(385 days)
K 042425
Not Found
No
The device description and performance studies focus on a standard enzyme immunoassay (EIA) method, which is a biochemical assay and does not inherently involve AI/ML. There are no mentions of AI, ML, or related concepts in the document.
No
The device is an in vitro diagnostic (IVD) test kit used for screening newborns for classical congenital adrenal hyperplasia (CAH) by measuring 17-OHP levels. It is not used for treatment or therapy.
Yes
The "Intended Use / Indications for Use" states that the results are used to "screen newborns for classical congenital adrenal hyperplasia (CAH)," which is a diagnostic purpose.
No
The device is a laboratory assay kit (EIA Kit) which is a physical product containing reagents and requiring laboratory equipment for its use, not a software-only device.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states it's for the "quantitative measurement of 17α-Hydroxyprogesterone (17-OHP) concentrations in neonatal blood samples... The results are used to screen newborns for classical congenital adrenal hyperplasia (CAH)." This clearly indicates the device is used to test samples taken from the human body (blood) to provide information about a medical condition (CAH).
- Device Description: The description details a "competitive solid phase enzyme immunoassay (EIA)" which is a common laboratory technique used to measure substances in biological samples.
- Sample Type: The device is designed to test "neonatal blood samples that have been collected onto Whatman 903® specimen collection paper," which are biological specimens.
- Clinical Application: The results are used for "screening newborns for classical congenital adrenal hyperplasia (CAH)," which is a diagnostic purpose.
All of these points align with the definition of an In Vitro Diagnostic device, which is a medical device intended for use in vitro for the examination of specimens derived from the human body solely or principally for the purpose of providing information concerning a physiological or pathological state, or congenital abnormality.
N/A
Intended Use / Indications for Use
The Accuwell" 17α-Hydroxyprogesterone EIA Kit is designed for the quantitative measurement of 17α-Hydroxyprogesterone (17-OHP) concentrations in neonatal blood samples that have been collected onto Whatman [previously known as Schleicher and Schuell (S&S®) 903"] specimen collection paper. The results are used to screen newborns for classical congenital adrenal hyperplasia (CAH).
Product codes (comma separated list FDA assigned to the subject device)
JLX
Device Description
The Accuwell" 17a-Hydroxyprogesterone EIA Kit is a competitive solid phase enzyme immunoassay (ElA) for the quantitative measurement of 17«-Hydroxyprogesterone (17-OHP) concentrations in neonatal blood samples that have been collected onto Whatman S&S 903" specimen collection paper. Standard control, and unknown dried blood spot sample discs are added to specified wells in a 90-well microplate coated with antiserum specific for 17-OHP.
Two procedures are presented.
The first procedure includes an elution step where blood is eluted from the dried blood spot sample. A 1/8 inch disc from standards, controls, and unknown dried blood spot samples are added to specified wells in an uncoated microplate. Saline is added to all wells and the plate is allowed to incubate until the blood has eluted from the paper. Immediately following a transfer of a portion of this eluate into the antibodycoated microplate a horseradish peroxidase enzyme conjugated to 17-OHP (17-OHP: HRP) is added to the wells. A competition begins between the endogenous 17-OHP from the unknown samples, standards and controls and the 17-OHP: HRP for the limited number of antibody binding sites in the wells. An inverse relationship develops between the concentration of endogenous 17-0HP and the amount of 17-OHP: HRP, which will bind to the coated microplate. After a period of incubation the excess 17-OHP: HRP is removed by washing. A color developer, containing a colorless 3, 3', 5, 5'tetramethylbenzidine (TMB) and peroxide (H2O2), is added to all wells. The peroxidase react and subsequently the TMB is converted from color. The color development is terminated through the addition of a stopping reagent and the absorbance is measured at 650 nm using a microplate absorbance reader. A standard curve is constructed using the known concentration of each standard plotted against the corresponding absorbance reading of that standard. The concentrations of unknown controls and samples are determined by comparison to this standard curve.
An alternate procedure is described where a 1/8 inch disc from each blood spot is placed directly into designated wells of the coated microplate. The enzyme conjugated 17-OHP is added and the competition proceeds concurrent with the elution step. Upon completion period, the excess 17-OHP:HRP and the discs are removed by decanting, or aspirating the discs from each well. The plate is then washed and processed using the same procedure as described above.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Neonatal / Newborns
Intended User / Care Setting
Clinical Laboratory Professionals
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
A retrospective study was conducted to compare the results obtained with the Accuwell 17-OHP EIA to those obtained by a currently marketed neonatal 17-OHP screening device. Test samples were submitted to the study as blinded neonatal dried blood spots collected in sequence under routine screening conditions from a U.S. department of public health laboratory. Original screening results for each sample using the predicate test kit were also obtained from the submitting laboratory for method comparison.
Sample sizes for different populations:
2500g b.w., 0-1 days old (n=133)
2500g b.w., 2-3 days old(n=133)
2500g b.w., >4 days old (n=197)
1400-2500g b.w., 1-6 days (n=30)
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Precision:
Precision studies were conducted in accordance with NCCLS EP5-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods"; Approved Guideline-Second Edition.
Testing included one run per day, with two sample aliquots per run, performed on 20 different days using each of three different assay procedures. The three procedures were designated as: 1) "ON Eluate", 2) "3 Hr Eluate" and 3) "ON Direct DBS", respectively. The resulting data were used to estimate repeatability, between-day and within-device precision as described by the standard, for each procedure.
