=====15.SOP FOR RETICULIN STAIN===== 1.** PURPOSE OF EXAMINATION:** To lay down the procedure for Reticulin stain in Histo-pathology section at NCHLS Surat. 2.** PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION:** Reticulin fibers have affinity for silver solution. So, they must treated with potassium permanganate to produce sensitized sites on fiber where silver deposition can be initiated. This appears dark black in color after reduction by formalin. 3.** STAFF RESPONSIBLE:** lab. Technician under supervision of resident doctor/tutor. 4.** PERFORMANCE CHARACTERISTICS:** NA 5.** TYPE OF SAMPLE: **Histopathology section slide 6.** PATIENT PREPARATION:** NA 7.** TYPE OF CONTAINER AND ADDITIVES: ** 8.** POSITIVE CONTROL:** Liver tissue with cirrhosis. 9.** REQUIRED EQUIPMENT AND REAGENT:** **EQUIPMENTS:** Distilled water cleaned glasswares, Coplin jars. **REAGENT:** Potassium permanganate 1 %( 1gm in 100ml DW) Oxalic acid 1%(1gm in 100ml DW), Iron alum 2.5%(2.5gm in100ml DW), Aqueous formalin 10%(10ml in 100ml DW), Sodium thiosulphate 5%(5gm in 100ml DW),10% silver nitrate solution, Ammonia, 10% formalin, absolute alcohol, Xylene, DPX. 10.** ENVIRONMENTAL AND SAFETY CONTROL: ** Universal safety precautions 11.** CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):**NA 12.** PROCEDURE(STEPS):** **Silver solution preparation** • Take 10ml of 10% aqueous silver nitrate solution add concentrated ammonia drop by drop until the precipitates first formed dissolve, taking care to avoid any excess of ammonia. • Add 10ml of 3.1% sodium hydroxide solution. • Redissolve the precipates by addition of concentrated ammonia drop by drop until the solution retaining a trace of opaque. • If at this stage any excess of ammonia is present indicated by the absence of opalescence add a few drops of 10%silver nitrate solution to produce a light precipitates. • Make up the volume to 100ml with DW before use. • Make fresh before use. **Staining procedure** • Dewax section by keeping in oven for 5 minute, clear with 2 changes of xylene each for 15 minute, dehydrate with graded alcohol 2 changes each for 2 minute • Bring section to water. • Oxidize with 1% acidified potassium permanganate solution for 5 minutes. • Wash in tap water and bleach with 1% oxalic acid solution for 1min. • Wash in running tap water then Rinse in distilled water. • Sensitize in 2.5% iron alum solution for 20 minutes. • Wash well in several changes of distilled water. • Impregnate with ammoniacal silver solution for 15 minute in Coplin jars • Wash well in several changes of distilled water • Reduce in 10% formalin for 2 minute • Rinse in tap water. • Treat with 5% sodium thiosulphate solution for 3 minutes. • Rinse in tap water. • Dehydrate, clear and mount by DPX. **Result:** *Reticulin fibers: Black *Collagen fibers: Yellow –Brown 13.** QUALITY CONTROL PROCEDURE:** The newer batch of stain is tested before being put in to use by checking their efficiency with the batch of stain already in use. 14.** INTERFERENCE:** Inadequate quality/improper dilution 15.** PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING, WHERE RELEVENT, THE MEASUREMENT OF UNCERTAINITY OF MEASURED QUANTITY VALUES:**NA 16.** BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES:**NA 17.** REPORTABLE INTERVAL OF EXAMINATION RESULTS:** NA 18.** INSTRUCTION FOR DETERMINING QUANTITATIVE RESULTS WHEN RESULTS IS NOT WITHIN THE MEASUREMENT INTERVAL:** NA 19.** ALERT / CRITICAL VALUES, WHERE APPROPRIATE:** NA 20.** LABORATORY CLINICAL INTERPRETATION:** interpret test result after true positivity of control slide. 21.** POTENTIAL SOURCE OF VARIATION:** NA 22.** REFERENCE:** Histopathological laboratory techniques by Culling’s.