i-FOB Turbidimetric - Linear

i-FOB TurbidimetricREFCONTENTS3100025 i-FOB Turbidimetric 1 x 50 mL3100030 i-FOB Turbidimetric 2 x 50 mLFor in vitro diagnostic use onlyi-FOB TurbidimetricLatex TurbidimetryPRINCIPLESTORAGE AND STABILITYThe latex particles coated with anti-human hemoglobin areagglutinated when they react with feces samples containinghuman hemoglobin. The latex particles agglutination isproportional to the concentration of the hemoglobin in thesample and can be measured by turbidimetry. 1REAGENTS COMPOSITIONR1 Diluent. Glycine buffer, 100 mmol/L, pH 10.R2 Latex. Latex particles coated with polyclonal antihumanhemoglobin antibodies, pH 8.2.CAL Calibrator. Ref 3900005 1 x 2 mL. Humanhemoglobin. Hemoglobin concentration is statedon the label vial and it is traceable to theReference Material CRM 522(IRMM).Precautions: The reagents contain sodium azide 0.95 g/L.Avoid any contact with skin or mucous. The reagents fromhuman donors have been given negative results to anti-HIV1/2, HBsAg and anti-HCV. Handle cautiously isrecommended.1. The reagents will remain stable until the expiration dateprinted on the label, when stored tightly closed at 2-8ºC andcontaminations are prevented during their use. Do not usethe reagents after the expiration date. Do not interchangereagents from different lots.2. Reagent deterioration: Presence of particles, turbidity andincrement of blank reagent.SAMPLESFor collecting feces sample (Note 1), use only the Stool CollectionTube ref. 3101050 (1x100 tubes), following the instructions includedin the kit.Keep the specimen at 2-8ºC for maximum period of 9 days from itscollection.MATERIAL REQUIRED- Thermostatic bath at 37ºC.- Spectrophotometer or photometer thermostatable at 37ºC witha 650 ± 20 nm filter.PROCEDURER1R2CALREAGENT PREPARATIONReady to use.Ready to use. Shake gently the vial before use.Ready to use.Sample procesingRemove the aluminium seal from the collection tube (Note 2).When using an analyzer for the assay transfer tha sample bymeans of a pipette to one of the sample tubes (cuvettes) of theinstrument. Aleternatively, insert the collection tubes into thoseinstruments provided with this distinctive feature.Calibration curve: Prepare dilutions of the Calibrator usingNaCl 9 g/L as diluent. Multiply the concentration of theCalibrator by the corresponding factor indicated in the tablebelow to obtain the hemoglobin concentration of each pointof the curve.Dilution 1 2 3 4 5i-FOB CAL (µL) -- 25 25 50 100NaCl 9 g/L (µL) 100 175 75 50 --Factor 0.0 0.125 0.25 0.5 1.0Preliminary ProcedurePrewarm the reagents and the photometer (cuvette holder) to 37ºC(Note 3).Analitycal Procedure1. Using distilled water, zero de intrument at 650 nm2. Pipette into a cuvette:Diluent (R1)0.8 mLSample/Calibrator/Water (Blank) 50 µLLatex (R2)0.2 mL3. Mix well and incubate at 37ºC for 1 minute.4. At the end of this period, read the absorbance (A 1 ) at 650 nmand after 4 minutes (A 2 ) from the first reading.QUALITY SYSTEM CERTIFIEDISO 9001 ISO 13485LINEAR CHEMICALS S.L. Joaquim Costa 18 2ª planta. 08390 Montgat, Barcelona, SPAINTelf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es

CalculationCalculate the absorbance difference (A 2 -A 1 ) of each pointof the calibration curve and plot the values obtainedagainst the hemoglobin concentration of each calibratordilution. Hemoglobin concentration in the sample iscalculated by interpolation of its (A 2 -A 1 ) in the calibrationcurve.QUALITY CONTROLSHemoglobin Controls are recommended to monitor theperformance of manual and automated assayprocedures. It is recommended to use i-FOB Control(ref: 3900040).Each laboratory should establish its own QualityControl scheme and corrective actions if controls donot meet the acceptable tolerances.REFERENCE VALUESIt is recommended that each laboratory establish its cut –off value, according to the population and purposes.Bibliography 2 shows different results of sensitivity andspecificity according the cut-off level. As a general role:Cut – off (µg/L) Sensitivity (%) Specificity (%)50 79.4 89.7100 76.5 95.3150 70.6 95.9Physiological intestinal bleeding is estimated to be 0.1 –0.3 mg hemoglobin/g stool. 