Microsoft word - dcm004-6 metodica 17oh progesterone ce

17OH PROGESTERONE
for routine analysis
Direct immunoenzymatic determination of 17OH Progesterone in human serum or plasma.

REF DKO004

INTENDED USE
2. PRINCIPLE
Competitive immunoenzymatic colorimetric method for 17OH Progesterone (antigen) in the sample competes quantitative determination of 17OH Progesterone with horseradish peroxidase 17OH Progesterone (enzyme-label ed antigen) for binding onto the limited The 17OH Progesterone kit is intended for
number of anti- 17OH Progesterone coated on the laboratory use only.
After incubation, the bound/free separation is 1. CLINICAL SIGNIFICANCE
performed by a simple solid-phase washing. 17-Hydroxyprogesterone (17OH Progesterone or 17α- The enzyme substrate (H2O2) and theTMB-substrate OHP or 17-OHP) is a C-21 steroid hormone produced (TMB) are added. After an appropriate time has in the adrenal gland and gonads, during the synthesis elapsed for maximum colour development, the of glucocorticoids and sex steroids. It is derived from enzyme reaction is stopped and the absorbance are progesterone via 17-hydroxylase, a P450c17 enzyme, or from 17-hydroxypregnenolone via 3β-hydroxysteroid 17OH Progesterone concentration in the sample is calculated based on a series by a set of standard. 17-OHP has no defined physiologic role except as a The colour intensity is inversely proportional to the 17OH Progesterone concentration in the sample. Serum 17-OHP levels are age-dependent, with peak levels observed during fetal life and the immediate 3. REAGENTS, MATERIALS AND INSTRUMENTATION
postnatal period. During the first week of life, serum 17 -OHP levels fal ~50-fold as compared to cord blood 3.1. Reagents and materials supplied in the kit
values. A smal transient increase occurs in male 1. 17OH Progesterone Standards (6x1 vial = 1 mL) infants 30-60 days postnatal y. Levels for both sexes REF DCE002/0406-0
remain at constant low levels during childhood, and REF DCE002/0407-0
then progressively increase during puberty reaching adult levels of ~100 ng/dL (~3.03 nmol/L). As with REF DCE002/0408-0
cortisol, serum 17-OHP levels normal y have an REF DCE002/0409-0
ACTH-dependent diurnal variation, with peak levels in REF DCE002/0410-0
the morning and a nadir at night. In addition, ovarian production of 17-OHP increases during the luteal REF DCE002/0411-0
17-hydroxyprogesterone is a natural progestin and in pregnancy increases in the third trimester primarily REF DCE002/0402-0
3. Coated Microplate (1 microplate breakable coated Normal levels are 3-90 ng/dl in children, and in women, 15-70 ng/dl prior to ovulation, and 35-290 REF DCE002/0403-0
Measurements of levels of 17-hydroxyprogesterone are useful in the evaluation of patients with suspected H2O2-TMB 0.26 g/L (avoid any skin contact) congenital adrenal hyperplasia as the typical enzymes REF DCE004-0
that are defective, namely 21-hydroxylase and 11β- hydroxilase, lead to a build-up of 17-OHP. In contrast, Sulphuric acid 0.15 mol/L (avoid any skin contact) have very low or undetectable levels of 17-OHP. REF DCE005-0
Elevated serum 17-OHP levels at baseline and/or after ACTH stimulation have also been reported in other 3.2. Reagents necessary not supplied in the kit
Standard
3.3. Auxiliary materials and instrumentation
Notes
Store all reagents at 2÷8°C in the dark. Open the bag of reagent 3 (COATED MICROPLATE) only when it is at room temperature and close Remove the contents from each wel ; wash the wel s Do not remove the adesive sheets on the unutilised with 300 µL of distil ed water. Repeat the washing procedure by draining the water completely. 4. PRECAUTIONS
The reagent contain Proclin 300R as preservative. Avoid the exposure of reagent TMB/H2O2 to Incubate at room temperature 22÷28°C for 15 minutes in Maximum precision is required for reconstitution This method al ows the determination of 17OH Read the absorbance (E) at 450 nm against Blank. Progesterone from 0.2 ng/mL to 19.2 ng/mL. The clinical significance of the determination of 6. QUALITY CONTROL
17OH Progesterone can be invalidated if the Each laboratory should assay controls at normal, high patient was treated with cortisone or natural or and low levels range of 17OH Progesterone for monitoring assay performance. These controls should be treated as unknowns and values determined in 5. PROCEDURE
every test procedure performed. Quality control charts should be maintained to fol ow the performance of the 5.1. Preparation
