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Quality assessment of prenatal cytogenetic diagnosisSikkema-Raddatz, Birgit

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B.Sikkemaproefschrift2.doc

Quality assessment of prenatalcytogenetic diagnosis:

Some guidelines for handling amniotic fluid andchorionic villus material


The printing of the thesis was financially supported by

Cambrex Bio Science

Printing: Ponsen & Looyen BV, Wageningen

Cover design: Y. Swart

ISBN: 90-367-2206-3

© Copyright by B. Sikkema – Raddatz


RIJKSUNIVERSITEIT GRONINGEN

Quality assessment of prenatal cytogenetic diagnosis:

Some guidelines for handling amniotic fluid and chorionic villus

material

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. F. Zwarts,

in het openbaar te verdedigen op

maandag 21 februari 2005

om 16.15 uur

door

Birgit Sikkema-Raddatz

geboren op 28 februari 1965

te Güstrow (Duitsland)


Promotor: Prof. dr. C.H.C.M. Buys

Copromotor: Dr. G.J. te Meerman

Beoordelingscommissie: Prof. dr. M.A. Ferguson-Smith

Prof. dr. M.J. Heineman

Prof. dr. N.J. Leschot


Habe nun, ach! Philosophie,

Juristerei und Medizin,

Und leider auch Theologie!

Durchaus studiert, mit heissem Bemühn.

Da steh ich nun ich armer Tor!

Und bin so klug wie nie zuvor;

Bilde mir nicht ein, was Rechts zu wissen,

Bilde mir nicht ein, ich könnte was lehren,

Die Menschen zu bessern und zu bekehren.

Auch hab ich weder Gut noch Geld,

Noch Ehr und Herrlichkeit der Welt;

Drum hab ich mich der Magie ergeben,

Dass ich nicht mehr, mit saurem Schweiss,

Zu sagen brauche, was ich nicht weiss;

Dass ich erkenne, was die Welt

Im Innersten zusammenhält.

(Aus Faust von J.W. Goethe)

Für Heike


Contents:

Chapter 1 Introduction...........................................................................................9

Chapter 1.1 Prenatal cytogenetic diagnosis………………………………………… 10

Chapter 1.2 Quality control and assessment in prenatal cytogenetic diagnosis… 16

Chapter 1.3 Scope of the thesis……………………………………………………… 27

Chapter 2 Cell culture of amniotic fluid cells .....................................................35

Chapter 2.1 Effect of variation in amniotic fluid specimens and of type of culturemedium on culture time and number of cell clones of primary in-situ cultures ofamniocytes ..............................................................................................................37

Chapter 2.2 Minimal volume of amniotic fluid for reliable prenatal cytogeneticdiagnosis..................................................................................................................49

Chapter 2.3 Absolute procedure to test the growth potential of medium in primaryamniotic fluid cell cultures and the influence of decreased oxygen tension............55

Chapter 3 Slide preparation of in-situ cell cultures from amniotic fluid cellsand chorionic villi ..................................................................................................71

Assessment of the influence of fixative, temperature, humidity and air flow onmetaphase quality in preparations from primary in-situ cultures of amniotic fluidcells and chorionic villi .............................................................................................73


Chapter 4 Chromosomal analysis in preparations from chorionic villi andamniotic fluid cell cultures ...................................................................................89

Chapter 4.1 A 46,XX,der(13;14)(q10;q10),+21 child born after a 45,der(13;14)(q10;q10) chromosomal finding in CVS...................................................................91

Chapter 4.2 Four years’ cytogenetic experience with the culture of chorionic villi..97

Chapter 4.3 Probability tables for exclusion of mosaicism in prenatal diagnosis..113

Chapter 5 Summary, future prospects, and suggested guidelines ...............121

Chapter 5.1 Summary………………………………………………………………… 122

Chapter 5.2 Future prospects…………………………………………………………124

Chapter 5.3 Suggested guidelines……………………………………………………129

Samenvatting .......................................................................................................135

Curriculum Vitae..................................................................................................145

List of publications..............................................................................................147

Dankwoord ...........................................................................................................149

Introduction


9

Chapter 1

Introduction

Chapter 1


10

1.1 Prenatal cytogenetic diagnosis

In many parts of the world prenatal diagnosis has been part of antenatal care for

already thirty to forty years. The purpose of prenatal diagnosis is to determine

whether a foetus, believed to be at risk for a certain disorder, actually has that

disorder and to inform the parents about the possible course, seriousness and

treatment of the disorder. On the basis of that information they can decide to

continue the pregnancy or not. In the Netherlands prenatal diagnosis is performed

in 5% -10 % of all pregnancies (Niermeijer et al., 1997; Nagel et al., 2004). Women

with an increased risk of bearing a foetus with a chromosomal abnormality can opt

for a prenatal cytogenetic diagnosis. The purpose is to detect or exclude a

chromosomal abnormality of the foetus. For that, sufficient metaphase

chromosomes of an appropriate quality are needed. Current routine procedures to

obtain cells for prenatal cytogenetic diagnosis are amniocentesis (AC) and

chorionic villus biopsy (CVB). In the Netherlands, 8370 ACs and 3230 CVBs were

performed in 2002 (Dutch Prenatal Working Party, 2002). Thus, second trimester

amniocentesis, normally performed between 16 – 18 weeks of pregnancy, when 15

–20 ml amniotic fluid is drawn under direct ultrasound guidance, is the preferred

technique in most centres for prenatal diagnosis. There is an estimated procedure-

related risk of abortion of 0.5%- 1% (NICHD, 1976). Because of the introduction of

first trimester serum screening combined with an early cytogenetic analysis in

Groningen, CVB is there, in contrast to other centres, the most used invasive

prenatal procedure for cytogenetic diagnosis. CVB is usually performed

transcervically or transabdominally between 10 –12 weeks of pregnancy, when

under ultrasound guidance 20 –100 mg chorion villi are obtained for karyotyping.

There is an estimated risk of procedure related abortion of 0.5% - 1.0% (Brambati

et al., 1991), i.e. similar as the risk associated with AC.

Introduction


11

1.1.1 Procedure of cytogenetic diagnosis on amniotic fluid cells

Amniocentesis was first used in Germany in the early 1880s to treat hydramnions

(Lambl, 1881). In the 1950s amniotic fluid was used for monitoring bilirubin levels

in foetuses with Rhesus isoimmunization (Bevis, 1952). This development was

followed by the determination of foetal sex (Fuchs and Riis, 1956) in amniotic fluid

(AF) cells. The first chromosomal analysis was performed in 1966 by Steele and

Breg, and the first chromosomal abnormality, a balanced translocation, was

detected by Jacobson and Barter in 1967. In 1968, both Valenti et al. and Nadler et

al. reported in utero detection of Down syndrome, with confirmation after elective

abortion. The relative safety and accuracy of the procedure was demonstrated in a

study on 142 patients during 155 pregnancies (Nadler and Gerbie, 1970). In the

same period, AF was used for prenatal detection of biochemical abnormalities

(Galjaard, 1972). Brock and Sutcliff (1972) found an association of a raised

concentration of alpha- foetoprotein in AF and open neural tube defect.

AF is suggested to be a mixture of transudate of the maternal plasma,

urine and other body secretions from the foetus, accumulating into the AF space

(Behrman et al., 1967). It contains cells that can be precipitated by low-speed

centrifugation. The cell pellet consists of at least three cell types that can be

morphologically distinguished in culture; fibroblast-like (F) cells, epithelioid (E) cells

and amniotic fluid (AF) type cells (Hoehn et al., 1975). The precise foetal origins of

these cells have still not been resolved. AF cells are thought to originate from

trophoblast tissue as it has been demonstrated that these cells produce chorionic

gonadotropin (Laundon et al., 1981), a product of trophoblast tissue. E- type

cultured cells probably come from foetal skin and AF- type cells are from

mesenchymal origin. Also kidney tissue might contribute to cells in amniotic fluid,

since it has been implicated as a source of trisomy 20 cells (Hsu el al., 1991). For

cell culture either the in-situ method or the flask method are applied as basic

techniques. For in-situ culture, cells are grown on coverslips and harvested in-situ,

on average after 6-14 days. The in-situ harvest method has first been described by

Cox et al. (1974), who used clones of AF cells grown and harvested in Petri dishes.

Chapter 1


12

The sides of the dish were removed and the bottom on which the cells were

growing was taped on a slide for microscopy. Peakman et al. (1977) modified this

method and grew cells on coverslips inside Petri dishes. The coverslips were

‘harvested in-situ’ in the dishes, removed and attached to slides for microscopy.

Cells cultured according to the flask method are harvested from a cell suspension,

which is obtained by trypsinisation of the cells growing attached to the bottom of

the flask. Since a proportion of cells is lost in this procedure, more cells are

needed, which implies a longer culture time.

Whatever the culture method, before harvesting cultures are treated

overnight with the metaphase arrester colcemid to obtain adequate numbers of

cells in metaphase. Hypotonic treatment is performed the following day and cells

are fixed several times with a mixture of methanol and glacial acetic acid. At last,

the fixative is removed and the cells are allowed to dry under specific climate

conditions to achieve well-spread metaphase chromosomes. After "ageing" either

overnight or for a short period at 900C, the slides are ready for histochemical

treatment in order to produce discriminating banding patterns on the

chromosomes.

From the stained slides a certain number of metaphases will be analysed. For

preparations from in-situ cultures a single metaphase per clone is used for

analysis.

1.1.2 Procedure of cytogenetic diagnosis on chorionic villi

In 1968, Mohr reported a new method as an alternative to second trimester

amniocentesis. In this method chorionic villi were aspirated transcervically. A test

series was made prior to elective abortion. Only half of the samples, however, were

found to contain chorionic tissue. Complications as puncture of the amniotic sac

and membranes aspirated instead of villi were encountered. Reports on larger

series (Hahnemann, 1974; Kullander and Sandahl, 1973) had to contend with

maternal bleeding and infections, absence of real time ultrasound and low success

Introduction


13

rate of chorionic villus cell culture. Still, interest in first trimester diagnosis and

improvement of ultrasound machines resulted in 1982 in the first report of placental

biopsies using ultrasound guidance (Kazy et al., 1982), followed by a publication of

Old et al. (1982) on genetic diagnosis of hemoglobinopathies. Simoni et al., (1983)

developed a method for a rapid, direct preparation of chromosomes that took

advantage of spontaneous mitoses in the rapidly dividing villi. This direct method

yielded a cytogenetic diagnosis within a couple of hours after sampling. Moreover,

maternal cell contamination, often encountered in chorionic tissue culture, was not

a problem anymore.

Chorionic villi have a central mesenchymal core with foetal blood vessels.

This core is covered by a trophoblastic epithelium which consists of two layers; an

inner layer of single cells, which is called the cytotrophoblast and a

"multinucleated" outer layer, which is called the syncytiotrophoblast. The

trophoblast outer layer already exists four to six days after fertilisation as a layer

that surrounds the blastocyst. After embedding of the blastocyst in the

endometrium, trophoblast cells will fuse, forming the syncytiotrophoblast layer.

Non-fusing trophoblast cells will grow into this syncytiotrophoblast layer. Stromal

cells originating from the extraembryonal mesoderm will invade the cytotrophoblast

after about 13 days. From this stage on, new placental tissue is formed by

proliferation and branching (Figure 1).

After aspiration of the chorionic villi, the biopsy is checked under a

microscope for quality and quantity of the cell material and carefully cleaned from

maternal decidua. Chorionic villi can either be cultured or used for direct

preparation.

Chapter 1


14

Figure 1: Early embryonic development, according to Crane and Cheung, 1988

Direct preparation of chorionic villi cells

For direct preparation, cells from the cytotrophoblast are used. The great

advantage is that culturing of trophoblast cells is not necessary for getting

metaphase cells. Thus, a cytogenetic result can be rapidly obtained. Colcemid is

added to the villi to obtain adequate numbers of cells in metaphase. After

approximately one hour, hypotonic treatment is performed and chorionic villi are

fixed by methanol and glacial acetic acid. The cells are released from the villi by a

brief exposure to 60% acetic acid. The suspension is spread over a slide using a

bent tip of a Pasteur pipette (Simoni et al., 1983). Since no culturing is involved, an

effect of maternal cell contamination is limited to a minimum. At least 10 mg

chorionic villi are needed. However, the banding quality of the chromosomes is

poor and sometimes not enough metaphases are available. Therefore, as an

Introduction


15

alternative to this method, a semi-direct preparation or short-term culture of

chorionic villi is used. The difference between direct and semi-direct methods is a

24h incubation of chorionic villi in fluorodeoxyuridine (Gibas et al., 1987). This

causes a block in DNA synthesis, which is subsequently released by adding

thymidine at high concentrations as well as colcemid, so that synchronisation is

reached. Although theoretically this should result in a higher yield of metaphases

and an improvement of the morphology of the chromosomes, the overall quality

improvement, however, appears to be minimal.

Culturing of chorionic villi cells

For culturing of chorionic villi, the mesenchymal core is used. After the first

attempts to culture trophoblast cells (Kullander et al., 1973), culture methods were

improved considerably by the introduction of hormone-supplemented medium

(Chang et al., 1982; Czepulkowski et al.,1986). To avoid maternal cell

contamination, the outer layer of chorionic villi is stripped with trypsin.

Mesenchymal cells are further desegregated by collagenase (Jackson, 1989) and

the resulting suspension is cultured. A cytogenetic diagnosis can be made after 7-

10 days. Very few mg of chorionic villi are sufficient for successful culturing.

However, for quality reasons at least four cultures in two different incubators

should be initiated. The percentage of maternal contamination turned out to be

relatively low, between 0.1% to 2.16% (Roberts et al., 1988; Ledbetter et al., 1990

and 1992). By the application of a long-term culture technique problems involved in

the direct method, namely poor banding quality and a low number of metaphases,

can be overcome.

For harvesting chorionic villus cultures, either the in-situ or the suspension

method can be used. The harvest procedure, as well as the procedures for slide

making, staining and analysis, are in principle the same as described for AF cell

cultures.

Chapter 1


16

1.2 Quality control and assessment in prenatal cytogenetic diagnosis

Quality assessment in cytogenetics is still somewhat in its formative stage. The

best developed quality control and assurance programmes can probably be found

in the United Kingdom and the United States, where every cytogenetic laboratory

must participate in external proficiency testing (Watson, 1997). This includes

inspection of the facilities and making cytogenetic diagnoses of different test

samples every other year. As a consequence, only qualified and certified personnel

are permitted to sign chromosome analysis reports. Moreover, in the United

Kingdom, the Association of Clinical Cytogeneticists (2001) has formulated

guidelines for cytogenetic diagnosis. In the Netherlands a report “Advice

concerning Genetic Counselling” published in 1977, described for the first time the

indications for post- and prenatal cytogenetic diagnosis and the requirements for

personnel and equipment. A report from the Working Party of the Dutch

Association of Clinical Cytogeneticists (Mellink et al., 2000) was published in 1995

and revised in 2003, formulating minimal standards for cytogenetic diagnosis. A

minimum number of metaphases for analysing and karyotyping, a minimum

number of chromosome bands, a maximum turn around time for different

indications and a minimum qualification of the personnel are prescribed. These

standards are, however, insufficient to cover all quality aspects of the cytogenetic

process. In particular, aspects of culturing and harvesting are missing in these

guidelines. Factors involved in handling material for cytogenetic diagnosis have

also not been spelled out, nor has the effect of such factors on cytogenetic quality

been considered. Nevertheless, such factors are essential in determining the

quality needed for adequate analysis. Therefore, the following gives an overview of

which factors are involved and what is their effect on quality during the process of

cell culturing, slide preparation and chromosomal analysis.

Introduction


17

1.2.1 Quality control of culturing AF cells and chorionic villi

Culture quality can be measured as the culture time required for slide preparation,

as the number of metaphases on a slide, as the number of culture failures and

more specifically for AF fluid cells; as the number of clones. Culture quality is

mainly dependent of the operator who performs AC or CVB, on the condition of the

submitted material and on the culture conditions. Although these aspects count for

AF cell cultures as well as for chorionic villus cultures, we will focus on the culture

of AF cells, because this has been more extensively investigated.

With respect to the operator, different studies (Silver et al., 1998; Horger et

al., 2001; Blackwell et al., 2002) have demonstrated that the caseload influences

the sampling efficiency and that successful aspiration of clear AF increases with

experience in amniocentesis. Therefore, only an experienced obstetrician should

perform amniocentesis (Elias et al., 1998). Failure to obtain AF is then less than 1

percent (Tabor et al., 1986). In order to be allowed to perform amniocentesis an

obstetrician in the Netherlands has to perform at least 30 amniocenteses per year

(Dutch Society of Obstetrics and Gynaecology, 1997). However, neither the

influence of the caseload of the operator on culture quality, nor the minimal amount

of AF necessary for a reliable cytogenetic diagnosis have been investigated. Well-

defined guidelines for the transport of AF (at which temperature and for how long)

are also missing.

For the submitted material, the gestational age at which the sample is

taken, the amount and appearance of AF as well as the reason for sampling might

influence the quality of culture. Analysis of the effect of these variations might

retrospectively explain culture failures or delay in culture time. Some publications

have appeared in which these aspects were investigated in relation to culture

quality. Although the total number of cells in AF increases with gestational age, the

number of viable cells decreases (Elejalde et al., 1990). Moreover, only a fraction

of these cells will form clones, on average no more than 10 cells/ ml AF (Hoehn et

al., 1974). At 7 to 9 weeks of gestation 7.7 to 12.2 clones were counted per ml AF

(Kennerknecht et al., 1992); at 14 to 16 weeks 3.1+/- 1.2 clones and at 24 to 32

weeks less than 1.5 clones were counted (Rischkind and Risch, 1990).

Chapter 1


18

A red or brown coloured appearance of AF due to the presence of haemoglobin

negatively influences cell culture quality according to Golbus et al. (1979), who

observed a higher percentage of foetal demise and culture failures among these

samples. In a series of Hankins et al. (1984), however, no advanced risk on

pregnancy nor foetal problems were detected. Chang and Jones (1991) as well as

Seguin and Palmer (1983) reported inhibition of cell growth in red coloured AF.

Persute and Lenke (1995) concluded that the risk of culture failure is higher in

cases of foetal aneuploidy. However, Reid et al. (1996) substantiated further that it

was not merely a higher percentage of foetal aneuploidies, but more in general the

reason of referral “structural abnormalities at ultrasound”, resulting in a higher

percentage of culture failures. However, for none of the above-mentioned

variations of the submitted AF the effect of these variations on cell culture quality

has been systematically investigated.

In most laboratories the in-situ method is used because of its reduced turn-

around time compared to the flask method. Furthermore, after harvesting one is

still dealing with clones, which is preferable for analysis. Various protocols describe

how many cultures need to be initiated, which type of culture medium has to be

used and what should be further environmental conditions during culturing (Priest

and Rao, 1997; Keagle et al.,1999; Saunders and Czepulkowski, 2001). Cell

culture quality has to be monitored at least by culture time and number of culture

failures. On average, 0.5% culture failure is considered as acceptable (Lam et al.,

1998).

Crucial for success and minimal duration of AF cell cultures is the (type of) culture

medium and its supplements, in particular the foetal calf serum. Mostly used are

commercial media, such as Chang medium, Amniochrome or Amniomax that

include growth factors and foetal calf serum. The growth potential of different lots

or types of medium can differ. To minimise culture failures, use of two different (lots

or types) of medium per sample is recommended, as well as culturing in separate

incubators. All the used reagents, particularly each lot of medium and serum, need

to be tested on sterility and ability to support growth (Priest and Rao, 1997).

However, an absolute and easy to use procedure for such testing is not available.

Introduction


19

In order to control all aspects that influence culture quality, protocols for collecting

AF and chorionic villi, guidelines for handling these and protocols for culturing are

necessary. To define such guidelines, knowledge and registration of the potential

influence of different factors on culture quality is essential. Some of these factors

have been well investigated and some guidelines are already existing. This is e.g.

the case for the influence of the operator on the material which is submitted and for

some factors during culturing. A systematic investigation of the effect of quality

variations in the submitted material on culture quality is missing, however, as are

guidelines for transport of the material and for testing the growth potential of the

culture medium. Therefore, there is a strong urge for investigating these factors in

order to formulate guidelines.

