Standards and Guidelines for Clinical Genetics Laboratories
2006 Edition.

E1 Cell Culture

See D1.

E2 Records
E2.1 Retention of Case Materials

In addition to the general guideline (C3.6) for duration of retention of case materials, the following are specific to cytogenetics.

E2.1.1 Slides used for diagnostic tests have a limited lifespan. If stained with a "permanent" banding method (G-, C- or R-banded, NOR), slides should be kept at least 3 years or in compliance with state regulations. Retention time of those with fluorochrome stained chromosomes is at the discretion of the laboratory director.
E2.1.2 Each laboratory should establish a policy to assure that any residual original patient specimens, cell cultures or pellets are retained until adequate metaphase preparations are available to complete the requested analysis.
E2.1.3 Processed patient specimens should be retained until the final report has been signed. Long-term retention time of those with abnormal results is at the discretion of the laboratory director.
E3 Procedural Guidelines
E3.1 General Analytical Standards
E3.1.1 Terminology

Chromosome counts are defined as the number of centric chromosomes per metaphase cell. During the establishment of the modal number for a study, all aneuploid metaphase cells should be characterized for specific gain/loss.

Analyzed cells are defined as banded metaphase cells in which the individual chromosomes are evaluated in their entirety, either at the microscope or from intact digitized images or photographic prints of intact cells.

Karyotyped cells are defined as the cutout and paired chromosomes from photograph(s) or computer-generated image(s) from a single cell following the format in An International System for Human Cytogenetic Nomenclature 1995 (ISCN 1995).

Scored cells refers to cells evaluated for the presence or absence of a specific cytogenetic feature, usually indicated by either a particular clinical history or by the finding of one or two abnormal cells during the course of a study. Numbers of cells to be scored in most situations are left to the discretion of the laboratory director, unless otherwise specified in the guidelines.

A colony is defined as a discrete focus or clone of cells that is harvested and stained while attached to the cell culture growth substrate.

An abnormal clone is present if at least two cells contain the same extra chromosome(s) or structural chromosome abnormality or if at least three cells have lost the same chromosome.

E3.1.2 Slide number and microscope stage coordinates should be recorded for all metaphases analyzed or counted. If additional cells are evaluated in questions of mosaicism, slide number should be recorded for all cells that are scored and slide coordinates should be recorded for all abnormal metaphases or suspected abnormal metaphases.
E3.1.3 All laboratories must be able to perform studies using G- and/or R-banding, in addition to special stains and/or FISH, to characterize polymorphisms, when indicated and at the discretion of the laboratory director.
E3.1.4 ISCN 1995 must be used to describe all karyotypes.
E3.1.5 A number of different objective methods have been described for the calculation of band stage of resolution [1,2,3]. One or more objective and reproducible method(s) must be used to assess banding level of resolution and must be formally described in laboratory standard operating procedures/protocol manual. Specific standards for resolution should be appropriate to the case and type of tissue studied. The 550-band stage should be the goal of all constitutional studies to rule out a structural abnormality, particularly in cases of mental retardation, birth defects, dysmorphology, or couples with recurrent pregnancy loss.
E3.1.6 Minimum standards established for the numbers of cells to count and/or analyze and karyotype during the "routine" component of a cytogenetic study are described in specific subsections appropriate to a specific tissue type, culture method and/or reason for referral. The numbers of cells to study in individual situations is dependent on the specific abnormality observed, the tissue being examined, whether the analysis involves prenatal diagnosis, etc. General recommendations are noted in the following subsections.
E3.1.6.1 Each laboratory should establish guidelines for procedures (e.g., numbers of cells to score) to follow for each general type of abnormality (hypodiploidy, hyperdiploidy and structural abnormality) with the recognition that uniformity among laboratories is not required.
E3.1.6.2 Guidelines should be based on current knowledge of the potential clinical significance of particular chromosome abnormalities and nonmodal cells.
E3.1.6.3 Fewer cells than indicated under analytical standards may be studied in circumstances in which screening for a specific abnormality is the indication for the study (e.g., checking for a known familial abnormality) or when an abnormality is detected but no more cells are available (see following section).
E3.1.7 Analyses should be performed and/or evaluated by at least two qualified individuals.
E3.2 Abbreviated, Focused or Limited Chromosome Studies
E3.2.1 General Considerations
E3.2.1.1 It is acknowledged that there are specific clinical circumstances for which an abbreviated or limited cytogenetic study may be appropriate. For example, in the tissue confirmation of an abnormal prenatal chromosome result or in peripheral blood chromosome studies on extended family members to exclude an identified chromosome rearrangement, limited analyses may be suitable.
E3.2.2 Analytical Standards

The laboratory should have established written criteria for which focused or abbreviated studies are permissible. Criteria should specifically address the rationale for such studies, the clinical reason for referral, the tissue type, and the minimum number of cells counted, analyzed and karyotyped under such circumstances.

E3.3 Maternal Cell Contamination (MCC)
E3.3.1 Maternal cell contamination of amniotic fluid and chorionic villi cell cultures is well documented and therefore represents a potential source of error in prenatal diagnosis. Adequate measures to minimize the inclusion of maternal cells in prenatal samples should be part of the laboratory quality assurance program.
E3.3.1.1 Amniotic Fluid

The overall frequency of MCC is approximately 0.5% of genetic amniocenteses [4]. Factors that increase the chance of MCC include the gauge of needle used for the amniocentesis procedure [5], the length of time in culture and the presence of blood in the sample.

It has also been documented that cultures initiated from the first 1-2 ml of amniotic fluid drawn at amniocentesis are at an increased risk for maternal cell contamination [5]. It is recommended that the first few milliliters of fluid be labeled appropriately and kept separate from the remaining sample to minimize inclusion of maternal cells. The initial aliquot should be used for cytogenetic analysis only if absolutely necessary.

E3.3.1.2 Chorionic Villi Sampling (CVS)

The risk for MCC in CVS is significantly higher than for amniocentesis samples (1-2%) [6]. A CVS specimen must be viewed under a dissecting microscope to allow for the gross identification and cleaning of villi from maternal decidua, blood vessels, membrane and other materials. It is recommendred that sterile instruments (e.g., probes, scissors, tweezers) be used to tease apart the sample to isolate the fetal chorionic villi from maternal decidua. It may be helpful to have two laboratory technologists clean or check the dissected tissue prior to initiating cultures.

E3.3.1.3 Products of Conception

Due to the manner in which abortus tissue and placenta samples are obtained and handled, there is a substantial risk of MCC, particularly in early fetal loss specimens. It is recommended that appropriate measures be taken to specifically identify fetal tissues and to dissect and culture only these tissues, as described above for prenatal CVS. Consultation with the referring physician may be warranted to determine the origin of the sample and/or the appropriateness of chromosome studies, particularly in cases for which the dissection of tissue appears to yield only maternal decidua.

E3.3.2 Analysis of Cultures with Known or Suspected MCC

Cultures with known or suspected MCC based on the condition of the specimen at receipt, or apparent maternal cells morphologically in culture, require variation in the normal analysis procedure. If XX cells are found in an otherwise XY study, the most likely explanation is MCC. Since the true fetal cells are probably represented by the XY complement, the full analysis and cell counts should be performed on these cells whenever possible. Counting and analyzing several cells with an XX constitution is recommended for documentation purposes. For prenatal testing, further studies may be warranted to exclude chimerism. Ultrasound examination to check the gender of the fetus, second amniocentesis or confirmatory amniocentesis after CVS and/or polymorphism studies (cytogenetic or molecular) between a maternal sample and the fetal sample may be required in the investigation.

If cell cultures that are initiated in the cytogenetics laboratory are to be used for molecular or biochemical testing, any serious concerns about MCC in those cultures must be conveyed to the molecular or biochemical testing laboratory. In addition, if direct prenatal samples are sent out for testing, it is recommended that back-up cultures be grown and maintained until the molecular or biochemical testing is complete and reported.

E3.3.3 MCC Reporting and Quality Assurance

Reporting of MCC is case-dependent and is at the discretion of the laboratory director. Consultation with the referring physician is recommended, when appropriate. Any significant observation of MCC in a prenatal diagnosis sample should be interpreted in consultation with the physician who performed the procedure. For samples with a significant risk for MCC that produce a normal female karyotype, a disclaimer should be added to the report suggesting that analysis of maternal cells due to MCC cannot be excluded.

Any time that MCC is suspected or confirmed, the laboratory director must ensure that an attempt to determine the cause is documented as part of the laboratory's quality assurance program. Additionally, it is recommended that the ratio of XX:XY cases be monitored as a quality control check for CVS and POC cases.

