Read the slide. Name every chromosome. Write the nomenclature.

The four content domains

Every item on the exam maps to one of four areas. The proportions below come from the published ASCP BOC content guideline category structure. Spend study time in proportion to weight, and remember that the largest area is also the one with the steepest skill curve.

Question styles you will meet

• Recall — definitions and facts ("Which group contains the acrocentric chromosomes 13, 14, 15?").
• Application — apply a rule to a scenario ("A metaphase has every chromosome separated but fuzzy banding — countable or analyzable?").
• Interpretation — read an image or result ("Three signals with an enumeration probe means…").
• Troubleshooting — diagnose a process failure ("Cultures grew but chromosomes are short and over-contracted — what changed?").

How to use the tools in this course

Four reasoning workflows to internalize early

These reappear throughout the course and are collected at the end for quick reference:

1. Image interpretation: assess quality → count → identify each chromosome → compare homologs → flag candidate abnormalities → confirm true vs. artifact.
2. ISCN writing: count → sex chromosomes → abnormality type → chromosome(s) involved → breakpoints → clone/cell numbers.
3. FISH scoring: confirm controls → identify probe strategy → score adequate nuclei/cells → compare to cutoff → interpret pattern.
4. Troubleshooting: define the defect → list process steps that produce it → change one variable → document.

Specimen types at a glance

Transport and stability

The aim is to deliver living, dividing-capable cells. General principles the exam expects:

• Keep it viable, not frozen. Cytogenetic specimens are transported at room temperature or refrigerated per SOP — never frozen, which destroys cells. (Freezing is for DNA/molecular aliquots, not culture.)
• Speed matters. Longer transit lowers viability and mitotic yield. Prolonged delay is a leading cause of culture failure.
• Sterility matters. Contamination introduced at collection or transport can overgrow a culture.
• Avoid clotting. A clotted blood or marrow specimen traps the cells you need.

Accessioning & chain of identity

Identity is everything in a result that drives clinical decisions. Universal principles:

• Use two unique patient identifiers (e.g., name + date of birth or medical record number) and verify the requisition matches the container label.
• Assign an accession/case number that follows the specimen through culture, harvest, slide making, analysis, and reporting.
• Label every tube, flask, slide, and image to preserve an unbroken chain of identity. An unlabeled flask in an incubator is a non-reportable specimen.
• Record specimen type, collection time, and clinical indication — the indication drives culture setup and analysis depth.

Test priority & triage

Not all specimens are equal in urgency or fragility:

• Clinical urgency: prenatal results, suspected acute leukemia at diagnosis, and pre-transplant studies are typically prioritized.
• Viability/limited material: products of conception and small biopsies may have one chance — set up promptly and conservatively.
• Indication drives setup: a constitutional blood needs mitogen-stimulated culture; a marrow for leukemia needs unstimulated cultures that capture the dividing clone.

Stimulated vs. unstimulated — the central decision

What is in the culture system

• Base medium (e.g., RPMI-type for blood/marrow) supplies nutrients and buffering.
• Serum (commonly fetal bovine serum) supplies growth factors.
• L-glutamine as an amino-acid source; antibiotics (e.g., penicillin/streptomycin) to limit contamination.
• Mitogen as appropriate (above).
• Buffering: bicarbonate systems need a CO₂ incubator to hold pH; some media use alternative buffers.

Incubator conditions

Standard mammalian culture conditions the exam expects you to associate with healthy growth:

• Temperature drift stalls or kills growth.
• CO₂ failure shifts pH (medium turns purple/alkaline or yellow/acidic), harming cells.
• Low humidity concentrates the medium by evaporation, stressing cells.

Culture failure & contamination

The harvest sequence

1. Colcemid (a spindle inhibitor) arrests dividing cells in metaphase by preventing spindle formation, accumulating metaphases.
2. Hypotonic solution (commonly ~0.075 M KCl) swells the cells so chromosomes can spread apart when dropped.
3. Fixative — Carnoy's, 3:1 methanol:glacial acetic acid — preserves and hardens chromosomes and removes cytoplasm; performed as several changes of fresh, cold fixative.
4. Slide making (dropping) releases fixed cells onto slides; controlled spreading produces analyzable metaphases.

Hypotonic and fixation failure modes

Slide making & the role of the environment

Spreading is famously sensitive to humidity and temperature. These are the variables techs adjust day to day:

• Higher humidity / warmth generally increases spreading (fixative evaporates slower, cells flatten more).
• Low humidity / cold/dry air tends to reduce spreading → overlapped chromosomes.
• Drop height, slide angle, slide temperature, and cell concentration all influence the result.