1.2.2 Quality control during slide preparation

Slide preparation quality can be measured as the degree of spreading of the

metaphase chromosomes and the presence or absence of cytoplasm over the

metaphases. It will likely depend mainly on conditions during harvesting (hypotonic

treatment and composition of fixative) and on climate conditions during the process

of slide preparation. Slide preparation is a rather complex process. Individual

laboratories have their own “tricks” to obtain appropriate metaphase quality.

Several authors have investigated factors involved in this process. Claussen et al.

(2002) found hypotonic treatment to be essential for obtaining well-spread

metaphase cells. Without such a treatment, chromosomes become preferentially

positioned close to each other in the cell centre. The slide preparation process

starts with a water-induced swelling of mitotic cells during evaporation of the

fixative from the slide (Claussen et al., 2002). Methanol from the fixative

evaporates early during drying. This is followed by a water-induced swelling

through the hygroscopic effect of the gradually evaporating acetic acid and leads to

a stretching of the chromosomes via their flattening (Hliscs et al., 1997). For this

process a relative humidity of at least 21% is needed, otherwise no swelling will

occur. Thus, from these studies it may be concluded that hypotonic treatment,

Chapter 1


20

evaporation of acetic acid and a certain percentage of relative humidity are

essential for well-spread metaphases.

Spurbeck et al. (1996) determined the optimal climate conditions for slide

preparation from cells from suspension and secondary AF cell cultures and found

that the largest metaphase area was reached at 55% relative humidity and a

temperature of 200C. Deng et al. (2003) demonstrated a strong influence of relative

humidity on spreading of cells from suspensions. However, in none of these

studies the effects of the investigated factors have been determined quantitatively.

Furthermore, in-situ cultures, for which the slide preparation process is much more

complicated than for cells from suspension due to variable retention of nuclear

matrix proteins over the metaphase plate, have not been investigated in these

studies. For primary in-situ cultures effects of different variables during harvesting

and slide making need, therefore, to be investigated in order to define optimal

harvest conditions and to establish rational guidelines.

1.2.3 Quality control of chromosomal analysis

The quality of analysis can be measured as sensitivity and specificity of diagnosis.

This in turn mainly depends on the banding resolution of the chromosomes and on

the number of analysed metaphases. The chromosomal analysis will reveal or

exclude structural and numerical abnormalities (in case of mosaicism with a given

likelihood for exclusion).

Some minimal banding resolution is one quality criterion for an accurate

analysis. The International System for Human Cytogenetic Nomenclature (ISCN)

defined a total number of bands in human metaphases in 1995. In the Netherlands,

the Working Party of the Dutch Association of Clinical Cytogeneticists formulated a

minimal banding quality for cytogenetic diagnosis. For prenatal diagnosis, the

minimum resolution has been determined at 400 bands (Mellink et al., 2003). This

is in general the resolution of metaphases from chorionic villus cultures.

Metaphases from direct preparations of chorionic villi usually have less than 400

bands. In metaphases from AF cell cultures up to 550 bands are visible.

Introduction


21

A chromosomal abnormality which is detected in only a proportion of the

investigated cells, is called chromosomal mosaicism. A mosaic pattern results from

an abnormal cell division, which leads to the presence of two or more cells with an

identical abnormality among the other (normal) cells. This can either reflect a true

foetal mosaicism or be confined to the extra embryonic tissue or represent a

culture artefact (pseudomosaicism). Pseudomosaicism is believed to usually

originate in vitro, without clinical consequences. Incidence and interpretation of

mosaicism is difficult and different for AF cells and chorionic villi.

Chromosomal analysis in AF cell cultures

Mosaicism can be excluded to a certain degree by analysing a given number of

metaphases. Hook (1977) published the number of cells that should be analysed

from peripheral blood cultures for exclusion of 10%, 20% and 30% mosaicism at

90%, 95% and 99% confidence levels, as derived from Newton's binomial formula.

Based on this model also the number of metaphases to be analysed from in-situ

cultures and flask cultures in order to exclude mosaicism in AF cell cultures were

similarly determined (Claussen et al., 1984; Richkind and Risch, 1990;

Featherstone et al., 1994). In current practice, for the flask method 20 cells from at

least two flasks and for the in-situ method 10 –15 cells from different clones should

be analysed. These guidelines allow exclusion of more than 20% mosaicism with a

confidence level of 95% to 99% (Rischkind and Risch; 1990).

In terms of a laboratory result, mosaicism can be classified at three

different levels (Hsu et al., 1992; Hsu and Benn, 1999): (1) a single cell is

abnormal; (2) two or more cells in the flask method, a single clone or two or more

clones within the same culture dish in in-situ cultures are abnormal; (3) two or more

cells/ clones from independent cultures are abnormal. Only the last situation is

considered to reflect true mosaicism. In AF cell cultures true mosaicism is rare with

an occurrence in 0.1% to 0.3 % of samples (Bui et al., 1984; Hsu; 1984; Worton

and Stern, 1984). The mosaic pattern is confirmed in the foetus in about 70% of

the cases. Pseudomosaicism of level (1) occurs in 2.47% to 7.10% and of level (2)

Chapter 1


22

in 0.64% to 1.10 % of cases, including more structural than numerical

abnormalities. In order to discriminate pseudomosaicism from a potentially true

mosaicism one can increase the number of analysed metaphases by analysing

metaphases in a culture dish different from the one in which the abnormal cells/

clones were present. Hsu et al. (1992; 1999) developed guidelines how to deal with

different chromosomal abnormalities. Depending on which abnormality was

detected, no analytical work up, moderate (12 extra clones) or extensive work up

(24 extra clones) should be done on metaphases in other dishes than the initial

dish in which the abnormal cells/ clones had been found (Table 1). The use of

these guidelines results in a reliable diagnosis of pseudomosaicism or true

mosaicism. In case of a work up sometimes, however, an insufficient number of

metaphases is available on the regularly harvested in-situ dishes and a back-up

culture needs to be trypsinised and analysed. In such a situation guidelines on how

many metaphases should be analysed from trypsinised back-up cultures are

missing.

Chromosomal analysis in chorionic villi

True mosaicism in chorionic villi can be confined to the placenta, to the foetus or

be present in both foetus and placenta, i.e. be a generalised mosaicism. Kalousek

and Dill (1983) demonstrated mosaicism confined to extra-embryonic tissue in two

out of 46 spontaneous abortions. Although foetus and placenta originate from the

same zygote, their chromosomal constitution can be different. Soon after the

introduction of CVB in routine practice, it turned out that the cytogenetic diagnosis

from direct preparations did not always reflect the chromosomal constitution of the

foetus. Reports appeared on false positive results (Bartels et al., 1986; Breed et

al., 1986; Leschot et al., 1987), followed by reports on false negative results

(Eichenbaum et al., 1986; Linton et al., 1986; Martin et al., 1986). Collaborative

studies (Ledbetter et al., 1992; ACC, 1994; Hahnemann and Vejerslev,1997)

showed mosaicism confined to the placenta in 1% to 2% of all cases.

Introduction


23

Table 1: Guidelines for work-up to exclude or detect pseudomosaicism or

mosaicism, from Hsu and Benn (1999)

Flask method In-situ method

A. Indications for extensive work-up1. Autosomal trisomy involving achromosome 21, 18, 13 or 2; 5, 8, 9, 12, 14, 1516, 20, 22 (SC, MC)

1. Autosomal trisomy involving a chromosome21, 18, 13 or 2; 5, 8, 9, 12, 14, 15 16, 20, 22(SCl, MCl)

2. Unbalanced structural rearrangements (MC) 2. Unbalanced structural rearrangements (MCl)

3. Marker chromosome (MC) 3. Marker chromosome (MCl)

B. Indications for moderate work-up4. Extra sex chromosome (SC, MC) 4. Extra sex chromosome (SCl, MCl)

5. Autosomal trisomy involving a chromosome 1,3, 4, 6, 7, 10, 11, 17, 19 (SC, MC)

5. Autosomal trisomy involving a chromosome 1,3, 4, 6, 7, 10, 11, 17, 19 (SCl, MCl)

6. 45,X (MC) 6. 45,X (SCl, MCl)

7. Monosomy (other than 45,X) (MC) 7. Monosomy (other than 45,X) (SCl, MCl)

8. Marker chromosome (MC) 8. Marker chromosome (MCl)

9. Balanced structural rearrangement (MC) 9. Balanced structural rearrangement (MCl)

C. Standard, no additional work-up10. 45,X (SC) 10. Unbalanced structural rearrangements (SCl)

11. Unbalanced structural rearrangements (SC) 12. Break at centromere with loss of one arm(SC)

13. Break at centromere with loss of one arm (SC) 13. All single- cell abnormalities

MC: multiple cells (single flask), MCl: multiple clones (single dish), SC: single cell, SCl: single clone

A model of Crane and Cheung (1988) can explain the discrepancy

between the chromosomal constitutions of foetus and placenta. According to their

model (Figure 2), from the first five cell divisions following fertilisation only two of

the 32 resulting cells will give rise to the formation of the embryo. All other cells are

responsible for the formation of the trophoblast and the extraembryonic tissues.

Chapter 1


24

Figure 2: Diagram of cell lineages arising from differentiation after the first five

divisions in a human embryo, according to Crane and Cheung (1988)

A possible failure (nondisjunction) in an early cell division may lead to an

abnormal cell in a specific cell line. A false positive result can be explained when in

a normal foetus a localised nondisjunction in the chorionic villi occurs, which may

cause an aberrant cell line in the direct preparation or culture, but not in the foetus.

A false negative result can be explained by the occurrence of a meiotic

nondisjunction, which may lead to a trisomic fertilised ovum, and a subsequent

mitotic nondisjunction in the chorionic villi leading to a normal diploid cell line in the

placenta, whereas the foetus remains fully trisomic.

Based on the position of the different cell lines and on the chromosomal

constitution of the foetus six major categories of mosaics can be distinguished

(Table 2).

Introduction


25

Table 2: Types and frequencies of placental-fetal discordance at chorionic villussampling, modified from Hahnemann and Verjerslev (1997)

Karyotype

Relativefrequencies (%)

Type Nature Trophoblast Villus core Embryo/foetusI CPM Abnormal Normal Normal 48

II CPM Normal Abnormal Normal 25

III CPM Abnormal Abnormal Normal 16

IV TFM Abnormal Normal Abnormal 0

V TFM Normal Abnormal Abnormal 4

VI TFM Abnormal Abnormal Abnormal 7

Trophoblast, preparations from direct/ short term culture; Villus core, preparations from long term

culture; CPM, confined placental mosaicism; TFM, true foetal mosaicism (generalised foetal-placental

mosaicism). An abnormal karyotype could be mosaic or non-mosaic. A normal embryonic/ foetal

karyotype could reflect normal biparental disomy or uniparental disomy.

This table also shows the relative frequencies of the different types of mosaicism

(Hahnemann and Vejerslev, 1997). The results of direct preparation alone (type I)

were less reliable than those of the method using culturing (type II). More cases of

mosaicism confined to the placenta were detected in the direct preparation alone,

which would in a number of cases lead to unnecessary amniocentesis as follow-up.

False negative cases occur at a very low frequency (Saura et al., 1998), but

(nearly) all of the cases were found in direct preparations (type IV versus type V).

The model of Crane and Cheung (1988) can also explain these differences.

Trophoblast cells (used in direct preparation) are forming the first line of

differentiation from totipotent cells of the morula and have therefore a greater

chance of failures of cell division. Mesodermal core cells (used in culture) reflect

the later segregating line of the extraembryonic mesoderm. These cells are closer

related to the cells that form the embryo than trophoblast cells. Therefore, the use

of a direct preparation alone should be avoided and reliability of the cytogenetic

diagnosis improved by the combined use of a direct preparation and a long-term

culture (Hahnemann and Vejerslev, 1997).

Chapter 1


26

Once an abnormality is detected, the question rises of the probability, i.e. a

calculated predictive value, of an abnormal foetal karyotype. In general, the

predictive value is high in case of a non-mosaic abnormality found in culture (83%-

100%), low in case of a mosaic abnormality in culture (24%) and zero in case of

a(n) (mosaic) abnormality detected only in a direct preparation (Hahnemann and

Vejerslev, 1997). Of special interest is the predictive value of a particular

chromosome abnormality for adequate counselling of the parents about follow-up

options, such as amniocentesis, ultrasound investigation or termination of

pregnancy. However, because of the small number of aberrant cases for most

chromosomes, predictive values are imprecise and additional cases are needed to

improve that.

Introduction


27

1.3 Scope of the thesis

The aim of prenatal cytogenetic diagnosis is to identify a possible chromosomal

abnormality of the foetus. The most important quality criteria for diagnosis are

sensitivity, i.e. the overall detection rate of abnormalities, and specificity, i.e. true

occurrence of the detected abnormality in the foetus. For a reliable result,

sensitivity and specificity need to be as close to 100%. Moreover, a result should

be given within the shortest time possible on a minimum of material and at the

lowest costs. To realise this, knowledge of all factors involved in the process of

diagnosis is needed, as is a determination of their effects. Based on this

knowledge, guidelines for handling prenatal material can be established. In

particular aspects of culturing and harvesting are missing in the previously

described cytogenetic procedures and factors involved in handling prenatal

material have not been spelled out nor has the effect of these factors on

cytogenetic quality been considered. This is particularly important in prenatal

cytogenetic diagnosis, where because of the risk for both the pregnant woman and

the foetus in obtaining the required material, the material is very precious.

Moreover, in case of a detected chromosomal abnormality, a following decision to

terminate or continue the pregnancy needs to be based on a reliable diagnosis.

The scope of the thesis is, therefore, to establish guidelines for quality controlled

processing of amniotic fluid cells and chorionic villi for purposes of prenatal

cytogenetic diagnosis. We will investigate which factors are of critical importance in

handling these materials and will quantify their influence. Depending on their

influence we will advice which factors need to be monitored carefully and we will

develop some new guidelines in case the existing ones are insufficient. Our goal is

to determine the limits between which reliable prenatal cytogenetic diagnosis is

possible. For this purpose we divide the cytogenetic process in three distinct main

parts, namely cell culture, slide preparation and chromosomal analysis.

Chapter 1


28

Culture quality can be measured as the culture time required for slide

preparation for analysis and for AFcell culture more specifically as the number of

clones and as the number of culture failures. The culture quality depends mainly on

the condition of the submitted material and on the culture conditions. Therefore, we

will first investigate the effect of variations in the submitted amniotic fluid (i.e.

volume, appearance) and in culture medium on cell culture quality (Chapter 2.1).

Second, we want to determine the minimal volume of AF required for a reliable

cytogenetic diagnosis, avoiding repeated amniocentesis (Chapter 2.2). Finally, we

want to develop an absolute procedure for testing the growth potential of reagents

involved in culturing and of other factors influencing cell culturing. Using such a

procedure, we want to test the influence of a decreased oxygen tension during

culturing and to determine the effect on culture quality (Chapter 2.3).

The quality of slide preparation can be measured as the degree of

spreading of the metaphase chromosomes and the presence or absence of

cytoplasm over the metaphases. It mainly depends on conditions during harvesting

(hypotonic treatment and composition of fixative) and the process of slide making

(climate conditions). We will investigate the effect of these variables on primary in

situ cultures (Chapter 3). Once we know the quantitative effects of ambient

temperature, relative humidity, air flow and composition of fixative, we will be able

to give some recommendations on how to achieve a consistent high quality of

preparations.

The quality of analysis can be measured as sensitivity and specificity of

diagnosis. It mainly depends on the banding resolution of the chromosomes and

the number of metaphases analysed. For amniotic fluid culture, probability tables

exist to determine the number of metaphases that should be analysed from either

the in-situ or the flask method of culturing in order to exclude a certain degree of

mosaicism. For trypsinised in-situ cultures, however, such a table is missing. We

will, therefore, construct a probability table to determine the number of metaphases

that need to be analysed in order to detect mosaicism when using trypsinised

back-up cultures complementing in-situ AF cell culture analysis (Chapter 4.3).

Introduction


29

For chorionic villi analysis, sensitivity and specificity need careful consideration,

starting from the selection of the cell type which should be analysed from the villi;

trophoblast cells in direct preparations, mesenchymal core cells in culturing or a

combination of direct preparation and culturing. Since routinely we are using

culturing only, we will investigate the results of that method in terms of success

rate, maternal cell contamination, chromosomal aberrations observed and

predictive value (false positive results). These results will be compared with those

from the literature on direct preparations alone and on the combination of the direct

preparation and the culturing method (Chapter 4.2).

Eventually, we will try to formulate guidelines for maintaining a good quality in

prenatal cytogenetic analysis of amniotic fluid culture and chorionic villi and

suggest possible further investigations (Chapter 5).

Chapter 1


30

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Claussen U, Schafer H, Trampisch HJ. Exclusion of chromosomal mosaicism in prenatal diagnosis.Hum Genet. 1984; 67(1):23-8.

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Cox DM, Niewczas-Late V, Riffell MI, Hamerton JL. Chromosomal mosaicism in diagnostic amnioticfluid cell cultures. Pediatr Res. 1974 ; 8(6):679-83.

Crane JP, Cheung SW. An embryogenic model to explain cytogenetic inconsistencies observed inchorionic villus versus fetal tissue. Prenat Diagn. 1988; 8(2):119-29.

Czepulkowski BH, Heaton DE, Kearney LU, Rodeck CH, Coleman DV. Chorionic villus culture for firsttrimester diagnosis of chromosome defects: evaluation by two London centres. Prenat Diagn. 1986;6(4):271-82.

Dutch Society of Obstetrics and Gynaecology. 1997. Guidelines for invasive prenatal diagnosis.http://www.nvog.nl

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Introduction


31

Eichenbaum SZ, Krumins EJ, Fortune DW, Duke J. False negative finding on chorionic villus sampling.Lancet 1986; 2: 391.

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Fuchs F. and Riis P. Antenatal sex determination. Nature 1956; 177: 330.

Galjaard H. Possibilities of quantitative histochemical analysis in prenatal detection and geneticclassification of hereditary metabolic disorders. In: Histochemistry and cytochemistry, T. Takeuchi (ed).Nakamishi Printing Kyoto 1972; 182-3.

Gibas LM, Grujic S, Barr MA, Jackson LG. A simple technique for obtaining high quality chromosomepreparations from chorionic villus samples using FdU synchronization. Prenat Diagn. 1987; 7(5):323-7.

Golbus MS, Loughman WD, Epstein CJ, Halbasch G, Stephens JD, Hall BD. Prenatal genetic diagnosisin 3000 amniocenteses. N Engl J Med. 1979; 300(4):157-63.

Hahnemann N. Early prenatal diagnosis; a study of biopsy techniques and cell culturing fromextraembryonic membranes. Clin Genet. 1974; 6(4):294-306.

Hahnemann JM, Vejerslev LO. European collaborative research on mosaicism in CVS (EUCROMIC)--foetal and extrafoetal cell lineages in 192 gestations with CVS mosaicism involving single autosomaltrisomy. Am J Med Genet. 1997; 70(2):179-87.

Hahnemann JM, Vejerslev LO. Accuracy of cytogenetic findings on chorionic villus sampling (CVS)--diagnostic consequences of CVS mosaicism and non-mosaic discrepancy in centres contributing toEUCROMIC 1986-1992. Prenat Diagn. 1997; 17(9):801-20.

Hliscs R, Muhlig P, Claussen U. The spreading of metaphases is a slow process which leads to astretching of chromosomes. Cytogenet Cell Genet. 1997; 76(3-4):167-71.