E4 Prenatal Diagnosis
E4.1 Amniotic Fluid, Chorionic Villi and Percutaneous Umbilical Blood Sampling (PUBS)
E4.1.1 General Standards
E4.1.1.l At least two independent cell cultures must be initiated and grown in separate incubators.
E4.1.1.2 With the exception of PUBS, there must be a plan for maintaining back-up cell culture(s) pending the need for additional studies.
E4.1.1.3 If studies of parental chromosomes are necessary to help identify a fetal chromosome abnormality or heteromorphism, the same laboratory should perform these, if possible and reasonable under the specific constraints of the clinical indications.
E4.1.1.4 The number of test failures (defined as failure to obtain final results from a submitted specimen) should not exceed 1 per 100 consecutive samples (1%).
E4.1.1.5 Efforts must be made to determine the cause of all test failures. These records and records of corrective actions taken must be available for external review and kept for at least 1 year.
E4.1.1.6 With the exception of PUBS, at least 90% of final results must be completed and reported (verbal or written) within 14 calendar days from receipt of specimen, unless additional studies are necessary.
E4.1.1.7 Laboratories failing to meet these standards should send samples to another laboratory until the problems are resolved.
E4.1.1.8 Abnormal diagnostic results should be confirmed by follow-up cytogenetic studies, to the extent possible.
E4.1.2 Amniotic Fluid: Processing Standards
E4.1.2.1 If little or no cell pellet is apparent in the sample, the laboratory should consider the use of a method (e.g., assays for pH, protein, glucose, etc.) that will help to distinguish amniotic from other fluids.
E4.1.2.2 Notification of inadequate cell culture growth should be made within 14 days of the amniocentesis procedure.
E4.1.2.3 A laboratory planning to establish amniotic fluid cytogenetic testing must arrange to split and successfully analyze at least 50 consecutive specimens with a laboratory performing such studies by established standards.
E4.1.3 Amniotic Fluid: Analytical Standards (see also E3.1.6)
E4.1.3.1 Flask Technique
  1. Count: a minimum of 20 cells, distributed as equally as possible between at least 2 independently established cultures. Document any numerical/structural aberrations observed.
  2. Analyze: 5 cells, distributed between at least 2 independently established cultures. Resolution should be appropriate to the reason for testing.
  3. Karyotype: 2 cells. If more than 1 clone (as defined in Section E3.1.1) is found, karyotype 1 cell representative of each clone identified.
E4.1.3.2 In Situ Technique
  1. Count: a minimum of 15 cells from at least 15 colonies (15 cells from at least 10 colonies if 15 colonies are not available), distributed as equally as possible between at least 2 independently established cultures. Document any numerical/structural aberrations observed.
  2. Analyze: 5 cells, each from a different colony, preferably from at least 2 independently established cultures. Resolution should be appropriate to the reason for testing.
  3. Karyotype: 2 cells. If more than 1 clone (as defined in Section E3.1.1) is found, karyotype 1 cell representative of each clone.
E4.1.4 Chorionic Villus Sample (CVS): Processing Standards
E4.1.4.1 When direct (uncultured) preparations are used clinically, a cell culture technique (defined as longer than 48 hours) must also be used.
E4.1.4.2 Final written reports should include a summary of the results of the cultured cells.
E4.1.4.3 A laboratory planning to establish CVS cytogenetics should already be testing amniotic fluid cells by established standards and methods. Prior to independent CVS analysis, the laboratory must:
  1. split and confirm at least 25 consecutive samples with a laboratory already performing CVS cytogenetics by established standards and methods, or
  2. perform CVS cytogenetics on 50 consecutive samples not used for diagnostic purposes and confirm that the karyotype of the abortus was identical to that of the CVS study, or
  3. a combination of a) and b) above.
E4.1.5 Chorionic Villi: Analytical Standards (see also E3.1.6)
E4.1.5.1 Direct (Uncultured) Preparations: not recommended for exclusive use in obtaining final results. (See Section E4.1.5.3 below.)
E4.1.5.2 Cultured Preparations, (Flask or Coverslip) Technique
  1. Count: a minimum of 20 cells distributed as equally as possible between at least 2 independently established cultures. Document any numerical/structural aberrations observed.
  2. Analyze: 5 cells, preferably from 2 independently established cultures. Resolution should be appropriate to the reason for testing.
  3. Karyotype: 2 cells. If more than 1 clone (as defined in section E3.1.1) is found, karyotype 1 cell representative of each clone.
E4.1.5.3 Combination of Direct Preparation and Culture Technique
  1. Count: a minimum of 20 cells, at least 10 of which come from cultured preparations. Document any numerical/structural aberrations observed.
  2. Analyze: 5 cells, preferably at least 4 cells from cultured preparations. Resolution should be appropriate to the reason for testing.
  3. Karyotype: 2 cells. If more than 1 clone (as defined in Section E3.1.1) is found, karyotype 1 cell representative of each clone.
E4.2 Fetal Blood: Percutaneous Blood Sampling (PUBS)
E4.2.1 Processing Standards
E4.2.1.1 Results of assays confirming the fetal origin of such specimens should be available to the laboratory.
E4.2.1.2 A minimum of 2 cultures should be established.
E4.2.1.3 Processing after 48 and 72 hours in culture is recommended.
E4.2.1.4 Final reports (verbal or written) should be available within 7 calendar days.
E4.2.2 Analytical Standards (see also E3.1.6)
  1. Count: a minimum of 20 cells.
  2. Analyze: 5 cells. Resolution should be appropriate to the reason for testing.
  3. Karyotype: 2 cells. If more than 1 clone (as defined in section E3.1.1) is found, karyotype 1 cell representative of each clone.
E5 Peripheral Blood and Solid Tissue Constitutional Chromosome Study
E5.1 Peripheral Blood (Stimulated Lymphocytes): Routine Studies
E5.1.1 Processing Standards
E5.1.1.1 At least 2 cultures should be established for each specimen.
E5.1.1.2 At least 90% of all routine peripheral blood analyses must have final written reports completed within 28 calendar days (21 calendar days is recommended) from receipt of the specimen. Clinical indications may dictate more rapid turn-around time. Specialized stains and studies may take longer.
E5.1.1.3 Test failures should not exceed 2% per year.
E5.1.1.4 The 550-band stage should be the goal of all constitutional studies to rule out a structural abnormality, particularly in cases of mental retardation, birth defects, dysmorphology, or couples with recurrent pregnancy loss.
E5.1.2 Analytical Standards (see also E3.1.6)
  1. Count: a minimum of 20 cells, documenting any numerical/structural abnormalities observed.
  2. Analyze: 5 cells. Resolution should be appropriate to the reason for testing.
  3. Karyotype: 2 cells. If more than 1 clone (as defined in Section E3.1.1) is found, karyotype 1 cell representative of each clone.
E5.1.2.2 Cases being studied for possible sex chromosome abnormalities, in which mosaicism is common, should include a minimum of 30 cells counted.
E5.2 Peripheral Blood (Stimulated Lymphocytes): Focused High

Resolution Analysis

E5.2.1 Analytical Standards
E5.2.1.1 Focused high resolution analysis should be reserved for cases in which a specific microabnormality syndrome is being considered, the diagnosis of which generally requires chromosomes above the 650-band stage (resolution at the 850 level is recommended). In addition, it can be applied to cases requiring the improved characterization of an identified chromosome abnormality. In many situations, in situ hybridization may be a supplemental approach to specific microdeletion analysis.
E5.2.1.2 General processing and analytical standards for routine peripheral blood studies apply. In addition, in a focused analysis, the primary analytical standard must be that the specific chromosomal region in question is visible and clearly separated from adjoining bands in both homologues, or in abnormal cases, in the normal homolog.
E5.3 Peripheral Blood (Stimulated Lymphocytes): Complete High Resolution Analysis
E5.3.1 Analytical Standards
E5.3.1.1 General processing and analytical standards for routine peripheral blood studies apply. In addition, complete high resolution chromosome analysis should always include a minimum of 3 pairs of each chromosome at a level of resolution above the 650-band stage (resolution at the 850 level is recommended).
E5.4 Peripheral Blood (Stimulated Lymphocytes): Heritable Fragile Sites (Including Fragile X)

This section initially provided guidelines for the evaluation of patients for fragile X syndrome using the cytogenetic expression of the Xq27.3 (FRAXA) fragile site. Such chromosome testing has been largely replaced by molecular genetic DNA evaluation of the FMR1 locus, and specific College recommendations have been published to cover such testing (see Section FX, "Technical Standards and Guidelines for Fragile X"). However, because some laboratories may still include fragile site screening in the chromosome study portion of fragile X testing, and cytogenetic testing for other heritable fragile sites is requested under some clinical circumstances, the following guidelines are recommended.

E5.4.1 Processing Standards
E5.4.1.1 Fragile site expression should be assessed using at least two different culture induction systems; i.e., low folate and an anti-metabolite for folate-sensitive fragile sites, BrdU and distamycin A for BrdU-inducible sites.
E5.4.2 Analytical Standards
E5.4.2.1 Scoring for a fragile site is analogous to the exclusion of chromosomal mosaicism, and at least 59 cells total from the two cultures should be scored.
E5.4.2.2 In reporting of results, fragile site nomenclature must follow ISCN 1995, and it is recommended that the report contain both the methods of induction and frequency of fragile site expression.
E5.4.2.3 Where possible, for clinically significant fragile sites, i.e., FRAXA, all fragile site positive individuals should be confirmed with molecular genetic testing. (Fragile site expression must not be used for fragile X syndrome [FRAXA] premutation carrier evaluation.)
E5.5 Solid Tissues (Skin, Organs, Products of Conception, etc.)
E5.5.1 Processing Standards
E5.5.1.1 Tissue biopsy specimens and small specimens should be transported in sterile cell culture medium with or without serum. Sterile saline solution may be used if medium is not available. Larger specimens should be transported according to written guidelines in each laboratory.
E5.5.1.2 At least two independent cultures should be established (three are recommended for resolving questions of mosaicism). These can be from explants of tissue grown in flasks or from enzyme-dissociated cells that can be processed in flasks or in situ.
E5.5.2 Analytical Standards

See amniotic fluid guidelines (E4.1.3) for analytical standards.

