LABFLOW IN STAINING

• After cell culture and harvest (with subsequent cell synchrony and chromosome extension), the chromosomes can be stained using conventional banding techniques, or undergo FISH or PCR

  
• Chromosome banding techniques


  • G staining
  • Q staining
  • C staining
  • R staining
  • T-staining
  • NOR staining
  • CD staining

  
• Prenatal testing samples


  • Amniotic fluid
  • CVS
• Prenatal testing purpose


  • Early genetic sex testing (using CVS)
  • Genetic abnormalities (for informed parental decisions)
• Constitutional chromosome analysis samples

  • Blood
  • Bone marrow
  • Biopsies
• Constitutional chromosome analysis purpose

  • Testing suspected genetic diseases (leukemias, etc)
  • Testing for familial traits (Adult-onset diseases: Huntington's, Alzheimer's, schizophrenia; cancer)

  
• Chromosome analysis is done after staining. Manual analysis using photomicrography has phased out and automated chromosome analysis machines have been employed in recent years

  
• Metafer 4


  • Modern technology allows machine-automated capturing of interphase and metaphase samples for study

  
• In combination, chromosomes can also be individually captured and arranged
• Specific abnormalities can also be automatically identified
• Manual karyotyping is easier with machine-assisted AI
• Chromosome analysis is requested for referring suspected diseases

  
• Chromosomes are arranged and identified based on size and banding patterns. Ideograms established by the ISCN are used as a standard

  
• Banding may differ based on how condensed chromosomes are. As such, it is important to be aware of any synchrony or extension procedures performed
• Indicator chromosomes


  • The dark bands of the chromosome regions such as X, 10, 11p, 12q and 17q can be used as quality indicators
  • If these areas are adequately banded, the finer bands display a wide range of gray values and appear distinct and separate

  
• Disadvantages of chromosome banding

  • Chromosome banding requires fresh tissue. Formaldehyde damages DNA through fragmentation, base modification, and cross-linkages within the DNA itself or between different DNA
  • Balanced translocations that are passed onto offspring tend to become unbalanced translocations. Unbalanced translocations are therefore difficult to detect without prior identification of the parents' karyotype. Use of comparative genomic hybridization (CGH) is preferred for unbalanced translocations
  • Labor intensive. Without automation, routine cytogenetic testing requires highly trained professionals. Cell culture is also very difficult, and is open to failure

  
• Other disadvantages of chromosome banding

  • Difficult for diagnoses of diseases involving low mitotic index. Cells in low-grade tumors or tumors with low proliferation fraction can make it difficult to harvest cells (CLL, multiple myeloma, etc)
  • Long turn-around time (TAT). Chromosome analysis (karyotyping) can take up to 14 days for most specimen. Peripheral blood samples can take ≤21 days, solid tumor biopsies can take ≤30 days, tumors can take ≤42 days
  • Technical difficulties can arise, and microscopists must keep an eye out for complex chromosome patterns. This can include patients with mosaicism (ie 47,XX/48,XXX), patients with multiple diseases, and samples with multiple abnormal cell lines
  • Even with cell synchrony and cell extension methods, some minute deletions (microdeletions) can be difficult to observe
• FISH allows for the study of genetic aberrations that are too small to visualize by routine cytogenetic studies and too large to detect using standard DNA sequencing

  
• FISH


  Involves the binding, or annealing, of fluorescence-labeled, target-specific nucleic acid probes to their complementary DNA or RNA sequences and the subsequent visualization of these probes within cells in the tissue of interest

  
• Algorithm for FISH analysis

  1. DNA or RNA sequences from the tissue of interest are allowed to denature to become single stranded

  2. A FISH probe is selected and applied

  3. Fluorescence labeling of the probe can be done either directly or indirectly

  4. The fluorescent-labeled probe and the target DNA or RNA sequences are brought together in the hybridization process

  5. Posthybridization washings remove excessive unbound probe

  6. The slide is then read under a fluorescent microscope

  
• Direct fluorescence labeling


  The fluorochrome(s) to be detected by the fluorescence microscope is directly bound to the probe DNA
• Indirect labeling


  A hapten, which is not visible under a fluorescence microscope, is incorporated into the probe DNA. The hapten is then detected immunohistochemically by a fluorophore-tagged antibody directed against the hapten