Hankins GD, Rowe J, Quirk JG Jr, Trubey R, Strickland DM. Significance of brown and/or greenamniotic fluid at the time of second trimester genetic amniocentesis. Obstet Gynecol. 1984; 64(3):353-8

Hoehn H, Bryant EM, Karp LE, Martin GM. Cultivated cells from diagnostic amniocentesis in secondtrimester pregnancies. I. Clonal morphology and growth potential. Pediatr Res. 1974; 8(8):746-54.

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Hook EB. Exclusion of chromosomal mosaicism: tables of 90%, 95% and 99% confidence limits andcomments on use. Am J Hum Genet. 1977; 29(1):94-7.

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Hsu LY, Kaffe S, Jenkins EC, Alonso L, Benn PA, David K, Hirschhorn K, Lieber E, Shanske A, ShapiroLR, et al. Proposed guidelines for diagnosis of chromosome mosaicism in amniocytes based on dataderived from chromosome mosaicism and pseudomosaicism studies. Prenat Diagn. 1992; 12(7):555-73

Chapter 1


32

Hsu LY, Benn PA. Revised guidelines for the diagnosis of mosaicism in amniocytes. Prenat Diagn.1999; 19(11):1081-82.

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Introduction


33

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Chapter 1


34

Cell culture of amniotic fluid cells


35

Chapter 2


Chapter 2


36



37

Chapter 2.1

Effect of variation in amniotic fluidspecimens and of type of culturemedium on culture time and numberof cell clones of primary in-situcultures of amniocytes

B.Sikkema-Raddatz, R.Suijkerbuijk, K.Bouman, C.H.C.M. Buys, G.J. te Meerman

Department of Medical Genetics, University of Groningen, The Netherlands

Chapter 2


38

2.1.1 Abstract

For a series of 579 consecutive amniotic fluid (AF) specimens received for prenatal

cytogenetic analysis within a year, we registered for each specimen the following

parameters: the indication for amniocentesis, which gynaecologist performed the

amniocentesis, the gestational age at amniocentesis, the volume and the sample

appearance (clear, with some blood, bloody or brown), the size of the cell pellet,

the lot and type of culture medium and the number of cell clones observed at the

first check after 5 or 6 days of culturing. Slides for cytogenetic analysis were made

when about 80% of the cell clones reached a medium size. The number of days

between initiation and end of the culture (total culture time) was registered, as was

the result of the cytogenetic analysis. The total number of clones at first inspection

and the total culture time as measures of cell culture quality were used as

dependent parameters. The influence of these parameters on cell culture quality

was statistically analysed by analysis of variance. The number of clones is

significantly influenced by the variability of admitted AF (volume, size of the pellet,

appearance). However, provided that at least 0.8 ml AF per culture dish is

admitted, this variability will in general not affect the procedure, since the number

of clones routinely found exceeds the number considered as minimal for a reliable

cytogenetic diagnosis. Culture time is only influenced by the type of culture

medium and the appearance of AF. Bloody or brown AF increased the culture

time, from on average 8.7 days to 11.5 days.

2.1.2 Introduction

Prenatal cytogenetic diagnosis by amniocentesis requires cell culture from amniotic

fluid (AF). The success rate of AF cell culture has dramatically increased over the

years, so that culture failure currently occurs in no more than 0.5% of the cases

(Lam et al., 1998). Introduction of the in situ method and optimisation of culture

media (Chang et al., 1982) have strongly reduced the time required for cytogenetic

diagnosis. The volume of the admitted AF specimen is rarely critical, as 3.2 ml

appear sufficient for a reliable cytogenetic diagnosis (Sikkema et al., 2002).



39

Normally, the specimens obtained consist of clear amniotic fluid, but sometimes

admixture of blood causes a bloody or brown appearance. It is speculated that this

might adversely influence cell culture quality as measured by the number of cell

clones and the total culture time (Seguin et al., 1983). Other factors that may affect

the quality of the cell culture are the experience of the gynaecologist performing

the amniocentesis, the gestational age at amniocentesis and the culture medium.

However, for none of these parameters the influence on cell culture quality has

been systematically investigated. Therefore, we retrospectively investigated the

effect of these parameters on the outcome of our standard AF cell culture

procedure for a period of one year by analysis of variance.

2.1.3 Materials and MethodsCulture procedure

Per patient approximately 16 ml AF was drawn and divided over two tubes. The

volume of AF was measured, the appearance was categorised as clear, containing

some blood, bloody or brown. After centrifugation the height of the pellet in a 15 ml

conical-based tube was ranked as small (< 1mm), normal (1 – 4 mm) or large

(>4mm). Amniotic fluid cells were cultured according to the in-situ method on 30

mm coverslips in 35 mm Petri dishes under 5% CO2 at 37 0C. From each tube two

cultures were started, using two different types of culture media. In one system

Amniomax (Invitrogen – Life Technologies, Breda, The Netherlands) from three

different lots was used. In the other system Amniochrome (Cambrex Company,

Verviers, Belgium) was used pure as well as diluted with foetal calf serum and α-

MEM (Cambrex Company, Verviers, Belgium). The latter system included three

different lots of Amniochrome and three different lots of serum. In total we have

used seven different compositions of culture medium by combining each lot of

Amniochrome with two different lots of serum and one lot of pure Amniochrome.

The medium was changed after 5 or 6 days and the cultures were checked

for cell growth under a phase contrast microscope (Zeiss, Germany). The number

Chapter 2


40

of clones was counted and the size of the clones was classified as "a cell group"

(from a minimum of 5 cells up to 50 cells), "medium" (more than 50 cells, but all

cells visible within one view at 65x magnification) and "large" (not all cells visible

within one view at 65x magnification). Cultures were checked every other day and

prepared for analysis of mitotic cells when about 80% of the clones were of

medium size. One of the four cultures per patient was not prepared for analysis

and kept as back-up until a chromosomal diagnosis was obtained.

Data registration

Data from 579 AF samples, received in a one-year period, were stored in a

datafile, with as independent variables per culture:

- gynaecologist who performed amniocentesis (N=9)

- indication for amniocentesis

- weeks of gestation (14 – 36 weeks)

- volume of AF from each tube (2ml – 15 ml )

- appearance of the AF (clear, some blood, bloody, brown)

- height of the pellet after centrifugation (small: < 1mm, cells yet visible; normal:

1 - 4mm; large: > 4mm in a 15 ml conical-based tube)

- culture medium: type and lot number of medium and serum

- chromosomal diagnosis

As dependent variables we used:

- the total number of clones at the first check (after 5 or 6 days of culture)

- total culture time (time between initiation of culture and preparation for

analysis)



41

Statistical analysis

Cell culture quality was defined per specimen as the total number of clones at the

first check and as the total culture time. For both parameters, analysis of variance

was used to assess the influence of the admitted AF, the gynaecologist and the

composition and lot of the culture medium. The data were analysed in SPSS10.

Analysis of variance and regression was used to assess the quantitative effect of

the different parameters.

2.1.4 ResultsVariance in the number of clones after the first check

The variance in the total number of clones at the first check is minor, but

significantly influenced by the diversity of the admitted AF (Table 1a). The same

result was calculated for the total number of clones. Of the variance of the number

of clones after the first check 4.7 % is explained by which gynaecologist performed

the amniocentesis. Different appearance of AF resulted in about 3% of the

variance in the number of clones. Size of the pellet and volume of AF explained

approximately 1.5% of the variance in the number of clones, whereas duration of

gestation had no significant influence. The total number of clones was significantly

different between the different lots of culture media, explaining up to 4.5% of the

variance (Table 1b). There was no significant difference in the total number of

clones between the different types of culture medium.

Chapter 2


42

Table 1a: Separate effect analysis of variance for the number of clones on thediversity of the admitted amniotic fluid

Effect Explainedvariance

(%)

Sum ofsquares

DF Meansquare

F ratio P (twotailed)

gynaecologist 4.7 3746.3 6 624.4 6.4 0.00

Error 79070.9 782 97.1

weeks of gestation 0.4 328.9 1 328.9 3.3 0.072

Error 76643.6 756 101.1

volume of AF 1.3 1045.1 2 522.6 5.2 0.006

Error 79070.9 782 100.0

size of the pellet 1.4 1084.8 2 542.4 5.4 0.005

Error 79070.9 782 99.9

appearance of the AF 2.9 2283.2 2 1141.6 11.6 0.00

Error 79070.9 782 98.4

Table 1b: Analysis of variance for the total number of clones on different lots andtypes of medium


(%)

Sum ofsquares

DF Meansquare

Fratio

P (twotailed)

Amniomax versusAmniochrome with serum 1.60 401.6 1 401.6 3.9 0.048

Error 24471.2 241 101.5

Amniomax (differentbatches) 3.50 3326.4 2 1663.2 14.9 0.00

Error 102291.4 919 111.3

Amniochrome (differentbatches with/withoutserum)

4.50 4734.2 6 789.0 7.2 0.00

Error 100883.6 915 110.3

Amniochrome pure versusAmniochrome with serum 2.10 2193.1 1 2193.1 19.5 0.00

Error 103424.7 920 112.4



43

Variance in the total culture time

Approximately 14% of the variance in culture time is explained by the appearance

of the AF (Table 2). For bloody or brown AF (Table 3) three extra days of culturing

appeared necessary. Although the culture time became extended, the appearance

of AF had a minor influence on the number of clones.

About 6 % of variance in culture time is explained by duration of gestation

(Table 2). However, this effect is minor, as it appears statistically to be not

significant. We received 40 AF samples drawn after 24 weeks of gestation. After

sub-grouping the duration of gestation in less or more than 24 weeks, effects on

culture time and on number of clones were still not significant, though a minor

increase in culture time was detectable: 9.2 days for specimens taken after 24

weeks of gestation versus 8.5 days for specimens taken before 24 weeks of

gestations. The reason for amniocentesis for the specimens taken after 24 weeks

of gestation, were ultrasound abnormalities. Ten of them were chromosomally

abnormal and in one case a total culture failure occurred.

Of all the 579 specimens, 36 showed an abnormal karyotype (29 with unbalanced

and 7 with balanced abnormalities). Culture time appeared not to be influenced by

an abnormal chromosomal diagnosis (2% of the variance explained, statistically

not significant; data not shown).

Chapter 2


44

Table 2: Single effect analysis of variance for the total culture time on the diversityof the admitted amniotic fluid


(%)

Sum ofsquares

DF Meansquare


gynaecologist 4.75 52.1 8 6.5 2.3 0.021

Error 1044.3 367 2.8

weeks ofgestation

6.10 66.8 21 3.2 1.1 0.351

Error 1029.5 354 2.9

volume of AF 5.30 57.9 13 4.5 1.6 0.097

Error 1038.5 362 2.9

size of the pellet 1.80 20.1 3 6.7 2.3 0.075

Error 1076.3 372 2.9

appearance ofthe AF 14.0 151.6 4 37.9 14.9 0.00

Error 944.8 371 2.5

Table 3: Mean effects on culture time

Appearance of amniotic fluid Number of samples Culture time in

days (±SD)clear 264 8.68 (± 1.4)

some blood 71 8.64 (± 1.2)

bloody 28 10.35 (± 3.1)

brown 11 11.45 (± 2.4)

SD, Standard deviation

A significant difference in culture time was present related to the type of

medium (Table 4), which explained 2.6% of the variance. This resulted in a one-

day delay in culture time for Amniochrome diluted with foetal calf serum and α-

MEM compared with Amniomax (Table 5). Therefore, more cultures were

harvested from the culture system with Amniomax. The total culture time is not

influenced by a lot effect for different batches of medium (Table 4).



45

Table 4: Analysis of variance for the total culture time on different lots and types ofmedium


(%)

Sum ofsquares

DF Meansquare


Amniomax versusAmniochrome withserum

2.6 55.3 1 55.3 23.6 0.000

Error 1999.3 852 2.3

Amniomax (differentbatches) 0.6 7.1 2 3.5 1.2 0.301

Error 1089.3 372 2.9

Amniochrome(different batcheswith/without serum)

1.5 16.7 6 2.8 0.9 0.461

Error 1079.7 368 2.9

Amniochrome pureversus Amniochromewith serum

0.1 1.0 1 1.0 0.3 0.566

Error 1095.4 373 2.9

Table 5: Comparison between culture media

Amniomax Amniochrome with serum

Average culture time(days ±SD) 8.45 (± 2.1) 8.88 (± 1.7)

No. of harvested slides 478 376

Number of clones (±SD) 18.95 (± 9.5) 17.22 (± 11.0)

No. of clones at day 5 (±SD) 11.9 (± 7.5) 9.99 (± 7.6)



Growth maximum at day 7 day 8

SD, standard deviation

Chapter 2


46

2.1.5 Discussion

In this study we have systematically examined the influence of differences in AF

sample characteristics and culture medium on cell culture quality. For admitted AF,

bloody or brown AF had a significant effect (14% explained variance) on culture

time. Seguin et al. (1983) suggested from their results that bloody or brown AF

inhibited cell growth by restriction of clone size. Chang and Jones (1991) showed

that a grossly bloody specimen could inhibit growth by 30 percent. Our results

show that bloody or brown AF delayed the growth for three days. However, the

number of clones was minimally affected.

The volume of AF did not affect total culture time and affected the number

of clones to only a minimal extent. Our former results (Sikkema et al, 2002) already

demonstrated that the same number of clones resulted from 0.8 ml to 1.5 ml of AF

per in-situ dish as from using 5 ml or more. Seguin et al. (1983) determined that

the inoculating concentration of cells which was most efficient corresponded to

about 0.8 to 2.5 ml AF for flask cultures. They found that for producing clones small

sample volumes are more efficient. This saturation effect might explain the fact that

only small differences were measured in our results, since the minimal amount of

AF received was 2 ml.

The gestational age at amniocentesis had no influence on culture quality. This is in

line with results of Elejalde et al. (1990). Although the total number of cells

increases with gestational age, the number of viable cells decreases and only a

fraction of these cells is forming clones. According to our results, the size of the

pellet correlated positively with the duration of gestation. We therefore conclude,

that the number of clone-forming cells remains stable during the second and third

trimester of gestation.

Reports in the literature (Reid et al., 1996; Persutte et al., 1995) suggest an

association between culture failure and karyotype abnormality. Among our cases

there was only a single case of culture failure (IUGR at 35 weeks, karyotype in

fibroblasts 46,XY, del(4p)). Because of this near absence of failures, the

association could not be investigated in this study. We therefore measured the



47

variance in culture time for patients with normal and abnormal chromosomal

diagnosis. Since we did not find any difference between these two groups, we

conclude that culture delay or even culture failures are not associated with

chromosomal abnormalities. Lam et al. (1998) conclude that rather than being

associated with an abnormal karyotype, culture failure is associated with foetal

structural defects found by ultrasound in advanced pregnancies. Our results are in

line with these data. Although there was no significant variance in culture time

before or after 24 weeks of gestation, culture time was delayed to a minor extent in

the group of patients with AF sampling after 24 weeks. All of them had foetal

abnormalities at ultrasound. An abnormal karyotype occurred in 25%.

The number of clones was only slightly affected under all investigated

conditions. Obviously, the quality of AF culture medium has been so much

optimised by enrichment with growth factors (Chang et al., 1985) that when a

minimum of AF is cultured, most of the living cells in the fluid develop into a clone.

This idea is supported by experiments of Chang and Jones (1991) in which growth

saturation occurred after enrichment of culture medium with cell attachment

factors.

For the type of culture medium a significant difference in culture time was

found between Amniomax and Amniochrome (with serum). For Amniochrome, one

day delay of culture time was measured. Since the exact compositions of the

culture media are not available, we cannot identify what causes this difference.

Although culture time for Amniochrome is extended, we keep using this medium in

our routine diagnostic procedures in order to have two different and independent

culture systems, thus contributing to the overall reliability of the diagnosis.

In summary, current procedures of amniocentesis and of culturing of

amniocytes are sufficient for a reliable and consistent culture quality. The number

of clones is significantly influenced by the variability of admitted AF (volume, size of

the pellet), but provided that at least 0.8 ml AF per culture dish is admitted, this is

hardly of importance since the number of clones generally exceeds the number

considered as minimal for a reliable cytogenetic diagnosis. Culture time is only

Chapter 2


48

influenced by the type of culture medium and the appearance of AF. Bloody or

brown AF increases culture time for three days.

2.1.6 Literature

Chang HC, Jones OW, Masui H. Human amniotic fluid cells grown in a hormone-supplemented

medium: suitability for prenatal diagnosis. Proc Natl Acad Sci U S A. 1982 ;79(15):4795-9.

Chang HC, Jones OW. Reduction of sera requirements in amniotic fluid cell culture. Prenat Diagn.

1985;5(5):305-12.

Chang HC, Jones OW. Enhancement of amniocyte growth on a precoated surface. Prenat Diagn.

1991;11(6):357-70.

Elejalde BR, de Elejalde MM, Acuna JM, Thelen D, Trujillo C, Karrmann M. Prospective study of

amniocentesis performed between weeks 9 and 16 of gestation: its feasibility, risks, complications and

use in early genetic prenatal diagnosis. Am J Med Genet. 1990;35(2):188-96.

Lam YH, Tang MHY, Sin SY, Ghosh A. Clinical significance of amniotic-fluid-cell culture failure. Prenat

Diagn. 1998;18(4):343-7.

Persutte WH, Lenke RR. Failure of amniotic-fluid-cell growth: is it related to foetal aneuploidy? Lancet.

1995;14;345:96-7.

Reid R, Sepulveda W, Kyle PM, Davies G. Amniotic fluid culture failure: clinical significance and

association with aneuploidy. Obstet Gynecol. 1996;87(4):588-92.

Seguin LR, Palmer CG. Variables influencing growth and morphology of colonies of cells from human

amniotic fluid. Prenat Diagn. 1983;3(2):107-16.

Sikkema-Raddatz B, van Echten J, van der Vlag J, Buys CHCM, te Meerman GJ. Minimal volume of

amniotic fluid for reliable prenatal cytogenetic diagnosis. Prenat Diagn. 2002;22(2):164-5.



49

Chapter 2.2

Minimal volume of amniotic fluid forreliable prenatal cytogenetic diagnosis

B. Sikkema-Raddatz, J. van Echten, J. van der Vlag, C.H.C.M. Buys and G.J. te

Meerman


Prenat. Diagn 2002; Vol. 22: 164-165

Chapter 2


50

Letter to the editor:

Second trimester amniocentesis and amniotic fluid (AF) culture is a well-known

method of prenatal cytogenetic diagnosis. Introduction of the in situ method and

optimised culture media has strongly reduced the culture time. In addition, culture

failures have been reduced to less than 0.5%. In current amniocentesis practice,

20 ml AF is drawn for prenatal cytogenetic diagnosis and usually at least three

cultures are established from this volume (Priest and Rao, 1997). However, in a

published amniocentesis series (Winsor et al.,1999), a volume of less than 8 ml

was obtained in 13% of the cases. Repeated amniocentesis is recommended when

less than 5 ml of AF is obtained (Priest and Rao, 1997).

This prompted us to investigate the minimal volume of AF required for a

reliable diagnosis, in order that repeated amniocentesis can be avoided.

In a pilot study we pippetted directly 0.25 ml, 0.50 ml, 0.75 ml, 1.0 ml and

1.5 ml AF of five different samples on Ø30 mm coverslips in Ø35 mm Petri dishes

and immediately added 2 ml of culture medium (Amniomax TM C-100, Life

Technologies B.V., The Netherlands). It appeared that from a volume of 0.25 ml AF

only one or two clones developed. A volume of 0.75 ml yielded more clones, but

fewer than eight, too few for a reliable diagnosis.

Therefore, volumes of 0.8 ml, 1.0 ml and 1.5 ml of AF were used for

culturing on Ø30 mm coverslips in Ø35 mm Petri dishes. From each of 12 samples,

randomly chosen within a certain time interval, these various volumes were directly

pipetted onto coverslips and 2 ml culture medium (Amniomax TM C-100, Life

Technologies) was immediately added. As a control, our standard procedure was

used, in which four cultures with an equivalent of about 5 ml AF are established.

