Experiment 04Clinical CytogeneticsG-Banding & Karyotyping
Human Karyotype Analysis & Metaphase Chromosome G-Banding (GTG) for Aneuploidy Diagnosis
Lab Duration: 72h Culture + 4h Harvesting & Karyogram Assembly
🎯 Aim: To perform short-term culture of human peripheral blood lymphocytes with phytohemagglutinin (PHA), arrest dividing cells at metaphase with colchicine, harvest and prepare metaphase chromosome spreads via hypotonic shock and Carnoy’s fixation, stain with Trypsin-Giemsa (GTG banding), arrange chromosomes into an International System for Human Cytogenomic Nomenclature (ISCN) karyogram, and diagnose constitutional numerical and structural chromosomal aberrations (Down syndrome $47,XX,+21$, Turner syndrome $45,X$, Klinefelter syndrome $47,XXY$).
🔬 Principle & Cytogenetic Architecture
Human peripheral blood T-lymphocytes are quiescent in the $G_0$ phase of the cell cycle. Introduction of the lectin Phytohemagglutinin (PHA-M) cross-links T-cell receptors and activates mitogenic signaling, inducing transformation into dividing lymphoblasts that enter active mitosis after approximately 48 to 72 hours of incubation at $37^circtext{C}$.
To obtain well-spread, condensed metaphase chromosomes suitable for band analysis:
- Colchicine / Colcemid ($0.1,mutext{g/mL}$): Binds to tubulin heterodimers and prevents their polymerization into mitotic spindle microtubules, arresting dividing cells at the metaphase stage where chromosomes reach maximal condensation.
- Hypotonic Treatment ($0.075,text{M KCl}$ at $37^circtext{C}$): Induces controlled endosmotic water influx, causing cell swelling and lysing red blood cells without bursting lymphocyte membranes. This spreads the metaphase chromosomes evenly across a 2-dimensional plane.
- Carnoy’s Fixation (3:1 Methanol : Glacial Acetic Acid): Methanol dehydrates and coagulates proteins; acetic acid rapidly penetrates and fixes nucleoproteins, rendering chromosomes receptive to subsequent trypsin digestion.
- G-Banding with Trypsin & Giemsa (GTG Banding): Brief proteolytic digestion with trypsin removes acidic non-histone proteins, relaxing chromatin. Subsequent staining with Giemsa produces reproducible dark and light bands along the chromosome arms. Dark G-bands (positive bands) represent condensed, late-replicating, AT-rich heterochromatin with few active genes. Light bands represent GC-rich, early-replicating euchromatin containing transcriptionally active house-keeping genes.
📋 Step-by-Step Harvesting & Staining Protocol
- Lymphocyte Inoculation (Day 0): Under a laminar flow hood, aseptically collect $5,text{mL}$ of peripheral venous blood in a heparinized vacutainer tube (never EDTA, which chelates divalent cations essential for PHA mitogenesis). Inoculate $0.5,text{mL}$ of whole blood into $5,text{mL}$ of RPMI-1640 culture medium supplemented with $15%$ fetal bovine serum (FBS), $1%$ penicillin-streptomycin, and $0.1,text{mL}$ of PHA ($10,mutext{g/mL}$). Incubate vertically at $37^circtext{C}$ with $5%,text{CO}_2$ for 71 hours.
- Metaphase Arrest (Day 3, 71h): Add $0.1,text{mL}$ of Colcemid solution ($10,mutext{g/mL}$, final concentration $0.1,mutext{g/mL}$) to the culture flask. Gently mix and re-incubate for exactly 60 minutes.
- Cell Harvesting & Hypotonic Shock: Centrifuge culture at $1000,text{rpm}$ ($200times g$) for 10 minutes; aspirate supernatant. Resuspend pellet in $8,text{mL}$ of pre-warmed ($37^circtext{C}$) hypotonic $0.075,text{M KCl}$ solution added dropwise with gentle vortexing. Incubate in a $37^circtext{C}$ water bath for exactly 20 minutes.
- Fixation: Add $1,text{mL}$ of freshly prepared ice-cold Carnoy’s fixative (3 parts absolute methanol : 1 part glacial acetic acid) to stop hypotonic action. Centrifuge at $1000,text{rpm}$ for 10 minutes. Aspirate supernatant, add $8,text{mL}$ fresh Carnoy’s fixative, let stand for 30 minutes, and centrifuge. Repeat fixative washes 3 times until the pellet is chalky white.
- Slide Preparation: Resuspend final cell pellet in $0.5,text{mL}$ fresh fixative. Hold a pre-chilled, wet glass slide at a $45^circ$ angle and drop 2-3 drops of cell suspension from a height of $30-40,text{cm}$ to burst the swollen cell membranes and spread chromosomes. Allow slides to air dry.
- Trypsin-Giemsa (GTG) Banding: Age slides at $60^circtext{C}$ overnight. Immerse slide in $0.05%$ Trypsin-EDTA in PBS for 15 to 45 seconds (titrate based on slide age), rinse immediately in cold PBS, and stain in $4%$ Giemsa in Gurr buffer (pH 6.8) for 5 minutes. Rinse in distilled water, dry, and mount.
- Karyotype Assembly: Locate well-spread metaphase plates under $100times$ oil immersion objective. Count chromosomes (normal diploid = 46). Capture digital images, measure p and q arm lengths, compute the centromeric index ($CI = frac{p}{p+q}$), and classify chromosomes into the 7 standard Denver groups (A through G plus sex chromosomes).
📊 Denver Classification of Human Chromosomes
| Group | Chromosome Pairs | Centromere Position | Distinguishing Cytogenetic Characteristics |
|---|---|---|---|
| Group A | 1, 2, 3 | Metacentric (1, 3); Submetacentric (2) | Largest autosomes in the human complement |
| Group B | 4, 5 | Submetacentric | Large autosomes; short p-arm deleted in Cri-du-chat ($5p-$) |
| Group C | 6 to 12 + X | Submetacentric | Medium-sized; X chromosome is part of Group C |
| Group D | 13, 14, 15 | Acrocentric with Satellites | Medium acrocentric; Robertsonian translocations |
| Group E | 16, 17, 18 | Metacentric (16); Submetacentric (17, 18) | Small; Trisomy 18 is Edwards syndrome |
| Group F | 19, 20 | Metacentric | Small metacentric chromosomes |
| Group G | 21, 22 + Y | Acrocentric with Satellites (21, 22) | Smallest autosomes; Trisomy 21 = Down syndrome; Y lacks satellites |