E5.5.2.1 When sex chromosome anomalies are suspected, at least 30 cells should be counted and scored for sex chromosome complement.
E5.5.3 Except for products of conception (POC), test failure rates should not exceed 5% per year, in total. It is suggested that periodic monitoring of POCs be done to assure that the ratio of 46,XX: 46,XY results approximates 1:1.
E5.6 Bone marrow studies for constitutional disorders for detection of a lethal abnormality (e.g., trisomy 13) in a newborn may be performed. However, these studies have largely been replaced by direct preparation or short term cultures of peripheral blood or interphase FISH analysis. When bone marrow studies are undertaken, these studies should include a routine peripheral blood evaluation unless bone marrow study achieves the 400-band level or a resolution level appropriate to the clinical indication for testing.
E5.7 Chromosome Instability Syndromes: Peripheral Blood Breakage Analyses
E5.7.1 General Standards

The rarity of chromosome instability syndromes requires that inexperienced laboratories should refer cases to reference laboratories with experience in diagnosing such disorders. Additionally, as research leads to the identification and cloning of the putative disease genes, molecular testing is recommended to supplement cytogenetic analysis.

E5.7.1.1 G-banded or unbanded preparations may be applied, depending upon the particular goal of the study. Unbanded preparations are acceptable only if there is no need to identify abnormalities such as translocations or inversions that will not be visible in unbanded preparations.

All abnormalities should be recorded using appropriate ISCN 1995 designations.

E5.7.2 Fanconi Anemia

Cytogenetic evaluation for Fanconi anemia (FA) should include analysis of crosslinking agent (e.g., mitomycin C [MMC], diepoxybutane [DEB]) induction of breakage in addition to baseline chromosome breakage.

E5.7.2.1 Culture Conditions

Each laboratory should have well-established negative control (non-Fanconi) and positive control (Fanconi) ranges for each culture (with and without mutagen) condition. Each new lot number of crosslinking agent should be appropriately quality controlled for its efficacy and potency for inducing chromosomal breakage. Given variability between drug lots, and the need to routinely prepare fresh stock and working solutions for most of the crosslinking agents, parallel testing of control specimens is recommended, as necessary. When a sufficient amount of blood specimen (and cell count) is available, two drug-treated cultures (e.g., either two different concentrations of either DEB or MMC, or one culture each of MMC and DEB) are recommended.

E5.7.2.2 Chromosome Breakage Analysis

Optimally, 50 metaphase cells (banded or unbanded) should be scored from each culture condition. The average rate of chromosomal aberrations per cell or the distribution of aberrations among cells should be compared to negative and positive control reference ranges. The percentage of cells demonstrating aberrations should be reported to enable identification of those patients who are mosaic for mutant and wild type cells.

E5.7.3 Bloom Syndrome

Cytogenetic evaluation for Bloom syndrome must include assessment of baseline sister chromatid exchange (SCE) rates.

As the Bloom syndrome gene BLM has been cloned, molecular evaluation to identify the mutation may be possible to supplement a positive cytogenetic result.

E5.7.3.1 Culture Conditions

Each laboratory should have well-established negative (non-Bloom syndrome) and positive (Bloom syndrome) control SCE ranges. Duplicate cultures should be established both from the patient and a concurrently processed negative control. Cultures should be supplemented with bromodeoxyuridine and maintained in darkness prior to harvesting. Cells should be cultured for the period of time predetermined by the laboratory to yield a high percentage of cells in second division. At the discretion of the laboratory director, cultures for evaluation of baseline chromosome breakage may also be established.

E5.7.3.2 Analysis

For confirmation of a negative result, fifty metaphase cells (25 from each of the two duplicate cultures) should be evaluated. For a positive result, fewer cells may be evaluated. However, it is recommended that both cultures from the patient and at least one from the concurrent control be sampled in the evaluation.

E5.7.4 Ataxia Telangiectasia and Nijmegen Breakage Syndrome

Cytogenetic evaluation for ataxia telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) should include specific screening for rearrangements involving chromosomes 7 and/or 14.

Evaluation for A-T and NBS should include evaluation of sensitivity to radiation. Although such sensitivity can be assessed by cytogenetic methods, it generally is evaluated by survival assays on lymphoblastoid or fibroblast cells.

As the (A-T) gene (ATM) and the NBS gene (NBS1) have been cloned, molecular evaluation may be helpful for confirming the diagnosis in patients who have positive cytogenetic and/or radiosensitivity assays.

E5.7.4.1 Culture Conditions

Each laboratory should have well-established negative (non-A-T, non-NBS) and positive (A-T or NBS) control chromosome 7/14 rearrangement and chromosome breakage rates. Duplicate stimulated cultures should be established for each patient case, and cultured for 48-96 hours identical to the conditions utilized for the establishment of the laboratory's control ranges.

E5.7.4.2 Analysis

Analysis should include 50 G-banded metaphase cells screened for rearrangements involving chromosomes 7 and/or 14. Overall breakage and rearrangement rates may also be helpful. It should be noted that, among confirmed A-T patients, the frequency of 7/14 rearrangements increases with age. Thus, negative results should be interpreted with caution. Among healthy controls, rearrangements of chromosomes 7 and 14 are well documented in PHA-stimulated cultures. Thus, comparison to laboratory norms for age-matched controls is essential.

E5.7.5 Miscellaneous

Cytogenetic evaluation of chromosome breakage may also be undertaken for other reasons, e.g., prior exposure to clastogens. The specific culture methods utilized (e.g., timing of cultures) and the methods of analysis (G-banded vs. unbanded chromosomes) should be appropriate to the referral. The laboratory should have well established positive and negative control ranges for the specific analyses being conducted.


Chromosome Studies for Acquired Abnormalities

Most cytogenetic analyses to detect and characterize acquired chromosomal abnormalities are performed to diagnose or monitor a premalignant or malignant disorder.

General Considerations
E6 (a) A patient with an acquired clonal chromosomal abnormality or one who is at high risk for developing such an abnormality may have multiple cytogenetic studies during the course of his or her disease.
E6 (b) Tissue processing, analytical variables and turn-around times should be determined by the laboratory based on the indication for the cytogenetic referral (e.g., initial diagnosis versus follow-up studies; pre- versus post-transplant studies) and the clinical application of the cytogenetic results (e.g., selection of therapy).
E6 (c) All results should, to the extent possible, be interpreted in the context of other clinical and laboratory findings.
E6 (d) The laboratory director and staff should be familiar with the various recurrent chromosome abnormalities associated with specific subtypes of malignancies that may be crucial for differential diagnosis.1,2
E6 (e) Monitoring the percentage of cases for which an abnormal clone has been identified is recommended for quality assurance assessment for the laboratory. Such monitoring is particularly valuable for cases of documented acute leukemia, in which the expected percentage with abnormalities is greater than 50%; a lower than expected rate can alert the laboratory to issues such as tissue processing or analysis guidelines.
E6 (f) For studies performed, the cytogenetics laboratory should obtain, at the time of receipt of the specimen, as much information as possible about the suspected pathologic diagnosis, preliminary morphologic, immunophenotypic and clinical findings, results of any previous cytogenetic testing and indication for the requested study.

Bone Marrow/Blood: Specimen Procurement and Processing

Only those cells involved in the disease process will harbor the abnormalities being sought. The culture techniques and methodologies utilized should optimize the probability of detecting an abnormal clone.


In most cases, bone marrow is the tissue of choice for analysis of suspected premalignant or malignant hematologic disorders.

In circumstances in which adequate bone marrow aspirate can not be obtained, alternative specimens may include:

E6.1.1 (a) Peripheral blood: (Blood specimens may yield informative results when the circulating blast count is higher than 10% to 20%.) In general, the abnormal clone can be identified in such specimens, albeit not as often as in bone marrow.3
E6.1.1 (b) Bone marrow core biopsy (discussed under specimen collection, Section E6.1.2):
E6.1.2 Specimen Collection
E6.1.2 (a) Bone marrow and blood specimens should be collected under sterile conditions in sodium heparin. The concentration of sodium heparin should be approximately 20 units per ml of specimen (per either bone marrow volume alone, or per total volume of bone marrow and transport medium combined). If collected in a green top tube, the smaller sized sodium heparin tube should be used.
E6.1.2 (b) The volume of bone marrow available will differ for adults and children. An approximate specimen of 1 to 3 ml should be requested when possible. During the procurement of the specimen, several draws are likely to be taken from the patient. As the first draw is more concentrated with blasts, it is recommended that cytogenetics receive first or second draw whenever possible.
E6.1.2 (c) Specimens should be received by the laboratory as soon as possible (ideally within 24 hours).
E6.1.2 (d) It is generally recommended that specimens be maintained at ambient temperature during transit. Extreme temperatures should be avoided.