G-banding (GTG): the workhorse

GTG = G-bands by Trypsin using Giemsa. Slides are briefly digested with trypsin (a protease) and then stained with Giemsa. The result is the familiar pattern of dark and light transverse bands that lets you tell chromosomes apart.

• Dark G-bands — AT-rich, gene-poor, late-replicating chromatin.
• Light (pale) G-bands — GC-rich, gene-rich, early-replicating chromatin.

The major banding / staining methods

Magnification & the scanning workflow

• Low power (e.g., 10×) — scan the slide to locate metaphases and judge spread density/quality. You cannot analyze bands here.
• High power (100× oil immersion) — resolve individual bands for analysis and karyotyping. Oil matches the refractive index of glass to preserve resolution.
• Workflow: low-power scan → record coordinates of good metaphases → return at high power to count, analyze, and capture.

Brightfield vs. fluorescence; automated systems

Brightfield microscopy is used for G-banded analysis. Fluorescence microscopy is used for FISH: a high-intensity source (mercury, metal-halide, or LED) excites fluorophores, and filter cubes matched to each fluorophore pass only its emission wavelength. Using the wrong filter makes a real signal invisible.

Digital imaging / karyotyping systems capture the metaphase and help separate and arrange chromosomes into a karyogram; automated metaphase finders scan slides and flag candidate metaphases. Both are assistive—the technologist confirms counts, corrects pairings, and makes the final call.

Imaging troubleshooting quick map

The three quality tiers (a nested hierarchy)

They are nested: every karyotypable cell is analyzable, and every analyzable cell is countable—but not the reverse. A short, fuzzy spread may be countable but not analyzable; a good spread with one stubborn overlap may be analyzable but not ideal to karyotype.

Quality factors you are grading

• Spreading — separated (good) vs. overlapping (touching/crossing) vs. scattered (blown apart, chromosomes lost).
• Length / contraction — longer chromosomes = more bands = higher resolution; short/contracted = fewer bands.
• Band sharpness — crisp, well-differentiated bands vs. fuzzy/washed-out (trypsin or contraction issues).
• Completeness — a full complement present; random missing chromosomes suggest artifactual loss.
• Cleanliness — minimal cytoplasm, debris, or overlapping nuclei.

How many cells? (concept, not a universal number)

The number of cells counted, analyzed, and karyotyped depends on case type, indication, accreditation requirements, and laboratory SOP. Conceptually, a routine constitutional study counts a screening set, analyzes a subset in detail, and karyotypes a few representative cells—with more cells scored when mosaicism is suspected or when a clone must be confirmed or excluded. Do not memorize a single "magic number"; recognize that suspected mosaicism increases the count.

Karyogram vs. karyotype

The karyogram (or idiogram-style layout) is the picture—chromosomes cut out and arranged in order. The karyotype is the ISCN description of that picture (e.g., 46,XY). The image is built so the description can be verified by anyone who looks at it.

Figure 8.1 — Normal human male karyotype (G-banded), 46,XY

[Image did not load] A normal male karyogram: 22 autosome pairs in size/group order (A–G) plus X and Y, each p-arm up. View it at the source link below.

Arrangement conventions

• Order: autosomes 1→22 grouped A–G (largest to smallest within each group), then the sex chromosomes (X, then Y).
• Orientation: short p arm up, long q arm down, centromeres aligned on a common axis.
• Pairing: homologs placed side by side; band patterns should match band-for-band.

Build sequence & review

1. Separate each chromosome from the captured metaphase (software-assisted; tech corrects boundaries).
2. Classify by size, centromere position, and banding pattern.
3. Pair homologs; orient p-up; arrange in standard order.
4. Review: compare the karyogram back to the original metaphase—no chromosome added, lost, clipped, or duplicated—and confirm any abnormality is present in the cell itself.

The identification triad

1. Size — overall length relative to the rest of the spread.
2. Centromere position — metacentric (central), submetacentric (off-center), or acrocentric (near one end).
3. Banding pattern — the reproducible dark/light signature that confirms the exact chromosome.

Groups A–G (Denver classification)

Landmarks: constrictions & satellites

• Secondary constrictions (qh): heterochromatic regions at 1q, 9q, 16q—sizes vary as normal variants.
• Satellites & stalks: on the short arms of the acrocentrics (13, 14, 15, 21, 22)—the NOR-bearing regions. The Y is acrocentric-shaped but lacks satellites.

The classic mix-ups (and how to break them)

Aneuploidy vs. polyploidy

• Aneuploidy — gain or loss of individual chromosomes. Trisomy (+1 copy), monosomy (−1 copy). Usual mechanism: nondisjunction (meiotic → constitutional; mitotic → mosaic) or anaphase lag.
• Polyploidy — whole extra haploid sets: triploidy = 69, tetraploidy = 92.

