been directly observed.550 It is not known why their number in a given fluid is so highly variable and whether this reflects the wellbeing of the fetus, for which other properties of the AF may be more predictive.551 Since the change of the fetal skin from a simple two‐layered structure to mature stratified epithelium takes place around the 16th week and occurs at different rates in different body zones, minor differences in gestational age might account for comparatively large differences in overall cornification and desquamation.552 Classic cytology and transmission or scanning electron microscopy have attempted a subdivision of cells in midtrimester fluids.550, 553–555
A variable number of cells attach to the culture substrate within 6–72 hours after incubation but the number of colony‐forming cells rarely exceeds 10 cells/mL fluid.556, 557 Cells that attach in less than 24 hours (rapidly adhering or RA cells), if present in clear fluids in large quantities, may indicate an NTD.551 Such cells often take on the characteristic elongated spindle‐like appearance of neural crest cells in monolayer culture. In AFC cultures from NTD pregnancies, the RA cells include monocytic cells that have phagocytic activity and cells of glial origin that lack phagocytic activity and stain positive for synaptophysin and neuron‐specific enolase.558, 559 Rapidly adhering, phagocytic, esterase‐ and Fc receptor‐positive cells are also found in AF from normal fetuses, albeit in more moderate quantities.560
In cases of abdominal wall defects, macrophage‐like cells and even lymphocyte‐like cells responding to PHA have been described.561 AF from distressed fetuses may likewise contain macrophage‐like cells, termed fetal distress (FD) cells.562 Such FD cells occur in spontaneous abortion, severe intrauterine growth retardation, and preeclampsia. They may originate from the placenta.563
Colony‐forming cells: morphology and nomenclature
Multiple approaches have been used to characterize and classify colony‐forming cell types. Specific antibodies to the intermediate filament components of the mammalian cell cytoskeleton564 provide the means for establishing tentative correlations between cell types in culture and their presumptive in vivo counterparts.565
A synopsis of the current classification of human AFCs in culture is provided in Table 3.7, which also summarizes criteria used for classification and the various nomenclatures to which they have led (for a more extensive compilation of the properties of AFCs, see the review by Gosden563). Morphologic criteria were applied first. They quickly led to the realization that a high degree of cytoplasmic and nuclear pleomorphism is the hallmark of cultured AFCs. In contrast to what is known from postnatally derived human skin fibroblast cultures, multinucleation is a frequent and distinctive feature of cultivated AFCs. One report describes 7 percent of AFCs having two nuclei and 1 percent showing three or more nuclei.566 Within the clonal progeny of a single AF specimen, considerably more cells appear to be of epithelial than fibroblast origin. A cell type that looks very much like a prototype fibroblast‐like cell at the individual cell level (Figure 3.4) was distinguished by Hoehn et al.556 from classic fibroblasts on the basis of its “bull's‐eye” colony pattern. Such a pattern is never observed with classic skin or embryonic lung fibroblasts. Figure 3.5 shows that the typical bull's‐eye colony pattern is displayed by epithelioid (E) and by AF‐type cells. The clonal pattern of F‐type cells exhibits a whorl‐like center and parallel arrays of spindle‐shaped cells. Since shapes of individual cells and clonal units are influenced by culture conditions, these features change during long‐term culture.567
Table 3.7 The classification of human second‐trimester amniotic fluid cells in culture (excluding RA cells)
| Reference | Melancon et al. 593 Gerbie et al. 594 | Sutherland et al.725 | Hoehn et al. 556, 588 | Priest et al. 566, 569, 571 | Virtanen et al. 583 | Cremer et al.584 Ochs et al. 587 |
| Criteria | Morphology, enzyme production | Morphology, growth behavior | Morphology, clone patterns, longevity, cytogenetics | Collagen and gonadotropin production, ultrastructure | IIF, intermediate filaments | Intermediate filaments, prokeratin peptides |
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RA, rapidly adhering; AF, amniotic fluid‐specific; E, epithelioid; ED, epithelial and densely packed; 566 F, fibroblastoid; IIF, indirect immunofluorescence microscopy. Dotted lines indicate correspondence between the various nomenclatures (e.g. E3 corresponds to AF and E1).
See also review by Gosden. 563
Figure 3.4 Examples of living F‐, AF‐, and E‐type cells observed by phase‐contrast microscopy. Note the relative homogeneity of F‐type, in comparison to the pleomorphism of AF‐ and E‐type cells.
Figure 3.5 Examples of fixed colonies of F, AF and E clonal types at 2 weeks after plating. The AF‐ and E‐type colonies display typical “bull's‐eye” patterns. Compared with AF clones, the E‐type clones display wider growth margins around the darkly stained central core. The examples of AF and E clones are from primary platings of uncentrifuged amniotic fluid at 17 weeks gestational age. The F‐clone examples are subclones derived from a single F‐type primary clone isolated by a steel cloning cylinder and subsequent dilute plating on 2 × 3 inch glass slides. Crystal violet stain, 4/5 of actual size. Reproduced at 90 percent.
Biochemical characterization
The distinctiveness of the AFC types received support in a series of ultrastructural and cell secretion studies.566, 568–572 Hormones such as hCG, estrogen and progesterone are produced by AF‐type cells, some of which must originate from (placental) trophoblast tissue.572, 573 In contrast, F‐type AFCs failed to show hormone production, which is consistent with their likely mesenchymal origin.571,