E6.1.2 (e) If a bone marrow core biopsy is obtained, it should be minced to generate cell suspensions. This can be done mechanically or enzymatically (e.g. with collagenase), if it is resistant to mechanical dissociation. Culture conditions are the same as those for bone marrow aspirates.
E6.1.2.2 Culture conditions should be optimized for the specific hematologic disorder suspected:
E6.1.2.2 (a)

Acute leukemias: Unstimulated short-term cultures are recommended. If sufficient specimen is received, at least two cultures should be initiated, one of which should be designated as a 24-hour culture.
E6.1.2.2 (b) Aplastic anemia and chronic myeloproliferative disorders: Same as acute leukemias above.
E6.1.2.2 (c) Chronic lymphoproliferative disorders: Depending on the immunophenotype, additional cultures with B- or T-cell mitogens may be helpful.
E6.1.2.2 (d) Well-differentiated T-cell disorders (e.g., T-cell leukemia, T-cell lymphoma, Sezary syndrome, mycosis fungoides): T-cell mitogens may be helpful.
E6.1.2.2 (e) Mature B-cell disorders (e.g., plasma cell leukemia, multiple myeloma): Although there is no consensus on this point, some laboratories have had success in identifying abnormal clones with the addition of B-cell mitogens.
E6.2 Analytical Standards

Cell Selection

Cells selected for analysis should not be selected solely on the basis of good chromosome morphology. In general, the technologist should select an area of a slide to begin the analysis, and then examine cells as they appear consecutively in the microscope field, only skipping cells for which extremely poor morphology precludes chromosome identification.

When cells are skipped because of poor morphology, it is important to attempt to count the number of chromosomes (particularly for possible hyper- or hypodiploidy relevant in pediatric ALL). In addition, attempts should be made to identify possible structurally abnormal chromosomes, particularly if the disease under consideration is associated with a specific recurring chromosome abnormality such as t(9;22) in CML.


Number of Cells Evaluated

The number of metaphase cells analyzed versus the number of metaphase cells counted or scored should be appropriate to the type of study (e.g., initial diagnosis versus follow-up); the purpose of the study (e.g., detection of residual disease or response to therapy, monitoring for cytogenetic evolution or monitoring of engraftment of allogeneic transplant); and/or the characteristics of the specific chromosomal abnormalities present or suspected.


Initial Diagnostic Studies
E6.2.2.1 (a)

G-band analysis


Analyze a minimum of 20 cells from unstimulated cultures. A combination of unstimulated and mitogen-stimulated cultures may be appropriate for the mature B- and T-cell disorders as described above (Section E6.1.2.2). For all other diagnoses (e.g., acute leukemias), only when the abnormal clone is identified in the mitogen-stimulated cultures can the G-band analysis include less than 20 metaphases from the unstimulated cultures.


For the abnormal cells:

If only one abnormal clone is present: two karyotypes.

If more than one related abnormal clone is present: Two karyotypes of the stemline and one of each sideline.

If unrelated clones are present: Two karyotypes for each stemline and one for each associated pertinent sideline.

For the normal cells:

If only normal cells are present: two karyotypes.

If normal and abnormal cells are present: one karyotype of a normal cell.

E6.2.2.1 (b)

Supplemental FISH Analysis

Studies using FISH may be indicated to (1) provide a rapid result that will aid in the differential diagnosis or for planning induction therapy, or (2) to rule out a cryptic abnormality. Characterization of the initial diagnostic FISH pattern will also allow for future monitoring of the patient’s disease. Examples of such cases include, but are not limited to:

  1. Acute promyelocytic leukemia (AML-M3): FISH with PML/RARA gene probes to detect t(15;17).
  2. Chronic myelogenous leukemia: FISH with BCR/ABL gene probes for t(9;22) or variants thereof.
  3. Acute myelomonocytic leukemia with abnormal eosinophils (AML-M4eo): FISH with CBFB gene probe or other informative probe for inv(16) or t(16;16).
  4. Infant or childhood acute leukemia with high risk features: FISH with BCR/ABL and/or MLL gene probes.
  5. Precursor B-cell acute lymphoblastic leukemia most frequently seen in children ages 1 - 10 years: FISH with TEL/AML1 gene probes to detect a cryptic 12;21 translocation.
  6. Suspected Burkitt leukemia/lymphoma (acute lymphoblastic leukemia L3), FISH with CMYC/IgH gene probes or others to detect t(8;14) or variant translocations.
  7. B-cell CLL when a normal cytogenetic result is obtained by analysis of unstimulated and B-cell mitogen stimulated cultures to detect
    +12, del(13)(q14), deletions of the ATM (11q22.3) and p53(17p13.1) genes.

Other applications of FISH include (but are not limited to) interphase FISH when there is insufficient material for G-banded analysis and there is a suspected pathologic diagnosis associated with a recurring chromosomal abnormality; metaphase or interphase FISH to investigate the presence of a variant translocation suspected in the G-banding analysis by an abnormality involving a recurring chromosomal breakpoint.


Documentation of FISH results should be in accord with Sections E9 and E10 of these Standards and Guidelines for Clinical Genetics Laboratories.


Follow-up studies of patients who have had a previous cytogenetic study

For the following analytic guidelines, it is assumed that the laboratory has documentation of the patient’s previous cytogenetic results. If the study has been performed elsewhere, and there is minimal information available, it is recommended that, except for patients seen for the first time post-transplant, the analysis be considered the same as an initial diagnostic workup (see above).

E6.2.2.2 (a)

Patients who have NOT received allogeneic hematopoietic cell transplant (e.g., history of previous radiation and/or chemotherapy, autologous transplant, no prior therapy, etc.):


Analyze 20 cells. If all cells are normal, additional cells may be scored for a specific abnormality by G-banding or FISH.

*Note: For some patients, follow-up cytogenetic study is ordered to rule out a therapy-associated malignancy (e.g., MDS) rather than disease recurrence.


For cases with both normal and abnormal cells or only abnormal cells:

One karyotype of a normal cell, if such a karyotype was not documented in a previous study by the laboratory; otherwise, one normal metaphase spread.

One or two karyotypes from each abnormal clone for a minimum total of two karyotypes.

For cases with all normal cells: Two karyotypes.

E6.2.2.2 (b)

Patients who are post-allogeneic hematopoietic cell transplant for whom donor versus recipient origin of cells can be determined (by sex chromosome complement or cytogenetic heteromorphisms):

For studies aimed solely at determining engraftment status, molecular methods and/or interphase FISH (in the case of opposite sex transplant) are more sensitive than G-banded analysis and are the preferred methodologies. Therefore, in consultation with the referring physician, cancellation of test requests for G-band analysis for engraftment status should be considered.

During the course of the cytogenetic analysis it will become evident whether there is chimerism for donor and recipient cells. It is expected that there will be different approaches used by different laboratories to address these studies.

If only donor cells are present:


Analyze 20 cells.


Document two karyotypes for each cell line. (In such cases, one is documenting either the constitutional karyotype (normal or abnormal of the donor) or the rare event of a malignant process arising in a donor cell.)

If donor and recipient cells are present:


Analyze recipient cells completely for previously identified clonal chromosome abnormalities and any newly acquired abnormalities. In some cases there may be structural chromosomal abnormalities secondary to chromosome breakage or rearrangement induced by the pretransplant conditioning regimen. The laboratory should distinguish clonal from nonclonal changes and determine the significance of new abnormalities as possible.

Analyze all recipient cells present out of 20 cells. Evaluate each recipient cell for the presence of the abnormality present prior to transplant (i.e., the diagnostic abnormality). Depending on the number of recipient cells present among the initial 20 metaphase cells scored, additional recipient cells may be analyzed completely and/or scored for the presence of the diagnostic abnormality.

Donor cells: Analyze two donor cells if donor cells have not been analyzed in previous studies. Otherwise simply score these cells as being of donor origin, and count.


For the recipient cells: Two karyotypes of the stem line and one of each sideline.

For the donor cells: If donor cells have been documented previously, then provide a single metaphase spread. If donor cells have not been documented previously, provide two karyotypes.

If only recipient cells are present:


Analyze 20 cells, following the guidelines set forth above with respect to the characterization of secondary abnormalities.


Same as above for abnormal recipient cells.

E6.2.2.2 (c)

Patients post-hematopoietic transplant for whom donor and recipient cells cannot be differentiated by G-banding:


Analyze 20 cells. As in case scenarios outlined above, follow guidelines for recipient cells as set forth above.


Turnaround time (TAT) should be appropriate for clinical utility.

It should generally be recognized that under certain clinical circumstances, specific chromosome abnormalities, or lack thereof, may be crucial in establishing a diagnosis and have direct relevance to specific treatment offered to the patient. Every effort should be made to expedite conveying such chromosome information. It is strongly recommended that the cytogenetics laboratory director have a written policy describing how cases are prioritized in the laboratory, with associated TATs, and addressing the particular specimen processing and analytical needs.

E6.3.1 For initial diagnostic work-up, to rule out, confirm, or further characterize a diagnosis of acute lymphoblastic leukemia, acute myeloid leukemia, or chronic myelogenous leukemia, it is strongly recommended that these studies have preliminary results reported within 7 calendar days. Final results should be reported out within 21 calendar days.
E.6.3.2 In the initial diagnostic work-up of other hematologic disorders, and for longitudinal studies on a patient, it is strongly recommended that final results be reported within 21 calendar days.
E6.3.3 Preliminary verbal reports and dates such results were given should be clearly documented in the final report. The content of the preliminary report should also be documented if it differs from that of the final report.