All precision testing was performed by Neo-Genesis' Portland, OR manufacturing facility. Sources of variability included the use of different: operators, days and reagent lot numbers.
| Sample ID: | CDC 451* (25+ ng/ml) | CDC 452 (50+ ng/ml) | CDC 453 (100+ ng/ml) | |
|---|---|---|---|---|
| ON Eluate | Count | 40 | 40 | 40 |
| Mean | 28.2 | 56.1 | 108.1 | |
| SD | 3.0 | 4.9 | 12.3 | |
| %CV | 10.7 | 8.8 | 11.4 | |
| Within-Run Standard Deviation: | Sr | 1.0 | 2.1 | 2.9 |
| Between-Day Standard Deviation: | Sdd | 2.9 | 4.5 | 12.1 |
| Within Device Standard Deviation: | ST | 3.1 | 5.0 | 12.5 |
| 3 HR Eluate | Count | 40 | 40 | 40 |
| Mean | 26.2 | 53.0 | 104.6 | |
| SD | 3.4 | 4.1 | 11.9 | |
| %CV | 12.9 | 7.7 | 11.3 | |
| Within-Run Standard Deviation: | Sr | 1.9 | 2.2 | 5.4 |
| Between-Day Standard Deviation: | Sdd | 2.9 | 3.4 | 10.7 |
| Within Device Standard Deviation: | ST | 3.4 | 4.1 | 12.0 |
| ON Direct DBS | Count | 40 | 40 | 40 |
| Mean | 29.5 | 57.9 | 108.6 | |
| SD | 4.2 | 5.8 | 13.2 | |
| %CV | 14.1 | 10.0 | 12.2 | |
| Within-Run Standard Deviation: | Sr | 1.5 | 4.4 | 11.7 |
| Between-Day Standard Deviation: | Sdd | 3.9 | 3.9 | 6.2 |
| Within Device Standard Deviation: | ST | 4.2 | 5.8 | 13.3 |
Analytical Sensitivity:
The analytical sensitivity was determined for each procedure including Overnight Eluate, 3 hour Eluate, and the Overnight Direct. Sensitivity was defined by the calculated concentration that corresponds to the mean of the absorbance of the zero standard (N=26) minus two times the standard deviation of those same absorbance measurements.
The zero standard was tested multiple time (N=26) in one assay. Testing was performed in each of the two procedures, eluate and direct, and spanning the range of allowable elution times, three hours and overnight for the eluate procedure.
| Overnight Eluate | 3 Hour Eluate | Overnight Direct | |
|---|---|---|---|
| Absorbance Mean | 2.506 | 1.648 | 2.604 |
| Standard Deviation | 0.04 | 0.02 | 0.06 |
| Absorbance -2 SD | 2.426 | 1.608 | 2.484 |
| Calculated Concentration (ng/ml) | 2.2 | 1.5 | 2.4 |
Linearity and Recovery:
Dried blood spots representing a range of sample concentrations were prepared for the study. The sample analyte levels in ng/ml were: 10, 24.5, 39, 68, 97, 126, 155, 184, 213, 242, 271 and 300 (n = 12 concentrations).
Four aliquots of each sample of the range of samples prepared, were tested in each of two runs using each of three different assay procedures. The three procedures were designated as:1) "ON Eluate", 2) "3 Hr Eluate" and 3) "ON Direct DBS", respectively. The mean of the total number of values obtained for each sample (n=8) within each procedure, was compared to the samples expected value.
Linear Regression Analysis:
ON Eluate: y = 1.0036x - 0.3475, R^2 = 0.9994
3 Hr Eluate: y = 1.1236x - 9.3625, R^2 = 0.992
ON Direct DBS: y = 1.1362x - 9.5154, R^2 = 0.99
Average Recovery-Overall:
ON Eluate: 99.7%
3 Hr Eluate: 102.5%
ON Direct DBS: 103.1%
Specificity - Cross Reactivity:
Cross reactivity study performed for various substances. Progesterone showed 0.5% cross reactivity, 21-Desoxycortisol 2.4%, 16alpha-Hydroxyprogestrone 1.2%. Other substances showed 2500g b.w., 0-1 days old (n=133):
Accuwell ON Direct: mean =16.5 ng/ml, range 3.7 to 85.6 ng/ml. Predicate: mean =22.6 ng/ml, range 10.1 to 86.0 ng/ml. Correlation: y (Predicate) = 0.970 (Accuwell ON Direct) + 6.569, R = 0.9570
Accuwell ON Eluate: mean =16.7 ng/ml, range 5.3 to 65.6 ng/ml. Predicate: mean =22.6 ng/ml, range 10.1 to 86.0 ng/ml. Correlation: y (Predicate) = 1.049 (Accuwell ON Direct) + 5.066, R = 0.9257
Accuwell 3Hr Eluate: mean =17.3 ng/ml, range 5.1 to 71.1 ng/ml. Predicate: mean =22.6 ng/ml, range 10.1 to 86.0 ng/ml. Correlation: y (Predicate) = 0.998 (Accuwell ON Direct) + 5.410, R = 0.9291
2500g b.w., 2-3 days old (n=133):
Accuwell ON Direct: mean =11.3 ng/ml, range 3.4 to 81.1 ng/ml. Predicate: mean =16.2 ng/ml, range 6.9 to 81.0 ng/ml. Correlation: y (Predicate) = 1.045 (Accuwell ON Direct) + 4.392, R = 0.9785
Accuwell ON Eluate: mean =12.2 ng/ml, range 3.4 to 67.5 ng/ml. Predicate: mean =16.2 ng/ml, range 6.9 to 81.0 ng/ml. Correlation: y (Predicate) = 1.156 (Accuwell ON Eluate) + 2.030, R = 0.9729
