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Immunochemical FOBT (iFOBT) use antibodies to detect presence in stool of the whole globin, or some of its lumenally-derived degradation products, portion of human hemoglobin. For to collect stool in toilet house, proceed such as following by we suggested : a) twice flush the toilet for to eliminate detergent and deodorant substance; b) lay toilet paper in anterior area toilet bowl; c) reverse sitting position (reverse squatting) is need be adopted to allow for stool to collect on the toilet paper to simplify collection of the stool sample. This if the drain it’s back, while if it’s front , such as German toilet, to position the toilet paper and the squatting in regular position. After bowel movement, brushing the surface of the stool with spatula and to dip it in fecal-dissolving liquid. Contrary to chemical tests (e.g. guaiac) dietary restrictions are not required, because the tests do not react to hemoglobin in meat or plant peroxidase. A limitation of the tests is that globin is degraded by upper GI enzymes and thus iFOBT does not detect blood from upper GI bleeding, but also may not detect a lower intestinal tumor that is not bleeding. In addition, in a testing solution, human hemoglobin is gradually denatured, and its antigenicity is decreased. Also, in a testing solution, storage conditions such as storage temperature often accelerates denaturation of human hemoglobin, or bacteria and digestive enzymes in feces often degrade human hemoglobin. Such denaturation and degradation destroy human hemoglobin conformation, resulting in decrease of antigenicity. Therefore, in an immunological method for measuring human hemoglobin, denaturation and degradation of the hemoglobin involve an incorrect diagnosis. In Africa, but also in mediterranean countries, feces are often collected by subjects themselves at their places, and are provided for the test with a closed container, by dissolving the feces in a feces dissolving solution in the container. In such cases, human hemoglobin in feces is often remained in the solution for several days, or placed in a high temperature (especially in summer period) when utilizing a transportation method such as a “Clinical mobile”. Also, even when feces are collected in a clinical laboratory, it sometime takes longer until carrying out the fecal occult blood test because other tests are also performed. Under such circumstances, an accurate measurement is disturbed by denaturation and degradation of human hemoglobin as described above. To prevent such denaturation and degradation of human hemoglobin in a solution, specific dissolving solution have been realized but, today, any safety of absolute stability exists.
When a diluted fecal suspension, containing Hb, is mixed with the gold colloid particles, Hb antigen will be aggregated to cause color change from red-violet to gray.
Feces-dissolving liquid : dissolve 0.0123 mM Brillant Blue FCF in diluent solution.
Diluent solution : 30 mM MES (2-Morpholinoethanesulfonic acid, monohydrate at pH 6.3), 0.9 % NaCl, 0.2 % BSA, 0.2 % boric acid, 1.8 % polyethylene glycol 20000, 0.1 % NaN3.
Gold Conjugate Reagent : dilute 130 µl/ml antihuman hemoglobin from rabbit gold colloid-label in conjugate diluent (5 mM TES [N-Tris(hydroxymethyl)methyl-2-aminoethanesulfolic acid], 0.1 % BSA, 3 % mannitol, 0.025 % xanthurenic acid, 0.05 % EDTA (Ethylenediamminetetraacetic acid)•2Na, and 0.05 % NaN3)
Microplate (MTP) : available BIOHIT MTP
Feces sampling eppendorf tube : containing 1 ml of feces-dissolving liquid
Collect stool such as above descrived and dip the spatula in liquid contained feces sampling eppendorf tube, cleose to tightly and vigorously shaking for obtained an homogenous suspension and to stabilize hemoglobin present in the sample. This is stable 7 days at room temperature. When tube to get at laboratory, centrifuge at 10900 rpm for 3 min. at room temperature for to eliminate the debris in suspension. In MTP dispense 100 µl of diluent solution in each wells used for tests, calibrators, controls and samples, then dispense 10 µl of centrifuged fecal suspension in feces-dissolving liquid, and mix together on shaker plate. Dispense 50 µl of gold conjugate reagent mix on shaker plate for 30 sec. and read at 1 min. the Abs (A1) at 540 and 660 nm on reader plate. Incubate for 7 min. and to measure again Abs (A2) at same wavelength. For to calculate the Hb present in sample to apply the following formula : ΔA = A2-A1, and this goes in equation to hemoglobin standards (50, 100 and 500 ng/ml). This complete reagent has been applied on Hemotect NS-Plus C Analyzer from Alfresa Pharma Co. (JP).