Autosomal trisomies seen in liveborns: 21 (Down), 18 (Edwards), 13 (Patau). Sex-chromosome aneuploidies: 45,X (Turner), 47,XXY (Klinefelter), 47,XXX, 47,XYY.

Figure 10.1 — Trisomy 21 (Down syndrome) karyotype, 47,XY,+21

[Image did not load] A karyotype with three copies of chromosome 21 (trisomy 21). View it at the Wellcome Collection link below.

ISCN for numerical findings

More examples:

The within-chromosome rearrangements

Reading a deletion string

Two breakpoints in the same arm mean the segment between them is gone (interstitial). A single breakpoint—e.g., del(5)(p15.2)—means everything distal to it is lost (terminal).

Isochromosome logic

An i(17q) has two long arms of 17 and no short arm—so it produces gain of 17q and loss of 17p at once. (i(17q) is a recurring finding in some myeloid malignancies; isochromosome Xq appears in some Turner variants.)

Between-chromosome rearrangements

Reciprocal translocation & Robertsonian

The Robertsonian fuses two acrocentric long arms into one chromosome and discards the small short arms—so the total count drops to 45 while almost all genetic material is retained. Carriers are usually healthy but have reproductive risk (e.g., translocation trisomy in offspring). rob(13;14) is the most common in humans.

Inversions at a glance

inv(2)(p21q31) is pericentric—breakpoints in both arms, so the inverted segment spans the centromere. inv(3)(q21q26) is paracentric—both breakpoints on the q arm, centromere untouched. Pericentric inversions are easier to spot because they can alter the centromere position/arm ratio; paracentric inversions are subtle and easy to miss.

Rings & markers

A ring forms when both ends are lost and the broken ends join; rings replicate unstably, producing secondary-cell variation (a form of dynamic mosaicism). A marker is extra material that banding can't assign—reported as +mar and then characterized with FISH (centromere/region probes) or microarray. A small supernumerary marker is an "SMC/sSMC."

Cancer (acquired) cytogenetics

• The Philadelphia (Ph) chromosome is the small der(22) from t(9;22)(q34;q11.2), creating the BCR::ABL1 fusion—hallmark of CML (and some ALL).
• Clone size is given in [brackets]: the number of cells with that karyotype.
• Stemline (sl) = the main clone; sideline (sdl) = a related subclone from clonal evolution; cp = composite karyotype when abnormalities are distributed across cells.
• Gene-fusion nomenclature uses a double colon (BCR::ABL1); chromosome nomenclature still uses the t(9;22) form.

Mosaicism vs. chimerism

After allogeneic bone-marrow transplant, a recipient's marrow may show the donor karyotype—a form of chimerism, not a new mosaic clone. Context (transplant history) is essential to interpret it.

Pseudomosaicism vs. true mosaicism (prenatal logic)

An abnormality seen in a single cell (or single colony) is usually pseudomosaicism—a culture/technical artifact. An abnormality in multiple colonies across independent cultures is more likely true mosaicism. Colony/in-situ analysis and counting across cultures drive this call. Beware confined placental mosaicism (CPM): an abnormal clone in placenta (CVS) that may not reflect the fetus.

Culture artifacts & pseudoclones

• Artifactual loss — random single-chromosome loss (mimics monosomy); the higher clone threshold for loss exists to guard against this.
• Endoreduplication / tetraploidy — can be an in-vitro artifact.
• In-vitro pseudoclones — culture-acquired changes (e.g., certain trisomies in long cultures, age-related loss of Y) that are not the patient's constitutional/disease karyotype.
• Scatter/breakage from harsh handling (Module 4) versus genuine instability.

Normal variants & instability

Benign heteromorphisms include inv(9)(p12q13) (a common pericentric inversion of chromosome 9), large 1qh/9qh/16qh, satellite-size variants, and Yqh length—none pathogenic. By contrast, chromosome-instability syndromes (exam concept) show excessive breakage/rearrangement—e.g., increased breaks with specific agents in Fanconi anemia, elevated sister-chromatid exchange in Bloom syndrome—and are assessed with breakage studies.

The grammar of an ISCN string

Read it as three slots: [modal number] , [sex chromosomes] , [abnormalities]. Abnormalities are listed in a set order: sex-chromosome abnormalities first, then autosomes in numerical order; within a given chromosome, numerical changes precede structural ones.

