The Karyotype

Why the karyotype matters

• It is the only test that sees the whole genome at once. A single G-banded metaphase spread surveys all 46 chromosomes for both count and gross structure. No PCR, no sequencing read, and no microarray gives you that panoramic view of every chromosome in one image — a karyotype catches a balanced translocation that a copy-number array would miss entirely because no DNA is gained or lost.
• It diagnoses the common aneuploidies. Trisomy 21 (Down syndrome, 47,+21), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), monosomy X (Turner syndrome, 45,X), and XXY (Klinefelter syndrome, 47,XXY) are all read directly off the arranged spread as an extra or missing chromosome.
• It founded medical cytogenetics. In 1959 Jérôme Lejeune, Marthe Gautier, and Raymond Turpin showed that Down syndrome patients carry 47 chromosomes — the first human disease ever traced to a chromosomal abnormality. Turner and Klinefelter syndromes were mapped to sex-chromosome counts the same year.
• It reveals cancer-driving translocations. The Philadelphia chromosome — a reciprocal t(9;22)(q34;q11) that fuses BCR to ABL1 — was spotted by Peter Nowell and David Hungerford in 1960 as an abnormally small chromosome 22 in chronic myeloid leukemia. That single cytogenetic observation eventually gave us imatinib (Gleevec).
• It anchors prenatal diagnosis. Amniocentesis (from about 15 weeks) and chorionic villus sampling (from about 10 to 13 weeks) supply fetal cells for karyotyping, the definitive confirmatory test after a positive maternal serum screen or non-invasive prenatal test (NIPT).
• It carries forensic and evolutionary weight. Comparing karyotypes across species shows that humans (2n = 46) have one fewer chromosome pair than the great apes (2n = 48) because ancestral chromosomes 2A and 2B fused end-to-end into human chromosome 2 — a fusion visible as a second, vestigial centromere and interstitial telomere repeats.
• It detects mosaicism. Because a lab scores 15 to 20 independent metaphase spreads, it can spot a person who is part 46,XX and part 45,X — mosaic Turner syndrome — a pattern a single bulk-DNA test can blur or miss.

How a karyotype is made, step by step

Chromosomes only become individually visible when they condense for cell division, so every karyotype begins with dividing cells. For a standard blood karyotype, peripheral lymphocytes are cultured for roughly 72 hours with the mitogen phytohaemagglutinin (PHA), which drives resting T cells back into the cycle. Prenatal samples use cultured amniocytes from amniotic fluid or trophoblast cells from chorionic villi; bone marrow is used directly for leukemia work.

The culture is then arrested in metaphase with colcemid, a colchicine derivative that binds tubulin and blocks spindle assembly. With no functioning spindle, cells enter mitosis, condense their chromosomes to maximum compaction, and stall — accumulating a population caught precisely at the stage where each chromosome is a discrete, countable object. Timing is a craft: too little colcemid and few cells arrest; too much and the chromosomes over-condense into short, poorly banded blobs.

Next comes the hypotonic shock. Cells are suspended in dilute (0.075 M) potassium chloride, which draws water in by osmosis and swells them so the 46 chromosomes drift apart instead of clumping. A methanol–acetic acid fixative hardens and cleans the preparation, and a drop of the suspension is released onto a chilled slide from a height; the impact bursts each cell and flattens its chromosomes into a spread metaphase plate — ideally one intact, non-overlapping spread of 46.

The slide is then G-banded: brief digestion with the protease trypsin partially strips chromosomal proteins, and staining with Giemsa dye produces the reproducible barcode. Dark bands are AT-rich, gene-poor, late-replicating heterochromatin; pale bands are GC-rich, gene-dense euchromatin. A trained analyst or automated imaging system photographs several well-spread metaphases, digitally cuts out each chromosome, and arranges them into the numbered ideogram — largest (chromosome 1) to smallest autosome (chromosome 21, which is actually slightly smaller than 22), with the sex chromosomes placed last. Every break is described with p (petit, short arm) and q (long arm) band coordinates under the ISCN nomenclature — for example, the Philadelphia breakpoint at 22q11.

Reading karyotype notation

Cytogenetic results follow the International System for Human Cytogenomic Nomenclature (ISCN): the total chromosome number, a comma, the sex chromosomes, then any abnormality. A plus or minus before a chromosome number means a whole extra or missing chromosome; letters like del, dup, inv, and t describe structural changes, with breakpoints in parentheses.