Specific exceptions to these guidelines are: Diagnostic studies for acute

promyelocytic leukemia should be approached on a STAT basis, as selection of induction chemotherapy (including or excluding the use of ATRA as part of the regimen) depends largely upon cytogenetic or molecular findings. Therefore, if APL is suspected in a patient, it is highly desirable for preliminary results of FISH and/or G-banding to be reported within 2 working days of receipt of the specimen.

E6.4 Lymph Nodes

Specimen Procurement and Processing

Common diagnoses include Hodgkin and non-Hodgkin lymphomas, including follicular, diffuse large B-cell, marginal zone, mantle cell, T-cell, and anaplastic large cell lymphoma.

E6.4.1 (a) Information regarding the pathologic examination of the tissue (e.g., whether or not a malignant process was identified, any subtyping that is available, including immunophenotype) can provide valuable information regarding the processing and analytical procedures to be followed.
E6.4.1 (b)

Lymphoma may initially present as a mediastinal or other mass. Therefore, pathologic information should be pursued at the time of receipt of the tissue in the laboratory. If lymphoma is in the differential diagnosis, the cultures initiated should include at least one non-stimulated suspension culture as follows:

  1. In general, short-term (to include a 24-hour unstimulated culture) are preferred, with the number of cultures established being consistent with the apparent cellularity of the tissue specimen.
  2. Of note, in suspected lymphoma cases, bone marrow aspirate is frequently sent for staging purposes, but if it shows no or limited involvement by lymphoma, conventional cytogenetics will most often yield normal chromosome findings. Therefore the laboratory should be sure to discuss with the clinicians the need to get a biopsy specimen from the involved lymph node or mass.
E.6.4.2 Analytical Standards
E.6.4.2 (a) Selection of cells to analyze: As with bone marrow and blood specimens, cells selected for analysis should not be chosen based on good chromosome morphology. After an area of a slide is chosen to begin analysis, the technologist should examine cells as they appear consecutively in the microscope field.
E.6.4.2 (b)


Analyze 10-20 G-banded metaphase cells. (Fewer than 20 metaphases is acceptable as long as the abnormal clone has been identified and characterized.)

Note: Some lymphomas have highly complex karyotypes. In such cases, it may not be practical to analyze 20 metaphases. Therefore, in such studies, it is recommended that a sufficient number of metaphases (generally at least 10) be analyzed to permit characterization of the abnormal clone(s). The laboratory director should ensure that in the case of performing an abbreviated study, the CPT codes billed are consistent with the work performed as described in Section E3.2 of the Standards and Guidelines for Clinical Genetics Laboratories.

Rationale for the "Note": Unlike a study for aplastic anemia or myelodyplastic syndrome wherein the proportion of abnormal versus normal metaphase cells may have clinical significance, in the analysis of a lymph node or solid tumor, the sole clinical importance is the presence or absence of an abnormal clone, and the characterization of the chromosomal abnormalities involved. In a tissue in which the laboratory finds only abnormal cells, all with highly complex karyotypes, the goal should be to analyze a sufficient number to permit characterization of the clone. Extending the study to 20 cells could be very time- and resource-consuming, without any real added value to the diagnosis of the patient.


Follow guidelines for initial diagnostic workup of blood and bone marrow (Section E6.2).

If there is confirmed pathologic diagnosis of lymphoma, and analysis of 20 metaphases yields only normal cells, additional screening for specific lymphoma-associated chromosomal rearrangements (e.g., the 2;5, 8;14, 11;14, or 14;18 translocations) can be performed by G-banding or FISH.

E6.4.3 Turnaround Time
E6.4.3 (a) It is recommended that 90% of specimens have final reports completed within 21 calendar days.
E6.4.3 (b) Certain patient cases may need preliminary and/or STAT verbal or written reports. The laboratory should obtain sufficient information at the time of specimen receipt to permit prioritization in the laboratory in accord with clinical need.

Solid Tumors

It is critical that upon receipt in the laboratory of solid tumor material, as much information as possible be obtained regarding clinical information about the patient and suspected pathologic diagnosis.

E6.5.1 Specimen Collection and Processing
E6.5.1 (a) Specimens should be transported to the laboratory in sterile medium. If sterile medium is not available, sterile saline may be used. Optimally, 1 cm3 of specimen should be submitted for cytogenetic analysis.
E6.5.1 (b) Upon its receipt in the laboratory, the specimen should be examined under sterile conditions, and any obvious fat or necrotic tissue should be removed. The tissue should be minced, or enzymatically digested if tissue is resistant to mechanical dissociation.
E6.5.1 (c)

To the extent possible, culture processing should be tailored to the suspected pathologic diagnosis.

Among solid tumors there is great diversity in the culture conditions that are optimal for growth of the malignant clone. For some, the malignant tumor will grow in suspension cultures; others will grow best in flask or coverslip cultures. For example:

  1. The small round blue cells tumors of childhood (Ewing/PNET, neuroblastoma, Wilms) present a group of solid tumor studies for which cytogenetic findings can be critical for differential diagnosis. Either unstimulated suspension cultures or monolayer (coverslip) cultures have been reported to yield successful results.
  2. For sarcomas and germ cell tumors, monolayer or coverslip cultures are likely to be most successful.

Some of these solid tissue specimens may represent lymphomas, and should be processed accordingly (Section E6.4). Others may represent extramedullary hematopoietic malignancies, and should be processed according to the bone marrow procedures (Section E6.3). This emphasizes the importance of obtaining as much information as possible regarding the differential diagnosis at the time of specimen receipt.

E6.5.1 (d) It is generally recommended that the monolayer or coverslip cultures initiated be harvested as soon as sufficient mitotic activity is present. There is some risk of overgrowth of normal tissue elements in long-term tissue culture.

Analytical Methods

Analyze 10-20 metaphase cells by G-banding as described above for lymphomas. If the karyotype of the tumor is complex, 10 metaphases are likely to be sufficient to characterize the abnormal clone.


Follow guidelines for initial diagnostic workup of blood and bone marrow (Section E6.2).


Turnaround Time

It is recommended that 90% of cases have final reports out within 21 calendar days. The laboratory director should be apprised of special clinical issues that may require a shorter TAT for impacting patient care



1. Jaffee ES, Harris NL, Stein H, Vardiman JW, editors. World Health Organization classification of tumours: pathology and genetics of tumours of hematopoietic and lymphoid tissues, vol. 3. Lyon: IARC Press, 2001.

2. Mitelman Database of Chromosome Aberrations in Cancer (2004). Mitelman F, Johansson B and Mertens F (editors),

3. Weinkauff R, Estey EH, Starostik P, Hayes K, Huh YO, Hirsch-Ginsberg C, Andreeff M, Keating M, Kantarjian HM, Freireich EJ, Albitar M. Use of peripheral blood blasts versus bone marrow blasts for diagnosis of acute leukemia. Am J Clin Pathol 1999;111:733-740.

E7 Sex Chromatin

General Standards

E7.1 The indirect nature of sex chromatin analysis has rendered the test obsolete. Any patient in whom the question of sex chromosome abnormality is being considered should have complete chromosome analysis.
E8 Reporting Standards

Final written reports of the results of diagnostic testing should include the following information:

E8.1 Case identification includes name (or other first identifier), date of birth of patient (or other second identifier), date of collection and/or receipt of specimen, laboratory accession number(s), type of specimen and name(s) of physician(s) to whom report is sent.
E8.2 Specific details of the study to be reported should include:
  1. Indication for study.
  2. Numbers of cells in which chromosomes were counted, analyzed and karyotyped.
  3. Cell culture times and conditions and banding methods employed, when they bear on the cytogenetic interpretation.
  4. Banding method, level of resolution and current ISCN karyotype designation(s) of cells analyzed.
  5. A statement of additional work done to resolve questions of mosaicism.
  6. Correlation with previous studies.
  7. When parallel controls are used for comparative purposes in a study, the results of those controls.
  8. Interpretation of results to include: correlation with clinical information, indication of an abnormal result where applicable, recommendations for additional laboratory genetic studies for the patient and/or family, and a discussion of the significance of the findings, when appropriate. When appropriate, recommendations for genetic counseling should be made. The interpretation should be clear to a nongeneticist physician.
  9. When investigational procedures are employed, the investigational nature of the testing.
  10. Cautions as to possible inaccuracies and test limitations.
  11. Individuals qualified as under B3.1 must sign all final reports.
  12. Specifics of any preliminary results given including what the preliminary result was, the date and the person to whom the report was given
E8.3 Laboratory identification includes name, address, and phone number of the laboratory in which the study was performed.
E9 Metaphase Fluorescence in Situ Hybridization (FISH)
E9.1 General Considerations

Regulatory requirements may differ depending on the origin and FDA approval status of molecular probes used in FISH. The FDA regulates manufacturers who market medical devices, including in vitro diagnostic devices and certain reagents, such as molecular probes, used by laboratories to develop in-house tests. CMS (formerly called HCFA), through CLIA '88, regulates laboratories and their practices.

