Amniotic Fluid Cell Culture

The one present exception to this is hlstidase, an enzyme characteristic of epithelial cells. In this instance, it has been possible to select clones of epithelioid cells from the mixed cell population of the amniotic fluid cell cultures and to analyse them for this enzyme (55). 

It has been demonstrated that such controls are not valid and that there may be significant differences in enzyme concentrations between two cell types (56). For this reason, normal amniotic fluid cells themselves must be used as controls for amniotic fluid cell cultures being subjected to enzymologic inves t igat ion. 

Biochemical Analysis> The second principal use of cultured amniotic fluid cells is to determine the biochemical status of the fetus. Again, it should be pointed out that the individual biochemical disorders are quite low in incidence, but many of them are very serious in their manifestations and can occur with relatively high frequencies in specific families. If two parents are carriers of a gene for an autosomal recessive condition, then the risk to each child of having the disorder is 25%. 

There are important methodologic considerations which apply to the use of cultured amniotic fluid cells for the detection of biochemical disorders. The first is that the enzymes which can be sampled are those which are usually present in fibroblasts or fibroblast-like cells. Therefore, conditions such as phenylketonuria and glycogen storage disease type I, which are associated with deficiencies of enzymes present only in liver and kidney, are not amenable to this approach. The same also pertains to enzyme deficiencies affecting other specific tissues. 

The second methodologic consideration relates to the type of cells which are used as controls for the biochemical determinations. While less a problem now, there was a tendency in some early investigations to use cultured skin fibroblasts or similar cells as controls for the cultured amniotic fluid cells. 

Prenatal analysis Chorionic villus sampling or cultured amniotic fluid cells... 

Prenatal diagnosis of the disorder may be made (A15). Mothers carrying an affected fetus excrete large quantities of methylmalonic acid. Free methylmalonic acid is also present in the amniotic fluid and cultures of the am-niotic cells enable the nature of the defect to be determined. In the vitamin B12-responsive disorder, treatment of the mother with large doses of vitamin B12 was found to reduce the maternal excretion of methylmalonic acid. 

Biotinidase activity can be measured in cultured amniotic fluid cells and in amniotic fluid. Therefore, prenatal diagnosis of biotinidase deficiency is possible. Prenatal diagnosis has been performed in two at-risk pregnancies in which amniocentesis was performed because of advanced maternal age. The fetuses were found to be unaffected, and this was confirmed after birth. In addition to enzyme determination in amniocytes, a fetus was correctly shown to be a heterozygote by molecular mutation analysis in an at-risk pregnancy. Because treatment is so effective in this disorder, some laboratories are now performing prenatal diagnosis. 

Prenatal diagnosis of I-cell disease has been based on greatly reduced phosphotransferase activity (cf. Biochemical Perspectives section) and abnormal intracellular-extracellular distribution of lysosomal enzymes in cultured amni-otic fluid cells (Table 17-3).As indicated in Table 17-3, amniotic fluid cells secrete large amounts of lysosomal enzymes into the extracellular medium. Decreased levels of lysosomal enzymes in chorionic villi obtained by biopsy have also been observed in I-cell disease however, the characteristic secondary effect (i.e.,increased levels of lysosomal enzymes in the extracellular compartment) is only partially expressed or not expressed at all in chorionic villi, suggesting an alternative mechanism for the transport of lysosomal proteins. Although... 

Where no effective treatment exists at present for some inborn errors of metabolism, and where these disorders are associated with severe illness, mental retardation, or early death, the prenatal diagnosis of the diseased fetus by amniotic fluid enzyme analysis is now possible (MIO, Mil, M12). These new developments in the field of transabdominal amniocentesis, coupled with more liberal abortion laws, can serve to reassure families with a previous history of a fatal disease that their offspring will be physiologically normal. As applied to the field of amino acid disorders, enzyme analysis of cultured amniotic fluid cells has been used to diagnosis potential cases of homocystinuria (Fig. 51) and... 

Interests in prenatal diagnosis of genetic hyperglycosaminoglycanuria have prompted the examination of amniotic fluid cells via in vitro culture for enzyme deletions (H3 see also Section 7.6.5, p. 73), but data are not yet available. Peripheral leukocytes from patients with Sanfilippo A syndrome are deflcient in heparan sulfate sulfamidase (K40). 

H3. Hall, C. W., and Neufeld, E. F., a-i.-Iduronidase activity in cultured skin fibroblasts and amniotic fluid cells. Arch. Biochem. Biophys. 158, 817-821... 

