LEUKO-TEST INTENDED USE The LEUKO-TEST is a latex agglutination test for the detection of lactoferrin released from fecal leukocytes in diarrheal stool specimens. The test can be used as a screening test to detect the presence of elevated levels of lactoferrin in stool specimens from adult patients (16 years of age and older). A positive test result indicates as increased level of fecal leukocytes. I. PRINCIPLE Latex beads coated with antibodies against lactoferrin or with normal IgG are supplied with the kit. If lactoferrin is present at elevated levels in the clinical specimen, under the conditions recommended it will cross-link the latex beads containing antibodies against lactoferrin and give a positive agglutination reaction. This reaction indicates an increased number of leukocytes. The latex beads coated with normal IgG serve as a negative control to identify any nonspecific reactions. II. SPECIMEN COLLECTION 1. Standard stool collection and handling procedures used in house are appropriate. Stool specimens that have been preserved in 10% formalin, MF, SAF, PVA, or other fixatives cannot be used. Stool specimens that are in transport media such as Cary Blair or C&S may be used; however, these samples arrive diluted (1:5) and this initial dilution factor should be taken into account when making the final test dilution of 1:50. 2. Specimens should be transported as soon as possible and stored between 2° and 8°C. 3. Whenever possible, test stool specimens which are less than 48 hours old. 4. Make sure that specimens are thoroughly mixed prior to performing the assay. This includes complete mixing of the specimen prior to transfer to Diluent as well as complete mixing of the diluted specimen prior to performing the assay. III. REAGENTS, SUPPLIES AND EQUIPMENT For In Vitro Diagnostic Use The kit should be stored between 2° and 8°C. Do not freeze the reagents. 1. Diluent, 2 bottles, 65 mL each (buffered protein solution containing 0.1% sodium azide) 2. Sensitized Latex, 6 mL (coated latex beads are in buffer containing 0.1% sodium azide) 3. Negative Control Latex, 3 mL (coated latex beads are in buffer containing 0.1% sodium azide) 4. Positive Control Reagent, 3 mL (purified lactoferrin in buffered protein solution containing 0.1% sodium azide) 5. 100 disposable plastic pipettes (1 drop contains 50 µL) 6. 25 disposable latex agglutination cards MATERIALS NOT PROVIDED 1. 2. 3. 4. Plastic test tubes for preparing dilutions Timer Pipettes for dispensing Diluent into tubes Clinical rotator (optional) REAGENT AND SPECIMEN HANDLING PRECAUTIONARY NOTES 1. Stool specimens that have been preserved in 10% formalin, MF, SAF, PVA, or other fixatives cannot be used. Stool specimens that are in transport media such as Cary Blair or C&S may be used; however, these samples arrive diluted (1:5) and this initial dilution factor should be taken into account when making the final test dilution of 1:50. 2. Bring all reagents to room temperature before using them. 3. Gently mix all reagents before dispensing. 4. Caps and tips are color-coded; do not mix. 5. Hold dropper bottles vertically to ensure proper delivery. 6. Positive control, stool samples and cards should be handled and disposed of as potential biohazards after use. Wear disposable gloves when doing the test. 7. Reagents contain sodium azide as a preservative and should be handled with normal laboratory caution. 8. Reagents from different kits should not be mixed. Do not use the kit past the expiration date. 9. Use the dilutions of stool specimen as recommended in the kit. Normal stool specimens contain low levels of lactoferrin and the dilutions recommended in the kit are designed to detect an increase in lactoferrin over background levels. 10. Do not freeze the reagents. The kit should be stored between 2° and 8°C. 11. The positive control contains lactoferrin which is a human derived material. Plasma from each donor has been tested and found negative for antibody to HIV-1, HIV-2, HCV, and HbsAg. No known test method can offer complete assurance that infectious agents are absent. ALL HUMAN SOURCE PRODUCTS SHOULD BE HANDLED AS POTENTIALLY INFECTIOUS MATERIAL. A procedure for handling biohazards is published in the CDC/NIH Manual of Biosafety in Microbiology & Biomedical Laboratories. IV. CALIBRATION – N/A V. QUALITY CONTROL 1. A positive control must be run with each series of test specimens. The Positive Control should give at least 2+ agglutination reaction. 