Karyotype

Karyotype (conventional cytogenetic analysis) examines the number and structure of chromosomes in dividing cells, stained by G-banding (Giemsa trypsin), at a resolution of 400 to 550 bands in each haploid set (5 to 10 megabases).

Cells are cultured from peripheral blood lymphocytes (stimulated with phytohaemagglutinin for 72 hours), amniotic fluid, chorionic villi, products of conception, skin fibroblasts, bone marrow (direct or short term culture for malignancy), or tissue, and arrested in metaphase with colcemid.

Analysis of at least 15 to 20 cells (30 for mosaicism, 50 or more for suspected low level mosaicism) is done, with counting of 20 cells and analysis of 5 cells in routine constitutional work, reported using the International System for Human Cytogenomic Nomenclature (for example, 46,XY or 47,XX,+21).

Chromosomal abnormalities occur in 0.6 to 0.7% of live births (about 1 in 150), in 50% of first trimester miscarriages, 6 to 11% of stillbirths and neonatal deaths, and in many couples with infertility and recurrent pregnancy loss.

Common abnormalities are trisomy 21 (Down syndrome, 1 in 700 births), trisomy 18 (1 in 5000) and 13 (1 in 10 000), Turner syndrome (45,X, 1 in 2500 females), Klinefelter syndrome (47,XXY, 1 in 600 males), and balanced translocations (1 in 500 people).

In haematological malignancy, tumour karyotype defines diagnosis, risk stratification and treatment, including the Philadelphia chromosome t(9;22) in chronic myeloid leukaemia (95%) and t(15;17) in acute promyelocytic leukaemia.

Indications. Constitutional: multiple congenital anomalies, dysmorphism, developmental delay or intellectual disability (usually microarray first), growth failure and short stature in girls (Turner), delayed or absent puberty, ambiguous genitalia, primary amenorrhoea, infertility (azoospermia, oligospermia), recurrent miscarriage (two or more) in either partner, stillbirth,

suspected aneuploidy (Down, Edwards, Patau, Klinefelter, Turner), family history of chromosomal rearrangement, abnormal prenatal screening or ultrasound.

Prenatal: advanced maternal age, abnormal screening, abnormal ultrasound, previous affected child, parental translocation. Acquired: leukaemia, lymphoma, myelodysplasia, myeloproliferative neoplasms, myeloma at diagnosis and relapse; solid tumours in selected cases; chromosome breakage syndromes (Fanconi anaemia, ataxia telangiectasia).

Specimen. Peripheral blood 2 to 5 mL (sodium heparin, never EDTA, which prevents culture), kept at room temperature and delivered within 24 to 48 hours. Amniotic fluid 15 to 20 mL (from 15 weeks), chorionic villus sample 10 to 20 mg (from 11 weeks), products of conception in sterile saline or culture medium (not formalin), skin biopsy (fibroblasts) in culture medium.

Bone marrow aspirate 1 to 2 mL in sodium heparin, with first pull sample preferred. Request form records the indication, family history, and suspected syndrome, so that the laboratory chooses the right culture and extra studies (for example, 100 cells for Turner mosaicism).

Reference values. Normal female karyotype 46,XX, normal male karyotype 46,XY, with no structural abnormality at the band resolution achieved. Reported with the number of cells counted and analysed and band level.

Interpretation.

• Numerical abnormalities: trisomy (47,XX,+21), monosomy (45,X), sex chromosome aneuploidies (47,XXY; 47,XYY; 47,XXX) and polyploidy (triploidy 69, tetraploidy 92, mostly in miscarriages).
• Structural abnormalities: balanced translocations (reciprocal or Robertsonian, carriers are usually normal but have risk of unbalanced gametes, miscarriage and affected children), unbalanced translocations, deletions, duplications, inversions, rings, isochromosomes and marker chromosomes.
• Mosaicism: two or more cell lines (45,X/46,XX), with the proportion varying between tissues; 30 cells give 90% confidence of excluding 8% mosaicism.
• Philadelphia chromosome t(9;22)(q34;q11): chronic myeloid leukaemia or Philadelphia positive acute lymphoblastic leukaemia, with confirmation by BCR-ABL1 PCR.
• Other leukaemia findings: t(15;17) in acute promyelocytic leukaemia, inv(16) or t(8;21) in favourable risk AML, monosomy 7 and complex karyotype in high risk disease, hyperdiploidy and t(12;21) in childhood ALL, del(5q) in myelodysplasia.
• Normal karyotype with a clinical phenotype: submicroscopic changes (microdeletions, copy number variants, single gene disorders) are not seen, and chromosomal microarray or sequencing is the next step.
• Culture failure occurs in 1 to 2% of blood and up to 10% of products of conception and bone marrow; maternal cell contamination can cause a false 46,XX result in miscarriage samples.
• Pseudomosaicism and culture artefact in amniotic fluid are interpreted by examining multiple colonies and flasks, and confirmatory testing in foetal blood or placenta in discrepant cases.

