Free PSA iFlash

Immunoassay Analyzer

 A direct relationship exists between the amount of Free

REF C86001 2×50 Tests 0123 PSA in the sample and the RLUs detected by the
iFlash optical system.
INTENDED USE  Results are determined via a calibration curve, which
is instrument-specifically generated by 3-point
The iFlash-Free PSA assay is a paramagnetic particle calibration and a master curve provided via the
chemiluminescent immunoassay (CLIA) for quantitative reagent QR code.
determination of free prostate specific antigen (FPSA) in
human serum and plasma using the iFlash Immunoassay REAGENTS
Analyzer. Reagent kit, 100 tests, 2 packs, 50 tests/pack
Anti-PSA coated microparticles, 3.5 mL/pack,
SUMMARY AND EXPLANATION R1
0.05% ProClin 300.
Prostate specific antigen (PSA) is a glycoprotein acting as
Anti-PSA acridinium-labeled conjugate; 4.0
a serine protease. The proteolytic activity of PSA in blood R2
mL/pack; 0.05% ProClin 300.
is inhibited by the irreversible formation of complexes with
proteinase inhibitors such as alpha-1-antichymotrypsin, Calibrator 1, 1 bottle, 1.0 mL, Tris buffer with
alpha-2-macroglobulin and other acute phase proteins. In CAL1 protein stabilizers, goat serum,0.05% ProClin
addition to being present in these complexes, PSA is also 300.
present in blood in the free form, but is proteolytically Calibrator 2, 1 bottle, 1.0 mL, Free PSA in
inactive. Elevated concentrations of PSA in serum are Tris buffer with protein stabilizers, goat
CAL2
generally indicative of a pathologic condition of the serum, 0.05% ProClin 300.
prostate (prostatitis, benign hyperplasia or carcinoma).
Calibrator 3, 1 bottle, 1.0 mL, Free PSA in
Early diagnosis of carcinoma of the prostate is hindered
CAL3 Tris buffer with protein stabilizers, goat
by the lack of symptoms in men with localized tumors.
Therefore, early detection requires a simple, safe, and serum,0.05% ProClin 300.
inexpensive test for the disease in asymptomatic men.
MATERIALS REQUIRED (BUT NOT PROVIDED)
PSA testing can have significant value in detecting
metastatic or persistent disease in patients following REF C89999/C89959/C89949, iFlash Pre-Trigger
surgical or medical treatment of prostate cancer. Solution: hydrogen peroxide solution.
Persistent elevation of PSA following treatment, or an REF C89998/ C89958/ C89948, iFlash Trigger Solution:
increase in a post-treatment PSA level is indicative of sodium hydroxide solution.
recurrent or residual disease. PSA testing is widely REF C89997, iFlash Wash Buffer: phosphate buffered
accepted as an adjunctive test in the management of saline solution with 0.05% ProClin 300.
prostate cancer patients. REF C80001, iFlash Wash Buffer (10×): phosphate
PSA tests lack sufficient sensitivity and specificity to be buffered saline solution with 0.05% ProClin 300.
considered ideal or absolutely diagnostic for screening or REF C89996, reaction vessels.
early detection because PSA is not specific for prostate
Controls: Commercial controls could be used.
cancer. A number of studies have found that the % Free
PSA (FPSA) was significantly lower in patients having WARNINGS AND PRECAUTIONS
prostate cancer than those with benign disease or normal IVD For in vitro diagnostic use
controls. The ratio FPSA/TPSA has been demonstrated to
 No known test method can offer the complete
improve the sensitivity and specificity in patients with
assurance that products derived from human sources
TPSA values in the “gray zone” of 4-10 ng/mL.
will not transmit infection. Therefore, all human
ASSAY PRINCIPLE sourced materials should be considered potentially
The iFlash-Free PSA assay is a sandwich immunoassay. infectious.

