIG Purified human IgG, used as a surrogate for IVIG, was tested for interference in the PakPlus assay. At serum
concentrations > 200 mg/dL significant interference in the assay occurred. Uninterpretable (pan-reactive) or false
positive results were observed. Testing with serum concentrations < 200 mg/dL was not performed. Trough levels
22
in patients receiving IVIG therapy are targeted to be higher than the lowest concentration tested.
Results from individuals on IVIG therapy should be evaluated carefully in light of this issue.

REFERENCES

1. Rozman P. Platelet antigens. The role of human platelet alloantigens (HPA) in blood transfusion and transplantation.
Transpl.Immunol. 2002;10:165-181.
2. Metcalfe P et al. Nomenclature of human platelet antigens. Vox Sang. 2003; 85:240.
3. Santoso S. Human platelet alloantigens. Transfus.Apher.Sci. 2003;28:227-236.
4. Saw CL, Szykoluk H, Curtis BR et al. Two cases of platelet transfusion refractoriness associated with anti-CD36. Transfusion
2010;50:2638-2642.
5. Klein J, Sato A. The HLA system. N Eng J Med. 2000; 343:702.
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7. 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.
8. Brand A. Immunological aspects of blood transfusions. Transpl.Immunol. 2002;10:183-190.

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9. McFarland JG. Detection and identification of platelet antibodies in clinical disorders. Transfus.Apher.Sci. 2003;28:297-305.
10. 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.
11. Rebulla P. A mini-review on platelet refractoriness. Haematologica 2005;90:247-253.
12. Kanhai HH, Porcelijn L, Engelfriet CP et al. Management of alloimmune thrombocytopenia. Vox Sang. 2007;93:370-385.
13. Stroncek DF, Rebulla P. Platelet transfusions. Lancet 2007;370:427-438.
14. Arnold DM, Smith JW, Kelton JG. Diagnosis and management of neonatal alloimmune thrombocytopenia. Transfus.Med.Rev.
2008;22:255-267.
15. Bussel JB, Primiani A. Fetal and neonatal alloimmune thrombocytopenia: progress and ongoing debates. Blood Rev. 2008;22:33-
52.
16. Curtis BR, McFarland JG. Detection and identification of platelet antibodies and antigens in the clinical laboratory.
Immunohematology. 2009;25:125-135.
17. Vassallo RR. Recognition and management of antibodies to human platelet antigens in platelet transfusion-refractory patients.
Immunohematology. 2009;25:119-124.
18. Kaplan C, Ni H, Freedman J. Alloimmune Thrombocytopenia. In: Michelson A eds. Platelets 3rd Edition. Academic Press –
Elsevier. 2012; 46:953-970.
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.
22. Goddard EA. Intravenous Immunoglobulin. Current Allergy & Clinical Immunology, March 2008 Vol 21, No. 1. 2008:26-31.

EC REP
Immucor GTI Diagnostics, Inc. Immucor Medizinische Diagnostik GmbH
20925 Crossroads Circle Robert-Bosch-Strasse 32
Waukesha, WI 53186 USA 63303 Dreieich
Germany

US and International Contact Information:

Technical Support : waukeshatechsupport@immucor.com

www.immucor.com

© 1995-2017 Immucor GTI Diagnostics, Inc.

303469.IFUEN Rev H
2017-07-25

Warning Warning
Danger Danger
H302 Harmful if swallowed
H318 Causes serious eye damage
H412 Harmful to aquatic life with long lasting effects
EUH032 Contact with acids liberates very toxic gas
P264 Wash hands thoroughly after handling
P270 Do not eat, drink or smoke when using this product
P273 Avoid release to the environment
P280 Wear protective gloves/protective clothing /eye protection/face protection
P301 + P312 IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell
P305 + P351 + P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to
do. Continue rinsing
P310 Immediately call a POISON CENTER or doctor/physician
P330 Rinse mouth

PakPlus assay 10 303469.IFUEN REV H

TRANSLATION OF SYMBOLS

SYMBOL ENGLISH CHINESE DEUTSCH FRANҪAIS GREEK ITALIANO PORTUGUÊS ESPANOL JAPANESE
(EN) (ZH) (DE) (FR) (EL) (IT) (PT) (ES) (JA)
Tableaux de Tabela de
RS Recording Sheet 记录单 Protokollbogen Φύλλο Καταγραφής Foglio di lavoro Tabla de Resultados 記録用紙
résultats resultados
No
Identification 识别编 Identifikations- Αριθμός Numero di Número de
ID # identification Nº de identificación 検体ID
Number 号 nummer Ταυτότητας identificazione identificação
du patient
读取日 Fecha
DATE Date Read Datum Date Ημερομηνία Data Data da leitura 検査日
期
TECH Technologist 技师 Untersucher Technologue Τεχνολόγος Tecnico Técnico Analista 検査者
阴性对 Negative Contrôle Αρνητικός Ορός Controllo Control 陰性コント
NC Negative Control Controlonegativo
照 Kontrolle négatif (A) ελέγχου negativo negativo ロール
阳性对 Positive Contrôle Θετικός Ορός Controllo Control 陽性コント
PC Positive Control Controlopositivo
照 Kontrolle positif ελέγχου positivo positivo ロール
Pocillo del
BLANK Blank Well 空白孔 Blank Puitsvierge ΤΥΦΛΟ Bianco Poço do Branco Blanco ブランク

