Components

PCP DFA Reagent - (Catalog No. 5195). One (1) 2 mL dropper vial of a mixture of two monoclonal antibodies against Pneumocystis carinii directly conjugated to FITC in a solution containing counterstain, stabilizers, and 0.1% sodium azide (NaN3).PBS Buffer Salts - (Catalog No. 5087). One (1) packet of phosphate-buffered saline salts yield 1 liter when dissolved in distilled water. Store in a clean closed container at room temperature.Tween 20 / Sodium Azide Solution (100x) - (Catalog No. 5037). One (1) 10 mL vial of polyoxyethylene sorbitan monolaurate (Tween 20) and sodium azide (NaN3) concentrate to be diluted 1:100 in PBS.Mounting Medium - (Catalog No. 5013). One (1) 10 mL dropper vial containing tris buffer, glycerin, fluorescence enhancer, and sodium azide as preservative. Store at room temperature.

Disclaimer

CE MarkFor in vitro Diagnostic Use

General description

Diagnostics Kit

The Light Diagnostics Pneumocystis carinii DFA kit is an in vitro procedure for the detection of Pneumocystis carinii cysts and trophozoites in direct smears of respiratory tract specimens including sputum, bronchoaveolar lavage, and lung biopsy.

For in vitro diagnostic use.

Test Principle:

The Light Diagnostics Pneumocystis carinii DFA kit is a direct fluorescent antibody test for the detection of Pneumocystis carinii in direct smears of respiratory tract specimens including induced sputum, bronchoalveolar lavage (BAL), and lung biopsy specimens. The anti-Pneumocystis carinii monoclonal antibodies bind to both cysts and trophozoites of Pneumocystis carinii present in a respiratory tract specimen. These monoclonal antibodies are labeled with fluorescein isothiocyanate (FITC). FITC exhibits an apple-green fluorescence when illuminated by ultraviolet light allowing visualization of the complex by fluorescence microscopy. Observation of fluorescence and the characteristic morphology of Pneumocystis carinii cysts and trophozoites indicate the presence of Pneumocystis carinii and constitutes a positive result. If the specimen contains no Pneumocystis carinii cysts and trophozoites, no fluorescent immune complex is formed and the specimen is considered negative.

Summary and Explanation:

Pneumocystis carinii is an opportunistic pulmonary protozoan pathogen which is composed of two basic forms (6):

a) The trophozoite is a small (1-4 micron) pleomorphic form which can be stained and identified in lung imprint smears.

b) The cyst, which is relatively larger (5-7 micron), is less common. The cyst has a thick wall and contains up to 8 daughter forms, termed sporozoites. The cyst can be identified by stains that are selective for its cell wall.

In addition, some intermediate forms and extracystic material can be observed when using the immunofluorescence method (14).

Pneumocystis carinii causes interstitial plasma cell pneumonia and occurs in malnourished, debilitated, premature infants and children with primary immunodeficiency diseases. It develops in patients of all ages receiving immunosuppressive agents for treatment of cancer, organ transplants, and other disorders (1-4). Recently, Pneumocystis carinii has emerged as the most frequent opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). Pneumocystis carinii pneumonia (PCP) accounts for 43% to 85% of pulmonary infection associated with AIDS (4,14). Typical symptoms include prolonged breathlessness, dry cough, fever, and an inability to take a deep breath.

The presenting manifestation of PCP can be subtle and non-specific. Rapid early detection of PCP improves prognosis through early therapeutic intervention. Pneumocystis carinii has been detected by methenamine silver, Giemsa, Gram-Weigert, and toluidine blue O stains (7-13). The organism has also been identified with indirect fluorescent monoclonal antibody (14,15).

The Light Diagnostics Pneumocystis carinii DFA kit utilized directly conjugated fluorescent antibodies to provide clear, easy to interpret results in only 15 minutes.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Storage and Stability

Kit components should be stored protected from light at 2-8°C, until the expiration date of the kit. Kit components must be brought to room temperature prior to use.

During incubation, slides should be protected from light and kept in a humid chamber at the recommended temperature.

A marked decrease in fluorescence may indicate antibody deterioration. A positive control should be tested with each specimen to ensure proper functioning of these reagents and proper staining procedure. If after appropriate analysis there is a decrease in staining intensity, discontinue use of the reagents.

Warnings and Precautions:

⋅ Do not mix components from different kit lots.

⋅ Do not use kit components beyond expiration date.

⋅ Certain reagents in the Light Diagnostics Pneumocystis carinii DFA kit contain sodium azide which may react with lead or copper plumbing to form potentially explosive metal azides. When disposing of such reagents, always flush with copious amounts of water to prevent build up.

⋅ Patient specimens can contain infectious agents, especially those obtained from AIDS patients. All specimens should be handled with extreme caution and should be regarded as potentially infectious.

⋅ Gloves should be worn throughout the acetone fixation step and preferably throughout the entire procedure. Wash hands thoroughly after completion of test procedure.

⋅ Do not mouth pipette.

⋅ Avoid exposing the fluorescent antibody reagent to bright light during storage or incubation.

⋅ Avoid scratching the fixed specimens on the surface of the slide when adding reagents to the slide wells.

⋅ Do not allow the microscope slide to dry at any time during the staining procedure.