590 Moreover, these urine‐derived clones resemble AF‐derived clones.597, 601 Using specific antibodies against urothelium, von Koskull et al.602 provided results that tend to affirm the urinary origin of some types of AFCs. Although native AF at 16–18 weeks of gestation contains around 18 percent cells of colonic mucosal origin (as defined by a specific monoclonal antibody), none of the adherent cells appear to belong to this category.76
Cell culture and cell harvest
Colony‐forming cells
The number of cells per milliliter of AF increases with gestational age: approximately 9,000 cells/mL of fluid at 9 weeks of gestation, 100,000 cells at 13 weeks and >200,000 cells/mL at 16 weeks.5 The number of colony‐forming cells is much lower. Figure 3.9 shows that in platings of 16‐ to 18‐week fluids, an average of 3.5 clones/mL are typically scored at day 12. Only 1.5 colonies per mL reach a clone size of at least 106 cells. Other laboratories report similar values.603 In a series of 14‐ to 16‐week amniocentesis specimens, Hoehn et al.604 observed 3.1 colonies per mL but most were large colonies at day 12. Kennerknecht et al.605 reported high clone counts in 7‐ to 9‐week AF, ranging from 7.9 to 12.2 colonies per mL. Late pregnancy fluids show cloning efficiency of less than 1.5 colonies per mL.
Figure 3.9 Cloning efficiency of 20 consecutive amniotic fluid specimens (18 weeks gestational age). Fewer than half of the colonies grew to more than 106 cells per clone (more than 20 cumulative population doublings (CPD)).
Since human AF contains traces of growth and attachment factors such as epidermal growth factor, interleukin‐1, tumor necrosis factor α, fibronectin, and endothelin‐1,606 a 1 : 1 mixture of native fluid and growth medium has been recommended.607 Cell growth inhibitors (e.g. IGFBP‐1, an insulin‐like growth factor binding protein) have also been found in human AF.608 Although the proportion of erythrocytes may vary from 103 to 108 cells/mL, only the most severe blood contamination significantly retards or prevents cell growth. Such specimens can be treated before culture with 0.7 percent sodium citrate hypotonic solution or ammonium chloride lysing reagent (BD Biosciences).
Culture methods
The principal difference between the flask and in situ methods resides in the trypsinization step required for a suspension‐type harvest. The resulting metaphase spreads can no longer be traced back to an individual parental colony. In contrast, the in situ method leaves colonies intact. When large numbers of cells are required for biochemical or molecular diagnostic studies, culture in T25 flasks remains the method of choice.
In situ procedure
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
Enhancement of amniotic fluid cell growth
Enrichment techniques
Since cloning efficiency is low, it would be advantageous to increase the number of viable cells introduced into the cell cultures. Two studies have examined whether maternal activity or uterine agitation before amniocentesis affect the concentration of viable amniocytes in the fluid.636, 637 After correcting for gestational age, both studies were negative. Enrichment of the cell culture innoculum has been attempted via centrifugation of fluids through isopycnic gradients.638 With bloody specimens, these methods might be of some help but they are impractical for routine use. This limitation also holds for an enrichment technique by which the AF is returned to the fetus after aspiration, filtration, and reinjection of AF as early as 12.5