Band addresses & breakpoints

A locus is read chromosome → arm → region → band → .sub-band: in 17q21.31 → chromosome 17, long arm, region 2, band 1, sub-bands 3 then 1. Use pter/qter for arm tips and cen for centromere. Single breakpoint = terminal event; two breakpoints in one parenthesis = interstitial; chromosomes separated by ; in interchromosomal events.

Constitutional & mosaic

Mosaic constitutional: mos 47,XXY[12]/46,XY[18]. Benign variant on a normal background: 46,XX,inv(9)(p12q13) (report as a heteromorphism, not pathology).

Acquired (clonal) with clone sizes

Subclones from clonal evolution are written with the stemline/sideline relationship; a composite karyotype (cp) is used when abnormalities are distributed across cells rather than all present together in each cell.

FISH nomenclature

• Interphase, normal dual-color: nuc ish(ABL1,BCR)x2 — two signals of each, no fusion.
• Interphase, fusion present: uses con (connected/fused) to show the abnormal juxtaposition of the two loci (BCR::ABL1-type pattern). Exact signal counts depend on probe design (e.g., dual-fusion).
• Break-apart positive: uses sep — the two colors of one probe are separated, indicating a rearrangement at that locus.
• Metaphase deletion: ish del(7)(q31q31)(D7S486-) — the locus-specific probe is absent (−); + denotes present.
• Amplification: amp denotes many copies of a target.

Microarray (exam-level)

Microarray results use arr with the genome build, region, and copy number: e.g., a single-copy loss reads as a region ×1 and a gain as ×3; long stretches of homozygosity (ROH/AOH) are reported separately. The exact coordinate format is build- and lab-convention dependent—recognize the arr prefix, the copy-number call, and that microarray gives dosage, not breakpoint mechanism.

How FISH works; interphase vs. metaphase

A fluorescently labeled DNA probe hybridizes to its complementary target; after denaturation, hybridization, washing, and a DAPI counterstain, signals are read under fluorescence.

Probe designs & their signal logic

Artifacts, cutoffs & controls

• Coincidental overlap — in interphase, two signals can randomly sit together and mimic a fusion. Cutoffs (from normal controls, commonly mean + 3 SD) define the false-positive threshold a result must exceed.
• Split signals — a replicated locus appears as a doublet and can be miscounted as an extra copy.
• Nuclear truncation (FFPE) — sectioning cuts through nuclei, so a signal can be physically absent—mimicking a deletion. Adequate section thickness and controls guard against this.
• Controls & scoring — positive and negative controls each run; score a defined number of intact, non-overlapping nuclei (count is SOP/probe-dependent); often two independent scorers.

Special situations

• Plasma-cell myeloma: malignant plasma cells are sparse, so FISH sensitivity drops unless you enrich (e.g., CD138 selection) or target plasma cells with cytoplasmic-immunoglobulin FISH (cIg-FISH). Unenriched myeloma FISH can be falsely negative.
• FFPE workflow: deparaffinize and pretreat before hybridization; interpret with the truncation caveat above.
• Validation: every probe/assay is validated (localization, cutoffs, performance) before clinical use, per CAP/CLIA expectations.

Chromosomal microarray (CMA): power and blind spots

Microarray scans the genome for copy-number gains and losses at far higher resolution than banding. SNP arrays additionally reveal regions of homozygosity (ROH/AOH), flagging possible uniparental disomy or consanguinity. But CMA reports dosage, not mechanism, and it has hard limits:

Safety on a cytogenetics bench

Specimen identity & chain of identity

Use two independent patient identifiers at collection and label-check at every step. Identity must be traceable and unbroken from accessioning → culture → slide → image → karyotype → report. A break anywhere can invalidate the result.

Equipment, reagent & environmental monitoring

• Incubators: temperature (~37°C) and CO₂ (~5%) monitored/logged; humidity maintained.
• Cold storage: refrigerator/freezer temperatures logged; reagents/probes stored to spec.
• Devices: biosafety cabinet certification, centrifuge checks, pipette calibration, water-bath temperature.
• Reagents/probes: lot tracking, expiration, and QC before clinical use.

Quality indicators & CQI

Labs track metrics such as turnaround time (TAT), culture-failure rate, band resolution achieved, analytical error rate, probe/hybridization-failure rate, and specimen-rejection rate. Out-of-range indicators trigger corrective action and root-cause analysis within a continuous-quality-improvement (CQI) cycle.

Proficiency testing & regulation

Confidentiality, competency & ethics

• HIPAA / privacy: protect patient information; share results only with authorized parties.
• Competency assessment: personnel competency is evaluated on a defined schedule for high-complexity testing.
• Ethics & integrity: report accurately; never fabricate or alter results or images; handle incidental findings per policy; maintain professional conduct.