E9.1.1 Molecular probes are of three general types:
  1. Those probes regulated by FDA as analyte specific reagents (ASRs) for use in clinical settings. They are of two types:
    1. Class II and III ASRs which are sold as components of tests or kits (in vitro diagnostics) that have been cleared or approved by the FDA in conjunction with review of the Class II or Class III ASRs ("ASR kits"). The clinical laboratory is responsible for ensuring that the test system is operating within the performance specifications stated in the product insert.
    2. Class I ASRs are exempt from FDA premarket approval or notification, and Class II and Class III ASRs are marketed or used as independent components, separate from an approved test or kit ("ASRs"). ASRs may be sold to clinical laboratories regulated under CLIA '88 as qualified to perform high complexity testing (or clinical laboratories regulated under Veterans Health Administration directive 1106). 21 CFR §809.30(a)(2). FDA regulations require the inclusion of a disclaimer on all reports of results of in-house tests using ASRs, 21 CFR §809.30(e), and restrict the ordering of such tests, 21 CFR §809.30(f). See Section E9.7.
  2. Probes developed and used exclusively in-house, and not sold to other laboratories ("home-brew probes"), are not currently regulated by the FDA. Clinical laboratories using such probes must verify or establish, for each specific use of each probe, the performance specifications for applicable performance characteristics, e.g., accuracy, precision, analytical sensitivity and specificity, etc. (42 CFR §493.1213). A laboratory making its own probes should meet the standards set forth under Section G (Clinical Molecular Genetics).
  3. Probes labeled "for research use only" (RUO) or "for investigational use only" (IUO) are subject to FDA approval but have not been approved by the FDA for clinical use. Laboratories may consider whether such probes may be used under the practice of medicine exemption or an investigative device exemption (IDE). Clinical laboratories using such probes must verify or establish performance standards as provided above. (42 CFR §493.1213). In overview, both probe validation, including sensitivity and specificity, and assay analytical validation, including sensitivity and specificity, must be documented for the use of any molecular probe (42 CFR §493.1213).
E9.1.2 Indications for use of metaphase FISH include evaluation of:
  1. marker chromosomes
  2. unknown material attached to a chromosome
  3. rearranged chromosomes (including cryptic translocations)
  4. suspected gain or loss of a chromosome segment
  5. mosaicism
E9.1.2.1 Under most circumstances, metaphase FISH should be considered an adjunct to conventional cytogenetic studies.
E9.2 Documentation of test or analytic validation is required under CLIA '88 for any new test being placed into clinical service after September 1994. In the present context, a "test" is defined by the specific use of a probe, rather than by the generic "FISH" technology. Subsequent biannual calibration verification of the test system is also required under CLIA '88. (See 42 CFR §493.1217 and Section E9.3.)
E9.2.1 Validation requirements vary with the regulatory status of the test/device (see above). Note that there may be alternative approaches to validation and the following is presented as a consensus method.
E9.2.1.1 Unique sequence FISH probes not approved as Class II or III ASR kits (ASRs, home-brews, IUOs and RUOs) must be validated in two ways:
  1. Probe validation/localization should be confirmed by:

    Scoring of a minimum of 5 metaphase cells to verify that each probe hybridizes to the appropriate chromosome target(s) and to no other chromosomes. Care should be taken in evaluation of potential probe contamination, as the contaminating probe may be present in a dilute concentration, thus hybridizing more weakly than the probe of interest.

    One of the following methods should be used to determine chromosomal localization:

    1. inverted DAPI, sequential G-/R-/or Q- to FISH or other banding method
    2. use of a cell line containing the region of chromosome of interest as an independently identifiable target on a solid stained chromosome (e.g., structural rearrangements, trisomy, etc.)
    3. other methods that localize the probe at a level of resolution appropriate to the intended chromosome target

    The source of the metaphase nuclei preparation may be any tissue type as the substrate (matrix) in question is the chromosome.

    Localization should ensure that:

    1. the tested probe is the intended probe
    2. no unknown probe is contaminating that lot

    Localization should also identify any cross-hybridization inherent to that probe. Probes with significant cross-hybridization should not be used.

  2. Analytical validation: sensitivity (percentage of scorable metaphase cells with appropriate number of distinct signals) and specificity (percentage of signals from nontarget sites) must be established for each new probe.
    1. Assays using commercially available probes approved as ASR kits must meet the sensitivity and specificity parameters stated in package inserts provided by the manufacturer.
    2. For probes that are not FDA approved for "in vitro diagnostic use," the laboratory must establish analytical sensitivity, specificity and reportable ranges of results.

      Analytical sensitivity and specificity should be established by analysis of the hybridization of the probe to the chromosomes of interest (genomic target). Sensitivity is defined as the percentage of metaphases with the expected signal pattern at the correct chromosomal location. Specificity is defined by the percentage of signals that hybridize to the correct locus and no other location. An adequate number of cells and loci should be scored to ensure that the probe is sensitive and specific for the clinical testing being performed.

      Analytical sensitivity and specificity should be established by analysis of the hybridization of the probe to chromosomes representing at least 200 distinct genomic targets. For instance, a target sequence for which the hybridization signals from each of the chromatids is separable would require analysis of 50 cells (4 targets per metaphase). If the target sequence is at or near the centromere such that the hybridization signals are not clearly separable, the analysis would require 100 cells (2 targets per metaphase). Cells should be from 5 chromosomally characterized individuals (aneuploid cell lines can maximize target number). Pooling of cells from these 5 individuals (presuming comparable mitotic indices) is acceptable. However, all cells should have the same number of potential targets, if pooled. Discordance may require that the individual cell lines be tested separately. Greater discriminatory power may be needed to distinguish mosaicism.

    3. Comparable analytic sensitivity and specificity must be established for each new lot of probe. This may be accomplished by analyzing a patient sample simultaneously with old and new lots to document consistency.
E9.2.1.2 When using unique sequence FISH probes that are approved as Class II or III devices, the laboratory must document that the assay's performance characteristics are the same as or better than those stated by the manufacturer in the package insert.
E9.2.2 Repeat Sequence or Whole Chromosome Probes
E9.2.2.1 Probe localization validation should be as described in Section E9.2.1.1a.
E9.2.2.2 Analytical sensitivity of:
  1. whole chromosome probes is internally controlled and depends on the detection of hybridization to the normal chromosome target(s) in the cell.
  2. repeated sequence probes is a function of the target size on the chromosome and therefore is both an individual and population based determination. Therefore, care in the interpretation of negative results is required. Hybridization to normal chromosome target sequences provides internal controls.
E9.2.2.3 Analytical specificity is determined by calculating the proportion of probe bound to the target vs. the proportion bound to other chromosome regions. For analytical specificity,
  1. whole chromosome probes should hybridize to nontarget sequences (background signals of greater than or equal intensity to signals at actual targets) in less than 2% of cells.
  2. repeated sequence probes should hybridize to nontarget sequences (background signals of intensity greater than or equal to signals at actual targets) in less than 2% of cells. Because specificity is a function of homology to other sequences and the stringency of hybridization, test conditions may need to be modified.
E9.3 Biannual (twice per year) calibration or continuous quality monitoring verification is required (42 CFR §493.1217) in the use of all FISH probes. This can be accomplished through a method of continuous monitoring of test results. For example, important test characteristics to monitor might include: (1) correct number of signals (i.e., no contamination of probe, no degradation of probe) and (2) no excess background or other technical problem that would preclude interpretation. Most likely, these factors are monitored for every FISH assay. However, documentation of this monitoring process is required.
E9.4 Target tissues include any tissue from which adequate numbers of dividing cells can be obtained. Although the above analytical validation considerations should be specific to the intended tissue of use, in tests that target metaphase chromosomes as the test matrix, the considerations are no different than those of standard cytogenetic analysis. Particular tissues and/or disease states may lead to chromosomes of poor morphologic quality and possibly to reduced signal intensity.
E9.5 Processing standards appropriate to particular tissues are as specified earlier. The impact of slide/cell age on FISH probe hybridization efficiency should be considered.
E9.6 Analytical Standards

It is recommended that hybridization test systems (excluding whole chromosome probes) include control probes to tag the chromosome of interest. Such probes also afford a limited level of quality control by providing an internal control of the hybridization efficiency. However, the target sequence on a normal chromosome serves as a better control of hybridization efficiency.

If a probe is used that does not have an inherent control signal (i.e., an X or Y chromosome probe in a male), another sample that is known to have the probe target should be run in parallel with the patient sample.

The following analytical standards for testing presume that sensitivity and specificity are at least 98%. If lower, a corresponding increase in the number of cells scored to attain comparable confidence levels is required. (See Table 1 at end of Section E12.)

E9.6.1 Cell selection for analysis should be based on the observed hybridization of the control probe(s) and the target-specific probe to metaphase chromosome(s).

Cells showing chromosome-bound background (hybridization signals from nontarget sites) should not be scored.

E9.6.2 Although a single slide is sufficient for analysis, any suspicion of mosaicism should be confirmed in cells from an additional slide(s). Additional tissue/cell culture-specific considerations apply.
E9.6.3 For characterization of nonmosaic marker chromosomes or unknown chromosome regions in derivative chromosomes, a laboratory that has attained ³ 98% sensitivity of the assay during its internal test validation must analyze a minimum of 5 metaphase cells.
E9.6.4 For nonmosaic microdeletion/duplication analyses, a minimum of 10 metaphase cells should be analyzed. If any cells are discordant, additional cells from a second slide should be analyzed to attain confidence limits of mosaicism exclusion comparable to that of routine cytogenetic analysis.
E9.6.5 Results should be confirmed by at least two experienced individuals, one of whom may be the laboratory director.
E9.6.6 A minimum of two images (either photographic or digital) should be preserved in the FISH records of the case file.
E9.7 Ordering and Reporting
E9.7.1 Tests using ASRs may be ordered only by physicians and other persons authorized by applicable state law. 21 CFR §809.30(f).
E9.7.2 The manufacturing source and identification of the probe(s) used, (either gene symbol or locus symbol), the number of cells evaluated, and detailed hybridization results must be reported.
E9.7.3 Limitations of the assay must be stated, some of which may be described in the manufacturer's package insert.
E9.7.3.1 Pursuant to 21 CFR §809.30(e), the following specific disclaimer must be included in test reports of all FISH testing using ASRs:

"This test was developed and its performance characteristics determined by [laboratory name] as required by CLIA '88 regulations. It has not been cleared or approved for specific uses by the U.S. Food and Drug Administration."