Briefly, approximately 20 ml of AF is collected in two tubes and centrifuged at 1000

rpm for 10 min. Supernatants are removed until a volume of 0.3 ml remains. Then,

0.5 ml culture medium (Amniomax TM C-100, Life Technologies) is added. The mix

per tube is divided over two Ø35 mm Petri dishes containing Ø30 mm coverslips.

The following day, 2 ml medium is added to the culture.



51

In order to exclude changes in the proportion of AF and culture medium, a

second series of 0.8 ml, 1.0 ml and 1.5 ml volumes from 12 other AF samples was

studied. After centrifugation at 1000 rpm for 10 min, supernatants were removed

until a volume of 0.8 ml remained. This was pipetted onto Ø30 mm coverslips in

Ø35 mm Petri dishes. Than 2 ml culture medium was added. As a control, about 5

ml of AF was centrifuged as described earlier. The supernatant was removed until

0.8 ml remained. This was pipetted directly on a coverslip and 2 ml culture medium

was added.

After 6 days, both test and standard cultures were blindly checked for cell

growth. Based upon the number of clones, the appropriate day for in-situ

cytogenetic analysis was determined.

In both series we found on average 14 clones per Petri dish with 0.8 ml AF

(Table 1), sufficient for chromosomal diagnosis. Although the proportion of AF and

culture medium differed between the two series, this did not affect the number of

resulting clones. The appropriate day for analysis, however, varied with the amount

of AF. Culturing of 1.5 ml AF was comparable with the standard procedure,

whereas culturing of 0.8 ml resulted in a delay of two days for the appropriate day

for analysis.

The reliability of cytogenetic diagnosis was confirmed in a series of 52 samples. On

average 13 ±6 clones were obtained that could be analysed at Day 11 (controls

16±5 clones at Day 9). No culture failure was obtained.

Chapter 2


52

Table 1: Number of clones and appropriate day of analyses resulting from different

volumes of amniotic fluid (AF)

AF (ml) Culturing with different endvolumes (n= 12)

Culturing with a fixed end volume(n= 12)

Number ofclones (SD)

Day of analyses(SD)

Number of clones(SD)

Day of analyses(SD)

0.8 14 (±7.6) 11.4 (±1.8) 14 (±7.2) 12.1 (±1.6)

1.0 16 (±5.1) 11.3 (±1.8) 14 (±5.7) 11.3 (±1.7)

1.5 16 (±3.7) 11.5 (±1.5) 17 (±7.5) 9.9 (±1.6)

Control(approximately5ml)

15 (±5.6) 8.6 (±0.7) 18 (±4.9) 9.0 (±0.8)

SD, standard deviation; n, number of samples

Whereas Richkind and Risch (1990) obtained on average no more than

two clones per millilitre of AF, about four times more cells grew out to analysable

clones under our much more marginal conditions. The finding that an

approximately equal number of clones grow from about 1ml AF in the present

experiments and from 5 ml in the standard procedure might be attributed to an

inhibitory effect. Substances derived from dead cells, present in the amniotic fluid,

might inhibit the viable cells to grow out to clones. This effect would be stronger

when a higher number of dead cells is present.

The present study shows that using four cultures per diagnosis, repeated

amniocentesis will not be necessary as long as more than 3.2 ml (4x 0.8 ml) of AF

is available. Since the number of clones obtained in second trimester

amniocentesis in the present experiments is comparable to the number obtained

per millilitre in early amniocentesis as reported by Kennerknecht et al. (1992), it

might be worthwhile investigating the feasibility of also performing early diagnosis

with smaller volumes of AF.



53

Literature

Kennerknecht I, Baur-Aubele S, Grab D, Terinde R. First trimester amniocentesis between the seventh

and 13th weeks: evaluation of the earliest possible genetic diagnosis. Prenat Diagn. 1992;12(7):595-

601.

Priest JH and Rao KW. Prenatal chromosome diagnosis. The AGT Cytogenetic Laboratory Manual (3rd

edn), Barch MJ, Knutsen T and Spurbeck J (eds.) Lippincott- Raven: Philadelphia, PA 1997; Chapter 5,

206.

Richkind KE, Risch NJ. Sensitivity of chromosomal mosaicism detection by different tissue culture

methods. Prenat Diagn. 1990;10(8):519-27.

Winsor EJ, Tomkins DJ, Kalousek D, Farrell S, Wyatt P, Fan YS, Carter R, Wang H, Dallaire L, Eydoux

P, Welch JP, Dawson A, Lin JC, Singer J, Johnson J, Wilson RD. Cytogenetic aspects of the Canadian

early and mid-trimester amniotic fluid trial (CEMAT). Prenat Diagn. 1999;19(7):620-7.

Chapter 2


54



55

Chapter 2.3

Absolute procedure to test the growthpotential of medium in primaryamniotic fluid cell cultures and theinfluence of decreased oxygen tension

B. Sikkema-Raddatz, R. Suijkerbuijk, J. van der Vlag, M. Stoepker, C.H.C.M. Buys,

G.J. te Meerman


Chapter 2


56

2.3.1 Abstract

We have developed an absolute procedure to test the performance of culture

medium in amniotic fluid in-situ cell cultures. By reducing the concentration of

medium until a 50% growth reduction was achieved, we expect that under these

sub-optimal conditions differences in culture quality will be more pronounced

(stress testing). Our test system is based on three cultures of primary amniotic fluid

(AF) cells from each of 10 individual patients (a total of 30 cultures), initiated with

medium diluted to a 30% end concentration. Absolute thresholds for acceptance

or rejection of new lots of medium were determined from 174 individual in-situ

cultures with different types of medium. We set the threshold for acceptance to at

least 22 successful cultures out of 30 and to an average number of 3 clones or

more. With these criteria the test system has a probability of less than 0.01 of false

rejection of a good lot of medium and sufficient power to detect downward

deviations in quality. We verified the validity of our stress testing system by

evaluating the effects of decreased oxygen tension during culturing and found that

decreased oxygen tension leads to an increase of the size of a clone and the

number of cells it contains, but with minimal effect on the number of clones only.

Maximal increase in cell number was observed under 5% oxygen tension

measured by growth curves.

2.3.2 Introduction

The quality of culture medium is crucial for the success and speed of a prenatal

chromosomal diagnosis. A milestone in AF cell culture was the introduction of

media supplemented with growth factors by Chang et al. (1982). The quality of the

cell cultures increased dramatically; the culture time was reduced from 20.3 days

to 12.8 days and culture failure from 1.53% to 0.33% (Salk et al., 1983). Each type

of AF cell culture medium contains a certain amount of serum, either included in a

commercial medium or added separately to a basic culture medium. The growth

potential of serum can vary between different lots (Pye, 1975), because serum

contains a complex mixture of growth promoting factors, since it is produced in

vivo. To maintain cell culture quality and to avoid culture failures, each lot of serum



57

and medium has to be tested for its growth potential (Priest and Rao, 1997). This is

particularly important in prenatal cytogenetic diagnosis, in which repeated

amniocentesis needs to be avoided.

To date, use of parallel cultures with new and old medium is the recommended

procedure (Priest and Rao, 1997). In our laboratory, growth of pooled secondary

AF cell cultures is compared between new and old medium by making growth

curves over several days. A new lot is only introduced in routine diagnostic practice

if growth in the new medium is better than or approximately equal to that in the old

medium. This type of testing is in principle subject to drift. It also may not yield the

most relevant quality indication, as the test is based on cells that have been in

culture already. We, therefore, wanted to develop an absolute test for primary AF

in-situ cell cultures which could be applied to left-over small fractions of patient

samples. Among individual AF cell cultures there is a high variability in number of

clones and culture time to harvest. The growth potential of serum is in particular

important for cultures with a minimal number of clones and slow growth.

Our absolute test for primary AF cell cultures should have fixed thresholds,

defined as a minimal number of clones and a minimal number of successful

cultures after certain days. Our starting point for developing the test is that

differences in quality of culture medium might be better detectable under sub-

optimal conditions, i.e. stress testing. The criterion for rejection should then be

based on the observed variability in culture success and number of clones, such

that false rejection should occur only rarely, e.g. in 1% of cases. Here we describe

the successful development of such a test. Our stress testing procedure is not only

suitable for testing the growth potential of culture medium, but can also be used to

investigate the influence on growth potential of any other factor involved in cell

culture. As an example we demonstrate its application to monitor the influence of

decreased oxygen tension on growth potential during culturing.

Chapter 2


58

2.3.3 Materials and MethodsStress testing

Amniotic fluid was obtained by second trimester amniocentesis between 15 weeks

and 20 weeks of gestation. Routine primary in situ cultures of AF cells were

initiated, leaving a small fraction (approximately 2.5ml) of the AF for stress testing.

We evaluated the effect of the following forms of different potential stress:

- Storage of AF; before culturing, AF was stored for 2 days at room temperature

(RT) or at 40C or AF was centrifuged and the pellet resuspended in PBS. This

mixture was stored either at RT or at 40C.

- Heat shock; before culturing, the received AF was placed in a waterbath at 400C

for 30 min or 60 min.

Storing or heat shock of AF before culturing showed no growth reduction (results

not shown).

- Dilution of culture medium; in order to have a comparable quality of AF in different

tests, pooled AF was used. A pool consisted of AF from at least three different

patients. This enabled us to test several conditions within a short time. In a first

series of experiments ten cultures were established from pooled AF. Culture

medium was diluted with PBS to end concentrations of 20%, 40%, 60% and 80%,

respectively.

Primary AF cell cultures were initiated from 0.8 ml AF and supplied with 2

ml diluted culture medium as described previously (Sikkema et al., 2002). Cultures

initiated with undiluted medium served as controls. Culture medium was refreshed

on days 6 and 8. The number of clones was counted under a phase contrast

microscope on days 6, 8 and 10 and compared with control cultures. The size of

the clones was classified as a " cell group" (from a minimum of 5 cells up to 50

cells), "medium" (more than 50 cells, but all cells visible within one view at 65x

magnification) and "large" (not all cells visible within one view at 65x magnification).

Growth reduction was defined as the reduction of the number of clones compared

with that in control cultures.



59

Finally, to obtain a good estimate of dilutions resulting in approximately 50%

growth reduction, end concentrations of culture medium of 60% and 30% were

tested with AF from 18 different pools (54 cultures).

Stress testing and determining thresholds for individual AF cell cultures

AF was used from all individuals from whom a minimum of 18ml fluid was received.

Experimental series, consisting of three cultures from each of 10 individual

patients, were initiated with 0.8 ml AF supplied with 2ml culture medium diluted to

an end concentration of 30%. Amniomax C-100 TM (Invitrogen – Life

Technologies, Breda, The Netherlands) and different lots of Amniochrome

(Cambrex Company, Verviers, Belgium) were tested. Media had been accepted

previously through growth curve testing and used in our routine diagnostic practice.

For acceptance or rejection of new lots of serum or medium, absolute thresholds

are defined as a minimal average number of clones and a minimal number of

successful cultures at 8 days. In total, six different series of 30 cultures resulting in

180 individual cultures were used. Six cultures were excluded from analysis

because of non-clonal growth of rapidly adhering cells. This left 174 cultures. The

number of clones was counted on days 6 and 8 and the number of successful

cultures was determined on day 8. Routine AF cell cultures from the same patients

served as a control for failures not due to stress testing. No culture failures were

observed under normal conditions. For validation of the thresholds, other media

with different growth potential were equally stress tested (Table 1). Medium with

known inferior quality as determined by growth curve testing (F10 supplemented

with 20% foetal calf serum), medium of unknown quality (Amniochrome 4MB0020)

and medium accepted in our routine practice were used in stress testing.

Chapter 2


60

Growth curve testing

The growth curve is used to compare a new lot of medium with the lot in use and is

based on secondary AFC cultures. About 14 days after initiating routine (primary)

cultures, five different back-up cultures are trypsinised. Cell suspensions of these

cultures are pooled and the number of cells is counted on a cell counter (Sysmex

F-820, Charles Goffin, Tiel, The Netherlands). The suspension is split to initiate 24

secondary cultures, of which12 are cultured on a new lot of medium and the other

12 on the lot in use. The medium is changed on day 6. Three cultures of each lot

are harvested on days 5, 6, 7 and 8. The number of cells in each culture is counted

and the mean and standard deviation is calculated. The new lot of medium is only

introduced into routine diagnostic practice if growth of the new lot is equal (within 1

standard deviation) or better than the lot in use. This indicates that drift of the

acceptance criterion towards higher and eventually insurmountable thresholds may

occur, as well as gradual worsening of quality.

Stress testing of decreased oxygen tension during culturing

The decreased oxygen tension was set at 2.5% and 5%, compared to the normal,

approximately 20% in the atmosphere. Stress testing was performed as described

before with individual AF cell cultures at 370C and a CO2 concentration of 5% in a

waterjacketed CO2 incubator (Forma Scientific, Thermo Electron Corporation,

USA). Parallel cultures from the same individuals were incubated at standard

atmospheric (20%) oxygen in a Steri- Cycle CO2 incubator.

A single lot number of Amniochrome, previously accepted in the routine, was used

for all cultures. The number of clones grown under 2.5% and 5% oxygen tension

was counted on days 6 and 8 and compared with the number of clones from

control cultures. Furthermore, growth curves were initiated as described before at

2.5%, 5%, and 10% and 20% oxygen tension (control condition), respectively, with

undiluted medium and with medium diluted to an end concentration of 30%. The

number of cells was counted and compared with the number of cells grown under

the control condition. To test for possible differences in growth potential between



61

both incubators, a series of 30 parallel cultures was initiated. No significant

differences in number of clones and/ or number of successful cultures were

detected.

2.3.4 ResultsMedium dilution as stress condition

Dilution of culture medium resulted in a proportional reduction of growth. In a series

of 18 pools (56 cultures), a 50% growth reduction was achieved at a dilution of

culture medium to an end concentration of 30%. The number of clones at day 8

was reduced from 10.1 ± 5.0 (mean ± S.D.) in controls to 5.3 ±3.2 (mean ± S.D.) in

the diluted medium. A dilution of culture medium to 30% end concentration has

therefore been used as the appropriate stress condition in the following

experiments.

Determining thresholds for stress testing of individual AF cell cultures

Thresholds for stress testing were determined from 174 individual primary in-situ

cultures from 6 different series, cultured in medium diluted to an end concentration

of 30%. On average 5.2 ± 5.0 (mean ± S.D.) clones were counted after 8 days of

culturing. Sixteen of the 174 cultures failed to grow, i.e. a 9.2% culture failure. The

average number of culture failures was 2.6 out of 30 within the 6 experimental

series. If we set the threshold for the minimally acceptable number of successful

cultures to 23 out of 30, the observed variability indicates that this would lead to

less than 1.0% wrongly rejected lots. In order to determine a threshold for the

minimal mean number of clones, we will accept that 1% of the rejected lots of

medium will be rejected wrongly. Considering the variability among the cultures,

this results in a threshold of on average 3 clones in 30 cultures. For the exact

derivation the reader is referred to the Appendix.

These thresholds were validated with different types and lots of medium

(Table 1). Basic medium F10 supplemented with 20% foetal calf serum was

Chapter 2


62

rejected under stress testing, as expected. The other media were tested as

acceptable.

Table 1: Results of stress testing of individual amniotic fluid in-situ cell cultures

from different types and lots of medium

Culture medium Day ofanalysis

No. of clones(mean ± S.D.)

No. of culturefailures

Acceptedfor routine

Amniomax (lot 1198269) day 6 3.1 ± 2.45 2

day 8 4.0 ± 2.9 1 yes

Amniochrome (lot 3MB0203) day 6 2.1 ± 1.3 2

day 8 3.0 ± 1.9 3 yes


day 8 6.7 ± 5.3 0 yes


day 8 5.0 ± 4.0 1 yes

F10 with 20% FCS day 6 1.0 ± 0.98 12

day 8 1.4 ±1.4 11 no

Stress testing of decreased oxygen tension during culturing

Using stress testing, the average number of clones at day 8 when cultured at 2.5%

O2 was 5.8 versus 4.8 for control cultures. This difference is significant (P< 0.036,

paired T- test, two sided). For 5% O2 we observed 7.7 clones versus 7.0 clones in

control cultures. This difference was not significant (P> 0.429, paired T- test, two

sided). The number of successful cultures at 2.5% O2 was 29 versus 27 in control

cultures and at 5% O2 28 versus 26 in control cultures under 20% O2. The number

of clones of medium or large size was higher under decreased oxygen tensions of

both, 2.5% and 5%.



63

Table 2: Analysis of variance for the effect of decreased oxygen tension on thegrowth of amniotic fluid cells

Dependent variable Explainedvariance

(%) 1)

Effect Sum ofsquares

DF Meansquare

F- ratio P (twotailed)

Growth (undilutedmedium)

Culturetime

22.0 3 7.3 128.6 0.000

20.0 O2 tension 5.7 1 5.7 100.5 0.000

Error 1.1 19 0.06

Total 28.8 23

Growth (medium of30% end-concentration)

Culturetime

12.1 3 4.0 52.3 0.00

36.0 O2 tension 7.8 1 7.8 101.1 0.000

Error 1.5 19 0.08

Total 21.4 231)variance in difference between number of cells cultured under 5% and 20% oxygen tension

To measure the increase in number of cells over time, growth curves were

made, both with decreased oxygen tension (2.5%; 5% and 10%) and 20% oxygen

(control concentration). A significant increase in the number of cells was measured

at all investigated conditions of decreased oxygen. At 5% (Figure 1a and b) the

effect was most pronounced, with a 2.5 fold average increase, explaining 20% of

the variance (Table 2). Within this condition, the largest increase was seen at days

5 and 6 (Figure 1a). In the same experiment, but using diluted medium with end

concentration of 30%, the increase was even 4.5 fold, explaining 36% of the

variance (Figure 1b).

Chapter 2


64

Figure 1a: Growth curves of secondary amniotic fluid cells with undiluted medium

0 5 6 7 8Day

0

10

20

30

40

Num

ber o

f cel

ls /m

l x 1

0.00

0

20%5%

Standard deviation I

Oxygen tension

Figure 1 b: Growth curves of secondary amniotic fluid cells with medium diluted to 30%

0 5 6 7 8Day

0

5

10

15

Num

ber o

f cel

ls /m

l x 1

0.00

0

5%20%

Standard deviation I

Oxygen tension



65

2.3.5 Discussion

In this study we describe an absolute method for testing the influence of any factor

involved in in-situ cultures of primary amniotic fluid cells on the growth potential.

Stress testing appears more sensitive to detect differences in growth potential than

testing without a stress condition, as is apparent from the stronger response of

cultures to a decreased oxygen tension. This supports the validity of using stress

testing as a method to determine acceptance or not of different lots of medium for

cell culture for prenatal diagnosis.

Whereas other absolute procedures to test the growth potential of medium,

such as plating efficiency (Goodheart et al., 1973), incorporation of radionuclides

during cell growth (Dvorakova and Starek, 1980) or an end point dilution method

(Shu-de and Hampson, 1985) require a high degree of technical skill or elaborate

equipment, the method we propose is easy to incorporate in daily routine.

Our stress testing is based on absolute thresholds for the minimum number

of clones and for the minimum number of successful cultures after 8 days of

culturing. This implies a high probability that no medium will be accepted with less

than the required quality, while rejection of lots that would have had sufficient

quality if tested more extensively, will be minimal. The acceptance criterion is

absolute and not subject to any drift. We recommend to use at least 30 cultures

from at least 10 different patients. This should be sufficient to detect important

differences in quality.