Accuwell 3Hr Eluate: mean =12.8 ng/ml, range 4.5 to 69.6 ng/ml. Predicate: mean =16.2 ng/ml, range 6.9 to 81.0 ng/ml. Correlation: y (Predicate) = 1.150 (Accuwell ON Direct) + 1.495, R = 0.9691
2500g b.w., >4 days old (n=197):
Accuwell ON Direct: mean =7.3 ng/ml, range 2.2 to 22.6 ng/ml. Predicate: mean =9.5 ng/ml, range 2.7 to 31.0 ng/ml. Correlation: y (Predicate) = 1.090 (Accuwell ON Direct) + 1.565, R = 0.9538
Accuwell ON Eluate: mean =7.8 ng/ml, range 1.4 to 26.5 ng/ml. Predicate: mean =9.5 ng/ml, range 2.7 to 31.0 ng/ml. Correlation: y (Predicate) = 1.070 (Accuwell ON Eluate) + 1.172, R = 0.9142
Accuwell 3Hr Eluate: mean =8.3 ng/ml, range 2.2 to 28.4 ng/ml. Predicate: mean =9.5 ng/ml, range 2.7 to 31.0 ng/ml. Correlation: y (Predicate) = 1.024 (Accuwell 3Hr Eluate) + 1.081, R = 0.9350
1400-2500g b.w., 1-6 days (n=30):
Accuwell ON Direct: mean =19.3 ng/ml, range 6.5 to 47.8 ng/ml. Predicate: mean =25.5 ng/ml, range 9.0 to 65.0 ng/ml. Correlation: y (Predicate) = 1.22 (Accuwell ON Direct) + 1.971, R = 0.9556
Accuwell ON Eluate: mean =19.2 ng/ml, range 6.7 to 40.2 ng/ml. Predicate: mean =25.5 ng/ml, range 9.0 to 65.0 ng/ml. Correlation: y (Predicate) = 1.234 (Accuwell ON Direct) + 1.812, R = 0.8770
Accuwell 3Hr Eluate: mean =19.3 ng/ml, range 6.6 to 39.2 ng/ml. Predicate: mean =25.5 ng/ml, range 9.0 to 65.0 ng/ml. Correlation: y (Predicate) = 1.174 (Accuwell 3Hr Eluate) + 2.795, R = 0.8421
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Analytical Sensitivity:
Overnight Eluate: 2.2 ng/ml
3 Hour Eluate: 1.5 ng/ml
Overnight Direct: 2.4 ng/ml
Precision:
Within Run Precision (%CV): 3.5% to 14.1%
Between Run Precision (%CV): 3.3% to 10.3%
Total Precision (%CV): 8.9% to 12.0%
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
K 042425
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 862.1395 17-Hydroxyprogesterone test system.
(a)
Identification. A 17-hydroxyprogesterone test system is a device intended to measure 17-hydroxyprogesterone (a steroid) in plasma and serum. Measurements of 17-hydroxyprogesterone are used in the diagnosis and treatment of various disorders of the adrenal glands or the ovaries.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 862.9.
0
Accuwell 17-alpha-Hydroxyprogesterone EIA 510(k) Summary
| 1) Submitter Name: | Genesis Northwest (dba) Neo-Genesis |
|---|---|
| Address: | 15140 S.E. 82 Drive, Suite 270 Clackamas, OR 97015 USA |
| Telephone Number: | 503-657-8000 |
| Contact Person: | Jannet Baldwin, President |
| Date Prepared: | 02/22/2007 |
MAR 13 2007
| 2) Device Trade or Proprietary Name: | Accuwell 17-a-hydroxyprogesterone (OHP) enzyme immunoassay (EIA) |
|---|---|
| Device Common or Usual Name: | 17-a-hydroxyprogesterone (OHP) neonatal screening enzyme immunoassay (EIA) |
| Device Classification Name: | Radioimmunoassay, 17-hydroxyprogesterone (JLX) |
| 3) Legally Marketed Predicate Device: | AutoDELFIA Neonatal 17a-OH-progesterone, Model B048-112 |
4 ) Device Description:
Summary and Explanation of the Assay
Congenital Adrenal Hyperplasia (CAH) is a family of genetic disorders caused by reduced enzyme activities, which are required in the biosynthesis pathway leading to the production of cortisol in the adrenal cortex. The most common of these disorders is the 21-hydroxylase deficiency and accounts for more than 90% of CAH cases. 1, 2, 3
The incidence of classical 21-hydroxylase deficiencies widely by population and efhnic group. In Japan the incidence is thought to be about 1 in 21,000, while in Europe and North America incidence ranges between 1 in 10,000 to 1 in 16,000. The Yupik Eskimos in Alaska have an extremely high incidence of around 1 in 300 4,5
Clinical symptoms of the 21-hydroxylase deficiency occur primarily due to the over production of precursors to the blocked enzymatic step. These precursors are shunted into the androgen biosynthesis pathway, which produces virilization in the female fetus, rapid postnatal growth with accelerated skeletal maturation, precocious puberty and short stature in both males and females. About 75% of the patients with classical CAH also have a defect in their ability to synthesize aldosterone, which can lead to a salt wasting crisis.
