Medical Genetics - V. M. Zaporozhan 2005

Diagnostic Methods for Hereditary Diseases
Cytogenetic Methods
Determination of Sex Chromatin

This is a rapid screening method used to determine the number of sex Chromosomes, though it requires subsequent karyotyping to confirm the Diagnosis.

Methods have been developed for detecting X-sex Chromatin (Barr bodies) and Y-sex chroma_tin.

X-sex chromatin. Barr bodies represent a condensed X chromosome. In female fetuses, one of the X chromosomes is inactivated and condenses on days 16–19 of embryonic development, while the other remains active. The condensed X chromosome is visible in somatic Cell nuclei as a dark, densely stained clump (Fig. 10.6). Barr bodies are detected in epithelial Cells from the oral mucosa (buccal scrapings).

The Procedure for determining sex chromatin in a buccal scraping is as follows. After rinsing the Mouth, an epithelial scraping is gently taken from the inner surface of the cheek near the molar Teeth using a dental spatula. The scraping is spread evenly onto a Glass slide, stained with aceto-orcein for 2 minutes, and covered with a coverslip. Excess stain is removed using filter paper. The number of sex chromatin bodies is counted under oil immersion in round or oval nuclei with intact nuclear membranes. In epithelial cells, sex chromatin clumps are typically located beneath the nuclear membrane. Normally, women exhibit sex chromatin in more than 20% of these cells, whereas it is absent in normal males.

Sex chromatin can also be identified in Blood smears stained with Wright-Giemsa. In neutrophil leukocytes, Barr bodies appear as drumsticks. Normally, drumsticks are found in 1–2% of leukocytes in females and are absent in males.

This method is utilized:

— for rapid diagnosis of Chromosomal Disorders associated with variations in the number of X chromosomes.

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Fig. 10.6. Sex chromatin in buccal mucosa epithelial cells (sex chromatin clumps are indicated by arrows)

The number of X chromosomes is one greater than the number of sex chromatin clumps and is determined by the formula:

N = n + 1,

where N is the number of X chromosomes and n is the number of sex chromatin clumps;

— as a rapid method for Sex Determination in cases of Hermaphroditism;

— in forensic medicine to determine the sex origin of human tissue remains (Barr bodies are exceptionally well-preserved in Cartilage tissue).

Y-sex chromatin. Y-chromatin is an intensely fluorescent region of the long arm of the Y chromosome in interphase nuclei. It can be identified in buccal scrapings and peripheral Blood Leukocytes using the following procedure. The preparation is stained with the fluorescent dye quinacrine mustard. Under a fluorescence Microscope, Y-chromatin appears within the Cell Nucleus as a bright spot with a diameter of 0.3–1.0 μm. Normal males exhibit a single Y-chromatin clump. This method is employed for the rapid diagnosis of Y polysomy syndromes.