A further advantage of this method is the use of non-pooled primary AF in-

situ cell cultures. Therefore, the growth behaviour of the cells during testing is likely

more representative for the conditions present in routine cultures. To demonstrate

that the absolute method can readily be used for an assessment of the effect of

other factors than culture medium influencing growth potential and to demonstrate

the high sensitivity of stress testing, we investigated the effect of a decreased

oxygen tension during culturing. Two early reports (Brackerts et al., 1983; Held and

Soennichsen et al., 1984) have appeared in which reduced oxygen supply during

Chapter 2


66

culturing resulted in an up to 25% increase in the number of clones and in a

significant increase of the number of cells. In stress testing we could only confirm

this for the lowest oxygen tension (2.5%), but we did find that the size of the clones

was larger under both conditions of decreased oxygen. This was in agreement with

the results of growth curve testing which showed that the number of cells was 2.5-

fold increased, explaining 20% of the variance and under stress conditions even

36% of the variance when culturing under a 5% oxygen tension. The difference in

number of clones between the former results and ours might be explained by our

use of hormone-supplemented medium as introduced by Chang et al. in 1982,

whereas in the earlier experiments a basic medium supplemented with foetal calf

serum only was used. The hormone-supplemented medium already maximises the

growth of the viable cells from AF, giving a maximum number of clones (Chang et

al., 1991). Thus, with respect to the number of clones, the effect of the culturing

medium is much stronger than that of a decreased oxygen tension. The strongest

effect had a decreased oxygen tension on cultures with a small number of clones

and on the increase in number of cells during the first days of culturing, as seen in

our series. This finding may suggest that cell culturing under a decreased oxygen

tension appears to be more successful.

In general, to assess the growth potential of different lots of culture medium

for acceptance or rejection in routine diagnosis, we recommend stress testing. To

test the growth potential of a new type of medium or any other factor involved in

culturing, stress testing should be performed first. In case of an increased growth

potential, the number of cells can subsequently be determined very precisely, if

desired, by making a growth curve.



67

2.3.6 Literature

Brackertz M, Kubbies M, Feige A, Salk D. Decreased oxygen supply enhances growth in culture of

human mid-trimester amniotic fluid cells. Hum Genet. 1983; 64(4):334-8.

Chang HC, Jones OW, Masui H. Human amniotic fluid cells grown in a hormone-supplemented

medium: suitability for prenatal diagnosis. Proc Natl Acad Sci U S A. 1982; 79(15):4795-9.

Chang HC, Jones OW. Enhancement of amniocyte growth on a precoated surface. Prenat Diagn.

1991;11(6):357-70.

Dvorakova M, Starek M. A method for testing the growth promoting property of tissue culture media

using radionuclides. J Biol Stand 1980; 8: 107- 113.

Goodheart CR, Casto BC, Zwiers A. Plating efficiency for primary hamster embryo cells as an index of

efficacy of foetal bovine serum for cell culture. Appl Microbiol 1973; 26: 525- 528.

Held KR, Sonnichsen S. The effect of oxygen tension on colony formation and cell proliferation of

amniotic fluid cells in vitro. Prenat Diagn. 1984; 4(3):171-9.

Hoehn H, Bryant EM, Karp LE, Martin GM. Cultivated cells from diagnostic amniocentesis in second

trimester pregnancies. II. Cytogenetic parameters as functions of clonal type and preparative technique.

Clin Genet. 1975; 7(1):29-36.

Priest JH, Rao KW. Prenatal chromosome diagnosis. Barch MJ, Knutsen T, Spurbeck (eds.) The AGT

Cytogenetics laboratory manual, Lippincott – Raven Publishers Philadelphia 1997.

Pye D. Simple and rapid evaluation of serum for cell culture. J Biol Stand. 1975; 3(1):83-7.

Salk D, Disteche C, Stenchever MR, Mitchell L, Rodriguez M. Routine use of Chang medium for

prenatal diagnosis: Improved growth and increased chromosomal breakage. Am J Hum Genet 1983;

35: 151A.

Shu-de J, Hampson AW. A simple endpoint dilution method for evaluating serum used for cell culture. J

Biol Stand 1985;13: 303-308.

Sikkema-Raddatz B, van Echten J, van der Vlag J, Buys CHCM, te Meerman GJ. Minimal volume of

amniotic fluid for reliable prenatal cytogenetic diagnosis. Prenat Diagn. 2002; 22(2):164-5.

Chapter 2


68

Appendix

Determination of the thresholds:

To set a threshold for the minimum mean number of clones, we will reject a new lot

of medium if we find significantly less than the average of 5.2 clones which are

found growing after 8 days of culture. We accept that 1% of the rejected lots of

medium has by chance less than this number.

Justification:

K is the minimum mean number of clones found growing in 30 separate culture

experiments.

We assume that the mean number of clones is distributed according to a T

distribution with 29 degrees of freedom. The one-sided threshold of finding a value

occurring in less than 1% of the cases = -2.462. This relates to the acceptable false

rejection rate.

The standard deviation of the number of clones is 5. The standard deviation of the

mean number of clones in 30 experiments is therefore 5/(sqrt(30). The following

equations show the derivation of the minimum acceptable mean number of clones

(K- 5.2) / (5/√30) > -2.462

(K- 5.2) x 1.09 > -2.462

K> 5.2 – 2.258

K > 2.94

Considering the variability among the cultures, this results in a threshold of an

average of 3 clones in 30 cultures.

To set a threshold for the minimum of successful cultures, we observed that on

average 2.6 cultures failed in a series of 30. We think that this number of failures is

on average acceptable. The number of actual culture failures might be higher than

expected due to culture failures by chance. We again accept that 1 of the 100 lots

will be rejected falsely when the real number of culture failures is 2.6 out of 30. In

30 experiments the number of failures follows a binomial distribution with



69

probability 0.0833. The probability that we find more than 7 failed cultures is then <

0.01.

If we set the threshold for the minimally acceptable number of successful cultures

to 23 out of 30, this would lead to less than 1.0% wrongly rejected lots.

Chapter 2


70

Slide preparation of in-situ cell cultures from amniotic fluid cells and chorionic villi


71

Chapter 3

Slide preparation of in-situ cellcultures from amniotic fluid cells andchorionic villi

Chapter 3


72


proefdruk1.doc

73

Assessment of the influence offixative, temperature, humidity and airflow on metaphase quality inpreparations from primary in-situcultures of amniotic fluid cells andchorionic villi

Birgit Sikkema- Raddatz1; Harry Moes 1;2; Marian Stoepker1; Bauke de Jong1;

Charles H.C.M. Buys 1and Gerard J. te Meerman1

1 Department of Medical Genetics, University of Groningen, The Netherlands

2Present address:

Department of Obstetrics and Gynaecology, University Hospital Groningen, The

Netherlands

Chapter 3


74

3.1 Abstract

The quality of metaphase chromosomes in preparations from amniotic fluid (AF)

and chorionic villus sample (CVS) in situ cell cultures for prenatal cytogenetic

diagnosis depends on various factors at harvesting. To determine the quantitative

effect of fixative, ambient temperature, relative humidity and air flow, primary AF

cell cultures and primary CVS in situ cultures were harvested under a range of

different conditions in a chromosome harvest chamber. We applied analysis of

variance and regression to establish the relation of these conditions with the

degree of metaphase spreading and the presence of cytoplasm. We found that

ambient temperature alone had no effect on metaphase quality. Relative humidity

explained 18.6% of the variance in the presence of cytoplasm and 7.5% of the

variance in the degree of metaphase spreading in AF cell cultures. In CVS

cultures, these percentages were 7.2% and 4.2 %, respectively. In AF cell cultures,

air flow had a limited influence on metaphase quality. In CVS cultures, however,

this factor was responsible for 29.9% of variance. Different compositions of fixative

influenced the spreading of metaphases for 13.7% in AF cell cultures and 4.7% in

CVS cultures. We conclude that metaphase quality in preparations from primary in

situ cultures is only to a limited degree affected by the investigated factors at

harvesting. Within reasonable limits, conditions cannot be considered as critical.

Keywords:

prenatal diagnosis, in situ culture, metaphase quality; chromosome preparation



75

3.2 Introduction

Proper techniques for harvesting dividing cells and preparing slides for

chromosome analysis are critical for attaining the quality needed for correct

karyotype analysis (Lawce and Brown, 1991). In prenatal cytogenetic diagnosis a

reliable quality of (in-situ) preparations is essential, since failure of the analysis and

repeated removal of amniotic fluid and chorionic villi bear an increased risk of

foetal loss. An understanding of the factors influencing the process of chromosome

preparation is important for maintaining a good quality. Hliscs et al. (1997)

demonstrated that spreading of metaphases is a slow process, which leads to a

stretching of chromosomes via flattening. The process of spreading involves a

significant water-induced swelling of mitotic cells during evaporation of the fixative

from the slide (Claussen et al., 2002). The degree of spreading of metaphases

depends on several factors, among which the evaporation time of the fixative is

considered an imported one (Spurbeck et al., 1996). Evaporation time depends on

the composition of the fixative, on relative humidity and ambient temperature

(Spurbeck et al., 1996). The last two factors can be controlled by the use of a

special climate room (Lundsteen et al., 1985) or a chromosome harvest chamber.

The effect of several factors on metaphase spreading has been determined

from cell suspensions from different primary cells and cell lines (Deng et al., 2003,

Claussen et al., 2002; Henegariu et al., 2001) and secondary AF cell cultures

(Spurbeck et al., 1996). For prenatal cytogenetic analyses, however, primary in situ

cultures are the most used and sensitivity of spreading for metaphases of these

cells is unknown. Therefore, we investigated the effect of relative humidity, of

ambient temperature, of air flow and of the composition of fixative at harvesting of

primary in-situ cell cultures (amniotic fluid (AF) cells and chorionic villus sample

(CVS) cultures). The quality of metaphases was determined using as parameters

the degree of spreading and the presence of cytoplasm.

Chapter 3


76

3.3 Materials and Methods

Culturing

Primary AFcells were cultured according to the in situ method on 30 mm coverslips

in 35 mm Petri dishes. The culture medium was Amniomax-C100 (Invitrogen – Life

Technologies, Breda, The Netherlands) and Chang A (Irvine Scientific, USA) with

α-MEM. Cultures were checked for cell growth after 5 days. They were harvested

when approximately 10 clones were observed, which occurred on average after 8

days.

Chorionic villi were treated with trypsin (Cambrex Company, Verviers, Belgium)

and collagenase (Sigma-Aldrich Chemie Gmbh; Steinheim, Germany) before being

cultured. The cell suspension was then transferred to coverslips (30 mm in 35 mm

Petri dishes) and cultured in Amniomax and Chang with α-MEM. Cultures were

checked for cell growth after 4 days. When growth was adequate, cells were

harvested, on average after 6 days.

Harvesting

Cultures were treated with BrdU (final concentration in culture, 36 µg/ml) and

colcemid (final concentration in culture, 0.015 μg/ml) for 16 hours (overnight).

Subsequently, cultures were harvested with a Tecan 5031 robotic harvester

(Tecan, Maennedorf, Switzerland) using 0.8% (w/v) sodium citrate as hypotonic

solution and methanol and glacial acetic acid (3:1, v/v) as fixative. During the last

fixation step the cultures were transferred to the chromosome harvest chamber

(C30, Percival Scientific, Inc., Perry Iowa, USA). The fixative was removed by

suction and the cultures were allowed to dry.

Harvesting conditions

To quantify the effects of ambient temperature, relative humidity and air flow at

harvesting, cultures were harvested under a range of conditions. The ambient

temperature ranged from 200C to 300C (intervals of 20C). The relative humidity

ranged from 25% to 60% (intervals of 5%). The effect of air flow (0.2; 0.4; 0.6 and



77

0.7 m/s) was tested at established optimal conditions (a temperature of 260C and

35% relative humidity, respectively). Because of the technical limits of the harvest

chamber it was not feasible to investigate more extreme conditions, e.g. 200C and

25% relative humidity (Table 1).

Table 1a: Summary of the results of metaphase quality scoring in AF cell culturesat different climate conditions

Relative humidity (%)

25 30 35 40 45 50 60

Tempera-ture (0C)

20 not real. not real. not real. not real. 54 metaph. not determ. 55 metaph.

44% 16%

22 not real. not real. not real. 72 metaph. 71 metaph. not determ. not determ.

47% 56%

24 not real. not real. 58 metaph. 88 metaph. 50 metaph. not determ. 55 metaph.

67% 33% 48% 20%

26 not real. 81 metaph. 101metaph. 72 metaph. 72 metaph. 67 metaph. not determ.

63% 78% 57% 16% 7%

28 not real. 69 metaph. 69 metaph. 70 metaph. 59 metaph. 57 metaph. not determ.

37% 58% 64% 2% 24%

30 56 metaph. 60 metaph. not determ. 70 metaph. not determ. not determ. 60 metaph.

41% 42% 47% 4%

metaph., metaphases; not real.; not realisable because of technical limits of the harvest chamber;

not determ., not determined

56 metaph. means that 56 metaphases were screened

41% means that 41% of the metaphases had an optimum quality (well-spread, no cytoplasm)

Chapter 3


78

Table 1b: Summary of the results of metaphase quality scoring in CVS cultures atdifferent climate conditions

Relative humidity (%)

25 30 35 40 45 50 60

Tempera-ture (0C)

20 not real. not real. not real. not real. 60 metaph. not determ. 60 metaph.

17% 26%

22 not real. not real. not real. 60 metaph. 58 metaph. not determ. not determ.

18% 12%

24 not real. not real. 58 metaph. 84 metaph. 72 metaph. not determ. 60 metaph.

47% 29% 45% 5%

26 not real. 67 metaph. 96 metaph. 72 metaph. 72 metaph. 47 metaph. not determ.

42% 38% 25% 10% 19%

28 not real. 72 metaph. 72 metaph. 72 metaph. not determ. 60 metaph. not determ.

34% 37% 18% 13%

30 57 metaph. 60 metaph. not determ. not determ. 72 metaph. not determ. 60 metaph.

26% 46% 20% 12%

metaph., metaphases; not real., not realisable because of technical limits of the harvest chamber;

not determ.; not determined

57 metaph. means that 57 metaphases were screened

26% means that 26% of the metaphases had an optimum quality (well-spread, no cytoplasm)

The different ambient temperatures, relative humidities and air flow conditions were

set by adjusting the control panel of the Percival harvest chamber. Relative

humidity was calibrated with a psychrometer. The air flow was measured with an

anemometer.

The culture slides were aged at 900C for one hour and stained with Giemsa after

treatment with 0.1% pancreatine (w/v) (Sigma-Aldrich Chemie Gmbh; Steinheim,

Germany) by an automatic staining device (Midas, Merck, Whitehouse Station,

USA).



79

Quality scoring of metaphases

Metaphase quality was determined by scoring the metaphases for the degree of

spreading of chromosomes and for the presence of cytoplasm under different

conditions.

For each condition of ambient temperature and relative humidity a minimum of

50 metaphases on a total of five different culture slides were scored. Per slide a

minimum of 10 metaphases were screened. For AF cell cultures, only one

metaphase (the best as judged by visual observation) per clone was selected

(Table 1). For the different ambient temperatures and relative humidities a total of

1371 metaphases were screened for AF cell cultures and 1391 for CVS cultures.

For each condition of air flow a minimum of 60 metaphases on a total of five

different slides was scored. Per slide a minimum of 12 metaphases were screened.

For AF cell cultures, only one metaphase (the best as judged by visual

observation) per clone was selected. For the effect of the air flow a total of 413

metaphases (AF cell cultures) and 423 metaphases (CVS cultures) were

examined.

Scoring for the degree of spreading of a metaphase was as follows;

1: good (a maximum of 5 overlaps of chromosomes), 2: over-spread (not al

chromosomes in one view by 1,000 times magnification) and 3: bad (more than 5

overlaps of chromosomes).

Scores for the degree of presence of cytoplasm were;

1: good (no visible cytoplasm), 2: moderate (haze of cytoplasm) and 3: bad (cell

membrane still visible).

These well-defined scores allowed an independent application of the scoring

system by different observers. Two persons independently scored all metaphases.

Chapter 3


80

Different compositions of fixative

The prenatal cultures were harvested as described before and dried at 260C, 35%

relative humidity and 0.6 m/s air flow. Methanol and methanol- glacial acetic acid in

proportions of 1:1(v/v); 1:2(v/v); 1:3(v/v); 1:4(v/v) and 1:5(v/v) were used as

fixatives. For each condition a minimum of at least 60 metaphases on a total of

five different culture slides was scored. Per slide a minimum of 12 metaphases

were screened. For AF cell cultures, only one metaphase (the best as judged by

visual observation) per clone was selected.

For each condition the degrees of metaphase spreading and presence of

cytoplasm were determined as described before.

Statistical analysis

Analyses of variance and regression were used to assess the quantitative effect of

the conditions. The software package Systat 5.01 was used for statistical analyses.

The combined influence of ambient temperature and relative humidity, the

quadratic effects of ambient temperature alone and relative humidity alone, the

quadratic effect of air flow and the quadratic effect of the composition of fixative

were analysed. A general linear model was used to evaluate the data.

3.4 Results

Quantitative effects of ambient temperature, relative humidity and air flow

The effects of ambient temperature, relative humidity (Table 2) and air flow (Table

3) on the degree of presence of cytoplasm and on the degree of metaphase

spreading showed differences between AF cell and CVS cultures. In CVS cultures

ambient temperature and relative humidity together explained 9.1% of the variation

in cytoplasm and 4.2% of the variation in spreading. In AF cell cultures these

percentages were 20.2% and 8.0%, respectively. Relative humidity alone showed

the greatest influence; 18.6% explained variance in AF cell cultures and 7.2% in



81

CVS cultures. Ambient temperature alone had a minimal effect on metaphase

spreading and presence of cytoplasm. The air flow was the most important quality

factor for CVS cultures; 29.9% of the variance in cytoplasm was explained by this

factor. In contrast, AF cell cultures were hardly influenced by air flow.

Metaphase quality

No clearly optimal harvest conditions exist, as the effects of ambient temperature,

relative humidity and air flow on metaphase quality are relatively minor (Table 1).

There is no statistically significant interaction between the investigated factors.

For AF cell cultures, a low relative humidity resulted in a haze of cytoplasm and

a bad spreading. A high relative humidity resulted in bad quality metaphases,

visibility of the cell membrane and/ or a haze of cytoplasm and bad spreading. The

best quality was obtained with a relative humidity between 30% and 40%. Ambient

temperature had little effect on metaphase quality. High or low ambient

temperatures resulted in a lesser quality.

For CVS cultures, regression analysis showed only a very limited influence of

ambient temperature and relative humidity. Only 4% to 9% of the variation (Table

2) was due to the experimental conditions. Therefore, these conditions can be

considered as not critical under the investigated range of conditions. Air flow, the

third factor, did not affect the metaphase quality in AF cell cultures. In contrast to

that, air flow did influence metaphase quality in CVS cultures. In preparations from

these cultures, presence of cytoplasm was strongly reduced at 0.6 m/s (T=260C;

RH=35%)(Table3).

Chapter 3


82

Table 2: Results of regression analysis of non linear effects of temperature and/ or relativehumidity on metaphase quality

AF cell-cultures (n = 1371) CVS-cultures (n = 1391)

Effect on cytoplasm Coefficient (SD) P (two tailed) Coefficient (SD) P (two tailed)

Temperature -1.418 (1.138) 0.213 -5.864 (1.095) 0.000

Temperature x Temperature -0.012 (0.198) 0.951 0.998 (0.189) 0.000

Humidity -2.247 (0.304) 0.000 -0.995 (0.300) 0.001

Humidity x Humidity 0.187 (0.020) 0.000 0.077 (0.019) 0.000

Temperature x Humidity 0.329 (0.067) 0.000 0.166 (0.066) 0.012

Explained variance (%) 20.2 9.1

Humidity -0.094 (0.014) 0.000 -0.054 (0.013) 0.000



Temperature -0.415 (0.098) 0.000 not determined not determined

Temperature x Temperature 0.008 (0.002) 0.000 not determined not determined

Explained variance (%) 1.4 not determined

Effect on spreading

Temperature -0.256 (0.926) 0.783 -0.547 (0.926) 0.555

Temperature x Temperature 0.117 (0.161) 0.466 0.069 (0.160) 0.666

Humidity -0.058 (0.247) 0.814 0.202 (0.253) 0.426

Humidity x Humidity 0.038 (0.016) 0.018 -0.024 (0.016) 0.138

Temperature x Humidity -0.047 (0.055) 0.391 0.041 (0.056) 0.460


Temperature -0.309 (0.074) 0.000 -0.080 (0.072) 0.272

Temperature x Temperature 0.006 (0.001) 0.000 0.001 (0.001) 0.362


Humidity -0.036 (0.011) 0.001 0.036 (0.011) 0.001



Metaphase quality was measured for several climate conditions during slide preparation. Ambienttemperature ranged from 200C-300C and relative humidity from 25%-60%. Analysis of regressionbetween these conditions was performed to determine the effect on the degree of metaphase spreadingand the presence of cytoplasm.