Alternatives include the isolation of foetal cells from maternal blood, foetal blood sampling, foetal tissue biopsy and somatic cell hybridization. The latter involves the formation of hybrids between deficient parenchymal cells and cultured amniotic fluid cells. 

Studies using different methods of harvesting cultured human fibroblasts from a patient exhibiting mucopolysaccharidosis, and amniotic fluid, have indicated that reliable prenatal diagnosis of mucopolysaccharidosis can be performed only if the pericellular pool of glycosaminoglycans is removed by pretreatment with trypsin. Thus amniotic fluid cells from a pregnancy carrying a foetus affected by Hurler s disease revealed the expected increase in the level of S04-incorporation into fibroblasts only after mild proteolytic treatment. 

Cell-free amniotic fluid and cultured amniocytes Iduronate 2-sulfatase... 

Hansen, T.L. and Christensen, E. (1979), Studies on pyruvate carboxylase from cultured human fibroblasts and amniotic fluid cells. J. Inher. Metah. Dis., 2,23. 

Hirai, C., Ichiba, H., Saito, M., Shintaku, H., Yamano, T., and Kusuda, S. (2002). Trophic effect of multiple growth factors in amniotic fluid or human milk on cultured human fetal small intestinal cells. /. Pediatr. Gastroenterol. Nutr. 34,524—528. 

Sources of DNA DNA may be obtained from white blood cells, amniotic fluid, or chorionic villi (Figure 32.15). For amniotic fluid, in the past, it was necessary to culture the cells in order to have suffi cient DNA for analysis. This took two to three weeks to grow a suffi cient number of cells. The development of the polymerase chain reaction (PCR, see below) has dramatically shortened the time needed for a DNA analysis. 

Cells are grown either in suspension in a free or immobilized form 102), or by adherence to a solid surface 100). Materials used for promoting surface-dependent cell growth are glasses, metals, plastics, carbohydrate polymers etc. the media used contain substances such as blood plasma, amniotic fluids, tissue extracts, etc.103). Recent developments in animal cell culture are aimed at the improvement of strains and culture techniques, medium optimization, and scale-up. In contrast to plant cell culture, animal cell culture has already found its technical application. Large-scale... 

In several laboratories it has been shown that normal liver and liver-derived cell lines in tissue culture release somatomedin binding proteins into the surrounding medium. In the rat, primary hepatocyte cultures (S22, S28), liver explants (B25) and isolated perfused liver (S9, S21) produce a protein of molecular mass 40-50,000. A binding species of similar size is released by the BRL 3A2 cell line (M24). Whereas somatomedin peptides could also be detected in all of these studies, human hepatoma (M22) and mammary carcinoma (B20) cell lines have been described which release the 40-50,000 molecular mass binding protein without any detectable low-molecular-mass somatomedins. Human amniotic fluid also contains a binding species of similar size (C8, D20) but, contrary to an original report that there was no detectable somatomedin (C8), it appears that both IGF-I and -II are also present (M14). 

Infectious materials include semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid visibly contaminated with blood, and all body fluids in situations where it is difficult or impossible to differentiate between body fluids. They also include any unfixed tissue or organ, other than intact skin, from a human (living or dead) any human immunodeficiency virus- (HIV-) containing cell or tissue cultures organ culture and HIV or hepatitis- (HBV-) containing culture media or other solutions as well as blood, organs, or other tissues from experimental animals infected with HIV or HBV. 

For prenatal diagnosis, amniotic fluid can be used to identify acylcarni-tines characteristic of certain branched-chain amino acid disorders. Cultured amniocytes or chorionic villous cells can be used with in-vitro substrate loading for diagnosis of fatty acid and branched-chain amino acid disorders as described earlier for fibroblasts [13]. 

The GCMS analysis of methylcitrate or methylmalonate in the amniotic fluid is of advantage over enzyme analysis because of its rapidity. Moreover, it avoids possible maternal contamination of amniocyte cultures. In one documented instance the fetus was diagnosed as affected by methylcitrate analysis and normal by enzyme analysis an affected infant was born and the cells ultimately available for analysis were later shown to be maternal. In families in which the mutation is known, prenatal diagnosis may be carried out by analysis of DNA, but the numbers of mutations are such that this is not generally available. 

Priest, R. E., K. M. Marimuthu, and J. H. Priest. 1978. Origin of cells in human amniotic fluid cultures Ultrastructural features. Lab Invest 39 106-109. 

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