Standard
supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual 0,S1,S2,S3,S4,S5)
The standard has the fol owing concentration of 17OH laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the standard curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established Stability of Standards: until the expiration date of the performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. When are open, the standards are stable six months Fresh reagents should be used to determine the 5.2. Preparation of the Sample
7. LIMITATIONS OF PROCEDURE
The determination of 17OH Progesterone can be performed in human plasma as wel as in serum. 7.1. Assay Performance.
Store the sample at -20°C if the determination is not Sample(s), which are contaminated microbiological y, performed on the same day of the sample connection. should not be used in the assay. Highly lipemeic or haemolysed specimen(s) should similarly not be used. 5.3. Procedure
It is important that the time of reaction in each wel is As it is necessary to perform the determination in held constant for reproducible results. Pipetting of duplicate, prepare two wel s for each of the six points samples should not extend beyond ten (10) minutes to of the standard curve (S0-S5), and for each sample, avoid assay drift. If more than one (1) plate is used, it is recommended to repeat the dose response curve. Addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction. Plate readers measure vertical y. Do not touch the bottom of the wel s. Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication 10.4. Specificity
The cross reaction of the antibody calculated at 50% according to Abraham are shown in the table: 7.2. Results interpretation
If computer control ed data reduction is used to calculate the results of the test, it is imperative that the predicted values for the calibrators fal within 10% of 8. RESULTS
10.5. Correlation with RIA
8.1. Mean Absorbance
The Dia.metra 17OH Progesterone ELISA was Calculate the mean of the absorbance (Em) for each compared to another commercial y available 17OH point of the standard curve and of each sample progesterone assay. 38 serum samples were analysed 8.2. Standard Curve
Plot the mean value of absorbance of the standards (17-OHP Diametra)=0.83*(17-OHP RIA)+0.17 (Em) against concentration. Draw the best-fit curve through the plotted points. (es: Four Parameter 11. WASTE MANAGEMENT
Reagents must be disposed off in accordance with 8.3. Calculation of Results
Interpolate the values of the samples on the standard curve to obtain the corresponding values of the BIBLIOGRAPHY
1. Wisdom, G.B. Clin. Chem. 22/8 1243 - 1255 9. REFERENCE VALUES
The serum or plasma 17OH Progesterone reference 2. De Vil a, G.O. et al. J.Clin. Endoc. Metob. 3. Hubl, W., et al Endokrinologie, 1982, 79 (2), 4. Arakawa, H., et al Chem. Pharm. Bul . Tokyo 30 (8) 5. D. Riad - Fanny, et al Endocr. Reviews, 3 (4) 304 CHILDREN
10. PERFORMANCE AND CHARACTERISTICS
10.1. Precision
Ed 06/2010
DCM004-6
Within run variation was determined by replicate DiaMetra S.r.l. Headquater: Via Garibaldi, 18 – 20090 measurements (16x) of two different control sera in SEGRATE (MI) Tel. 0039-02-2139184 – 02-26921595 one assay. The within assay variability is ≤ 7.4%. Manufact: Via Giustozzi (già via Bartolomei), 35 – Z.I Between run variation was determined by replicate Paciana – 06034 FOLIGNO (PG) ITALY. Tel. 0039- measurements of three different control sera in different lots. The between assay variability is ≤ 13%. 10.2. Accuracy
The recovery of 1 – 2 – 4 ng/mL of 17OH Progesterone added to a sample gave an average value (±SD) of 103.94% ± 2.78% with reference to the 10.3. Sensitivity
The lowest detectable concentration of 17OH Progesterone that can be distinguished from the zero standard is 0.09 ng/mL at the 95 % confidence limit. DIA.METRA SRL
PACKAGING INFORMATION SHEET
Spiegazione dei simboli Explanation of symbols Explication des symboles Significado de los simbolos
DE
Verwendete Symbole Explicaçao dos simbolos Producto sanitario para diagnóstico In vitro Dispositif medical de diagnostic in vitro Dispositivos medicos de diagnostico in vitro Establa hasta (usar antes de último día del mes) Utiliser avant (dernier jour du mois indiqué) Utilizzare prima del (ultimo giorno del mese) Contém o suficiente para “n” testes DIA.METRA SRL
PACKAGING INFORMATION SHEET