In classical 21-hydroxylase deficiencies the 17 α-hydroxyprogesterone (17-OHP) levels are markedly increased. However, the 17-OHP levels in normal infants are elevated in the first two to three days after birth. These high concentrations in normal infants decline rapidly to adult levels within the first seven to ten days.' Low birth-weight, premature and sick babies may continue to have higher than normal 17-OHP levels for an extended time frame. For this reason it has been recommended that a multi-tiered approach
1
be employed to setting the normal cut-off. Laboratories must be notified of either dexamethasone therapy or transfusion status. Due to the population differences it is also strongly recommended that each laboratory establish their own normal range and cutoff levels. Periodic review and possible adjustment if necessary to reduce the false positive rate is also recommended. Non-classical forms of CAH are not
reliably detected by newborn screening." 9.10.11
The Working Group on Neonatal Screening of the European Society of Pediatric Endocrinology (ESPE) has recommended that after a positive screening test for CAH, it is imperative that the diagnosis be validated with confirmatory testing. Confirmation methods vary depending on availability but include urine steroid analysis, ACTH stimulation and CY21 gene analysis.12, 13, 1
Principle of the Assay
The Accuwell" 17a-Hydroxyprogesterone EIA Kit is a competitive solid phase enzyme immunoassay (ElA) for the quantitative measurement of 17«-Hydroxyprogesterone (17-OHP) concentrations in neonatal blood samples that have been collected onto Whatman S&S 903" specimen collection paper. Standard control, and unknown dried blood spot sample discs are added to specified wells in a 90-well microplate coated with antiserum specific for 17-OHP.
Two procedures are presented.
The first procedure includes an elution step where blood is eluted from the dried blood spot sample. A 1/8 inch disc from standards, controls, and unknown dried blood spot samples are added to specified wells in an uncoated microplate. Saline is added to all wells and the plate is allowed to incubate until the blood has eluted from the paper. Immediately following a transfer of a portion of this eluate into the antibodycoated microplate a horseradish peroxidase enzyme conjugated to 17-OHP (17-OHP: HRP) is added to the wells. A competition begins between the endogenous 17-OHP from the unknown samples, standards and controls and the 17-OHP: HRP for the limited number of antibody binding sites in the wells. An inverse relationship develops between the concentration of endogenous 17-0HP and the amount of 17-OHP: HRP, which will bind to the coated microplate. After a period of incubation the excess 17-OHP: HRP is removed by washing. A color developer, containing a colorless 3, 3', 5, 5'tetramethylbenzidine (TMB) and peroxide (H2O2), is added to all wells. The peroxidase react and subsequently the TMB is converted from color. The color development is terminated through the addition of a stopping reagent and the absorbance is measured at 650 nm using a microplate absorbance reader. A standard curve is constructed using the known concentration of each standard plotted against the corresponding absorbance reading of that standard. The concentrations of unknown controls and samples are determined by comparison to this standard curve.
An alternate procedure is described where a 1/8 inch disc from each blood spot is placed directly into designated wells of the coated microplate. The enzyme conjugated 17-OHP is added and the competition proceeds concurrent with the elution step. Upon completion period, the excess 17-OHP:HRP and the discs are removed by decanting, or aspirating the discs from each well. The plate is then washed and processed using the same procedure as described above.
Kit Contents
Materials Supplied
| Quantity per Kit | |||
|---|---|---|---|
| Wells | 96 | 384 | 1920 |
| Catalog No. | 601501 | 601502 | 601503 |
| Coated Microplates | |||
| (Rabbit Anti-17-OHP) | 1 Strip plate | 4 plates | 20 plates (5 bags |
| of 4 plates each) | |||
| Enzyme Conjugate Concentrate | |||
| (17-OHP:HRP)* | 2 mls | 5 mls | 25 mls |
| Conjugate Diluent* | 20 mls | 50 mls | 250 mls |
2
| Wash Buffer Concentrate (20X) | 25 mls | 50 mls | 250 mls |
|---|---|---|---|
| Color Developer * | 20 mls | 55 mls | 250 mls |
| Stopping Reagent | 20 mls | 55 mls | 250 mls |
| Multi-Analyte Standards and | |||
| Controls | |||
| TSH/T4/17-OHP | 1 set | 1 set | 4 sets |
*These reagents are light sensitive. Avoid prolonged exposure to light.
NOTE: Do not use reagents or solutions that have become cloudy or discolored as these conditions indicate deterioration. The reagents should be stored in their original containers.
Do not exchange reagents from one kit with those of another kit unless the lot number and expiration of the reagent are the same.
Reagent Description
Coated Microplates (Rabbit Anti-17-OHP)
Microplates are coated with an antiserum produced in rabbits immunized with a 17-OHP hapten. The microplates are packaged in zipper-lock foil bags containing a desiccant. Store the unused microplates and/or strips from the strip plate in the zipper-lock bags with desiccant. Each microplate is labeled with a unique bar-code label.
Storage: Drv at 2-25° C
Expiration: Refer to the expiration date printed on the label
Enzyme Conjugate Concentrate (17-OHP: HRP)*
A 17-OHP derivative conjugated to horseradish peroxidase, danazol with an enzyme stabilizer.
*This reagent is light sensitive, plate store in the original brown confainer.
Enzyme Conjugate Concentrate must be diluted with Conjugate Diluent before use.
See Reagent Preparation Instructions section on page 11.
Storage: 2-8° C Protected From Light.
Expiration: Refer to the expiration date printed on the label.
Coniugate Diluent*
Tris-buffer, bovine serum and preservatives. * This reagent is light sensitive, store in the original brown container. Conjugate Diluent is used to dilute the conjugate concentrate before use. See Reagent Preparation Instructions section on page 11.
Storage: 2-8° C Protected from Light.
Expiration: Refer to the expiration date printed on the label.
Wash Buffer Concentrate (20X)
A concentrated solution of phosphate buffered saline containing a surfactant.
Wash Buffer Concentrate must be diluted with deionized or distilled water before use.
See Reagent Preparation Instructions section on page 11.