 Incubation: Free PSA in the sample, anti-PSA coated  Exercise the normal precautions required for handling
paramagnetic microparticles and anti-PSA all laboratory reagents.
acridinium-labeled conjugate react to form a sandwich  Disposal of all waste material should be in accordance
complex. with local guidelines.
 Wash: The unbound materials are washed away from  Wear gloves when handling specimens or reagents.
the solid phase in a magnetic field.  Clean and disinfect all spills of specimens or reagents
 Trigger of signal: The Pre-Trigger and Trigger using a suitable disinfectant.
Solutions are added to the reaction mixture. The  iFlash Trigger solution contains sodium hydroxide
resulting chemiluminescent reaction is measured as (NaOH) and should be avoided contact with eyes.
relative light units (RLUs).

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Free PSA iFlash
Immunoassay Analyzer

REAGENT HANDLING  Do not use heat-inactivated samples.

 The reagents may not be used after the stated  Ensure the patient samples, calibrators and controls
expiration date. are at ambient temperature (20–25°C) before
 Avoid the formation of foam with all reagents. measurement.
 The reagents in the pack and calibrators are ready for  Due to the possible evaporation, specimens and
use calibrators on the analyzers should be measured within
2 hours.
 Close the bottles of calibrator right after calibration and
store at 2–8°C. ASSAY PROCEDURE
 Do not pool reagents within a reagent kit or between  Refer to the system operating instruction or the online
reagent kits. help system for detailed information on preparing the
 Prior to loading the iFlash Free PSA reagent pack on the system.
system for the first time, resuspend the microparticles by  The test-specific parameters stored in barcode on the
inverting the reagent pack 30 times slightly reagent pack are read in. In cases the barcode cannot
 For further information on reagent handling precautions be read, enter the sequence numbers.
during system operation, refer to the iFlash system  Carry out calibration, if necessary.
operating instruction.
 Place the calibrators CAL1, CAL2, and CAL3 in the
STORAGE AND STABILITY calibrator rack in the sample zone. Only keep
calibrators open during calibration.
Storage:
 Test application.
 Store at 2~8°C in an upright position.
 Load samples.
 The kit may be used immediately after removal from
2~8°C storage.  Press RUN, the iFlash System performs all the
functions automatically and calculates the results.
Stability:
 Unopened at 2~8°C: up to the stated expiration date, 12 CALIBRATION
months.  Traceability: This assay has been standardized
 Opened at 2~8°C: 28 days. against the WHO Reference Standard 96/668 (100 %
 Store on-board: 28 days. Free PSA).
 Every iFlash-Free PSA reagent kit has a QR code
SPECIMEN COLLECTION AND PREPARATION
label containing the specific information for calibration
 Serum or plasma (lithium heparin, sodium heparin, of the particular reagent lot.
Dipotassium EDTA, and sodium citrate) are the  To perform an iFlash-Free PSA calibration, test CAL1.
recommended samples. Other anticoagulants have not
CAL2, and CAL3 in duplicate, and the predefined
been validated for use with the iFlash Free PSA assay.
master curve is adapted to the analyzer.
 Ensure that serum specimens to form complete clot
 Once an iFlash-Free PSA calibration is accepted and
prior to centrifugation. stored, all subsequent samples may be tested without
 Centrifuge the specimens. further calibration unless:
 Store specimens at room temperature (20 to 25°C) for  After 28 days when using the same reagent lot.
no longer than 8 hours.  A reagent kit with a new lot number is used
 If the testing will not be completed within 8 hours,  Controls are out of range
refrigerate the samples at 2 to 8°C.
 Required by pertinent regulations.
 If the testing will not be completed within 3 days, or for
shipment of samples, freeze at -20°C or colder. MEASURING RANGE
 Frozen specimens must be mixed thoroughly after  0.01 – 20 ng/mL
thawing.
QUALITY CONTROL
 The samples may be frozen for maximum 2 times.
Quality control materials should be run as single
 Centrifuge specimens with a lipid layer on the top, and
determinations at least once every 24 hours when the test
transfer only the clarified specimen without the lipemic
is in use, once per reagent kit and after every calibration.
material.
Include commercially available quality control materials
 Ensure that residual fibrin and cellular matter have that cover at least two levels of analyte. Follow
been removed prior to analysis. manufacturer’s instructions for reconstitution and storage.
 Use with caution in handling patient specimens to Each laboratory should establish mean values and
prevent cross-contamination. acceptable ranges to assure proper performance. Quality