Critical values and findings needing immediate action.

• Prenatal diagnosis of trisomy 13, 18, 21, triploidy or other major abnormality: urgent genetic counselling, discussion of options within the legal and gestational limits, and neonatal planning.
• Karyotype abnormality in a patient with acute leukaemia (t(15;17), Philadelphia chromosome): immediate specific therapy (all-trans retinoic acid for acute promyelocytic leukaemia, tyrosine kinase inhibitor for BCR-ABL1 positive disease), and monitoring of coagulopathy.
• Ambiguous genitalia in a newborn with karyotype pending: urgent endocrine assessment (salt losing congenital adrenal hyperplasia), cortisol, 17-hydroxyprogesterone, electrolytes and glucose.
• Parental balanced translocation found after an affected child: counselling of the extended family, prenatal testing, and reproductive options.
• Sex chromosome abnormalities with Y material in Turner syndrome (45,X/46,XY): gonadectomy discussion because of gonadoblastoma risk.
• Fanconi or other breakage syndrome result: haematology review, avoidance of unnecessary radiation, and cancer screening.

Interferences and pitfalls.

• Resolution is limited: deletions and duplications under 5 to 10 megabases, uniparental disomy, imprinting disorders, balanced cryptic rearrangements and single gene disease are not detected; chromosomal microarray detects pathogenic copy number changes in 15 to 20% of those with developmental delay, against 3 to 5% for karyotype.
• Samples with heparin free or EDTA tubes, clotted samples, delay, extreme temperatures, formalin fixation or contamination cause culture failure; repeat sampling delays the result.
• Results take 7 to 14 days for blood (prenatal samples 7 to 14 days, bone marrow 3 to 7 days); rapid aneuploidy testing by FISH or quantitative fluorescent PCR gives 24 to 48 hour answers for common trisomies.
• Maternal cell contamination, mosaicism limited to the placenta (confined placental mosaicism, in 1 to 2% of chorionic villus samples), and cultured cell changes complicate prenatal results.
• Variants of uncertain significance (heteromorphisms such as 1qh+, 9qh+, 16qh+, Yqh variants, and satellite changes) are normal and are not clinically meaningful.
• Cancer karyotypes are complex and require specialist analysis; low mitotic index and poor chromosome morphology reduce yield, and FISH or molecular tests are added.
• Interpretation should be integrated with phenotype, family studies (parental karyotype for any structural abnormality) and counselling, and the report should state recurrence risk.
• Informed consent, counselling before and after testing and attention to incidental findings (sex chromosome aneuploidy, unexpected parentage) are required.
• Karyotype after a stem cell transplant reflects the donor in blood.
• Access to cytogenetics and genetic counselling is limited in many low income settings.

Special groups.

• Newborns: dysmorphic features, hypotonia, congenital heart disease (trisomy 21), cleft lip and palate, and ambiguous genitalia; rapid FISH or quantitative PCR in urgent cases.
• Prenatal: after screening tests (cell free DNA, combined test) and ultrasound anomalies, with amniocentesis or chorionic villus sampling, miscarriage risk of about 0.1 to 0.5%.
• Couples with recurrent miscarriage (3 to 5% have a balanced translocation in one partner) or infertility (azoospermia, Klinefelter syndrome, 47,XYY, Y deletions).
• Girls with short stature and delayed puberty: Turner syndrome including mosaicism, with 30 cells or more counted.
• Haematology patients: bone marrow karyotype at diagnosis, relapse and in monitoring after transplantation.
• Regions: access depends on laboratory capacity, and prenatal diagnosis and genetic counselling are unavailable or restricted in some countries.

Follow up tests. Parental karyotypes, FISH for specific regions (microdeletion syndromes, rapid aneuploidy, cryptic translocations), chromosomal microarray, quantitative fluorescent PCR, sequencing panels or exome for normal karyotype with phenotype, BCR-ABL1 and other molecular tests in leukaemia, Y chromosome microdeletion testing in azoospermia, hormone studies (FSH, LH, testosterone, AMH, oestradiol),

echocardiography and renal ultrasound in Turner syndrome and Down syndrome, thyroid function and coeliac screening in Down and Turner syndromes, genetic counselling, and prenatal testing in future pregnancies.