2-25° C Storage:
Expiration: Concentrate: Refer to the expiration date printed on the label.
Diluted wash buffer is stable for 1 month when stored at 2-25° C.
Color Developer*
Bottle contains a colorless solution of 3, 3', 5'-Tetramethylbenzidine in a diluted organic solvent with citrate buffer and hydrogen peroxide. *This reagent is light sensitive, store in the original brown container. This reagent should remain colorless; if it has discolored, discard it.
Storage: 2-8° C. Protected from light.
Expiration: Refer to the expiration date printed on the label.
3
Stopping Reagent
Bottle contains a dilute solution of sodium fluoride (NaF) and a red dye. Storage: 2-8° C. Expiration: Refer to the expiration date printed on the label.
Multi-Analyte Standards and Controls
TSH, T4, 17-OHP
Multi-Analyte standards have been prepared from human whole blood, adjusted to a hematocrit of 55%. Each card of standards contains two circles each of six concentrations of 17-OHP at approximately zero. 10. 25. 50. 100 and 250 ng/ml serum equivalents. Control cards contain four circles each of three different concentrations of 17-OHP at approximately 15, 40, and 90 ng/ml serum equivalent. The standards and controls are spotted onto Whatman (previously known as Schleicher and Schuell (S&S®) 903") specimen collection paper. Refer to the information on the labels for the exact concentrations of the standards and acceptable ranges for the controls. Each set of standards and controls consists of two standard cards and one control card.
Drv at 2-8° C or below. Storage: Expiration: Refer to the expiration date printed on the label or Certificate of Analysis received with the kit.
Standard and control units are expressed in ng/ml. The values may be converted using the following formula.
1 nmol/l blood= 0.33 ng/ml blood
1 nmol/l blood= 0.73 ng/ml serum (at 55% hematocrit)
| Conversion Table | ||
|---|---|---|
| Concentration in | Concentration in | |
| Serum ng/ml | Blood nmol/l | |
| Standard Zero | 0 | 0 |
| Standard A | 10 | 13.7 |
| Standard B | 25 | 34.2 |
| Standard C | 50 | 68.5 |
| Standard D | 100 | 137 |
| Standard E | 250 | 342 |
| Control 1 | 15 | 20.6 |
| Control 2 | 40 | 54.8 |
| Control 3 | 90 | 123 |
This is an example only. Consult standard and control label or Certificate of Analysis received with the kit for exact concentrations.
Materials Required But Not Supplied for Both the Elution and Direct Procedures
- 1/8 inch (3 mm) diameter hole punch 1.
- Forceps or fine tweezers to pick up the punched sample discs 2.
-
- Pipettes to accurately dispense 100 ul volumes
-
- Multi-channel pipettes to dispense 300 ul volumes or an automated plate washer
-
- Microplate reader capable of reading at a wavelength of 650 nm
- Plate rotator capable of 100-120 RPM or plate shaker. ి.
-
- Microplate covers; plate sealers or low evaporation solid plastic microplate lids (e.g. NUNC lids with condensation rings and evaporation barriers)
-
- Graduated cylinders
- Deionized or distilled water တဲ
4
Additional Materials Required for the Elution Procedure Only
- Un-coated round bottom 96 well microplates
-
- Saline (0.85% NaCl).
-
- Multi-channel pipet with disposable tips to accurately transfer 15 µl of eluate.
Additional Materials Required for the Direct Procedure Only
- Aspiration device capable of removing discs from coated plates.
-
- Intended Use:
The Accuwell" 17α-Hydroxyprogesterone EIA Kit is designed for the quantitative measurement of 17cc-Hydroxyprogesterone (17-OHP) concentrations in neonatal blood samples that have been collected onto Whatman [previously known as Schleicher and Schuell (S&S®) 903"] specimen collection paper. The results are used to screen newborns for classical congenital adrenal hyperplasia (CAH).
- Intended Use:
| Characteristic: | Predicate Device: | Proposed Device: |
|---|---|---|
| K 042425; Model B048-112 | ||
| Wallac Oy AutoDelfia Neonatal | ||
| 17α-Hydroxyprogesterone | ||
| Time-resolved | ||
| flouoroimmunoassay | Neo-Genesis Accuwell | |
| 17α-Hydroxyprogesterone EIA | ||
| Intended User | Clinical Laboratory Professionals | Clinical Laboratory Professionals |
| Intended Use | Measure 17α- | |
| Hydroxyprogesterone (17- | ||
| OHP) levels in neonatal dried | ||
| blood spots | Measure 17α- | |
| Hydroxyprogesterone (17- | ||
| OHP) levels in neonatal dried | ||
| blood spots | ||
| Indications for Use | Screening for increased levels of | |
| 17-OHP in newborns | Screening for increased levels of | |
| 17-OHP in newborns | ||
| Chemical Principle | Competitive reaction between | |
| europium-labeled 17-OHP | ||
| and sample 17-OHP for a | ||
| limited number of binding | ||
| sites on 17-OHP specific | ||
| polyclonal antibodies derived | ||
| from rabbit | Competitive reaction between | |
| horseradish peroxidase | ||
| (HRP) labeled 17-OHP and | ||
| sample 17-OHP for a limited | ||
| number of binding sites on | ||
| 17-OHP specific polyclonal | ||
| antibody derived from rabbit | ||
| Assay Type | Time-resolved | |
| fluoroimmunoassay | Enzyme-immunoassay | |
| Detection Method | Europium ions dissociated from | |
| the labeled antiserum form | ||
| highly fluorescent chelates | ||
| with components of an | ||
| enhancement solution. | ||
| Fluorescence in each well is | ||
| then measured | Peroxidase which remains bound | |
| to micro-wells reacts with | ||
| peroxide and TMB | ||
| subsequently converting the | ||
| TMB from colorless to a blue | ||
| color which is measured | ||
| Assay Processing Method | Automated | Manual |
| Detection Equipment | Autodelfia - time resolved | |
| fluorometer | Microplate Spectrophotometer | |
| Sample Requirements | Newborn blood collected on | |
| S&S 903 filter paper or | ||
| equivalent collection cards | Newborn blood collected on | |
| S&S 903 filter paper |
-
- Summary of Technological Characteristics 1,2:
5
. .