Diluire con 使用前に希

使用前 Vor Gebrauch Diluer avant Αραιώστε Diluir antes de
DIL Dilute Before Use acquadeionizzata Dilución 釈してくだ
稀释 verdünnen utilisation πριντηνΧρήση prima dell’uso utilizar
さい
Moyenne des
平均阴 Μέση τιμή OD Media del
Mean Negative OD-Mittelwertder DO du DO Média do promedio de
MNCOD 性对照O Αρνητικού Controllo Negativo
Control OD NC contrôle Controlo Negativo la OD del CN
D μάρτυαρα OD
négatif
Moyenne des
平均阳 Media del
Mean Positive OD-Mittelwertder DO du Μέση τιμή OD DO Média do promedio de
MPCOD 性对照O Controllo Positivo
Control OD PC contrôle Θετικού μάρτυαρα Controlo Positivo la OD del CP
D OD
positif
Multiplicateur
乘数截 Πολλαπλασιαστής Moltiplicatore Multiplicador de
Multiplier Cutoff Multiplier Cutoff Multiplier du seuil de
止数值 cutoff Cutoff Cutoff
positivité
对照截 Seuil de
Control Cutoff CUTOFF der Μαρτυρας
CUTOFF 止数值 positivité du Controllo Cutoff Controlo Cutoff Cutoff (corte)
Kontrolle cutoff
contrôle
光密度 Densité Densidade Óptica Densidad
OD 1 Optical Density 1 Optische Dichte 1 Οπτική πυκνότητα 1 Densità ottica 1
1 optique 1 1 Optica 1
Densité Densidade Óptica Densidad
OD 2 Optical Density 2 光密度2 Optische Dicht 2 Οπτική πυκνότητα 2 Densità ottica 2
optique 2 2 óptica 2
Row A A行
ROW A Reihe A Rangée A Γραμμή Α Riga A Linha A Fila A

Row B B行
ROW B Reihe B Rangée B Γραμμή Β Riga B Linha B Fila B
Moyenne des
平均样 Mittelwert OD Media Campione DO Média da
MEAN Mean Sample OD DO de Μέση OD δείγματος Media
品 OD der Probe OD Amostra
l’échantillon
INTRP
Interpretation 说明 Interpretation Interprétation Ερμηνεία Interpretazione Interpretação Interpretacion

对照符 Les contrôles I controlli

Controlos
Controls meet rencontrent Ο έλεγχος πληρεί τα corrispondono ai Resultados de los
合有效 Kontrollen valide satisfazem os
VALID criteria for valid les critères de κριτήρια για έγκυρη requisiti richiesti controles para un
测定标 für den Test critérios de
assay validité de μέθοδο per la validazione ensayo valido
准 validação do teste
l’essai del test
Result 结果
RSLT Ergebnis Résultat Αποτέλεσμα Risultato Resultado Resultados

Variation des
Duplicate 可接受 Αποδεκτή Variazione
Doppelbes- analyses en Variação Duplicada Variación duplicada
DUP OK Variation 的重复 διακύμανση εις accettabile dei
timmung sind ok double Aceitável aceptable
Acceptable 变化 διπλούν duplicati
acceptable
TEMPLATE 平皿模 Modèle de la Template/
Plate Template Plattenbelegung Μήτρα πλάκας Modello di piastra Template
板 plaque Plantilla
CALCS Control 对照计 Berechnungen Calculs de Υπολογισμός Calculos
Calcoli di controllo Cálculos de control
calculations 算 der Kontrolle contrôle ελέγχου control
SAMPLE 样品结 Ergebnis der Résultats des Αποτελέσματα Risultati del Resultdos da
Sample Results Resultados
RESULTS 果 Probe échantillons δειγμάτων campione Amostra
SAMPLE Sample 样品 Probe Échantillon Δείγμα Campione Amostra Muestra
CONTROLS Controls 对照 Kontrollen Contrôles Μάρτυρες Controlli Controlos Controles
Gegengelesen
RVW Reviewed by 审核 Revu par Ανασκόπηση από Controllato da Revisto por Revisado por
durch

PakPlus assay 11 303469.IFUEN REV H