The Principles of Clinical Cytogenetics - Extra Materials - Springer

338 Solveig Pflueger
on embryonic development is needed in order to determine the frequency of their contribution to poor
pregnancy outcome.
SPECIMENS FOR CYTOGENETIC STUDIES
Although cytogenetic studies could be very helpful in managing patients with recurrent pregnancy
loss, fetal karyotypes are infrequently performed. Cowchock and colleagues reported a success rate
of 84% in a series of 100 samples, showing that chromosome analysis is indeed feasible in most
specimens (80). Chorionic villi are often the tissue of choice, as skin biopsies from deceased fetal
tissue can be associated with a higher failure rate. As previously mentioned, with spontaneous pregnancy
loss, it is frequently the case that the tissue is retained in utero for several days or even weeks
following embryonic or fetal demise. Because of this, fetal tissue is often autolyzed and is unlikely to
respond to standard culture methods, although chondrocytes appear to survive longer than skin and
other soft tissues following fetal demise and could offer a greater chance of success (94). Placental
tissue, on the other hand, often remains viable for a much longer period of time, because necessary
substrates for survival are provided by contact with the maternal blood supply. Ideally, both fetal and
placental sources should be utilized. The advantage of the fetal tissue is that there is little risk for
maternal cell contamination. If the fetus appears macerated, however, a high success rate is not to be
expected. Placental tissue usually grows well but adds the risk of maternal cell contamination. This
risk is reduced if the technologist processing the sample is experienced in the identification of membrane
and chorionic villi.
Direct preparations using the in situ method of tissue culture work well with cells derived
from spontaneously aborted tissues and have the advantage of rapid results with a high success
rate and minimal risk for maternal cell contamination (95,96). However, if maternal cells are
present in the original sample, trypsinization of slow-growing cultures to increase cell yield
appears to increase the risk for maternal cell overgrowth. Careful tissue selection and washing to
decrease the number of maternal cells might be helpful in decreasing the likelihood of maternal
cell contamination (97).
Fluorescence in situ hybridization (see Chapter 17) using either tissue sections or disaggregated
cells can be used in cases in which the tissue was accidentally fixed in formalin prior to receipt in the
cytogenetics laboratory because it does not require dividing cells (98). It must be remembered, however,
that this method will detect only those chromosome abnormalities for which specific probes are
available. FISH can be useful in diagnosing suspected aneuploidies, similar to its use in prenatal
screening of uncultured amniocytes, but the resulting information is limited to those specific chromosomal
regions for which probes are applied. Chromosomal rearrangements not involving numeric
changes are not generally amenable to this type of FISH analysis in interphase cells.
Flow cytometry can also provide useful information in cases that are not amenable to cell culture,
as it allows quantification of DNA (99). This can be especially useful for products of conception with
hydropic changes seen on histology, as it can differentiate between complete hydatidiform moles
(paternal diploids) and partial moles (usually triploid), an important distinction with regard to patient
management because of the risk for persistent trophoblastic disease with complete moles. DNA image
cytometry has also been shown to be useful in the diagnosis of molar pregnancies (100).
A newer methodology that has been proven useful for diagnosis of unbalanced karyotypes in cases
for which dividing cells are not available is comparative genomic hybridization (CGH) (101–103).
This method is dependent on DNA extraction but does not require viable or intact cells and thus can
be used for formalin-fixed frozen or paraffin-embedded tissues as well as fresh samples. Using different
fluorochromes, test specimen and reference DNA samples are hybridized to normal metaphase
chromosomes. The intensities between the test and reference samples are compared, enabling identification
of gains or losses of individual chromosomes or chromosomal regions (see Chapter 17). This
technique can detect unbalanced karyotypes such as trisomies, the largest group of chromosomally