The wording of the above statement is mandatory and should not be changed. However, because the statement may cause some confusion regarding whether such tests are clinically necessary and reimbursable, laboratories may wish to add clarifying language, such as the following, after the disclaimer:

"The FDA has determined that such clearance or approval is not necessary. This test is used for clinical diagnostic purposes. It should not be regarded as investigational or for research."

Laboratories also may wish to add language such as the following, if accurate:

"Pursuant to the requirements of CLIA '88, this laboratory has established and verified the test's accuracy and precision."

E9.7.4 Metaphase FISH analysis provides information only about the probe locus in question. It does not substitute for complete karyotypic analysis.
E9.7.5 Care must be taken in the interpretation of results from derivative chromosome regions of small size, because current whole chromosome probe cocktails may not be uniformly distributed across the full length of a target chromosome.
E9.7.6 Care must be taken in the interpretation of negative results from studies based on repeated sequence probes, due to rare individuals with small numbers of the repeated sequence target.
E9.7.7 When using metaphase FISH to document a microdeletion in which the probe is not the gene in question, care must be taken in interpreting results.
E9.7.8 Dependent on the robustness of the reference ranges established during analytical validation and the number of cells scored, appropriate comments as to the possibility of mosaicism should be considered.
E9.7.9 If a microduplication is suspected, caution should be exercised in interpreting metaphase FISH results and interphase FISH analysis is recommended for clinical interpretation (see E10 and E10.5.8 specifically). In addition, results should be confirmed by alternative methods (e.g., clinical information, molecular analysis) whenever possible.
E10 Interphase/Nuclear Fluorescence In Situ Hybridization
E10.1 General Considerations (see E9.1)
E10.1.1 These Standards and Guidelines are designed for use with standard clinical diagnostic tests using interphase in situ hybridization. These guidelines do not address all possible applications. Applications involving rare tissues (some solid tumors, etc.) should be considered as investigational tests until either more specific guidelines are established or sufficient experience is acquired to meet these guidelines.

These guidelines are not intended to address interphase FISH used in preimplantation genetics or in some fixed tissue section assays.

E10.1.2 Applications for use of interphase FISH include evaluation of:
  1. numerical abnormalities
  2. duplications
  3. deletions
  4. rearrangements
  5. sex chromosome constitution
  6. mosaicism
  7. gene amplification (not specifically addressed in this guideline)

Detection of a given chromosomal abnormality is dependent upon availability of an appropriate probe(s).

E10.1.3 Interphase FISH analysis provides information regarding only the specific probe locus used. It does not substitute for complete karyotypic analysis.
E10.1.4 Abnormalities detected by interphase FISH that are also considered reliably detectable by chromosome analysis should be confirmed by conventional cytogenetic analysis.
E10.1.4.1 If repeated interphase FISH analyses are performed in an attempt to detect the continued presence or absence of a previously identified abnormality, repeated adjunctive chromosomal analyses may not be necessary.
E10.1.5 Whole chromosome painting probes or whole p arm or q arm probes should not be used for interphase FISH analysis.
E10.1.6 It is the laboratory director's responsibility to ensure and document that technologists who perform interphase FISH tests are appropriately trained and have demonstrated consistent ability to score and interpret these cases. Color blindness testing is recommended.
E10.1.7 Prior to use of interphase FISH technology in clinical diagnostic cases, the following validation stages must be completed:
  1. Probe localization validation (E9.2.1.1a and E10.2)
  2. Assay analytical validation through establishment of a database of reportable reference ranges (E10.3).

Assay analytical validation must be completed for each new probe (including control probes within a multiprobe mix). The extent of validation differs between the various types of FDA-approved probes (analyte specific reagents (ASRs); the traditional Class II and III devices (ASR kits) obtained from commercial companies operating under good manufacturing practices; and the non-FDA cleared probes, those labeled RUO (research use only) and IUO (investigational use only) and home-brew probes. (See E9.1.1.)

Data collection for documenting analytical validation and establishing a database of reportable reference ranges is required only prior to the initial transfer of the test into clinical diagnostic use for each specific intended use and cell type. Incompletely validated probes may be used in exceptional clinical situations, but this must be stated in the final report.

E10.2 Probe Validation (See E9.2)
E10.2.1 Localization confirmation should follow guidelines for metaphase FISH probe validation described in Section E9.2.1.1a.
E10.2.2 Probe Sensitivity and Specificity (see E9.2.1.1b)
E10.2.3 Probe validation assessments should be based on each probe's performance separately (within the resolution of the different probe signals). The values obtained from different probes that are provided together in a cocktail (e.g., probes to 18, X, and Y) should not be averaged to determine probe validation values.
E10.3 Assay Analytical Validation and Establishment of a Database of Reportable Reference Ranges

Assay sensitivity and reportable ranges must be set in each laboratory based on the following database collection and analyses and/or statistical analyses. Results from samples used to establish reportable ranges should not be reported as test results.

Validation requirements vary among FDA-approved Class II and III ASR kits in which extensive analytical (and clinical) validation has been performed by the manufacturer. (See E9.1.1a and E9.2.1.)

E10.3.1 Database collection must be specific for an intended tissue type or cell population. For instance, database collection must distinguish between cultured versus uncultured amniocytes due to known differences of cell populations. This differentiation between tissue types and cell populations is at the discretion of the laboratory director.

The normal database should consist of an adequate number of cells from a group of control individuals (as determined by the director) who do not have abnormalities involving the target (and control) probes. At least 500 nuclei each from 20 control samples should be included in the normal database. When possible, an abnormal database should be established. This database should be limited to include only samples from individuals who have abnormality(ies) involving the specific target probe(s).

E10.3.2 Reportable Reference Ranges [8,9]

Reportable ranges for normal and abnormal results should be based on at least a 95% confidence interval achieved from the appropriate database.

A normal result can be considered valid if the percentage of cells that are normal is within the 95% confidence interval based on the database of normal individuals, or outside the 95% confidence interval (when available) for individuals with the abnormality.

An abnormal result can be considered valid if the percentage of nuclei that are abnormal (e.g., monosomy, trisomy) is outside of the 95% confidence interval based on the database of normal individuals, or within the 95% confidence interval (when available) for individuals with the abnormality.

It should be noted tha, while assays intended to detect nonmosaic abnormalities should have an analytical sensitivity equal to or exceeding 95%, as assay sensitivity increases, fewer cells may be evaluated (see Table 1).

E10.3.2.1 Detection of mosaicism should be based on results from a normal database. Quantitation of mosaicism can be determined by mixing of cells at known proportions from individuals with abnormalities and those without, and this may be informative as to the limitations of mosaicism identification. However, proportions of abnormal cells falling between the 95% confidence levels for normal and abnormal likely reflect true mosaicism.

To detect mosaicism >20%, probe analytical sensitivity should equal or exceed 95%. If detection of lower levels of mosaicism is desired, a higher analytical sensitivity percentage may be necessary to reach a comparable confidence interval (see Table 1).

E10.3.2.2 Commercially available probes may provide reportable ranges that can be used for guidance; however, each laboratory must establish its reportable ranges reflecting its own experience.
E10.4 Biannual (twice per year) calibration or continuous quality monitoring is required to ensure that assay analytical sensitivity and specificity remain at the levels established during initial validation. This can be accomplished through a method of continuous monitoring of test results (i.e., no contamination of probe, no excess background, results falling in reportable ranges). Most likely, these factors are monitored for every FISH assay (see Section E9.3); however, the process must be documented at least biannually.

Any test results that fall outside of the reportable range should be repeated. Continual test results that are outside of reportable ranges should initiate re-assessment of the test system. Documentation of this monitoring process is required.

E10.5 Analytical Standards

The following analytical standards for testing presume that the probe analysis sensitivity is at least 95% for those probes not intended to be used for mosaicism detection. See Table 1 to determine the numbers of cells needed to reach a 95% confidence level based on the analytical sensitivity of the assay in the laboratory.

E10.5.1 Selection of cell nuclei for analysis should be based on the observed hybridization of the probe(s). If utilizing a control probe, score only nuclei with the expected number of control probe signals.

Nuclei that are broken, overlapped, or have significant background "noise" should not be scored.