Notation	Meaning	Clinical condition
46,XX / 46,XY	Normal female / normal male	Typical (euploid)
47,XY,+21	Extra chromosome 21	Down syndrome (trisomy 21)
47,XX,+18	Extra chromosome 18	Edwards syndrome
45,X	One sex chromosome missing	Turner syndrome (monosomy X)
47,XXY	Extra X in a male	Klinefelter syndrome
46,XY,t(9;22)(q34;q11)	Reciprocal swap between 9 and 22	Philadelphia chromosome (CML)
45,XX,rob(14;21)(q10;q10)	Robertsonian fusion of 14 and 21	Balanced carrier (fertility/recurrence risk)
46,XX,del(5)(p15.2)	Deletion of distal 5p	Cri-du-chat syndrome

Common misconceptions

• A karyotype reads your genes. It does not. Karyotyping resolves changes at the scale of whole chromosomes or large band-sized segments (roughly 5 to 10 megabases at best); it is blind to single-gene mutations, small deletions below a band, and point changes. A person can have a perfectly normal 46,XX karyotype and still carry cystic fibrosis or sickle-cell disease. Sub-band changes need microarray or sequencing.
• Humans have always been known to have 46 chromosomes. For over three decades textbooks stated 48. Only in 1956 did Tjio and Levan, using an improved hypotonic-spread technique on cultured lung fibroblasts, count 46 reliably — and their result was so counter to dogma that they nearly withheld it. The prior miscount came from poorly spread, overlapping chromosomes.
• A balanced translocation makes a person sick. Usually not. In a balanced reciprocal or Robertsonian translocation no genetic material is net gained or lost, so the carrier is typically healthy. The risk is reproductive: during meiosis they can produce unbalanced gametes, raising the chance of miscarriage or a child with a chromosomal syndrome such as translocation Down syndrome.
• Down syndrome is always “an extra 21” from the mother. About 95% of cases are free trisomy 21 from meiotic nondisjunction (predominantly maternal, rising steeply after age 35), but roughly 3 to 4% arise from a Robertsonian translocation — which can be inherited from a balanced-carrier parent — and about 1 to 2% are mosaic, with only a fraction of cells trisomic.
• Spectral karyotyping replaced G-banding. They are complementary. SKY paints each chromosome a color and excels at complex or cryptic translocations, but it is color-blind to rearrangements within a single chromosome — a paracentric inversion keeps the same color and vanishes, while G-banding catches the shifted band order. Most labs still start with G-banding.
• Karyotyping is obsolete now that we have NIPT. Non-invasive prenatal testing (cell-free fetal DNA in maternal blood) is a highly sensitive screen, not a diagnosis. A positive NIPT is confirmed by a diagnostic karyotype or microarray on amniocytes or chorionic villi, because NIPT can be confounded by confined placental mosaicism, vanishing twins, or maternal copy-number variation.

Karyotyping vs FISH vs microarray vs sequencing

Feature	G-banded karyotype	FISH	Chromosomal microarray	Whole-genome sequencing
Resolution	~5–10 Mb (whole chromosome/band)	~100 kb–1 Mb (targeted)	~10–100 kb (copy number)	Single base pair
Whole-genome view	Yes, all 46 at once	No, only probed loci	Yes, copy number genome-wide	Yes, complete sequence
Balanced translocations	Detected	Detected if probed	Missed (no copy change)	Detected (breakpoint reads)
Aneuploidy	Detected	Detected (interphase)	Detected	Detected
Single-gene mutations	Missed	Missed	Missed	Detected
Needs dividing cells	Yes (metaphase)	No (interphase FISH)	No	No
Turnaround	Days (culture-limited)	1–2 days	3–7 days	Days–weeks
Mosaicism	Detected (cell-by-cell)	Detected (cell-by-cell)	Detected if >~10–20%	Detected (read fraction)

A famous history

• Tjio and Levan settle the number (1956). Working in Lund, Sweden, Joe Hin Tjio and Albert Levan applied hypotonic pretreatment and colchicine to cultured human fetal lung cells and counted a clean 46 chromosomes in spread after spread, overturning the entrenched figure of 48. The correction launched modern human cytogenetics; without an accurate baseline, no abnormality could be defined.
• Lejeune links Down syndrome to trisomy 21 (1959). In Paris, Jérôme Lejeune, Marthe Gautier, and Raymond Turpin karyotyped children with Down syndrome and found 47 chromosomes — an extra copy of the smallest autosome, chromosome 21. It was the first time a human clinical syndrome was tied to a specific chromosomal error, and it created the entire field of clinical cytogenetics almost overnight.
• Turner and Klinefelter mapped to sex-chromosome counts (1959). The same year, Charles Ford and colleagues showed that Turner syndrome patients carry a single X (45,X), while Patricia Jacobs and John Strong found that Klinefelter patients carry an extra X (47,XXY), proving that sex-chromosome dosage — not just autosomes — could underlie recognizable syndromes.
• The Philadelphia chromosome (1960). Peter Nowell and David Hungerford, in Philadelphia, noticed an abnormally small chromosome 22 recurring in chronic myeloid leukemia. In 1973 Janet Rowley used newly developed banding to show it was a reciprocal translocation, t(9;22), fusing BCR and ABL1 — the first consistent chromosomal abnormality tied to a specific cancer, and the target of the drug imatinib.
• Banding and painting (1970s–1990s). Torbjörn Caspersson introduced quinacrine Q-banding in 1970 and G-banding followed, turning uniform gray chromosomes into individually identifiable barcodes. In 1996 Evelin Schröck and Thomas Ried published spectral karyotyping (SKY), assigning each of the 24 human chromosomes a distinct spectral color to expose complex rearrangements in cancer genomes at a glance.