Introduction

Genetics is the study of how hereditary information is stored, transmitted, and expressed in living organisms. This information is encoded in the nucleotide sequence of deoxyribonucleic acid (DNA), which serves as the molecular foundation for all inherited traits. Although an individual's DNA sequence remains largely stable throughout life, DNA replication is not entirely error-free, allowing some nucleotide changes (mutations) to escape repair.[1]

Many genetic variants do not affect the phenotype, but others may cause congenital or acquired disease and can be inherited by future generations if they occur in germ cells. In addition to traditional Mendelian inheritance, gene expression is influenced by epigenetic mechanisms that alter phenotype without changing the DNA sequence. Together, these concepts provide the basis for understanding chromosome behavior and abnormalities and underpin cytogenetic testing.[2]

Cytogenetic testing involves examining chromosomes to detect abnormalities, including aneuploidy and structural abnormalities. A normal human cell contains 23 pairs of chromosomes, including 22 pairs of autosomes and a pair of sex chromosomes (XX or XY). (See Image. Human Male Karyotype). Aneuploidy is the presence of one or more extra chromosomes (47, XX,+21 or 48, XXXY) or having missing chromosomes (45, XO). The most common aneuploidies are Down syndrome (trisomy 21), Edward syndrome (trisomy 18), and Turner syndrome (monosomy X).

The types of structural abnormalities are:[2]

• Duplication: Part of a chromosome is repeated
• Deletion: Part of a chromosome is missing
• Translocation: Material between 2 different chromosomes is exchanged (this exchange may be balanced or unbalanced)
• Inversion: Part of the chromosome is inverted within the chromosome
• Insertion: Addition of material from another chromosome

Cytogenetic testing is used across a wide range of clinical scenarios, including the evaluation of congenital disorders, prenatal assessment after abnormal ultrasound or biochemical screening, recurrent miscarriage, and postnatal investigations for mosaicism, intellectual disability, autism spectrum disorder, and developmental delay. Cytogenetic testing is also a key tool in identifying both solid tumor and hematologic malignancies, where cytogenetic findings provide crucial diagnostic and prognostic information. Conventional cytogenetic analysis involves examining metaphase chromosomes using Giemsa staining (G-banding) to visualize A · T and G · C regions.[3] Karyotyping provides a genome-wide overview and detects large chromosomal abnormalities (> 5 Mb), such as aneuploidy, major deletions, and translocations. However, more minor or cryptic alterations require more sensitive molecular techniques for accurate detection.[4]

Specimen Collection

Specimen collection modalities for cytogenetic analysis include fine-needle aspiration cytology, fluid sampling with subsequent centrifugation, and tissue biopsies. For prenatal diagnosis, cytogenetic testing can be performed as early as 10 to 13 weeks of gestation using chorionic villus sampling, which analyzes cytotrophoblast cells and cultured mesenchymal core cells. Amniotic fluid analysis, typically performed between 15 and 18 weeks, allows culture of fetal cells and helps resolve suspected mosaicism identified on chorionic villus sampling.[5]

Fetal blood sampling (percutaneous umbilical blood sampling) may be performed between 18 and 20 weeks to obtain fetal lymphocytes for rapid karyotyping or targeted fluorescence in situ hybridization (FISH) studies.[6] In cases of pregnancy loss, fetal skin fibroblasts are preferred. Postnatally, peripheral blood lymphocytes or skin fibroblasts can be used for cytogenetic evaluation. For chromosomal microarray analysis (CMA), genomic DNA may be extracted from peripheral blood, cultured fibroblasts, or amniotic fluid.[7][8]

In acute leukemias, bone marrow aspirates are routinely obtained for conventional karyotyping and FISH at diagnosis because timely cytogenetic evaluation is essential for risk stratification and therapeutic decision-making. For solid tumors and lymphoproliferative disorders, FISH is typically performed on histopathological (often formalin-fixed, paraffin-embedded) tissue samples and provides important diagnostic and prognostic information that influences treatment selection.[9]

Procedures

Karyotyping is a preferred method for detecting both numerical and structural chromosomal abnormalities. Structural abnormalities include deletions, duplications, inversions, balanced or unbalanced translocations, and insertions. (See Image. Chromosomal Abnormaliities.) (See Image. Insertion and Translocation). Common numerical abnormalities include trisomies of chromosomes 13, 16, 18, or 21, monosomy X (Turner syndrome), and whole-genome anomalies such as triploidy or tetraploidy. Using G-banding, analysis of 20 metaphase cells is standard, whereas 30 to 50 metaphases should be examined if mosaicism is suspected.[10]