E10.5.2 If more than one probe is used simultaneously, different fluorochrome colors must be used to allow differentiation.
E10.5.3 As noted in section E9.2.2.2, care must be exercised in the interpretation of results from studies based on repeated sequence probes. Although rare, individuals exist who have a low copy number of a repeat on one homolog. This could result in misleading results due to reduced hybridization and/or signal intensity.
E10.5.4 Interphase FISH analyses attempting to detect mosaicism should be interpreted cautiously at the discretion of the laboratory director.
E10.5.5 The presence of contamination by maternal cells (in prenatal cases), bacteria, or fungus can lead to false positive or negative results. Routine processes to identify these contaminants are recommended, such as evaluating spun pellet for visible blood, which can indicate maternal cell contamination, or evaluating slides for nonspecific background signals that could indicate fungal or bacterial contamination.
E10.5.6 All analyses must be performed and/or evaluated by at least two qualified individuals (one of whom can be the laboratory director). The degree to which the scoring should be split between these individuals is at the discretion of the laboratory director.
E10.5.7 For analysis of constitutional numerical abnormalities or microduplications, a minimum of 50 total nuclei (25 per reader) should be scored.

If scores from the two individuals are significantly discrepant, a third analysis by another qualified individual should be pursued or the test repeated.

If a result does not meet laboratory established reporting ranges, the study should be repeated. If no additional material is available, a third analysis (at least 50 nuclei) by a qualified individual can be performed in an attempt to account for questionable results (e.g., potential poor hybridization/background on a portion of the slide).

If mosaicism is suspected for a constitutional abnormality, the number of nuclei scored should be sufficient to have ruled out with 95% confidence a result of nonmosaic normal, or abnormal, if clinically relevant (see Table 1).

E10.5.8 Interphase FISH may be used as an adjunctive test to assess levels of mosaicism/chimerism of cell lines with abnormalities previously established by standard banded chromosome and/or metaphase FISH analysis.

For analysis of acquired numerical abnormalities or microdeletions/duplications, the total number of nuclei scored should be sufficient to have excluded with 95% confidence either nonmosaic normal or abnormal (see Table 1).

For a given study, if presence of the abnormality is clearly detectable within the analysis of a total of 50 nuclei by two independent readers, it is up to the director's discretion to limit the study and report the results provided the reportable range used is based on a database of a similar number of analyses.

If repetitive sequence probes are used, an initial scoring of 10 metaphase cells for intensity of signal in the individual to be tested is recommended (see E10.5.4).

E10.5.9 For analysis of acquired abnormalities, a minimum of 50 total nuclei (25 per reader) should be scored per probe.

If an abnormal cell line is suspected, an appropriate number of nuclei should be scored (see Table 1).

E10.6 A minimum of two images (either photographic or digital) should be preserved for the records of each case.
E10.7 Ordering and Reporting
E10.7.1 Interphase FISH ordering and reporting standards and guidelines should follow those articulated in Section E9.7 for metaphase FISH. In addition, the following require special consideration.
E10.7.2 When using interphase FISH to detect a microdeletion or microduplication in which the probe is not the gene in question, in cases where a normal result is found, a disclaimer should be included in the written report stating the limitation of the test. Such a disclaimer may include information as given in the following example:

"The probe used, however, may give a normal result in cases that are due to very small deletions, point mutations or other genetic etiologies."

E10.7.3 Interphase FISH results that require confirmation by conventional chromosome analysis can be released prior to the cytogenetic analysis, although the results should be considered preliminary (see E10.1.4.1).
E10.8 Special Case Interphase Analysis: It is recognized that technology and probe development may proceed at such a rapid pace that the Standards and Guidelines may not specifically address all situations in entirety. It is the laboratory director's responsibility to ensure quality assurance and proper pre- and post-analytical practices that are consistent with the general guidelines presented here.
E10.8.1 Paraffin-embedded samples: Pre-hybridization specimen handling, including fixation, block cutting, deparaffination and protein digestion, are critical parameters that affect results and results interpretation. Each of these factors must be optimized for tissue type and preparation method.
E10.8.1.1 Selection of nuclei for analysis should be based on location of cells of interest (e.g., if there are cancer cells and normal stroma on the same section, caution must be taken to score the appropriate cell type) and optimal hybridization of the probe. In such instances, a consultation with a pathologist may be appropriate to ensure proper identification of nuclei.
E10.8.1.2 Signal scoring should involve focusing through the entire section to detect signals in different planes. The number and location of nuclei to be scored may vary due to the number of cells available in each tissue section. Scoring criteria for FDA-approved devices supercede those presented here.
E10.8.1.3 Appropriate control probes may not be readily available (e.g., amplification controls). In such cases, a negative (e.g., no amplification) and a positive (e.g., known amplification) control sample should be included in the analytical process.
E11 Multi-Target FISH Tests
E11.1 General Considerations

Multi-target FISH tests include assays developed to analyze multiple chromosome loci (>2) in a single test format.

Multi-target FISH tests provide information regarding only the specific probe loci used. This testing does not substitute for complete karyotype analysis.

Abnormalities detected by multi-target FISH should be confirmed by another methodology whenever possible (e.g., cytogenetic G-banding, locus-specific FISH).

It is the laboratory director's responsibility to ensure and document that technologists who perform multi-target FISH tests are appropriately trained and have demonstrated consistent ability to score and interpret these tests.

E11.2 Probe Validation

Probes used in these tests should be validated for localization, sensitivity and specificity as described in Section E9.2.1.1a.

Multi-target FISH tests that involve interphase FISH analysis should follow the previous guidelines given for interphase FISH probe validation, including establishing databases and reportable reference ranges (E10.2.2-E10.4), as well as biannual calibration.

Multi-target FISH testing on metaphase cells should generally follow the validation requirements outlined in E9.2.1.1a.

E11.3 Analytical Standards

Probes used for interphase multi-target analysis will require database generation to establish reportable ranges. Refer to the guidelines on interphase FISH for these tests (E10.3 and E10.5).

Probes used for metaphase analysis should follow metaphase FISH analytical standards (E9.6):

  1. For nonmosaic metaphase analysis, a total of 5 cells should be evaluated for repetitive and painting probes.
  2. For nonmosaic metaphase analysis, at least 5 cells should be evaluated for the majority of the unique sequence probes, whenever possible.

At least 30 metaphase cells should be studied for evaluation of mosaicism.

Image documentation should consist of at least one image for each locus or combination of loci (either photographic or digital) to be preserved for the records of the case. All abnormalities detected with one or more of the probes should be documented by at least one additional image.

E11.4 Ordering and Reporting

Follow the previous FISH reporting guidelines (including the mandatory FDA statement). For situations in which nomenclature has not been specifically addressed by ISCN 1995, and in which the reporting of all normal loci should be confusing for clinicians (e.g., subtelomere analysis), the director may establish a consistent and clinically meaningful reporting mechanism. This shorthand nomenclature or other reporting format should be documented in the laboratory standard operating procedures.

E12 References
  1. Stallard R, Johnson W. Am J Hum Genet 1983; 35/6: 155A.
  2. Yuan S et al. Am J Clin Pathol 1990; 93:765-770.
  3. Josifek K et al. Appl Cytogenet 1991;17/4:101-105.
  4. Hsu LYF. Prenatal diagnosis of chromosomal abnormalities through amniocentesis. In: Milunsky A, editor. Genetic disorders and the fetus: diagnosis, prevention and treatment, 3rd ed. Baltimore: Johns Hopkins University Press, 1992: 155.
  5. Ledbetter DH. Prenatal cytogenetics: indications, accuracy and future directions. In: Simpson JL and Elias S, editors. Essentials of prenatal diagnosis. New York: Churchill Livingstone, 1993.
  6. Ledbetter et al. Cytogenetic results from the U.S. Collaborative Study on CVS. Prenat Diag 1992;12:317.
  7. Maddalena A, Richards CS, McGinniss MJ, Brothman A, Desnick RJ, Grier RE, Hirsch B, Jacky P, McDowell GA, Popovich B, Watson M, Wolff, DJ. Technical standards and guidelines for fragile X: the first of a series of disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics. Genet in Med 2001;3(3):200-205.
  8. Dewald G, Stallard R, Alsaadi A, Arnold S, Blough R, Ceperich TM, Rafael Elejalde B, Fink J, Higgins JV, Higgins RR, et al. A multicenter investigation with D-FISH BCR/ABL1 probes. Cancer Genet Cytogenet 2000;116(2):97-104.
  9. Schad CR, Hanson CA, Paietta E, Casper J, Jalal SM, Dewald GW. Efficacy of fluorescence in situ hybridization for detecting PML/RARA gene fusion in treated and untreated acute promyelocytic leukemia. Mayo Clin Proc 1994; 69(11):1047-53.
  10. Dewald G, Stallard R, AlSaadi A, et al. A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y- chromosome probes. Am J Med Genet 1998;76/4:318-26.
One-sided test for a binomial proportion to determine the number of metaphases or interphase cells required to provide a sufficient confidence limit or power* (95 or 99%) to detect mosaicism or chimerism at varying levels of analytical sensitivity using interphase FISH. (See Section E12 for Dewald et al. 1998.)
  95% Power 99% Power
Frequency (%) of
2nd Cell Line >&
Analytical Sensitivity Analytical Sensitivity
90% 95% 99% 90% 95% 99%











































* Power is the probability that the test rejects the null hypothesis, H0, when, in fact, H0 is false.

&=Frequency equals 1- po and is defined parenthetically as analytical sensitivity

ID= indistinguishable from background

For example, to detect a level of 20% mosaicism in an assay of 90% analytical sensitivity, a total of 203 cells must be scored to obtain a result at a 99% confidence limit (power).