Karyotyping

Definition And Core Concepts

Laboratory technique for visualization and analysis of chromosomes in metaphase cells to determine number, size, shape, and banding pattern.
Produces a karyogram, a digital representation organizing the entire genome’s paired chromosomes.
Chromosomes are classified based on length (chromosome 1 is longest, 22 is smallest), banding pattern, and centromere position (metacentric, submetacentric, or acrocentric).
Standard resolution is 5-10 Megabases (Mb), allowing detection of numerical abnormalities (aneuploidies) and large structural rearrangements.

Sample Collection: Requires actively dividing cells sourced from peripheral blood lymphocytes, skin fibroblasts, bone marrow aspirates, chorionic villi, or amniotic fluid.
Cell Culture: Phytohemagglutinin (PHA) stimulation is utilized for T-lymphocytes over 72 hours.
Mitotic Arrest: Colchicine or colcemid is added to block spindle formation, arresting cells in metaphase or prometaphase.
Hypotonic Treatment: Swells cells and disrupts the nuclear membrane, facilitating proper dispersion of chromosomes on the slide.
Fixation: Utilizes a methanol and acetic acid mixture.
Banding And Staining: GTG banding (G bands by trypsin using Giemsa) is the most widely utilized method, producing a unique sequence of dark (G-positive) and light (G-negative) bands.
Microscopic Analysis: Standard metaphase spread provides 450-550 bands. High-resolution analysis utilizes less-condensed prometaphase chromosomes, yielding 550-850 bands. A minimum of 20-30 metaphases are analyzed to detect mosaicism.

Category	Specific Indications
Suspected Aneuploidy Syndromes	Clinical features of Down syndrome (Trisomy 21), Edwards syndrome (Trisomy 18), Patau syndrome (Trisomy 13), Turner syndrome (45,X), or Klinefelter syndrome (47,XXY).
Pediatric And Developmental	Multiple congenital anomalies, dysmorphic features, unexplained global developmental delay or intellectual disability.
Reproductive And Endocrine	Ambiguous genitalia, disorders of sexual differentiation, primary amenorrhea, and infertility.
Family History	Recurrent pregnancy losses ( $\geq$ 3), history of stillbirths/neonatal deaths, or family history of balanced structural chromosomal rearrangements.
Oncology	Surveillance for specific solid tumors and leukemias (e.g., Philadelphia chromosome) via bone marrow aspirates.
Prenatal Testing	Advanced maternal age, abnormal non-invasive prenatal testing (NIPT), or abnormal fetal ultrasound findings.

Results are reported using the International System for Human Cytogenomic Nomenclature (ISCN).
Normal female is designated 46,XX; normal male is 46,XY.

Symbol/Abbreviation	Definition	Clinical Example
+ / -	Gain or loss of a whole chromosome	47,XX,+21 (Female with Trisomy 21)
del	Deletion of a chromosome segment	46,XY,del(5p) (Male with Cri-du-chat syndrome)
t	Translocation (exchange between chromosomes)	t(2;8)(q33;q24.1) (Reciprocal translocation)
inv	Inversion of a chromosome segment	46,XY,inv(2)(p21q31) (Pericentric inversion)
mos / /	Mosaicism (presence of distinct cell lines)	mos 45,X/46,XX (Turner syndrome mosaicism)

Feature	Advantages	Limitations
Diagnostic Capabilities	Gold standard for identifying balanced structural rearrangements (reciprocal translocations, Robertsonian translocations, inversions) missed by Chromosomal Microarray (CMA).	Low resolution limit of 5-10 Mb. Cannot detect microdeletions, microduplications, single-gene point mutations, or epigenetic changes.
Tissue Requirements	Allows direct visual confirmation of the entire chromosomal complement.	Strictly requires actively dividing cells; culture failure occurs in 5-10% of samples.
Turnaround And Cost	Low cost and widely available.	Time-consuming process requiring 7-14 days compared to rapid molecular methods.
Mosaicism Detection	Evaluates multiple cell lines systematically.	May fail to detect low-level somatic mosaicism (typically less than 10-15%).