83

Table 3: Analysis of variance of the effect of air flow on metaphase quality.

Dependentvariable


(%)

Sum ofsquares

DF Meansquare

F -ratio

P (twotailed)

AFcell-cultures(n= 413)

Cytoplasm air flow 7.4 6.10 3 2.02 10.85 0.000

Error 76.17 409 0.19

Spreading air flow 1.9 1.96 3 0.65 2.66 0.048

Error 100.50 409 0.25

CVS-cultures(n = 423)

Cytoplasm air flow 29.9 38.59 3 12.86 59.64 0.000

Error 90.38 419 0.22

Spreading air flow 2.4 2.80 3 0.93 3.42 0.017

Error 114.45 419 0.27

Metaphase quality was measured for four different air flows during slide preparation; namely 0.2; 0.4;0.6 and 0.7m/s (at 35% relative humidity and 200C ambient temperature). Analysis of variance wasperformed between these conditions to determine the effect on the degrees of metaphase spreadingand presence of cytoplasm.

Different compositions of fixative

The effect of the composition of fixative on metaphase quality was different for AF

cell and CVS cultures (Table 4). In AF cell cultures, the composition of fixative

explained 13.7% of the variance in presence of cytoplasm and degree of

spreading. Spreading was most strongly affected, explaining 13.9% of the

variance. The presence of cytoplasm was not strongly dependent of these factors.

It explained only 1.5% of the variance. In CVS cultures, the composition of fixative

explained 4.7% of the variance in metaphase quality. Cytoplasm and spreading

were both minimally influenced (5% of the variance). The composition of fixative

was not critical for metaphase quality, although methanol alone gave a bad result.

Chapter 3


84

Table 4: Analysis of variance of the effect on the composition of fixative onmetaphase quality.

Dependentvariable


(%)

Sum ofsquares

DF Meansquare

F- ratio P (twotailed)

AFcell cultures(n=904)

Cytoplasm fixative 1.5 0.02 4 0.004 1.76 0.135

Error 1.97 896 0.002

Spreading fixative 13.9 91.22 4 22.81 36.18 0.000

Error 566.67 899 0.63

Cytoplasm +Spreading

fixative 13.7 90.67 4 22.67 35.67 0.000

Error 571.26 899 0.64

CVS cultures(n=880)

Cytoplasm fixative 5.3 4.88 4 1.22 5.30 0.000

Error 200.95 872 0.23

Spreading fixative 5.1 22.86 4 5.71 11.81 0.000

Error 423.14 875 0.48

Cytoplasm +Spreading

fixative 4.7 35.54 4 8.89 10.75 0.000

Error 723.09 875 0.83

Metaphase quality was measured for five different compositions of fixative during harvesting; namely

methanol- glacial acetic acid 1:1; 1:2; 1:3; 1:4 and 1:5 (v/v) (at 35% relative humidity and 200C ambient

temperature). Analysis of variance was performed between these conditions to determine the effect on

the degrees of metaphase spreading and presence of cytoplasm.

3.5 Discussion

In this study we have quantified the effects of ambient temperature, relative

humidity, air flow and composition of fixative on metaphase quality in preparations



85

of primary in-situ cultures of CVS and AF cells. We found that our design was

sufficiently sensitive to detect effects. Most effects appeared to be minor. Whereas

in AF cell cultures, fixative, ambient temperature and relative humidity together are

of greater influence, in CVS cultures the air flow is more important. In the absence

of a statistically significant interaction, the effects can be considered as additive.

Relative humidity appeared in our study as the most important factor for

the degree of presence of cytoplasm (18.6% explained variance) and to a minor

extent as a factor influencing the degree of spreading (7.5% explained variance)

for AF cell cultures. Deng et al. (2003) determined relative humidity as the most

important factor for spreading (degree of presence of cytoplasm was not

measured) as well. Increasing the relative humidity resulted in better metaphase

quality, at least up to a certain degree, after which higher humidity no longer

resulted in better spreading. The latter effect might be explained by a fast increase

in water content of the fixative, so that a dramatic rehydration takes place, too fast

to result in a good spreading of the chromosomes (Deng et al., 2003).

Ambient temperature alone had no effect on metaphase quality in our study. An

explanation might be that evaporation of methanol from the fixative leads to a

cooling of the remaining fluid on the slide, a process that cannot be totally

compensated for by controlling ambient temperature (Claussen et al., 2002).

The composition of fixative seems to be of minor importance, as seen in

our results (14% explained variance for AF cells). Use of methanol only led to

hardly spread metaphases. It is clear that the hygroscopic effect of acetic acid is

needed for water-induced swelling. Higher concentrations of acetic acid resulted in

a better spreading. We routinely add pure glacial acetic acid in case of badly

spread metaphases to improve spreading. This effect has, however, not been

systematically investigated in the present study.

In CVS cultures, air flow has a substantial effect (29.9%) on the presence

of cytoplasm, whereas it has no effect in AF cell cultures. In contrast to the latter,

cultures of primary chorionic villi show a much more dispersed growth, often

Chapter 3


86

including larger cell clusters, resulting in an uneven evaporation of the fixative

during harvesting and in variations in metaphase quality. Therefore, more extreme

forces, such as produced by the air flow from a fan might lead to a more dynamic

washing of the cells by evaporating fixative and a thinner and more equal layer of

fixative (Hliscs et al., 1997). This may result in a better overall metaphase quality.

According to the results of Claussen et al. (2002), the potential of swelling of

different tissues is related to the lengths of the resulting chromosomes.

Preparations of primary CVS cultures show short chromosomes, comparable to

those prepared from bone marrow. This explains the very limited influence of the

investigated factors on the degree of spreading.

The difference in influences of ambient temperature, relative humidity and

composition of fixative as found in previous reports and in our study may be related

to differences in the type of cell culture. In cell suspensions, Deng et al. (2003)

found a strong influence of the relative humidity on the spreading of metaphases.

Claussen et al. (2002) also demonstrated that in cell suspensions the composition

of fixative had a great effect on metaphase spreading. However, in these studies,

the magnitude of the effect on metaphase quality has not been quantified.

During slide preparation, cells in suspension stick to the glass surface after

they have been dropped onto the slide. Evaporation of methanol from the fixative

causes a shrinking of the cells. Subsequently, a water-induced swelling

(rehydration) of the cells occurs by the hygroscopic effect of acetic acid. During this

last step, cells are flattening and the remaining fixative evaporates. The process of

swelling is crucial for elongation and spreading of the chromosomes. Slide

preparation from in-situ culture follows in principle the same steps. However, cells

from in-situ cultures are already sticking onto the slide. Within an AF cell clone for

instance, they do not grow as a homogeneous layer and swelling of mitotic cells

can be hampered by lack of space. Cells harvested in-situ thus, have a more

limited capacity to rehydrate. The influence of different parameters on slide

preparation may, therefore, in general be more restricted for in-situ cultures than

for cells in suspension.



87

In our experiments, we could not find statistically significant optimum

conditions for harvesting, because of the limited influence of the factors analysed.

In our routine, we recommend a higher than the standard concentration (25% v/v)

of glacial acetic acid for a better metaphase spreading and a relative humidity of

30% – 40% for a minimal presence of cytoplasm in metaphase preparations from

primary prenatal in-situ cultures. Spurbeck et al. (1996) determined the optimal

harvest conditions for secondary AF cultures to be an ambient temperature of 200C

and a relative humidity of 55%, based on the criterion of the size of metaphase

area. However, they recommend to use in practice 250C and 50% relative humidity,

because they found fairly frequently an over-spreading, indicating that metaphase

area alone is not properly reflecting metaphase quality. The discrepancy in relative

humidity for optimal harvesting between the results of Spurbeck et al. (1996) and

ours might be explained by their use of secondary in-situ cultures contrary to our

use of primary cultures. As previously discussed, primary in-situ cultures may have

a lower capacity of swelling in general, leading to a reasonable spreading at a

lower relative humidity than in secondary in-situ cultures or cell suspensions.

According to their own figures, they did not find a clear optimum for different

harvest conditions, but only small differences between the various harvesting

conditions they used. In that respect, their results are consistent with ours.

In conclusion, in general, metaphase quality of prenatal in-situ cultures is

only to a limited extent affected by ambient temperature, relative humidity, air flow

and the composition of fixative. Harvesting conditions are therefore not critical

within the investigated range and for good metaphase quality expensive measures

for fine controlling of ambient temperature, relative humidity and air flow are not

required.

Chapter 3


88

3.6 Literature

Claussen U, Michel S, Muhlig P, Westermann M, Grummt UW, Kromeyer-Hauschild K, Liehr T.

Demystifying chromosome preparation and the implications for the concept of chromosome

condensation during mitosis. Cytogenet Genome Res. 2002; 98(2-3):136-46.

Deng W, Tsao SW, Lucas JN, Leung CS, Cheung AL. A new method for improving metaphase

chromosome spreading. Cytometry. 2003; 51A(1):46-51.

Henegariu O, Heerema NA, Lowe Wright L, Bray-Ward P, Ward DC, Vance GH. Improvements in

cytogenetic slide preparation: controlled chromosome spreading, chemical ageing and gradual

denaturing. Cytometry. 2001; 43(2):101-9.

Hliscs R, Muhlig P, Claussen U. The spreading of metaphases is a slow process which leads to a

stretching of chromosomes. Cytogenet Cell Genet. 1997; 76(3-4):167-71.

Lawce HJ and Brown MG. Harvesting, slide- making, and chromosome elongation techniques; In Barch

M J (ed): The AGT Cytogenetics Laboratory Manual. Second Edition, Raven Press New York 1991: 31-

66.

Lundsteen C, Lind AM. A test of a climate room for preparation of chromosome slides. Clin Genet.

1985; 28(3):260-2.

Spurbeck JL, Zinsmeister AR, Meyer KJ, Jalal SM. Dynamics of chromosome spreading. Am J MedGenet. 1996; 61(4):387-93.

Chromosomal analysis in preparations from chorionic villi and amniotic fluid cell cultures


89

Chapter 4

Chromosomal analysis in preparationsfrom chorionic villi and amniotic fluidcell cultures

Chapter 4


90



91

Chapter 4.1

A 46,XX,der(13;14)(q10;q10),+21child born after a 45,der(13;14)(q10;q10) chromosomal finding inCVS

B. Sikkema- Raddatz, C.C. Verschuuren- Bemelmans, M. Kloosterman* and B. de

Jong

Department of Medical Genetics, University of Groningen, Groningen, The

Netherlands

*Department of Prenatal Diagnosis, Rijnstate Hospital Arnhem, Arnhem,

The Netherlands

Prenat Diagn 1997; 17: 1085-1089

Chapter 4


92

Letter to the Editor:

A 38- year- old gravida 5, para 2 woman underwent chorionic villus sampling

(CVS) at 10+3 weeks of gestation because she is a carrier of a Robertsonian

translocation (13;14). Chromosome analysis was performed after direct

preparation. A female chromosome pattern with a Robertsonian translocation

45,XX, der(13;14)(q10;q10) was found in 15 GTG-banded metaphases.

The pregnancy continued uncomplicated and a female infant with clinical

manifestations of Down syndrome was born spontaneously at 38 weeks.

Cytogenetic analyses of PHA- stimulated lymphocyte culture showed 46,XX, der

(13;14)(q10;q10), +21 (5 metaphases). Fluorescence in situ hybridisation (FISH)

with the probe YAC259H11 showed 3 signals for chromosome 21 in 100

metaphases.

Slides of the direct chorionic villus preparation were subsequently re-

examined. An additional 112 metaphases were seen, all without a trisomy 21.

There was no chorionic villus culture available.

Discordant findings between CVS and the foetal tissue have been documented

before. The majority of the reported cases have been false-positive results (CVS

abnormal, child normal). In a very few instances, a false-negative result (CVS

normal, child abnormal) has been reported. There are several mechanisms by

which the false-negative results may have arisen, including:

- a post-zygotic non-disjunction in the inner cell mass during embryogenesis

(Crane and Cheung, 1988);

- a twin pregnancy, with loss and re-absorption of the normal foetus;

- a mosaic situation, undetected because of the low mitotic index; and

- contamination of the villi with normal maternal cells (Pindar et al.,1992).

In 1987 Simoni et al. made a rough estimation about the incidence (1 per

thousand) of a false-negative result. Lilford et al. (1991) suggest a frequency of



93

0.001per cent using the (semi)direct method. The case described here is the first

false-negative result in our series of 7000 CVS patients by the direct and 1000

patients by the culture method.

Table 1: False-negative results in CVS for sex-chromosomal abnormalities

Foetus Direct CVS Culture Indication ReferenceFibroblasts:

47,XYY[23]/46,XY[39] 46,XY[85] 47,XXY[43]/46,XY[7] ? Callen et al. (1988)

Blood:

47,XXY[20]/46,XY[58] 46,XY[85] 47,XXY[58]/46,XY[7] Maternal age Eichenbaum et al.(1986)

Blood:

47,XXY[?]/46,XY[?] 46,XY[10] 47,XXY[?] Maternal age Linton and Lilford(1986)

Blood:

47,XXY[7]/46,XY[13] 46,XY[15] 47,XXY[10]/46,XY[5] ? Miny et al. (1991)

Amniotic fluid:

45,X[28]/46,XY[32] 46,XY[12] 45,X[21]/46,XY[5] Maternal age Smidt-Jensen andLind (1987)

Different tissues:

45,X[9]/46,XX[281] 46,XX[50] 45,X[50] Previouschild +21

Schlesinger et al.(1990)

Blood:

47,XXX[47]/46,XX[3] 46,XX[35] --- Maternal age Verjaal et al.(1987)

Chapter 4

proefdruk1.doc

94

Tab

le2

– F

alse

-neg

ativ

ere

sults

inC

VS

for

tris

omie

s18

and

21

Fet

usD

irect

CV

SC

ultu

reIn

dica

tion

Ref

eren

ce

Dif

fere

nttis

sues

:47

,XY

,+18

[47]

46,X

Y[3

7]47

,XY

,+18

[30]

Mat

erna

lag

eM

artin

etal

.(1

986)

Am

niot

icflu

id:

47,X

Y,+

18[9

]46

,XY

[15]

47,X

Y,+

18[5

]?

Min

yet

al.

(199

1)B

lood

:47

,XX

,+18

[10]

/46,

XX

[2]

46,X

X[1

0]47

,XX

,+18

[15]

/46,

XX

[15]

US

abno

rmal

ities

at30

wee

ksLe

scho

teta

l.(1

988)

Blo

od:

47,X

X,+

18[5

6]46

,XX

[16]

–U

Sab

norm

aliti

esat

31w

eeks

Wirt

zet

al.

(198

8)F

ibro

blas

ts:

47,X

X,+

18[3

0]45

,XX

[4]

46,X

X[1

1]M

ater

nal

age

Pin

dar

etal

.(1

992)

Blo

od:

47,X

Y,+

21[9

9]46

,XY

[50]

–M

ater

nal

age

Bar

tels

etal

.(1

989)

Am

niot

icflu

id:

47,X

Y,+

21[1

5]/4

6,X

Y[7

2]46

,XY

[25]

47,X

Y,+

21[9

0]/4

6,X

Y[1

0]?

Cal

len

etal

.(1

988)

Dif

fere

nttis

sues

:47

,XY

,+21

[300

]46

,XY

[110

]47

,XY

,+21

[100

]M

ater

nal

age

Lilfo

rdet

al.

(199

1)A

mni

otic

fluid

:47

,XX

,+21

[12]

/46,

XX

[24]

46,X

X[2

5]47

,XX

,+21

[11]

Mat

erna

lag

eM

iny

etal

.(1

988)

Blo

od:

47,X

X,+

21[6

]/46,

XX

[94]

46,X

X[1

00]

47,X

X,+

21[8

]/46,

XX

[24]

Pre

viou

sch

ild+

21N

isan

iet

al.

(198

9)B

lood

:47

,XX

,+21

[17]

46,X

X[2

0]–

Mat

erna

lag

eS

imon

ieta

l.(1

987)

Am

niot

icflu

id:

47,X

X,+

21[?

]46

,XX

[?]

47,X

X,+

21[?

]?

Vej

ersl

ev&

Mik

kels

en(1

989)

Blo

od:

46,X

X,d

er(1

3;14

),+21

[105

]45

,XX

,der

(13;

14)[

129]

–M

othe

rde

r(13

;14)

Pre

sent

ed c

ase

(199

7)



95

To our knowledge, 20 cases (excluding structural abnormalities) of a false-

negative finding including our case have been published: seven cases with a sex-

chromosomal abnormality (Table 1); five cases with trisomy 18; and eight cases

with trisomy 21 (Table 2). In five cases, the (semi)direct method only was

performed. In most of the cases (11 times), the culture method gave the right result

whereas the (semi)direct method gave a false-negative result. In three cases, the

(semi)direct method was normal, the culture was non-mosaic abnormal, and

chromosome studies of the blood of the child showed a mosaic sexchromosomal

abnormality. In the case reported by Pindar et al. (1992), the (semi)direct as well

as the culture method gave a false-negative result (46,XX), whereas the child had

a 47,XX,+18 chromosomal pattern. By this new case it is again demonstrated that

the most favourable technique for accurate prediction is the combination of the

(semi)direct and culture method. According to these data, the culture method

alone is a very good second choice.

References

Bartels,I.; Hansmann,I.; Holland, U.; Zoll, B.; Rauskolb, R.(1989) Down syndrome at birth not detected

by first trimester chorionic villus sampling, Am. J. Med. Genet. 34, 606-607.

Callen, D.F.; Korban, G.; Dawson, G.; Gugasyan, L.; Krumins, E.J.M.; Eichenbaum,S.; Petrass,J.;

Purvis- Smith,S.; Smith, A.; Den Dulk, G.; Martin, N.(1988) Extra embryonic/ fetal karyotypic

discordance during diagnostic chorionic villus sampling, Prenat. Diagn. 8, 453- 460.

Crane, J.P., Cheung, S.W. (1988) An embryogenetic model to explain cytogenetic inconsistencies

observed in chorionic villus versus fetal tissue, Prenat. Diagn. 8, 119- 129.

Eichenbaum, S.Z.; Krumins, E.J.; Fortune, D.W.; Duke, J. (1986) False negative finding on chorionic

villus sampling, The Lancet, 391.

Leschot, N.L.; Wolf, G.; Weenink, G.H. (1988) False negative findings at third trimester chorionic villus

sampling, Clin. Genet. 34, 204- 205.

Lilford, R.J., Caine, A., Linton, G., Mason, G. (1991) Short term culture and false negative results for

Down's syndrome on chorionic villus sampling, The Lancet 337, 861.

Linton, G.; Lilford, R.J. (1986) False negative finding on chorionic villus sampling, The Lancet, 630.

Chapter 4


96

Martin, O.A.; Elias, S.; Rosinsky, B.; Bombard, A.T.; Simpson, J.L. (1986) False negative finding on

chorionic villus sampling, The Lancet, 391- 392.

Miny, P.; Basaran, S.; Horst, J.; Pawlowitzki, I.H.; Kim Nhan Ngo, T. (1988) False negative cytogenetic

result in direct preparations after CVS, Prenat. Diagn. 8, 633.

Miny, P.; Hammer, P.; Gerlach, B.; Tercanli, S.; Horst, J.; Holzgreve, W.; Eiben, B. (1991) Mosaicism

and accurancy of prenatal cytogenetic diagnosis after chorionic villus sampling and placental biopsies,

Prenat. Diagn. 11, 581-589.