2. Each diluted specimen must be tested with the Negative Control Latex. This negative control should not give any visible agglutination. 3. A positive agglutination reaction with diluted stool specimen and the Negative Control Latex indicates a nonspecific reaction and the results are invalid. 4. If the Positive Control does not react properly, contact TechLab, Inc. Technical Services at 1-800-832-4522. 5. Test results along with control reactions should be recorded and reported according to in-house procedures for future reference. VI. PROCEDURE 1. Prepare Diluted Specimen. a) Set up one plastic tube for each specimen to be tested. b) Add 2.5 mL Diluent to each tube. c) Using a plastic disposable pipette, add one drop (50 µL) of liquid stool to each specimen’s tube (1:50 dilution of the specimen) d) Mix well using a vortex mixer 2. Prepare Agglutination Card a) For each fecal sample you will need two black circles. b) In addition, you will need one black circle for a positive control for each group of fecal samples. Example: 5 stool samples = 10 circles Positive Control = 1 circle c) Cut any unused circles off the card before starting the test and save them for use in subsequent tests. 3. Mix Specimens and Reagents a) b) c) d) e) f) g) h) Vortex dilutions immediately prior to use! Label all testing areas accordingly. Place one drop of Sensitized Latex (yellow cap) onto each of two black circles on the card. One circle will be used for the positive control. Place one drop of the Negative Control Latex (blue cap) onto a separate black circle on the card. Place one drop (50 µL) of the 1:50 specimen dilution onto the drop of Negative Control Latex and mix well with a pipette. Place one drop (50 µL) of the 1:50 specimen dilution onto one of the drops of Sensitized Latex and mix well with the same pipette. Continue for each patient specimen to be tested. Place one drop of the Positive Control Reagent (red cap) on the other drop of Sensitized Latex for the control and mix well using a pipette. This is your positive control. 4. Incubate Specimen/Latex Mixtures. Place the card(s) on a rotator or rotate the cards by hand for three minutes at room temperature. 5. Record results VII. CALCULATIONS – N/A VIII. EXPECTED VALUES IX. REPORTING RESULTS 1. Positive Control: The positive control reaction (i.e. the mixture of the Sensitized Latex and the Positive Control Reagent) should have easily visible agglutination with a clearing background. The reaction should be at least 2+ reaction (see below). 2. Negative Control: The negative control reaction (i.e. the mixture of the Negative Control Latex and the 1:50 specimen dilution for each patient sample) should have no visible agglutination. 3. Sample Reactions: REACTION INTERPRETATION - No visible agglutination 1+ Definite, easily visible fine agglutination with a milky background 2+ Definite agglutination with a white ring starting to form at perimeter of liquid 3+ Greater agglutination with a clearing background and a more pronounced ring 4+ Highly pronounced agglutination with a clear background and highly pronounced ring around the perimeter Alternatively, any agglutination ranging from 1+ to 4+ reaction may simply be reported as positive without a numerical designation. X. LIMITATIONS AND PROCEDURAL NOTES 1. The LEUKO-TEST is a screening test that detects elevated levels of lactoferrin released from fecal leukocytes. The test may not be appropriate in immunocompromised patients. 2. The 1:50 dilution of stool specimen recommended in the brochure has been evaluated in clinical trials and has been found to be optimal for test results. The use of lower dilutions may result in positive agglutination reactions due to the presence of normal lactoferrin levels. Therefore, only the dilution recommended in the brochure should be used. 3. At this time, the LEUKO-TEST has not been clinically evaluated for use in the detection of leukocytes in other types of clinical specimens. It should only be used for the analysis of fecal specimens. 