3CLINICAL SIGNIFICANCEi-FOB turbidimetric is an immunoturbidimetric kit for thequantification of human hemoglobin in human feces.Hemoglobin is the major protein in blood red cells. Thepresence of hemoglobin in feces is indicative of thepresence of blood as a result of bleedings associated tosome pathologies of the gastrointestinal track (FecalOccult Blood, FOB). Such pathologies are colon polyps,adenomas, colorectal carcinoma, ulcerative colitis andCrohn’s disease.Colorectal cancer (CRC) 4 is third most common form ofcancer in Western world. CRC often causes nosymptoms until has reached a relatively advanced stage.Thus, many organisations recommend screening for thedisease with FOB-testing.Colorectal bleeding can be sometimes intermittent andso negative results do not exclude the disease.Colonoscopy is recommended as definitive test (Note 4).This method is worthless as screening test for diseasesof upper-gastrointestinal tract. 5ANALYTICAL PERFORMANCE- Linearity limit. Up to 1000 µg/L, under the describedassay conditions (Note 5). Samples with higherconcentrations should be diluted 1/5 en ClNa 9 g/Land retested again.- Detection limit. Values less than 19 µg/L give nonreproducibleresults.- Analytical sensitivity. 0.490 mAb / µg/L.- Prozone effect. Up to 15000 µg/L.- Precision.Mean (µg/L) CV (%)Intra-assay 130 5.51N = 10 357 1.94739 1.47Inter-assay 130 7.56N = 10 357 4.83739 2.64- Accuracy. Results obtained with this reagents did notshow systematic differences when compared withcommercial reagents of similar characteristics. Studies ofcomparison are available on request.- Interferences. Bovine hemoglobin (2.5 mg/L), goathemoglobin (2.5 mg/L), rabbit hemoglobin (2.5 mg/L, porkhemoglobin (2.5 mg/L), do not interfere. Ascorbic ac. (100mg/L), BSA (18 g/L), do not interfere. Lipemia (2 g/L),interferes. Other substances or drugs may interfere. 7NOTES1. Feces sample should no be collected during a menstrualperiod, or if patient suffers from bleeding hemorrhoids.Alcohol, aspirin and other drugs taken in excess may causegastrointestinal irritation producing occult bleeding. Avoidthese substances 48 hours before testing.Dietary restrictions are not necessary.2. Do not unscrew the bottom cap (red color), in order toavoid liquid spreading.3. This method may be used with different instruments. Anyapplication to an instrument should be validated todemonstrate that results meets the performancecharacteristics of the method. It is recommended tovalidate periodically the instrument. Contact to thedistributor for any question on the application method.4. Clinical diagnosis should not be made on findings of asingle test result, but should integrate both clinical andlaboratory data.5. The linearity limit depends on the sample/reagent ratio, aswell as the analyzer used. It will be higher by decreasingthe sample volume, although the sensitivity of the test willbe proportionally decreased.BIBLIOGRAPHY1. Newman DJ, et al. Ann Clin Biochem 29: 122-42 (1992).2. Vilkin A, et al. American Journal of Gastroenterology; 100(11):2519-2525 (2005).3. Nakama H, et al. Hepato-Gastroenterology. 46: 228-231 (1999).4. Mc Loughlin R, O’Morain CA. World Journal of Gastroenterology12(42): 6747 - 6750 (2006).5. Nakama H, et al. Hepato-Gastroenterology. 45: 752-754 (1998).6. Tietz Textbook of Clinical Chemistry, 3rd Ed. Burtis CA, AshwoodER. WB Saunders Co., (1999).7. Young DS. Effects of drugs on clinical laboratory tests. 3th ed.AACC Press (1997).T3100-02/0911R1.ingQUALITY SYSTEM CERTIFIEDISO 9001 ISO 13485LINEAR CHEMICALS S.L. Joaquim Costa 18 2ª planta. 08390 Montgat, Barcelona, SPAINTelf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es