SUGGERIMENTI PER LA RISOLUZIONE DEI PROBLEMI/TROUBLESHOOTING
ERRORE CAUSE POSSIBILI/ SUGGERIMENTI
Nessuna reazione colorimetrica del saggio
- mancata dispensazione del coniugato
- contaminazione del coniugato e/o del Substrato
- errori nell’esecuzione del saggio (es. Dispensazione accidentale dei reagenti in sequenza errata o provenienti da
flaconi sbagliati, etc.)
Reazione troppo blanda (OD troppo basse)
- coniugato non idoneo (es. non proveniente dal kit originale)
- tempo di incubazione troppo breve, temperatura di incubazione troppa bassa
Reazione troppo intensa (OD troppo alte)
- coniugato non idoneo (es. non proveniente dal kit originale)
- tempo di incubazione troppo lungo, temperatura di incubazione troppa alta
- qualità scadente dell’acqua usata per la soluzione di lavaggio (basso grado di deionizzazione)
- lavaggi insufficienti (coniugato non completamente rimosso)
Valori inspiegabilmente fuori scala
- contaminazione di pipette, puntali o contenitori- lavaggi insufficienti (coniugato non completamente rimosso)
CV% intrasaggio elevato
- reagenti e/o strip non portate a temperature ambiente prima dell’uso
- il lavatore per micropiastre non lava correttamente (suggerimento: pulire la testa del lavatore)
CV% intersaggio elevato
- condizioni di incubazione non costanti (tempo o temperatura)
- controlli e campioni non dispensati allo stesso tempo (con gli stessi intervalli) (controllare la sequenza di
dispensazione)
- variabilità intrinseca degli operatori
ERROR POSSIBLE CAUSES / SUGGESTIONS
No colorimetric reaction
- no conjugate pipetted reaction after addition
- contamination of conjugates and/or of substrate
- errors in performing the assay procedure (e.g. accidental pipetting of reagents in a wrong sequence or from the
wrong vial, etc.)
Too low reaction (too low ODs)
- incorrect conjugate (e.g. not from original kit)
- incubation time too short, incubation temperature too low
Too high reaction (too high ODs)
- incorrect conjugate (e.g. not from original kit)
- incubation time too long, incubation temperature too high
- water quality for wash buffer insufficient (low grade of deionization)
- insufficient washing (conjugates not properly removed)
Unexplainable outliers
- contamination of pipettes, tips or containers
insufficient washing (conjugates not properly removed) too high within-run
- reagents and/or strips not pre-warmed to CV% Room Temperature prior to use
- plate washer is not washing correctly (suggestion: clean washer head)
too high between-run - incubation conditions not constant (time, CV % temperature)
- controls and samples not dispensed at the same time (with the same intervals) (check pipetting order)
- person-related variation

Source: http://www.qualisyscanada.com/docs/IFU-17OH-progesterone-ENG.pdf