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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).