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Since the early 1980s, in situ culture and harvest has become the preferred method for cytogenetic studies.^609–614 The main advantages are: (i) earlier culture harvest leading to a faster diagnosis, (ii) clonal (or, more correctly, colony) analysis leading to an easier distinction between genuine mosaicism and pseudomosaicism, and (iii) recognition of maternal cell contamination on the basis of clonal morphology.

Maternal cell contamination (MCC) occurs in up to 0.5 percent of AFC cultures.^615–617 To minimize MCC, some laboratories prefer to discard the first 2 mL of AF. PCR‐detectable MCC of AF samples has been described as common (4–17 percent of samples) and probably represents contamination by maternal blood. This contamination can be an important consideration for biochemical or molecular genetic studies.^{618, 619} However, our local experience is that none of 66 direct AF samples have exhibited variable number of tandem repeat (VNTR)‐detectable MCC. This is consistent with the 0.5 percent rate of MCC in AFC cultures identified by PCR analysis by Smith et al.⁶²⁰ This is also consistent with our local experience of finding MCC in 21 of 5,108 (0.41 percent) consecutive AFC karyotype studies (i.e. one or two 46,XX colonies among 15 or more 46,XY colonies in the in situ harvests). MCC rarely confounds the interpretation of cytogenetic results.

Guidelines and tables are available detailing the number of metaphases to analyze by either suspension or in situ harvests, to exclude mosaicism at a desired confidence level.^{603, 621–624} A deficit of these calculations is that they ignore artifactual loss of chromosomes, which is more frequent with suspension than with in situ preparations. Environmental conditions (e.g. relative humidity and temperature) during drying of chromosome spreads can influence chromosome spreading, and artifactual aneuploidy is well documented.^{625, 626} Spurbeck has video‐documented the effects of temperature and humidity on metaphase cell spreading.⁶²⁷ To search for mosaicism, the number of colonies sampled is more informative than the number of metaphases analyzed.^{628, 629} Whether the gold standard should be a 15‐colony analysis has been the subject of some debate.^{630, 631} Guidelines issued by the American College of Medical Genetics⁶³² recommend for the flask technique counting 20 cells from at least two independently established cultures, analyzing five and karyotyping two. For the in situ method, counting a minimum of 15 cells from at least 15 colonies in at least two independently established cultures was recommended.

Laboratories using the in situ technique in conjunction with optimal culture media (e.g. Chang or AmnioMAX from Irvine Scientific or GIBCO, respectively) are able to karyotype most specimens in less than two weeks. Longer time intervals result from suboptimal cell growth conditions, adherence to a 5‐day work week, or from other administrative rather than biologic limitations. Many laboratories also employ a robotic harvesting system and an environmental control chamber to improve the number and quality of metaphase cells.^{625, 633, 634}

A typical AFC culture protocol was published by Miron in 2012.⁶³³ Automated harvesting for in situ chromosome analysis usually employs a Tecan or Scinomix Sci‐Prep robotic sample processor,⁶³⁵ which saves personnel time and improves consistency because the timing, rate, and quantity of aspiration and dispensing of media, hypotonic solution, and fixative are automated. An environmental room or chamber that controls temperature, humidity, and airflow is helpful to both optimize the quality of the metaphase spreading and reduce seasonal variations in harvest quality. The system must be optimized in each laboratory but generally provides high‐quality preparations in the range of 35–45 percent relative humidity at 25°C.⁶²⁵