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Free PSA iFlash
Immunoassay Analyzer

control results that do not fall within acceptable ranges high as 15000 ng/mL did not exhibit a hook effect in
may indicate invalid test results. the iFlash-Free PSA assay.
 The assay is unaffected by icterus (bilirubin < 30
RESULT
mg/dL), hemolysis (Hb < 1500 mg/dL), lipemia
Calculation:
(Intralipid < 1500 mg/dL) and total serum protein (< 10
The iFlash system automatically calculates the analyte g/dL).
concentration of each sample. The results are given in  No interference was observed from rheumatoid factors
ng/mL. up to a concentration of 2000 U/mL.
Expected Values:
 No interference was observed from anti-nuclear
A study of with iFlash Free PSA assay on samples from 290 antibodies up to a concentration of 500 U/mL.
apparently healthy males of various age groups yielded the  No interference was observed from HAMA up to a
following result: concentration of 600 ng/mL.
th
< 0.75 ng/mL (95 percentile)
PERFORMANCE CHARACTERISTICS
The probability of finding prostate cancer PCA with tPSA in
Below are the representative performance data. The
the gray zone (4–10 ng/mL) increases with increasing age
results obtained in different laboratories may differ due to
and with decreasing fPSA/tPSA ratios - see table.
difference in sex, age, race, etc.
Probability of finding PCA on needle
% fPSA ratio biopsy by age in years Precision
Two controls, consisting low, and median concentration of
50-60 61-75
Free PSA were assayed.
≤10% 51% 54%
The within run precision was determined by testing each
10.01%-20% 25% 32% sample in replicates of 10 (n = 10), and calculating percent
20.01%-25% 10% 18% coefficient of variation (%CV). The results of the study are
shown below:
＞25% 6% 13% Sample Mean (ng/mL) SD %CV
It is recommended that each laboratory establish its own 1 2.10 0.08 3.81
expected reference range for the population of interest. 2 9.93 0.51 5.14

LIMITATIONS The between run precision was determined by testing

 The iFlash Free PSA assay is limited to the each sample in duplicate, two separate runs daily for 20
determination of Free PSA in human serum or plasma days (n = 80), and calculating percent coefficient of
(lithium heparin, sodium heparin, potassium EDTA, variation (%CV). The results of the study are shown
and sodium citrate). It has not been validated for use below:
Sample Mean (ng/mL) SD %CV
with other types of plasma.
1 2.07 0.10 4.83
 The use of serum separator (gel) blood collection
tubes has been validated for use with this assay, 2 9.85 0.43 4.37
however it is not possible to survey all manufacturers
Analytical Sensitivity
or tube types.
The detection limit representing the lowest measurable
 The upper limit of the measuring range of this assay is
analyte level that can be distinguished from zero is 0.01
20 ng/mL. Over-range samples may be diluted with
ng/mL. It is calculated as the value lying two standard
negative human serum and re-tested to obtain an
deviations above that of the lowest standard of the master
estimate of the actual concentration.
curve (standard 1 + 2 SD, n = 20).
 If the results are inconsistent with clinical evidence,
Method comparison
additional testing is suggested to confirm the result.
A comparison of the iFlash Free PSA assay (y) with a
 For diagnostic purposes, the results should be commercially available Free PSA assay (x) using clinical
interpreted in light of the total clinical presentation of samples was performed, and the curve is fitted with Linear
the patient, including symptoms, clinical history results. regression)
 Specimens from heparinized patients may be partially y = 1.0021x - 0.015
coagulated and erroneous results could occur due to r = 0.995
the presence of fibrin. Sample concentration: 0.26 – 19.88 ng/mL
 The results from an alternative assays (i.e. EIA or RIA) Number of samples measured: 90
may not be equivalent and cannot be used
Risk assessment of patients
interchangeably.
Percent Free PSA may be used to determine the relative
 Samples containing an apparent Free PSA level as
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Free PSA iFlash
Immunoassay Analyzer