Nisani, R.; Chemke, J.; Voss, R.; Appelman, Z.; Caspi, B.; Lewin, A.; Dar, H.; Reiter, A. (1989) The

dilemma of chromosomal mosaicism in chorionic villus sampling - direct versus long term cultures,

Prenat. Diagn. 9, 223- 226.

Pindar, L.; Whitehouse, M.; Ocraft, D. (1992) A rare case of a false negative finding in both direct and

culture of a chorionic villus sample, Prenat. Diagn. 12, 525- 527.

Schlesinger, C.; Raabe, G.; Ngo,T.; Miller, K. (1990) Discordant findings in chorionic villus direct

preparation and long term culture- mosaicism in the fetus, Prenat. Diagn. 10, 609- 612.

Simoni, G.; Fraccaro, M.; Gimelli,G.; Maggi, F.; Dagna Bricarelli, F. (1987) False positive and false

negative findings on chorionic villus sampling, Prenat. Diagn. 7, 671- 672.

Smidt-Jensen, S.; Lind, A.M. (1987) A case of first trimester chromosomal mosa-icism confined to the

cultivation of the gestational products, Clin. Genet. 32, 133- 136.

Vejerslev, L.O.; Mikkelsen, M.(1989) The European Collaborative study on mosaicism in chorionic villus

sampling: data from 1986 to 1987, Prenat. Diagn. 9, 575-588.

Verjaal, M.; Leschot, N.J.; Wolf, H.; Treffers, P.E. (1887) Karyotyping differences between cells from

placenta and other fetal tissues, Prenat. Diagn. 7, 343- 348.

Wirtz, A.; Seidel, H.; Brusis E.; Murken, J.(1988) Another false- negative finding on placental sampling,

Prenat. Diagn. 8, 321.



97

Chapter 4.2

Four years’ cytogenetic experiencewith the culture of chorionic villi

B. Sikkema- Raddatz, K. Bouman, C.C. Verschuuren- Bemelmans, M. Stoepker, A.

Mantingh*, J.R. Beekhuis# and B. de Jong

Department of Medical Genetics, University of Groningen

*Department of Obstetrics and Gynaecology, Antenatal Diagnosis Unit, University

Hospital Groningen, Groningen, The Netherlands

# Department of Obstetrics and Gynaecology, Isala Klinieken, Zwolle, The

Netherlands

Prenat Diagn 2000; 20: 950-955.

Chapter 4


98

4.2.1 Summary

In 1958 chorionic villus samples, investigated by culture method, we found 137 (7%)

abnormalities. The abnormal results were classified in certain abnormal (generalised

abnormal at high probability) and uncertain abnormal (potentially confined to the

placenta) results. Certain abnormal were 73 cases (3.7%). Uncertain abnormal were

64 cases (3.3%), in which confirmation studies were done in 47 cases. In 12 cases of

these 47, the abnormality was confirmed and in 35 cases (1.8%) the abnormality was

confined to the placenta. Among the latter cases, poor pregnancy outcome [(16%

intrauterine death (IUD), 6% intrauterine growth retardation (IUGR)] was increased.

Total maternal cell contamination was not seen.

The positive predictive value of all confirmed abnormal cases was 66%. The positive

predictive value was 100% for indications ‘ultrasound abnormalities’ and ‘carrier’ and

between 50 and 60% for all other indications. Predictive value among uncertain

abnormal cases was low (26%). However, the positive predictive value depends of the

type of abnormality. Therefore we conclude that the culture method for chorionic villi is

a good test for indications ‘ultrasound abnormalities’ and ‘carrier’ and reliable for all

other indications. Whether or not follow-up investigations should be offered to the

parents depends of the type of abnormality. We conclude that the culture method is

reliable for prenatal diagnosis and can be used as the sole investigated method.

Key words:

chorionic villus sampling (CVS); confined placental mosaicism (CPM); culture method;

predictive value



99

4.2.2 Introduction

Chorionic villus sampling (CVS) is widely used as a method for the prenatal

detection of chromosome abnormalities. Two approaches were developed for

cytogenetic diagnosis: a (semi)direct method (Simoni et al., 1983) and a culture

method (Niazi et al., 1981; Heaton et al., 1984). However, soon after the

introduction of CVS it transpired that cytogenetic diagnosis did not always reflect

the chromosomal constitution of the foetus. In these cases the aberration was

confined to the placenta, so-called confined placental mosaicism (CPM) (Kalousek

and Dill, 1983). Reports on false positive and false negative results have been

published (Brambati et al., 1985; Breed et al., 1986; Eichenbaum et al., 1986).

Various (collaborative) studies (Ledbetter et al., 1992; Association of Clinical

Cytogeneticists Working Party on Chorionic Villi in Prenatal Diagnosis, 1994;

Hahnemann et al., 1997a; Smith et al., 1999) described cytogenetic findings and

pregnancy outcome of the (semi)direct and/ or culture method. These studies

reported on CPM (false negative and false positive findings), maternal cell

contamination (MCC) and calculations of the predictive value of the method

employed. A combination of the (semi)direct and culture method was recommended

to achieve the highest predictive value. When using only one method, the culture

method should be the method of choice because of fewer false negative and/ or false

positive findings (Ledbetter et al., 1992).

Formerly in our laboratory we used the direct method for routine cytogenetic

diagnosis. In case of abnormalities, the culture was also investigated (Breed et

al.,1990).

Based on the results of collaborative studies and on a false negative result in our

laboratory (Sikkema- Raddatz et al., 1997) we decided to change our policy. We

skipped the direct method and now use only the culture method, without increasing

turn around time of diagnosis. For economic reason it is not possible to use both

culture and direct method.

Chapter 4


100

In the present study we describe our results with the culture method over a 4-year

period. We describe the CVS success rate, MCC, the aberrations, and calculate the

predictive value (false positive results).

4.2.3 Materials and Methods

Between 1994 and 1998 we received 1958 chorionic villus samples. The majority of

the samples (1723) came from the University Hospital in Groningen. The other 235

samples came from the hospital "Isala Klinieken" in Zwolle.

CVS was performed transcervically between 10 and 12 weeks of gestation as

described previously (Breed et al., 1990). Exceptionally, CVS was performed

transabdominally in cases of more than 12 weeks’ gestation. Chorionic villi were

treated with trypsin and collagenase before being cultured (according to the method of

Jackson et al., 1989). The cell suspension was then transferred to coverslips and

cultured in Amniomax and Chang with α-mem. After adequate growth, cultures were

harvested after 6 days on average. Karyotyping was routinely performed after

Pancreatin- Giemsa staining. Other additional staining techniques, such as DA-DAPI-

staining, AgNOR- staining, C- banding and FISH were used, for confirming a suspect

chromosomal diagnosis after Pancreatin- Giemsa staining or when a rapid diagnosis

was required.

At least 15 cells were analysed. This number was extended to 29 cells in case of

a single cell with a trisomy 8, 9, 13, 18, 20, 21 or 22, a supernumerary marker

chromosome (ESAC) or a single cell with gain or loss of a sex chromosome.

A normal result was given in cases of 46,XX or 46,XY, in case of the common

inv(9)(p11q13), in cases of less than 50% of tetraploid cells and/ or in cases of one

cell exhibited any abnormality. All other results were considered abnormal.

Abnormal cytogenetic results

Between the abnormal results a distinction was made in non- mosaic cases and

mosaic cases.



101

A cytogenetic result was defined as mosaic when two or more cells showed a

karyotype different from the karyotype(s) in the other cells. Mosaic monosomy was

considered when three cells all lacked the same chromosome. In cases of a mosaic

result the parents were told that further investigation in amniotic fluid was required to

evaluate the result in chorionic villi.

The non-mosaic results were classified as structural and numerical abnormal

results. In cases of a structural abnormal result parental karyotyping was done, if the

structural abnormality was not known to be familial. In cases of a numerical abnormal

result further investigation in amniotic fluid was carried out to evaluate the result in

chorionic villi. An exception was made in cases of trisomy 21 and in cases of

ultrasound abnormalities in combination with a cytogenetic abnormality.

Certain abnormal cytogenetic results

Abnormal results were defined as certain when a non-mosaic trisomy 21, a non-

mosaic sex chromosomal trisomy, a non-mosaic triploidy, a non-mosaic structural

abnormality, or a non-mosaic ESAC was found. In addition, a non-mosaic 45,X

pattern and a trisomy 18 in combination with ultrasound abnormalities were defined as

certain abnormal. Trisomy 13 was not seen in this study, but would have been

counselled similar to trisomy 18.

Uncertain abnormal cytogenetic results

Abnormal results were defined as uncertain when a non-mosaic trisomy 18 and a

45,X pattern without ultrasound abnormalities, a non-mosaic autosomal trisomy other

than 13, 18 or 21 and all kinds of mosaic patterns (both numerical and structural) were

found.

Chapter 4


102

Predictive value

Predictive value was defined as the probability of an abnormal karyotype of the foetus,

given that CVS showed either a mosaic and/ or non-mosaic abnormality.

The 95% confidence intervals (CI) of the predictive value (p) with a standard error

SE(p) were calculated as follows:

CI(p)= p ± 1.96 * SE(p) and SE(p)= √p(1- p) / n.

Follow-up studies

In cases of uncertain abnormal results, follow-up studies (chromosomal study of

(un)cultured amniocytes, parental peripheral blood karyotyping or ultrasound

investigations) were offered to the parents.

Amniotic fluid cells were cultured according to the in situ method. Clones (n= 29)

were analysed to exclude at least 10% of a mosaic pattern (95% CI). In some cases

fluorescence in situ hybridisation (FISH) was performed on metaphase chromosomes

and/or on uncultured amniocytes confirming suspect chromosomal results and/ or for

rapid diagnosis. DNA studies were performed to investigate the possibility of

uniparental disomy (UPD) in abnormal CVS cases in which chromosome 15 was

involved.

Peripheral blood lymphocytes were prepared according to standard techniques

for karyotyping.

First and second trimester ultrasound investigations were performed by a

gynaecologist subspecialized in prenatal diagnosis with the use of third level

equipment (Acuson TM Corporation, Mountain View, CA, USA, 128xP10).

In cases of termination of pregnancy (TOP) we always tried to confirm the

cytogenetic diagnosis in foetal fibroblasts. Information about the course and outcome

of pregnancy was received from the woman and/or the referring midwife or physician.

Birth weights of babies were categorised according to the tables of Kloosterman

(1970).



103

4.2.4 Results

From the 1958 samples, a successful diagnosis was obtained in 99,7%. Of the six

failures, four involved small samples (≤5 mg). In one case cells did not attach on

coverslips. In the final case cell growth was very poor; only two cells could be exami-

ned.

Normal karyotypes were found in 1815 cases. Follow-up revealed no false

negative results. Abnormal karyotypes were found in 137 cases (7%) [Table 1]. Single

cell abnormalities were counted in 362 cases (18.5%).

Certain abnormal cytogenetic results

A total of 73 cases (3.7%) were certain abnormal (Table 2), including 34 cases of

structural abnormalities and 39 cases of numerical abnormalities. Confirmation

studies of the 73 certain abnormal cases were done in 58 cases.

Uncertain abnormal cytogenetic results

A total of 64 cases (3.3%) were uncertain abnormal (Table 3): 16 non-mosaic and 48

mosaic cases (2.45%). Confirmation studies were done in 47 cases. In 12 of these

cases the abnormality was confirmed. Thus of the 47 cases of expected CPM, 35

cases (1.8%) were indeed confined to the placenta. Not confirmed were two cases of

trisomy 16 and 2 cases with a 45,X. In one of the 45,X cases, a 46,XX pattern was

found in foetal tissue. Maternal contamination could not be excluded entirely in this

tissue. In the other case, a mos 45,X/46,XX pattern was found in foetal tissue after

TOP. In the confirmation studies of the mosaic abnormalities, 3/5 cases with a mosaic

sex chromosomal abnormality and 1/4 cases with a mosaic ESAC were confirmed.

Chapter 4

proefdruk1.doc

104

Tab

le1

–N

umbe

rof

abno

rmal

resu

ltsfo

rdi

ffere

ntin

dica

tions

Non

-mos

aic

case

sM

osai

cca

ses

Tris

-47

,XX

X69

,XX

XT

ris-

Str

uctu

ral

rear

rang

emen

tsA

utos

omal

tris

omy

Indi

catio

nN

umbe

rof

case

sN

orm

alca

ses

omy

2147

,XX

Y47

,XY

Y69

,XX

Y69

,XY

Yom

y18

45,X

Unu

sual

tris

omy

Bal

-an

ced

Un-

bala

nced

ES

AC

13;1

8;21

Oth

erS

ex-

chro

mos

omal

Str

uc-

tura

lE

SA

CT

etra

-pl

oid

MA

36-3

99

MA

>40

8U

Sab

na5

Car

rierb

1D

NA

/BIO

c

Oth

erd

2A

ll

1212

373

47 33 114

173

1952

1154

342

32 14 110

163

1815

25

3 1 4

1 6 7

4 4 1 9

3 2 1 2 8

2 2

8 3 13 1 3 28

1 1 2 4

2 2

1 3 4

12 5 1 1 19

5 1 6

4 2 1 1 8

1 3 1 1 6

2 2 1 5

aF

etal

abno

rmal

ities

atul

tra

soun

d.bP

aren

t al c

arr ie

rshi

p fo

r st

ruct

ural

rea

rran

gem

ent.

c Cyt

ogen

etic

inve

stig

atio

nse

cond

ary

to D

NA

orbi

oche

mic

alin

vest

igat

ion.

dP

revi

ous

child

with

chro

mos

omal

abno

rmal

ityor

oth

erre

ason

s fo

rpre

nata

lcyt

ogen

etic

dia

gnos

is.

MA

,M

ater

nala

ge(y

ears

).



105

Predictive value

Of all 137 abnormal cases, confirmation studies were done in 105 cases. Of the 105

cases (58 certain and 47 uncertain abnormal cases) the abnormality was confirmed in

70 cases. Therefore predictive value for all confirmed cases was 66% (95% CI: 57-

75%) (Table 4).

The predictive value of the 58 certain abnormal cases was 100%. The predictive

value of the 47 uncertain abnormal cases was 26% (95 % CI: 14%- 38%). Predictive

values differed for different abnormalities (Table 3).

Predictive values for different indications are shown in Table 4. The indication ‘carrier’

resulted in nearly 100% because of one case of a non-mosaic inv(7) in combination

with a mosaic +inv(7) pattern. The mosaic +inv(7) was confined to the placenta and

not confirmed in amniotic fluid.

Maternal cell contamination (MCC)

There was no case of incorrect sex prediction. In 71 cases (3.6%) a XX/XY admixture,

an indication of MCC, was found. All male foetuses were carefully screened for XX-

cells (on average 100 metaphases). In most of these cases (n=51) less than ten cells

of MCC were seen. In 16 cases MCC was between 10 and 50%. It was remarkable

that this high percentage was seen in only one of the two culture systems in eight

cases. The other culture system was without MCC. In four cases MCC was > 50%, in

one case it was up to 70%.

Chapter 4

proefdruk1.doc

106

Table 2 – Certain abnormal results (generalised abnormal at high probability)

Abnormality

Numberof cases(%)

Abnormality confirmed/number of confirmationstudies

Positive predictivevalue(%)

Autosomal aneuploidyTrisomy 21 25 (1.3) 14/14 100Trisomy 18 1 (0.05) 1/1 100

Sexchromosomal aneuploidy45,X 2 (0.09)47,XXX 2 (0.09) 1/1 10047,XXY 2 (0.09) 2/2 100

Triploidy 7 (0.36) 6/6 100Structural abnormality

Familial balanced 13 (0.7) 13/13 100Familial unbalanced 2 (0.09) 2/2 100Possible de novo

Balanced 15 (0.8) 15/15 100Unbalanced 2 (0.09) 2/2 100ESAC 2 (0.09) 2/2 100

Total 73 (3.7) 58/58 100

Table 4 – Positive predictive value of all confirmed cases for different indications

IndicationNumberof cases

Number ofabnormal cases(%)


Positive predictivevalue (%)(95% CIe)

MA 36 ≥ 39 1212 58 (4.79) 27/49 55 (41-69%)MA ≥ 40 373 31 (8.33) 10/19 53 (31-78%)US abnormalitiesa 47 15 (31.91) 11/11 100 (41-100%)Carrierb 33 19 (57.58) 17/18 94 (83-100%)DNA/BIO c 114 5 (4.51) 2/3 66 (12-100%)Otherd 173 10 (5.78) 3/5 60 (17-100%)Total 1952 137 (7.00) 70/105 66 (57-75%)

aFetal abnormalities at ultrasound.bParental carriership for structural rearrangement.cCytogenetic investigation secondary to DNA or biochemical investigation. dPreviouschild with chromosomal abnormality or other reasons for prenatal cytogenetic diagnosis.e95% Confidence interval.MA, Maternal age (years).

Table 3 – Uncertain abnormal results (potentially confined to the placenta)

Abnormality

Numberof cases(%)


Positive predictivevalue(%)

95%confidence

Non-mosaicsAutosomal

Trisomy 18 8 (0.4) 6/6 100 20-100%Trisomy 16 2 (0.1) 0/2 0

Sexchromosomal45,X 6 (0.3) 2/4 50 1-99%

MosaicsAutosomal 23 (1.2) 0/19 0Sexchromosomal 6 (0.3) 3/5 60 13-100%Tetraploidy 5 (0.3) 0/3 0Structural 14 (0.7) 1/8 12.50 0-35.5%

Total 64 (3.3) 12/47 26 13.5-38.5%



107

Pregnancy outcome of uncertain abnormal cytogenetic results

Of the 64 uncertain abnormal cases confirmation studies were done in 47 cases. In 35

cases the abnormality was not confirmed in cytogenetic follow-up studies. Of these

cases 26 (74%) had a normal pregnancy outcome, four (11%) ended in an intra

uterine death (IUD), one pregnancy (3%) was terminated, three (9%) had intrauterine

growth retardation (IUGR) and one (3%) developed HELLP (haemolysis, elevated

liver enzymes, low platelet count) syndrome. The cytogenetic results of the four

pregnancies that ended in IUD were 45,X, trisomy 16, a mosaic tetraploidy and a

mosaic trisomy 7. The cytogenetic results of the three pregnancies with IUGR were a

mosaic trisomy 2, a mosaic trisomy 21 and a mosaic 45X/46,XX.

4.2.5 Discussion

This study confirms the results of previous studies in terms of cytogenetic findings and

pregnancy outcome. The negative predictive value is 100% since we had no cases

with false negative results. The positive predictive value of 66% in this study is

comparable to the results of Breed et al. (1990), the US collaborative study (Ledbetter

et al.; 1992) and Los et al. (1998), which were 64%, 68.4% and 67.8% respectively.

The positive predictive value of 75% in the EUCROMIC study (Hahnemann and

Vejerslev,1997) is somewhat higher. This is by far the largest study: 62 865 cases.

Therefore, the predictive value from the EUCROMIC study might be the most

accurate. However, comparison of the accuracy should be done with great caution

since the method used and the indication for CVS may differ.

Furthermore in the present study, positive predictive values were calculated for

different abnormalities. For trisomy 18 the 100% predictive value in our study is

according to results in the literature (Smith et al., 1999). At least 70 cases of trisomy

18 have been confirmed. As far as we aware with the culture method only one

discrepancy (Breed, 1992) has been described. Therefore we conclude that trisomy

18, detected in the culture method, should be counselled as a certain abnormal

cytogenetic result. For the (semi)direct method, however, several discrepancies (false

positive findings) have been described. Hahnemann and Vejerslev (1997b), for

Chapter 4


108

example, found seven cases in the EUCROMIC study. Unfortunately they did not

mention the total number of all trisomy 18 cases detected in the study. Therefore it

was not possible to calculate the positive predictive value. Breed (1992) gave a

predictive value of 80% (95% CI: 52.3- 94.9%) for the direct method. These figures

indicate that trisomy 18, detected by the (semi)direct method, is still an uncertain

abnormal cytogenetic result.