Fluorescence in situ hybridization is a powerful complementary tool that overcomes some limitations of conventional karyotyping. FISH uses fluorescently labeled probes that bind to specific DNA sequences, allowing detection of inversions, insertions, translocations, microdeletions, and microduplications. The procedure can be performed on interphase or metaphase cells, but only the targeted sequences are analyzed; therefore, careful case evaluation and probe selection are required.[10][11] An example is the detection of inv(16)(p13q22), resulting in the CBFB-MYH11 fusion gene in acute myeloid leukemia.[11]

Chromosomal microarray analysis is a genome-wide method that improves diagnostic yield in patients without a clear syndromic phenotype, including those with developmental delay, intellectual disability, or autism spectrum disorder. Chromosomal microarray analysis detects copy number variations across the genome and is especially useful in suspected mosaicism.[8][12] Subtypes of CMA include microarray-based comparative genomic hybridization and single-nucleotide polymorphism arrays, which use fluorescently labeled probes to hybridize with patient DNA for comprehensive genome analysis.[8]

Indications

Cytogenetic testing is an important diagnostic tool in fetal and genetic medicine, oncology, and hematology. The main indications for karyotyping and FISH studies in neoplastic diseases include the following:[11]

• Diagnosis and classification of leukemias
• Estimation of prognosis
• Selection of appropriate treatment regimens
• Evaluation of response to treatment by confirming the elimination of cells carrying the abnormal genotype

The main indications of cytogenetic studies in congenital genetic diseases include the following:

• Abnormal ultrasound findings
• Abnormal biochemical results
• Recurrent miscarriage
• Advanced maternal age, particularly > 35 years old
• Family history
• Abnormal noninvasive prenatal test results (NIPT)
• Multiple congenital anomalies of undetermined significance

Abnormal biochemical findings in maternal serum may include decreased α-fetoprotein, which might indicate trisomy 21; decreased unconjugated estriol; increased human chorionic gonadotrophin; and abnormal inhibin-A levels. These findings are also associated with Down syndrome.[13] Additionally, in trisomy 18,α-fetoprotein, unconjugated estriol, and human chorionic gonadotropin levels are decreased.

While ultrasonographic findings are insufficient to confirm a diagnosis, short femoral length and thickened nuchal skin fold can raise suspicion for trisomy 21.[8][14] Results from several studies indicate that the risk of trisomy 13, 18, and 21 increases with advanced maternal age.[15][16] When biochemical tests, ultrasonography screening results, and maternal age are considered together, prenatal cytogenetic testing can be considered.[17] A pedigree that contains an individual with a chromosomal anomaly may also include other individuals with balanced translocations. In these cases, the parents should be tested because there may be an unbalanced rearrangement in the embryo.[18] Most of these pregnancies end in fetal loss during the first trimester. More than 2 pregnancy losses should alert the clinician to the possibility of a balanced translocation in one of the parents.[19] Chromosomal studies can diagnose an unbalanced translocation. Moreover, trisomy recurs in approximately 1% of subsequent pregnancies in couples diagnosed with trisomy in a previous pregnancy. The chromosomal analysis may be helpful in these cases.[20]

Noninvasive prenatal testing/screening (NIPT/NIPS) is a relatively new method that detects chromosomal anomalies by measuring circulating cell-free fetal DNA (cfDNA) in the mother's plasma.[21] High cfDNA from sex chromosomes and chromosomes 13, 18, and 21 is associated with the respective syndromes. Moreover, NIPT can reveal microdeletion syndromes by detecting an abnormal number of copies.[22]

Potential Diagnosis

As mentioned above, chromosomal analysis can be used to diagnose variable aneuploidies, such as monosomy X, trisomies, triploidy (3 haploid sets, 69 chromosomes, [3n/69]), and tetraploidy (4 haploid sets, 92 chromosomes [4n/92]). These tests are preferred for diagnosing balanced and unbalanced translocations, recurrent microdeletions such as the 22q11.2 deletion (DiGeorge syndrome), the 15q11.2q13.1 deletion (Prader-Willi/Angelman syndrome), and 5p15.3 microdeletion (cri-du-chat syndrome). In particular, CMA may be used to determine the etiology of developmental delay in patients without a known cause. CMA is also helpful in autism spectrum disorder or intellectual disability.[23]

Translocation between the long arms of chromosomes 9 and 22 t(9;22)(q34;q11) was the first demonstration that a malignancy could result from a genetic abnormality. This aberration was named the Philadelphia chromosome.[11][24] Chronic myeloid leukemia often has the Philadelphia chromosome, and it may also occur in acute lymphoblastic leukemia and acute myeloid leukemia. This translocation results in the BCR-ABL fusion protein, which has constitutive tyrosine kinase activity.[11]