| Specimen Rejection Criteria | Sample spot not uniformly
saturated with blood; sample
spots punched to close to the
edge of the blood spot;
poorly collected and
improperly dried specimens;
non-eluting blood spot;
contamination of blood spot
filter paper with foreign
material | | Sample spot not uniformly
saturated with blood; sample
spots punched to close to the
edge of the blood spot;
poorly collected and
improperly dried specimens | |
|--------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|
| Specimen | 1/8 inch spot (3.2mm) punched
from a standard collection
card | | 1/8 inch spot (3.2mm) punched
from a standard collection
card of S&S 903 paper | |
| Standard Configuration | Human blood with a hematocrit
of 50-55% and spotted on
S&S 903 filter paper.
Calibrated using gravimetric
methods. Seven levels | | Human whole blood adjusted to
55% hematocrit and spotted
on S&S 903 filter paper.
Calibrated using gravimetric
methods. Six levels | |
| Standard Range | 0 - 190 ng/ml approximate
serum equivalent | | 0 - 250 ng/ml approximate
serum equivalent | |
| Control Configuration | Human blood with a hematocrit
of 50-55% and spotted on
S&S 903 filter paper.
Calibrated using gravimetric
methods. Two levels | | Human whole blood adjusted to
55% hematocrit and spotted
on S&S 903 filter paper.
Calibrated using gravimetric
methods. Three levels | |
| Control Levels | 14 and 40 ng/ml approximate
serum equivalent | | 15, 40 and 90 ng/ml approximate
serum equivalent | |
| Calculation of Results-
Recommendations | Spline Smoothed regression
analysis is used to calibrate
each plate with the LOG of
the concentration on the X-
axis and B/Bmax on the Y-
axis (response). | | Five parameter logistic plot of
absorbance versus
concentration | |
| Analytical Sensitivity | 1.5 ng/ml | | 2.2 ng/ml | |
| Within Run Precision 3,4 | Mean (ng/ml)
% cv
Mean (ng/ml)
% cv
Mean (ng/ml)
% CV | 8.8
11.3
29.3
8.9
62.1
8.9 | Mean (ng/ml)
% CV
Mean (ng/ml)
% CV
Mean (ng/ml)
% CV | 28.2
3.5
56.1
3.7
108.1
2.7 |
| Between Run Precision 3,4 | Mean (ng/ml)
% CV
Mean (ng/ml)
% CV
Mean (ng/ml)
% CV | 8.8
4.0
29.3
3.3
62.1
2.4 | Mean (ng/ml)
% CV
Mean (ng/ml)
% CV
Mean (ng/ml)
% CV | 28.2
10.3
56.2
8.0
108.1
11.2 |
| Total Precision 3,4 | Mean (ng/ml)
% CV
Mean (ng/ml)
% CV
Mean (ng/ml)
% CV | 8.8
12.0
29.3
9.5
62.1
9.2 | Mean (ng/ml)
% CV
Mean (ng/ml)
% CV
Mean (ng/ml)
% CV | 28.1
11.0
56.1
8.9
108.1
11.6 |
11 - 11
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6
...
| Interfering Substances
Cross Reacting Substances
| Greater than 0.01 %: | EDTA or citrate blood | Lipemia |
|---|---|---|
| Progesterone | 3.60 % | 0.5 % |
| 21-Desoxycortisol | 1.75 % | 2.42 % |
| 16a-Hydroxyprogesterone | 0.3 % | 1.2 % |
| 11-Desoxycortisol | 0.4 % | 0.6 % |
| 11-Deoxy-17- | ||
| hydroxycorticosterone | 0.33 % | 2000g birthweight |
| (unknown percentile): | 1-3 days old, ≥ 2500g birthweight | |
| (99th percentile): | ||
| normal 44 ng/ml | ON Eluate: | |
| normal 4 days old, ≥ 2500g birthweight | ||
| (99th percentile): | ||
| ON Direct: | ||
| normal 73 ng/ml | ON Eluate: | |
| normal 2500g b.w., 0-1 days old (n=133)- | ||
| AutoDelfia Mean (ng/ml) =22.6 | ||
| Accuwell Means (ng/ml): | ||
| ON Direct = 16.5 | ||
| ON Eluate = 16.7 | ||
| 3Hr Eluate = 17.3 |
2500g b.w., 2-3 days old(n=133)-
AutoDelfia Mean (ng/ml) =16.2
Accuwell Means (ng/ml):
ON Direct = 11.3
ON Eluate = 12.2
3Hr Eluate = 12.8
2500g b.w., >4 days old (n=197)-
AutoDelfia Mean (ng/ml) =9.5
Accuwell Means (ng/ml):
ON Direct = 7.3
ON Eluate = 7.8
3Hr Eluate = 8.3
1400-2500g b.w., 1-6 days (n=30)-
AutoDelfia Mean (ng/ml) =25.5
Accuwell Means (ng/ml):
ON Direct = 19.3
ON Eluate = 19.2
3Hr Eluate = 19.3 |
.. .