risk of prostate cancer in individual men. Samples from 11. Thiel RP, Oesterling JE, Wojno KJ, et al. A multicenter
healthy subjects determined by non-suspicious digital comparison of the diagnostic performance of free
rectal examination and that from detecting prostate cancer prostate-specific antigen. Urology 1996;48(6A):45-50.
patients with needle biopsy were measured using iFlash
Total and Free PSA assays. SHENZHEN YHLO BIOTECH CO., LTD.
1st-4th Floor, No.5 Building, Lishan Industrial
The elevated PSA levels increase the risk of detectable
cancer, and the percent Free PSA can further stratify risk Area,Xinghai Road, Nanshan District, Shenzhen
for men with PSA values (4-10 ng/mL) and non-suspicious 518054, China
digital rectal examination results. Lower percent Free PSA
values indicate higher risk of cancer. Wellkang Ltd (www.CE-marking.eu)
Percent Free PSA and probability of prostate cancer Suit B, 29 Harley St., London W1G 9QR, UK
% FPSA ratio Probability of cancer (%)
ANNEX A:
≤ 10 57%
Explanation of abbreviation
10 – 15 29%
15 – 20 20% Abbreviation Explanation

20 – 25 18%
Product No.
REFERENCES
1. Tewari PC, Bluestein BI. Multiple forms of prostate Calibrator
specific antigen and the influences of immunoassay
design on their measurement in patient serum. J Clin
Ligand Assay, 18 1995;3:186-196. Reagent
2. Prestigiacomo AF, Stamey TA. Clinical usefulness of
free and complexed PSA. Clin Lab Invest Suppl Number of tests
1995;221:32-34.
3. Semjonow A, De Angelis G, Oberpenning F et al. The
Manufactured by
clinical impact of different assays for prostate specific
antigen. BJU Int. 2000; 86(5):590-7
4. Kuriyama M, Wang MC, Papsidero LD, et al. EU Representative
Quantitation of Prostate-Specific Antigen in Serum by
a Sensitive Enzyme Immunoassay. Cancer Res
EC Declaration of Conformity
1980;40:4658-62.
5. Oesterling JE. Prostate Specific Antigen: A Critical
Assessment of the Most Useful Tumor Marker for Caution
Adenocarcinoma of the Prostate. J Urol
1991;145:907-23.
Instructions for use
6. Kantoff PW, Talcott JA. The Prostate Specific Antigen.
Its Use as a Tumor Marker for Prostate Cancer In vitro diagnostic medical
Hematol Oncol Clin N Amer 1994;8:555-72.
device
7. Partin AW, Oesterling JE. The Clinical Usefulness of
Prostate Specific Antigen: Update 1994. J Urol Lot No.
1994;152:1358-68.
8. Bunting S. A Guide to the Interpretation of Serum
Date of manufacture
Prostate Specific Antigen Levels. Clin Biochem
1995;28:221-41.
9. Catalona WJ, Richie JP, Ahmann FR, Hudson MA, Expiry date
Scardino PT, Flanigan RC, et al. Comparison of digital
rectal examination and serum prostate-specific antigen
Biohazard Symbol
in the early detection of prostate cancer: results of
a multicenter clinical trial of 6,630 men. J Urology
1994;151(5):1283-1290. Pictograms for Caution
10. Chen YT, Luderer AA, Thiel RP, et al. Using
proportions of free to total prostate-specific antigen, Pictograms for Hazardous to the
age, and total prostate-specific antigen to predict the aquatic environment
probability of prostate cancer. Urology
1996;47:518-524.

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