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D DFA Chlamydiae Culture Confirmation Kit and Three Currently Marketed Culture Green Fluorescent Inclusions Are Observed in the Infected Cells

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D DFA Chlamydiae Culture Confirmation Kit and Three Currently Marketed Culture Green Fluorescent Inclusions Are Observed in the Infected Cells using iodine, Giemsa, or fluorescein-conjugated monoclonal antibodies. The use of iodine and Giemsa has been shown to be less sensitive than the use of fluorescein- conjugated monoclonal antibodies.1 III. PRINCIPLE OF THE PROCEDURE 3 The Diagnostic Hybrids, Inc. D3 DFA Chlamydiae Culture Confirmation Kit uses a blend of two specific murine MAbs that are directly labeled with fluorescein isothiocyanate D DFA (FITC) for the qualitative detection of Chlamydiae in inoculated cell cultures. Cell culture isolation of Chlamydiae utilizes either McCoy cells, a mouse fibroblast cell line, or Buffalo Green Monkey Kidney (BGMK) cells in shell vial or multi-well plate Chlamydiae Culture formats. The cells are overlaid with a Chlamydia isolation medium containing cycloheximide to enhance Chlamydial growth prior to inoculation of the clinical specimen. The inoculated cells are centrifuged for 60 minutes and incubated at 35° to 37°C for 48 to 72 hours. The cells are fixed in acetone and the Chlamydiae DFA Confirmation Kit Reagent is added to the cells to stain any Chlamydiae specific antigens that may be present following culture. After incubating for 15 to 30 minutes at 35° to 37°C, the stained cells are washed with the supplied Phosphate Buffered Saline (PBS). A drop of REF: 01-040000 the supplied Mounting Fluid is added to the multi-wells or to a clean microscope slide for mounting a coverslip. The cells are examined at 100 to 400X magnification using a For in vitro Diagnostic Use microscope equipped with the correct filter combination for fluorescein. Chlamydiae Please contact Diagnostic HYBRIDS Technical Services USA Toll-free: (866) 344-3477 infected cells will have bright, apple-green fluorescent inclusions while non-infected for technical assistance regarding this procedure. Direct: +1(740) 589-3300 cells will fluoresce red due to the Evans Blue counter-stain. Fax: +1(740) 592-9820 Email: [email protected] IV. REAGENTS Symbols Lexicon/Glossary A. Kit Components In Vitro Diagnostic Medical Batch code/lot number Device 1. Chlamydiae DFA Reagent, 5-mL. One dropper bottle containing fluorescein labeled murine monoclonal antibodies directed against Chlamydiae LPS. The Temperature limit Catalog number buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative. Consult instructions for use 2. Chlamydia Antigen Control Slides, 10 slides. Five individually packaged Use by YYYY-MON-DD control slides with wells containing cell culture-derived Chlamydia trachomatis positive cells and five individually packaged control slides with wells containing Do not reuse Manufacturer cell culture-derived negative cells. Each slide is intended to be stained only one time. 3. 40X PBS Concentrate, 25-mL. One bottle containing a 40X concentrate Patent Numbers consisting of 4% sodium azide (0.1% sodium azide after dilution to 1X using de- Authorized representative in mineralized water) in PBS. Contains sodium azide 4. Mounting Fluid, 7-mL. One dropper bottle containing an aqueous, buffer- the European Community stabilized solution of glycerol, and 0.1% sodium azide. Contains 4% sodium azide CE mark of conformity when undiluted (Conformité Européen) B. Warnings and Precautions For in vitro diagnostic use only. I. INTENDED USE For professional use only. 1. Tissue culture cells may have some potential to be hazardous. Personnel The Diagnostic Hybrids, Inc. D3 DFA Chlamydiae Culture Confirmation Kit is intended for working with these cultures must be properly trained in safe handling the qualitative identification of Chlamydiae lipopolysaccharide (LPS) in inoculated cell techniques2,3,4 and have experience with tissue culture before attempting this cultures by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). procedure. 2. All procedures must be conducted in accordance with the CDC 5th edition Performance has not been established with direct patient specimens. Biosafety in Microbiological and Biomedical Laboratories, 2007, and CLSI Approved Guideline M29-A, Protection of Laboratory Workers from Instrument II. SUMMARY AND EXPLANATION OF THE TEST Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue. The Chlamydiae are prokaryotic parasites of eukaryotic cells. These organisms contain 3. Acetone, a reagent that is required for the test but not provided in the kit, is a enzymes and are able to synthesize proteins; however, they lack a mechanism to flammable, volatile organic solvent. Use it in a well-ventilated area and keep generate their own ATP (adenosine-triphosphate) and must rely upon the host cell for away from flames and other sources of ignition. survival. 