  
• FISH is considered as the gold standard technique to confirm the diagnosis of microdeletion syndromes
• FISH advantages


  • FISH can be done on both dividing and non-dividing cells. Does not require culturing, but can be done on cultured cells
  • Allows for disease monitoring
  • The tissue of interest can either be formalin-fixed, paraffin-embedded sections or fresh-frozen tissue
  • Allows for correlation with morphology
  • Allows choice of a specific area for evaluation
  • Can detect minute deletions that karyotyping could not
  • Fast turn-around time
  • High-efficiency and specificity
  • More sensitive than karyotyping or CGH
  • High detection rate
  • Large number of cells can be analyzed
  • SKY (M-FISH) techniques provide an overall evaluation of the whole genome and help to direct further analysis
• Spectral karyotype (SKY), also known as Multicolor FISH (M-FISH), allows for the staining of entire chromosomes with a distinct color

  
• FISH disadvantages


  • It is NOT a screening test. Unless you are suspecting a specific disease, it is unlikely that you test every single probe for every single genetic disease to identify a genetic anomaly. Some probes even share the same fluorophore based on what the lab has
  • Requires specific probes. Most cytogenetic laboratories have probes for common genetic anomalies such as probes for the BCR/ABL gene for CML, or centromere probes for only a few chromosomes (commonly trisomy 1, 9, 13, 18, 21 and X)
  • Some probes have a low resolution. Although higher than conventional karyotyping. The resolution of FISH is in the range of 20-200Kb (kilobase) which is 5-10x higher than G-banding at 5-10Mb (megabase)
  • Less sensitive than PCR-based assays
  • Tumor aneuploidy may result in inappropriate signal counts
  • Susceptible to artifacts in sections from paraffin-embedded tissues
  • Cross-hybridization artifacts
  • Truncation artifacts (nuclei are not intact)

  
• Comparative genomic hybridisation (CGH) is a technique that permits the detection of chromosomal copy number changes without the need for cell culturing

  
• CGH


  • Provides a global overview of chromosomal gains and losses throughout the whole genome of a tumour
  • Has an improved resolution of 5–10Mb (megabases) compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized

  
• Microarray-based comparative genomic hybridization (aCGH)

  Can detect copy number changes down to 5-10Kb

  
• aCGH


  1. Reference (normal) DNA is labelled red while the test DNA is labelled green. Both labels are mixed with a 1:1 ratio

  2. The green and red labelled DNA fragments compete for hybridisation to their locus of origin on the chromosomes

  3. The green to red fluorescence ratio measured along the chromosomal axis represents loss or gain of genetic material in the tumor at that specific locus

  
• Using a fluorescence microscope and computer software, the differentially coloured fluorescent signals are then compared along the length of each chromosome for identification of chromosomal differences between the two sources

  
• A higher intensity of the test sample color in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample color indicates the loss of material in the test sample in that specific region
• aCGH advantages


  • The main advantage of CGH is that it can detect copy number variations as small as several hundred bases
  • Turnaround time is roughly the same as regular karyotyping, but with a higher degree of analysis
• aCGH disadvantages


  • CGH is only able to detect unbalanced chromosomal abnormalities: deletions, duplications, insertions
  • Balanced chromosomal abnormalities such as reciprocal translocations, inversions or ring chromosomes do not affect copy number. CGH cannot detect balanced translocations and mosaicism due to this
• To perform FISH, one must know what one is "FISHing" for
• Compared with FISH, CGH is more expensive and has a longer TAT. Paraffin block preparation is required because cells must be microdissected in CGH, unlike with FISH
• Polymerase chain reaction (PCR), developed by Mullis and Faloona, is a core technique for most tests used in molecular diagnostics
• PCR


  • Fresh tissue is the best sample source for PCR analysis, but fixed tissue (formalin, ethanol) may also be used
  • Three major steps: denaturation of double-stranded DNA into single-stranded DNA (ssDNA) at high temperature (95°C), hybridization (annealing) of oligonucleotide primers to both ends of a target sequence, synthesis achieved by addition of four nucleotide bases and a Taq polymerase

  
• PCR enables the detection of malignant cells below the threshold of karyotyping or morphology, even when combined with immunophenotyping
• PCR targets a segments of DNA or RNA (RNA PCR) and produces multiple copies of a DNA region of interest