Immunofluorescence

The Medical Illustration unit comprising the photomicrography unit as well as the graphics section works hand in hand with the immunohistochemistry laboratory to provide this service. Immunofluorescence microscopy has become a vital tool for the diagnosis of immunologically mediated diseases especially in kidneys, skin dermatoses, as well as other organs.

The Department of Pathology is well equipped with two epifluorescence microscopes for studying FITC antibody detections of C3 complement, IgA, IgG, IgM and other immune complex deposits in cryosections.

The microscopes are:

The Zeiss Large Universal Fluorescence Microscope has a HBO 200 watt mercury arc light source, and Neofluar objectives: 10x, 16x, 25x, 40x and 100x (oil). Dichroic mirror/filter cube excitation-emission specification for FITC immunofluorescence is at excitation wavelengths 450 – 490nm and emission 510nm, and is used in conjunction with a 530nm-barrier filter. An UV interchangeable filter cube for UV fluorescence is also available. It has a 35mm manual camera system mounted on the trinocular viewing tube for taking photomicrographs.

The Olympus BHS Microscope is fitted with BH2-RFC reflected-light attachment, 100-watt mercury arc light source; objectives combinations are DplanApo UV - 10x, 20x, 40x, 60x (dry) and 100x (oil), and an automatic 35mm camera system for photomircrography. It has a full range of filter cubes for wavelength excitation-emission characteristics for major applications in fluorescence microscopy.

For examples: Ultraviolet (U) peak excitations at 334 and 365nm for:

• Observation of autofluorescence
• DAPI stain for DNA study

UV immunofluorescence for general pathology, bacterial specimens, etc.

Violet (V) peak excitation at 405nm for:

• Observation of catecholamine and serotonin
• Tetracycline stain for studies of bone, teeth, etc.

Blue-violet (BV) at 405 - 435nm for:

• Quinacrine/quinacrine mustard stain for chromosome study
• Thioflavine S for limphocite study
• Acriflavine for nuclei acid

Blue (B) 435 - 450nm, and Interference Blue filter (IB) near 490nm for:

• FITC immunofluorescence (IB near 490nm)
• Acridine orange stain for nuclei of cells
• Auramine stain for tubercle bacilli, etc.

Screening of Immunofluorescence Preparations

Qualified laboratory officer (medical technologist) does the technical work of cutting and staining cyrosections for immunofluorescence microscopy. The officer is also trained to conduct the preliminary screening of immunofluorescence preparations to weed out the negatively stained slides. The pathologist will then interpret the sections with positive staining, and the results of the intensity and pattern of immunofluorescence are recorded. Colour photomircographs are taken for further study, publications, and clinico-pathological case discussions.

A laboratory officer conducts preliminary screening of immunofluorescence preparations. Inset: glomerulus stained with IgG, showing 3+ intensity staining in a renal biopsy of a patient with lupus nephritis (SLE).