Other significant abnormalities include translocation (15;17) in acute promyelocytic leukemia. This subset of acute myeloid leukemia responds well to all-trans retinoic acid (tretinoin), highlighting the role of cytogenetic testing in determining the appropriate treatment.[25] Additionally, characteristic translocations in lymphomas include t(8;14) in Burkitt lymphoma, t(14;18) in follicular lymphoma, and t(11;14) in mantle cell lymphoma. Translocation (12;21) in pediatric acute lymphoblastic leukemia and inversion (16) in acute myeloid leukemia are examples of aberrations that confer a favorable prognosis, whereas inversion (3) and a complex karyotype, defined as 3 or more concurrent chromosomal abnormalities, have an adverse prognosis in AML.[26]

Normal and Critical Findings

Humans typically have 23 pairs of chromosomes, consisting of 22 pairs of autosomes and one pair of sex chromosomes (XX in women, XY in men), for a total of 46 chromosomes per cell. In karyotyping, autosomes are arranged by size and banding patterns, which help identify structural features. Conventional karyotyping can detect numerical abnormalities (aneuploidies), such as missing or extra chromosomes, as well as structural abnormalities, including translocations, deletions, duplications, inversions, and insertions. These findings are essential for diagnosing genetic disorders, congenital syndromes, and hematologic or solid tumor malignancies.

Interfering Factors

An estimate of the pretest probability of any potential diagnosis enables judicious use of cytogenetic testing and avoids unnecessary, costly testing. Lack of an appropriate differential diagnosis or suspicion of a particular diagnosis can lead to inappropriate testing or confirmation bias when interpreting test results. For conventional karyotyping, obtaining metaphase-stage cells requires cell division in the cultured sample. Lack of adequate preparation or the absence of cell types that do not grow in vitro without specialized methods may impede adequate testing. Keeping these pitfalls in mind enables efficient use of cytogenetic analysis and maximizes the benefits.

Complications

Risks related to chorionic villi sampling are higher before 10 weeks of gestation. While the complication rate for chorionic villus sampling is approximately 1%, it is lower (0.5%) for amniocentesis. Chorionic villus sampling allows the patient to contemplate pregnancy termination earlier.[8][27] Amniocentesis may result in rupture of the amniotic sac, amniotic fluid leakage, and chorioamnionitis. 

Patient Safety and Education

Patient safety in cytogenetic testing begins with clear, comprehensive education. Patients should be informed about the small but possible risk of false-positive findings and counseled regarding the appropriate timing, preparation, and risks associated with sample collection procedures. This preparation ensures informed decision-making and helps set realistic expectations regarding test outcomes.[28]

A complete and high-quality genetic report is essential for patient safety. The report must include patient identifiers (name, medical record number, or date of birth), sex, and relevant race or ethnicity. The report should specify the specimen type, date of receipt, laboratory identification number, the test requested, and the names and addresses of both the testing laboratory and the referring clinician. The report date should also be clearly labeled.

Analytic interpretation should use standardized nomenclature and describe all detected variants. The report must provide details of the methodology used, references to supporting literature where appropriate, and information on assay sensitivity and specificity (eg, number of variants tested, proportion undetected, potential for genetic heterogeneity or recombination). All variants should be classified as pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. Reference sequence, genome build, and genomic coordinates must also be listed.[29]

A well-structured clinical interpretation is crucial for patient care. This section should contextualize the results with the patient’s clinical history, explaining their significance for the individual and their family. The report may include recurrence risk, genotype–phenotype correlations, penetrance, disease association, or carrier-risk evaluations. Citations should be provided where applicable. Importantly, the report must clearly state that genetic counseling is recommended to help patients understand the implications of their results.[30] Because many genetic tests are laboratory-developed and not FDA-approved, it is also mandatory to include a disclaimer. Providing transparent information, comprehensive reporting, and access to genetic counseling collectively ensures patient safety and promotes responsible use of cytogenetic findings in clinical care.[31]

Clinical Significance

Cytogenetic testing provides an opportunity to improve the management of congenital disorders, hematologic malignancies, and solid tumors. Parents can be counseled about expectations related to congenital disorders, review fetal and maternal risks, and consider continuation or termination of the pregnancy. The risks associated with pregnancy termination are higher without an early cytogenetic diagnosis. Appropriate intervention for a child with a congenital disorder may include screening for cardiac defects, hearing, and vision assessments, and specialized education, which may be delayed without a timely diagnosis.[10] Correct diagnosis, prognostic evaluation, and improved therapeutic options often require appropriate cytogenetic testing in acute leukemias and other forms of cancer. Broader availability of diagnostic tools is key to advancing cancer management and survivorship.

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Disclosure: Muhammad Zubair declares no relevant financial relationships with ineligible companies.

Disclosure: Marcelo Lacerda declares no relevant financial relationships with ineligible companies.