4. Data concerning Performance Characteristics, Cross-Reactivity, and Reproducibility and Precision can be found in the Package Insert. XI. CLINICAL SIGNIFICANCE Diarrheal diseases can be classified into inflammatory and non-inflammatory diarrhea. Non-inflammatory diarrheas include those caused by viruses and most parasites and are, for the most part, effectively treated with simple oral hydration therapy. Inflammatory diarrheas, on the other hand, tend to be more serious and need to be followed up by more extensive testing. This type of diarrhea is caused by pathogens such as Shigella, Salmonella, Campylobacter jejuni, and Clostridium difficile (5,6). In inflammatory diarrheas, fecal leukocytes are found in feces in large numbers. The determination of fecal leukocytes by microscopy is a procedure used by many clinical laboratories to identify inflammatory diarrheas. However, this method has disadvantages. Microscopy is not standardized and specimens must be examined soon after collection to be accurate (9). The test can be difficult to interpret and storage of specimens overnight before examination may result in lower sensitivity due to cell lysis. Some enteric pathogens, such as Clostridium difficile, produce toxins that lyse leukocytes and other cells (4). As a result, leukocytes may not be visible late in the infection even though there is severe inflammation. The method of collection also affects the sensitivity of the test. Cup specimens are often hard to collect but hey are more sensitive for leukocytes than swab specimens, which tend to destroy the morphology of the leukocytes (7). The LEUKO-TEST overcomes the problems of microscopy. It detects lactoferrin, a stable protein that serves as a marker for leukocytes. Lactoferrin is very stable and is not degraded during infections by the toxins of pathogens such as C. difficile (4). The test is rapid and can be completed within 5 minutes. In clinical investigations, the LEUKOTEST was found to have a high predictive negative value (94.9%) compared with microscopy for fecal leukocytes, indicating that it is useful as a screening test (8,10) for identifying specimens that require culture and follow-up. More discussion about clinical significance may be found in the Package Insert. XII. REFERENCES 1. Fan, K., A.J. Morris, and L.B. Reller. 1993. Application of rejection criteria for stool cultures for bacterial enteric pathogens. J. Clin Microbiol. 31:2233-2235. 2. Guerrant, R.L., J.M. Hughes, N.L. Lima, and J.Crane. 1990. Diarrhea in developed and developing countries: magnitude, special settings, and etiologies. Rev. Infect. Dis 12:S41-S50 3. Guerrant, R.L., D.S. Shields, S.M. Thornson, J.B. Schorling, and D.H.M. Groschel. 1985. Evaluation and diagnosis of acute infectious diarrhea. Am. J. Med. 78:91-98. 4. Guerrant, R.L., V. Araujo, E. Soares, K. Kotoff, A.A.M. Lima, W.H. Cooper, and A.G. Lee. 1992. Measurement of fecal lactoferrin as a marker for fecal leukocutes. J. Clin. Microbiol. 30:1238-1242 5. Harris, J.C., H.L. DuPont, and B.R. Hornick. 1971. Fecal leukocytes in diarrheal illness. Ann. Intern. Med. 76:697:703. 6. Koplan, J.P., H.V. Fineberg, M.J.B. Ferraro, and M.L. Rosenberg. 1990. Value of stool cultures, Lancet 2:13-16. 7. Korzeniowski, O.M., F.A. Barada, J.D. Rouse, and R.L. Guerrant. 1979. Value of examination for fecal leukocytes in the early diagnosis of Shigellosis. Am J. Trop. Med. Hyg. 28:1031-1035. 8. Okhuysen, P., E. Scerpella, J. Mathewson, C. Ericson, R. Guerrant, E. Latimer and D. Lyerly. 1992. Utility of a rapid latex agglutination test for lactoferrin for predicting infection due to invasive enteropathogens. Intersci. Conf. Antimicrob. Agents Chemother. 9. Tarr, P. 1991. Microbiology studies. In T. Yamada, D.H. Alpers, C. Owyang. D.W. Powell and F.E. Silverstein (ed.), Textbook of Gastroenterolofy, pp 2523-2538. J.B. Lipincott Company, Philadelphia. 10.Washington, J.A. and G.V. Doern. 1991. Assessement of new technology. In A. Balows, W.J. Microbiology, pp. 44-48. American Society for Microbiology. 11.Data on file.