8
| CDC Control Results | ||||
|---|---|---|---|---|
| CDC Lot 451 - Enriched with | ||||
| 25 ng/ml serum | Mean (ng/ml) | |||
| % cv | 30.0 | |||
| 11.3 | Mean (ng/ml) | |||
| % cv | 28.1 | |||
| 11.0 | ||||
| CDC Lot 452 - Enriched with | ||||
| 50 ng/ml serum | Mean (ng/ml) | |||
| % cv | 59.3 | |||
| 10.6 | Mean (ng/ml) | |||
| % cv | 56.1 | |||
| 8.9 | ||||
| CDC Lot 453 - Enriched with | ||||
| 100 ng/ml serum | Mean (ng/ml) | |||
| % cv | 120.2 | |||
| 10.1 | Mean (ng/ml) | |||
| % cv | 108.1 | |||
| 11.6 | ||||
| Intercept = | ||||
| Slope = | - 0.45 | |||
| 1.2049 | + 2.1 | |||
| 1.0629 |
1 Data displayed for the predicate device derived from the directions for use unless otherwise noted.
2 Data displayed for the Accuwell 17-OHP kit was derived using only the overnight eluate method unless otherwise noted.
- Results from the predicate device were obtained from their published directions. The method used or definitions were not 3 declared, therefore they may or may not use the same calculation method.
- 4 Accuwell 17-OHP precision studies were conducted in accordance with NCCLS EP5-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods"; Approved Guideline-Second Edition. All precision testing was r of orned by Neo-Genesis at Neo-Genesis' Portland, OR manufacturing facility. Sources of variability included the use of different; operators, days and reagent lot numbers.
- 5 Results from an in house study of randomly selected samples from newborns with birth weight >2200 grams. These results are presented as an example only and used strictly as guidance and should not be used to establish reference ranges.
- 6 Results from an in house study of randomly selected samples from newborns with birth weight ≤2200 grams. These results roodito for an in housed strictly as guidance and should not be used to establish reference ranges.
- Predicate kit results for 17-OHP were derived from CDC publication "Newborn Screening Quality Assurance Program 2005 7 Annual Summary Report, Vol. 23, Jan 2006. % CV was calculated using the reported mean and the average within laboratory standard deviation. Intercept and slope are as reported.
-
- Clinical and Non-Clinical Data:
Precision
Precision studies were conducted in accordance with NCCLS EP5-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods"; Approved Guideline-Second Edition.
Testing included one run per day, with two sample aliquots per run, performed on 20 different days using each of three different assay procedures. The three procedures were designated as: 1) "ON Eluate", 2) "3 Hr Eluate" and 3) "ON Direct DBS", respectively. The resulting data were used to estimate repeatability, between-day and within-device precision as described by the standard, for each procedure.
All precision testing was performed by Neo-Genesis' Portland, OR manufacturing facility. Sources of variability included the use of different: operators, days and reagent lot numbers.
9
| Sample ID: | ||||
|---|---|---|---|---|
| CDC 451* | ||||
| (25+ ng/ml) | CDC 452 | |||
| (50+ ng/ml) | CDC 453 | |||
| (100+ ng/ml) | ||||
| Count | 40 | 40 | 40 | |
| Mean | 28.2 | 56.1 | 108.1 | |
| ON Eluate | SD | 3.0 | 4.9 | 12.3 |
| %CV | 10.7 | 8.8 | 11.4 | |
| Within-Run Standard Deviation: | Sr | 1.0 | 2.1 | 2.9 |
| Between-Day Standard Deviation: | Sdd | 2.9 | 4.5 | 12.1 |
| Within Device Standard Deviation: | ST | 3.1 | 5.0 | 12.5 |
| Count | 40 | 40 | 40 | |
| Mean | 26.2 | 53.0 | 104.6 | |
| 3 HR Eluate | SD | 3.4 | 4.1 | 11.9 |
| %CV | 12.9 | 7.7 | 11.3 | |
| Within-Run Standard Deviation: | Sr | 1.9 | 2.2 | 5.4 |
| Between-Day Standard Deviation: | Sdd | 2.9 | 3.4 | 10.7 |
| Within Device Standard Deviation: | ST | 3.4 | 4.1 | 12.0 |
| ON Direct DBS | Count | 40 | 40 | 40 |
| Mean | 29.5 | 57.9 | 108.6 | |
| SD | 4.2 | 5.8 | 13.2 | |
| %CV | 14.1 | 10.0 | 12.2 | |
| Within-Run Standard Deviation: | Sr | 1.5 | 4.4 | 11.7 |
| Between-Day Standard Deviation: | Sdd | 3.9 | 3.9 | 6.2 |
| Within Device Standard Deviation: | ST | 4.2 | 5.8 | 13.3 |
| 3 ummary Results and Estimates of Precision for Accuwell 17-OHP EIA Kit |
|---|
| ------------------------------------------------------------------------- |
*Enriched as reported by Centers for Disease Control and Prevention. Newborn Screening Quality Assurance Program Annual Report 2005
Analytical Sensitivity
The analytical sensitivity was determined for each procedure including Overnight Eluate, 3 hour Eluate, and the Overnight Direct. Sensitivity was defined by the calculated concentration that corresponds to the mean of the absorbance of the zero standard (N=26) minus two times the standard deviation of those same absorbance measurements.
The zero standard was tested multiple time (N=26) in one assay. Testing was performed in each of the two procedures, eluate and direct, and spanning the range of allowable elution times, three hours and overnight for the eluate procedure.