4. Sodium azide is included in the PBS Concentrate at 4%, and in the other solutions in this kit at 0.1%. A MSDS for sodium azide or for Diagnostic Hybrids, The phylum Chlamydiae contains many species. Chlamydia trachomatis is primarily a Inc (DHI) reagents containing sodium azide is available by contacting Diagnostic human pathogen and is associated with endemic trachoma, inclusion conjunctivitis, Hybrids Technical Services. lymphogranuloma venereum, nongonococcal urethritis epididymitis (male), cervicitis, salpingitis (female) and procitis in either sex. Chlamydophila psittaci is an avian T: Sodium azide at very low levels causes damage to health. Chlamydia which is transmitted to humans by infected birds resulting in pneumonia. Chlamydophila pneumoniae can cause pneumonia similar to Mycoplasma pneumoniae and it has been implicated in upper respiratory infections (i.e., cough and sore throat). R28 Very toxic if swallowed R32 Contact with acids liberates very toxic gas Chlamydiae have a unique growth cycle exhibiting two life forms: the elementary body and the reticulocyte body. The elementary body, smaller in size, invades a susceptible S28 After contact with skin, wash immediately with plenty of water cell through a phagocytic process. Once inside the cell, the elementary body surrounds S45 In case of accident, or if you feel unwell, seek medical advice itself with a protective vacuole and differentiates into an actively metabolizing immediately reticulocyte body. The reticulocyte body divides through binary fission in the cell from S60 This material and its container must be disposed of as hazardous approximately the 8th hour to 18 to 24 hours. During this time the Chlamydiae show waste. their enhanced metabolic activity. At approximately 18 to 24 hours the reticulocyte N: Sodium azide may present an immediate or delayed danger bodies begin to differentiate into the smaller elementary bodies. The inclusions then to one or more components of the environment consist of both reticulocyte bodies and (predominantly) elementary bodies. This entire process takes place in the vacuole. Between 48 to 72 hours, the vacuole will rupture, releasing the infectious elementary bodies and the growth cycle will continue. R51/53 Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. Cell culture isolation of Chlamydiae utilizes cell types such as McCoy, a mouse S61 Avoid release to the environment. Refer to special fibroblast cell line, or BGMK (Buffalo Green Monkey Kidney), an African Green Monkey instructions/safety data sheets. cell line, in shell vial or multi-well plate formats. The cells are overlaid with a Chlamydia a. Reagents containing sodium azide should be considered a poison. If isolation medium containing cycloheximide to enhance Chlamydial growth prior to products containing sodium azide are swallowed, seek medical advice inoculation of the clinical specimen. The cultures are incubated for 48 to 72 hours to immediately and show product container or label. [Refer to NIOSH, allow for the formation of the vacuole described above. The presence of this National Institute for Occupational Safety and Health; CAS#: 2628-22-8; elementary body containing vacuole can be determined by many methods, i.e. staining Diagnostic HYBRIDS 1055 East State Street, Suite 100 1 MDSS GmbH Athens, Ohio 45701 USA Schiffgraben 41 REF PI-222en v2010APR07 30175 Hannover, Germany 0843 © 2008 Diagnostic HYBRIDS, Athens, OH 45701 USA EC# 247-852-1; and also to GHS, The Globally Harmonized System of b. Rotate the swab several times to obtain columnar epithelial cells and Classification and Labeling of Chemicals.] withdraw. b. Aqueous solutions of sodium azide, when mixed with acids, may liberate NOTE: Patient should not have urinated within one hour of collection. toxic gas (sodium azide in water exists in ionic equilibrium with hydrazoic 3. Eye acid, which when mixed with acid may liberate a toxic gas). a. Gently swab the lower conjunctiva with a sterile, fine-tipped polyester swab, c. Any reagents containing sodium azide should be evaluated for proper collecting patient mucous membrane cells. disposal. Sodium azide may react with lead and copper plumbing to form 4. Nasopharynx and Throat highly explosive metal azides. If products containing sodium azide are a. Gently insert a sterile nasopharyngeal fine-wire polyester swab into one or discarded into a drain, flush with a large volume of water to prevent azide both anterior nares to the posterior pharynx; rotate to collect mucous build-up. Check with regulatory agencies to determine at what membrane cells and withdraw. concentration sodium azide may cause a product to be regulated as b. Swab the posterior pharynx vigorously with a large-tipped, sterile polyester hazardous waste. swab. 5. Evans Blue counter-stain is a potential carcinogen.
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