Extrapolation beyond the upper and lower limits of the standard curve or beyond the limits of detection is not an acceptable laboratory practice. Therefore any sample result falling below these calculated concentrations should be reported as less than the limit of detection.
This data is provided for example only and should be confirmed by each laboratory and appropriate concentrations defined.
| Overnight Eluate | 3 Hour Eluate | Overnight Direct | |
|---|---|---|---|
| Absorbance Mean | 2.506 | 1.648 | 2.604 |
| Standard Deviation | 0.04 | 0.02 | 0.06 |
| Absorbance -2 SD | 2.426 | 1.608 | 2.484 |
| Calculated Concentration (ng/ml) | 2.2 | 1.5 | 2.4 |
10
Linearity and Recovery
The Accuwell 17-OHP Kit linear range and the measuring (reportable) range is approximately 2.0 ng/ml to 250 ng/ml based on study data described below, the sensitivity study described elsewhere and limited by the value assigned to the highest standard of the calibration curve.
Assay results obtained outside the measuring (reportable) range should be reported as either "less than" or "greater than" the established low or high limit, respectively, as applicable to the individual result.
Dried blood spots representing a range of sample concentrations were prepared for the study. The sample analyte levels in ng/ml were: 10, 24.5, 39, 68, 97, 126, 155, 184, 213, 242, 271 and 300 (n = 12 concentrations).
Four aliquots of each sample of the range of samples prepared, were tested in each of two runs using each of three different assay procedures. The three procedures were designated as:1) "ON Eluate", 2) "3 Hr Eluate" and 3) "ON Direct DBS", respectively. The mean of the total number of values obtained for each sample (n=8) within each procedure, was compared to the samples expected value. Results of the comparisons are described below.
| ON Eluate | 3 Hr Eluate | ON Direct DBS | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Linear Regression Analysis | $y = 1.0036x - 0.3475$ | ||||||||
| $R^2 = 0.9994$ | $y = 1.1236x - 9.3625$ | ||||||||
| $R^2 = 0.992$ | $y = 1.1362x - 9.5154$ | ||||||||
| $R^2 = 0.99$ | |||||||||
| Sample # | Expected | ||||||||
| (ng/ml) | Actual | ||||||||
| (ng/ml) | Recovery | ||||||||
| (%) | Actual | ||||||||
| (ng/ml) | Recovery | ||||||||
| (%) | Actual | ||||||||
| (ng/ml) | Recovery | ||||||||
| (%) | |||||||||
| 1 | 300 | 298.1 | 99.4 | 354.4 | 118.1 | 319.9 | 106.6 | ||
| 2 | 271 | 274.4 | 101.3 | 284.4 | 104.9 | 324.1 | 119.6 | ||
| 3 | 242 | 241.9 | 100.0 | 257.9 | 106.6 | 261.6 | 108.1 | ||
| 4 | 213 | 212.3 | 99.7 | 221.7 | 104.1 | 244.0 | 114.6 | ||
| 5 | 184 | 182.9 | 99.4 | 194.1 | 105.5 | 185.9 | 101.0 | ||
| 6 | 155 | 157.2 | 101.4 | 159.8 | 103.1 | 157.5 | 101.6 | ||
| 7 | 126 | 131.7 | 104.5 | 127.7 | 101.3 | 128.5 | 102.0 | ||
| 8 | 97 | 95.2 | 98.1 | 94.2 | 97.1 | 92.6 | 95.5 | ||
| 9 | 68 | 66.2 | 97.4 | 66.4 | 97.6 | 69.9 | 102.8 | ||
| 10 | 39 | 38.2 | 97.9 | 37.0 | 94.9 | 33.3 | 85.4 | ||
| 11 | 24.5 | 23.0 | 93.9 | 23.0 | 93.9 | 22.8 | 93.1 | ||
| 12 | 10 | 10.4 | 104.0 | 10.3 | 103.0 | 10.7 | 107.0 | ||
| Average Recovery-Overall | 99.7 | 102.5 | 103.1 | ||||||
| Minimum | 93.9 | 93.9 | 85.4 | ||||||
| Maximum | 104.5 | 118.1 | 119.6 |
Results of Assay Linearity and % Recovery Study for Accuwell 17-OHP EIA Kit
11
Specificity
Cross Reactivity
The cross reactivity percentage is determined by dividing the concentration of analyte (17-OHP) at 50% displacement by the concentration of the interferant at 50% displacement from the absorbance corresponding to the zero standard.
| Trivial Name | Cross
Reactivity | Trivial Name | Cross
Reactivity |
|------------------------------------------|---------------------|-------------------------------------------|---------------------|
| 21-Desoxycortisol | 2.4 % | Cholesterol | 2500 gm and 0-1 Days of Age at Sample Collection
| 99th Percentile | 97.5th Percentile | 95th Percentile | ||||||
|---|---|---|---|---|---|---|---|---|
| Accuwell Normal | Follow-up | Accuwell Normal | Follow-up | Accuwell Normal | Follow-up | |||
| Predicate | 2500 gm and 2-3 Days of Age at Sample Collection |
| 95th Percentile | ||
|---|---|---|
| Accuwell Normal | Follow-up | |
| Predicate | ||
| 2500 gm and 2-3 Days of Age at Sample Collection |
| 95th Percentile | ||||
|---|---|---|---|---|
| Accuwell | Follow-up | |||
| Predicate | Normal | |||
| 2500 gm and > 4 Days of Age at Sample Collection |
| 97.5th Percentile | 95th Percentile | ||||
|---|---|---|---|---|---|
| Accuwell Normal | Follow-up | Accuwell Normal | Follow-up | ||
| Predicate |