For 45,X we found at least a 50% positive predictive value. Since one case of

possible MCC and one case of a mosaic 45,X/45,XX pattern was not included in the

calculation, the predictive value might even be higher. The result of the present study

would then correspond with a predictive value of 78% for the culture method by

Pittalis et al. (1994). For the direct method 47% was given however.

For the mosaic cases the predictive value is very low in both the (semi)direct and

the culture method, although the number of mosaic cases seems to be lower in CVS

culture. Therefore in amniocytes in fewer cases a follow-up study is necessary. In the

present study in 35 cases (1.8%) a second prenatal test (amniocentesis) was

necessary. In the study of Los et al.(1998) this percentage was 2.1% for the

(semi)direct method.

Predicted value for indications ‘carrier’ and ‘US abnormalities’ have the expected

(nearly) 100%. All other indications have a predicted value of 50- 60%. Since we

found no differences in the predictive value for these indications, we do not advise

CVS only for women older than 40 years (Los et al., 1998) but for all woman of 36

years and older. Therefore we conclude that positive predictive value depends not on

the indication, but on the cytogenetic result found.

A CPM of 1.8% in the present study is consistent with frequencies of CPM of 1 -

2% in other studies (Association of Clinical Cytogeneticists Working Party on

Chorionic Villi in Prenatal Diagnosis, 1994; Leschot et al., 1996; Hahnemann and

Vejerslev, 1997; Los et al., 1998). We found seven cases with a mosaic structural

abnormality in the 35 CPM cases in our study. In these cases we speculate that the

aberration might be a culture artefact rather than a real abnormality confined to the

placenta. In such situations, especially in cases of balanced aberrations, in



109

combination with a pregnancy without any anomalies, it is arguable whether a follow-

up study in amniocytes should be advised, since the incidence of mosaic structural

abnormalities in the population is negligible (Leegte et al., 1998). Moreover, when a

mosaic structural abnormality is detected, the risk figure of mental retardation and/ or

congenital abnormalities is low. Taking this into consideration, in the present study

1.4% of CPM (28 cases) rather than 1.8% is the correct frequency.

In the present study, the overall frequency of MCC is high (3.6%) in comparison

to other studies. In the UK study (Association of Clinical Cytogeneticists Working

Party on Chorionic Villi in Prenatal Diagnosis,1994) 0.5% MCC and in the US study

(Ledbetter et al.,1992) 2.16% MCC at transcervical sampling was found. Only Smidt-

Jensen et al. (1993) found a higher percentage of MCC, namely 4.2%. The reason for

the high percentage in the present study could be that all male karyotypes were

carefully screened for XX cells. However, the percentage quoted in the collaborative

studies are not relevant for cytogenetic diagnosis. More important is the percentage of

MCC per patient. Only at a high percentage of MCC or total overgrowth of maternal

cells is it possible to miss the correct diagnosis in chorionic villi. In the present study

this was not the case. In the UK study, however, total MCC occurred in six individual

cases, while an overall percentage of 0.5% MCC was given.

In the present study we found IUD in 17% and IUGR in 5% of the CPM cases.

The risk of poor pregnancy outcome might be even higher if the mosaic structural

abnormal cases (probably culture artefacts) were excluded. This result confirms the

increased risk of poor pregnancy outcome described in the literature. Breed et

al.(1991) reported 16.6% IUD and Wapner et al. (1992) 8.6% IUD. However, Gold-

berg and Wohlferd (1997) were unable to correlate CPM with poor pregnancy

outcome. Since the number of CPM cases in the present study is small, the

percentage of poor pregnancy outcome is more an indication than a precise risk

figure. However, Morssink et al. (1996) measured elevated levels of maternal serum

human chorionic gonadotropin (MShCG) in pregnancies with CPM. This resulted in a

negative effect on placental function and foetal development.

Chapter 4


110

In conclusion, the reliability of the culture method alone is in the present study

comparable to the combination of (semi)direct and culture methods and more reliable

than the (semi)direct method alone. The predictive value is acceptable for all

indications. The predictive value for trisomy 18 and 45,X is higher in the culture

method than in the (semi)direct method alone. MCC should be closely investigated to

avoid maternal overgrowth. We therefore conclude that the culture method alone is

reliable for prenatal diagnosis and may be used as the sole investigative method.



111

4.2.6 References

Association of Clinical Cytogeneticists Working Party on Chorionic Villi in Prenatal Diagnosis .1994.

Cytogenetic analyses of chorionic villi for prenatal diagnosis: a collaborative study of U.K. data. Prenat Diagn

14: 363- 379.

Brambati B, Simoni G, Danesino C, Oldrini A, Ferrazzi E, Romitti L,Terzoli GL, Rosella F, Ferrari M,

Fraccaro M .1985. First trimester fetal diagnosis of genetic disorders: Clinical evaluation of 250 cases. J Med

Genet 22, 92- 99

Breed ASPM, Mantingh A, Govaerts L, Boogert A, Anders G, Laurini R. 1986. Abnormal karyotype in the

chorion, not confirmed in a subsequently aborted fetus. Prenat Diagn 6: 375- 377.

Breed ASPM, Mantingh A, Beekhuis JR, Kloosterman MD, Ten Bolscher H, Anders GJPA .1990. The

predictive value of cytogenetic diagnosis after CVS: 1500 cases: Prenat Diagn 10: 101 -110.

Breed ASPM, Mantingh A, Vosters J, Beekhuis JR, Lith JMM, Anders GJPA .1991. Follow-up and

pregnancy outcome after a diagnosis of mosaicism in CVS. Prenat Diagn 11: 577- 580.

Breed ASPM .1992. An evaluation of cytogenetic diagnosis by chorionic villus sampling: Reliability andimplications. STYX Publications

Eichenbaum SZ, Krumins EJ, Fortune DW, Duke J .1986. False negative finding on chorionic villus

sampling. Lancet 2: 391.

Goldberg JD, Wohlferd MM .1997. Incidence and outcome of chromosomal mosaicism found at the time of

chorionic villus sampling. AM J Obstet Gynecol 176: 1349- 1353.

Hahnemann JM and Vejerslev LO .1997. Accuracy of cytogenetic findings on chorionic villus sampling

(CVS)- Diagnostic consequences of CVS mosaicism and non- mosaic discrepancy in centres contributing to

EUCROMIC 1986- 1992. Prenat Diagn 17:9: 801- 820.

Hahnemann JM and Vejerslev LO .1997. European collaborative research on mosaicism in CVS

(EUCROMIC)- Fetal and extrafetal cell lineages in 192 gestations with cvs mosaicism involving single

autosomal trisomy. Am J Med Genet 70: 179- 187.

Heaton DE, Czepulkowski BH, Horwell DH, Coleman DV .1984. Chromosome analyses of first trimester

chorionic villus biopsies prepared by a maceration technique. Prenat Diagn 3: 279- 287

Jackson L, Gibas LM, Coutinho W.1989. Chorionic villus. In: Verma RS and Babu A (ed) Human

chromosomes, manual of basic techniques. Pergamon Press, Oxford: 19 - 26.

Kalousek DK, Dill FJ. 1983. Chromosomal mosaicism confined to the placenta in human conceptions.Science 221: 665- 667.

Chapter 4


112

Kloosterman GJ .1970. On intrauterine growth; the significance of prenatal care. Int J Gynaecol Obstet 8:

895- 912.

Ledbetter DH, Zachary JM, Simpson MS, Golbus MS, Pergament E, Jackson L, Mahoney MJ, Desnick R,

Schulman J, Copeland KL, Verlinsky Y, Yang-Feng T, Schonberg SA, Babu A, Tharapel A, Dorfman A,

Lubs HA, Rhodas GG, Fowler SE and de la Cruz F.1992. Cytogenetic results from the collaborative study on

CVS. Prenat Diagn 12: 137- 345.

Leegte B, Sikkema- Raddatz B, Hordijk R, Bouman K, van Essen, Castedo S, de Jong B .1998. Three casesof mosaicism for balanced reciprocal translocations. Am J Med Genet 79: 362- 365.

Leschot NJ, Schuring- Blom GH, van Prooijen- Knegt AC, Verjaal M, Hansson K, Wolf H, Kanhai HHH, van

Vugt JGM, Christiaens GCML .1996. The outcome of pregnancies with confined placental chromosome

mosaicism in cytotrophoblast cells. Prenat Diagn 16: 705- 712.

Los FJ, van den Berg C, van Opstal D, Noomen P, Braat APG, Galjaard RJH, Pijpers L, Cohen- Overbeek

TE, Wildschut HIJ and Brandenburg H .1998. Abnormal karyotypes in semi- direct chorionic villus

preparations of woman with different cytogenetic risks. Prenat Diagn18: 1023- 1040.

Morssink LP, Sikkema- Raddatz B, Beekhuis JR, de Wolf BTHM and Mantingh A .1996. Placental

mosaicism is associated with unexplained second – trimester elevation of MShCG levels, but not with

elevation of MSAFP levels. Prenat Diagn 16: 845- 851

Niazi M, Coleman DV, Loeffler FE .1981. Trophoblast sampling in early pregnancy. Culture of rapidly

dividing cells from immature placental villi. Br J Obtst Gynaecol 88: 1081- 1085

Pittalis MC, Dalpra L, Torricelli F, Rizzo N, Nocera G, Cariati E, Santarini L, Tibiletti MG, Agosti S, Bovicelli L,

Forabosco A. 1994. The predictive value of cytogenetic diagnosis after CVS based on 4860 cases with both

direct and culture methods. Prenat Diagn 14: 267- 278

Simoni G, Brambati B, Danesino C, Rosella F, Terzoli GL, Ferrari M, Fraccaro M, .1983. Efficient direct

chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of

pregnancy. Hum Genet 63: 349- 357.

Sikkema- Raddatz B, Verschuuren- Bemelmans CC, Kloosterman M and de Jong B. 1997. A

46,XX,der(13;14)(q10;q10),+21 child born after a 45,XX,der(13;14)(q10;q10) chromosomal finding in CVS.Prenat Diagn 17: 1085- 1089.

Smidt- Jensen S, Lind A-M, Permin M, Zachary JM, Lundsteen C, Philip J .1993. Cytogenetic analyses of

2928 CVS samples and 1075 amniocentesis from randomized studies. Prenat Diagn 13: 723- 740.

Wapner RJ, Simpson JL, Golbus MS, Zachary JM, Ledbetter DH, Desnick RJ, Fowler SE, Jackson LG, Lubs

H, Mahony RJ, Pergament E, Rhoads GG, Shulman JD, de la Cruz F .1992. Chorionic mosaicism:

association with fetal loss but not with adverse perinatal outcome. Prenat Diagn 12: 347- 355.



113

Chapter 4.3

Probability tables for exclusion ofmosaicism in prenatal diagnosis

Birgit Sikkema-Raddatz*, Sérgio Castedo*† and Gerard J. te Meerman*

*Department of Medical Genetics, University of Groningen, Groningen, The

Netherlands

† Department of Medical Genetics, IPATIUP, Medical Faculty of Porto, Portugal

Prenat Diagn 1997; 17 (2): 115-118

Chapter 4


114

4.3.1 Summary

The decision concerning the number of metaphases that need to be analysed to

detect mosaicism of a certain degree depends mainly, for the same confidence levels,

on the culture method used (in situ or flask methods). Several probability tables,

designed for either the in situ or the flask method, have been reported and can be

used to assist laboratories in making the decisions referred to above. However, there

are instances where part of the analysis is done using the in situ and flask methods. In

such situations, the previously published tables are of limited use. We have generated

new table that can be used in such situations, as well as in cases where only the flask

method is used.

Keywords:

amniotic fluid culture; chromosomal mosaicism; probability tables; prenatal diagnosis

4.3.2 Introduction

True chromosomal mosaicism is a rare finding in amniotic fluid cultures, but its

exclusion represents an obvious major concern for most laboratories involved in

prenatal diagnosis (Hsu et al., 1992).

Several tables have been generated to determine the optimum number of

metaphases to be counted to exclude (detect) chromosomal mosaicism for either the

in situ or flask culture method (Claussen et al., 1984; Cheung et al.,1990; Rischkind

and Risch; 1990; Featherstone et al., 1994). However, for laboratories (like ours)

using the in situ culture technique, there are instances where not enough colonies can

be analysed (or counted) to exclude mosaicism with the usual confidence. In these

situations, a back-up in situ culture is usually trypsinized and the respective cells

divided over culture flasks, from which chromosomal analysis will proceed further.

Although it is possible to count how many colonies were present in the coverslip

prior to trypsinization, after trypsinization it is no longer possible to know which



115

metaphases come from which colony. For the calculation of the number of cells that

need to be analysed from these cultures, the tables referred to above are of limited

use.

A similar difficulty can occur when using the flask method, in cases where a small

sample of amniotic fluid (hence with fewer cells than usual) is cultured.

We have calculated the number of metaphases that need to be analysed in order

to be 95 per cent certain that at least one cell of a desired number of colonies has

been observed. The same table can be used to determine the number of metaphases

required for accurate analyses using only the flask method.

4.3.3 Mathematical calculation

Assuming that a coverslip contains, prior to trypsinization, N equal-sized colonies of

cells, a sample of K cells is analysed after trypsinization and harvesting. The question

is: what is the probability that M colonies are represented by at least one cell in the

sample? For the exact derivation the reader is referred to the Appendix.

A Pascal program has been written to evaluate the recursive expression. The

result is stored as probability distributions for the number of colonies M from which

cells are observed, conditional on the number N of colonies present and the number

of cells K sampled. Then the number of colonies for which there is 95 per cent

certainty that they have been observed, given the number of cells sampled, is

computed. This computation is done downwards from the highest sample number

present, such that the lowest sample number is obtained for every entry in the table.

4.3.4 Results

Table 1 shows the number of metaphases that need to be analysed to be 95 per

cent certain that at least the horizontal number of different colonies have been

sampled, when the number of colonies present in the culture is given by the

vertical number.

Chapter 4

proefdruk1.doc

116

Tab

le1

– R

equi

red

num

ber

ofsa

mpl

esto

obse

rve

cells

from

Mdi

ffere

ntco

loni

esou

tof

Nco

loni

espr

esen

t.95

perc

ent

prob

abili

tyta

ble

No.

ofco

loni

esN

pres

ent

No.

ofco

loni

esM

need

ed

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

30

26

00

00

00

00

00

00

00

00

00

00

00

00

00

00

34

110

00

00

00

00

00

00

00

00

00

00

00

00

00

44

716

00

00

00

00

00

00

00

00

00

00

00

00

00

53

611

210

00

00

00

00

00

00

00

00

00

00

00

00

63

69

1427

00

00

00

00

00

00

00

00

00

00

00

00

73

58

1218

330

00

00

00

00

00

00

00

00

00

00

00

83

57

1115

2238

00

00

00

00

00

00

00

00

00

00

00

93

57

10*

1318

2644

00

00

00

00

00

00

00

00

00

00

010

35

79

1216

2231

510

00

00

00

00

00

00

00

00

00

011

35

79

1215

1925

3557

00

00

00

00

00

00

00

00

00

012

34

69

1114

1822

2939

630

00

00

00

00

00

00

00

00

013

34

68

1113

1620

2532

4470

00

00

00

00

00

00

00

00

014

34

68

1013

1619

2329

3648

760

00

00

00

00

00

00

00

015

34

68

1012

1518

2226

3240

5383

00

00

00

00

00

00

00

016

34

68

1012

1417

2024

2935

4458

900

00

00

00

00

00

00

017

34

68

912

1417

1923

2732

3948

630

00

00

00

00

00

00

018

34

67

911

1416

1922

2630

3542

5267

00

00

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117

4.3.5 Discussion

In an attempt to define the minimum number of metaphases which need to be

analysed for a reliable prenatal diagnosis, several calculations have been made taking

into account the culturing method used (in situ or flask methods) and the desired

confidence levels for exclusion of mosaicism (Claussen et al., 1984; Cheung et al.,

1990; Richkind and Risch, 1990; Featherstone et al., 1994).

Some of these calculations resulted in different probability tables, depending on

different assumptions concerning the culture method used. However, even for

cytogenetic laboratories (like ours) using the in situ method, there are instances where

not enough colonies can be analysed to allow exclusion of chromosomal mosaicism

with the usual confidence. In such situations, a back-up culture is usually trypsinized

and the cells are inoculated onto two new coverslips, one of which will be harvested

when a sufficient number of mitoses is observed.

Since this trypsinization step obviously disrupts colony integrity, the probability

tables used for the in situ method are no longer applicable to determine the number of

metaphases that still need to be analysed. On the other hand, since part of the

analysis has been done from an in situ culture, the published tables for the flask

method (refs.) are also not directly applicable in such circumstances.

We have therefore generated a new table that can be used to determine quickly

the number of metaphases that need to be analysed from a sample of amniotic fluid in

order to exclude chromosomal mosaicism with a predefined confidence level (95 per

cent). The table is of use for both the flask method alone and the situations referred to

above where part of the analysis is done from an in situ culture and part from a

trypsinized culture.

To illustrate the use of the table, we discuss a few applications. For a laboratory

using the in situ method and wishing to detect 20 per cent mosaicism with 95 per cent

confidence, 14 metaphases, derived from different colonies, need to be analysed

irrespective of the total fluid volume (Richkind and Risch, 1990). If, however, only ten

colonies could be analysed from the in situ culture, four additional colonies must be

sampled from the trypsinized culture to achieve a similar sensitivity level of mosaicism

Chapter 4


118

detection. The decision as to how many metaphases need to be analysed from the

trypsinized culture will obviously depend on the total number of colonies present prior

to trypsinization. This number can be obtained by counting the actual number of

colonies in the culture, or by using the estimation of Richkind and Risch (1990), i.e.,

assuming the formation of 1.5 or 2 colonies per ml of cultured amniotic fluid.

Supposing that there were eight colonies prior to trypsinization, seven metaphases

would need to be analysed to be 95 per cent confident that at least four different

colonies had been sampled.

The total sensitivity will be somewhat lower (90.25 per cent) since the two

sensitivities (95 per cent confidence to detect mosaicism and 95 per cent confidence

to sample different colonies) need to be multiplied.

Our calculations, like those of Claussen et al. (1984), Richkind and Risch (1990),

Cheung et al. (1990), and Featherstone et al. (1994), obviously assume equal colony

sizes and contributions to the pool of analysable metaphases in the trypsinized

culture. When there are colonies of different size, only medium or large-sized colonies

should be counted and used in the calculation since these are more likely to

contribute to the pool referred to above.

The probability table (Table 1) presented here can also be used in laboratory

setting using only the flask method. Accordingly, if one wants to be 95 per cent

confident that a mosaicism ≥ 20 per cent is detected, and if the total amount of fluid

collected was 20 ml, a total number of 23 metaphases have to be analysed, assuming

the presence of 30 "colony-forming" cells in the cultured sample (Richkind and Risch,

1990). Smaller amounts of amniotic fluid imply, for the same sensitivity and

confidence levels, the analysis of more metaphases.

The calculations presented here, and corresponding probability tables, provide

simple guidelines that can help laboratories standardise the confidence levels of

prenatal diagnosis, either when they rely on the flask method alone or when, despite

using the in situ method, they need to use trypsinized cultures to complete the

analysis.



119

4.3.6 References

Cheung S.W., Spitznagel E., Featherstone T., Crane J.P. (1990). Exclusion of chromosomal mosaicism in

amniotic fluid cultures: Efficacy of the in situ versus the flask techniques, Prenat. Diagn., 10, 41- 57

Claussen U., Schäfer H., Trampisch H.J. (1984). Exclusion of chromosomal mosaicism in prenatal

diagnosis, Hum. Genet., 67, 23- 28

Featherstone T., Cheung S.W., Spitznagel E., Peakman D. (1994). Exclusion of chromosomal mosaicism in

amniotic fluid cultures: determination of number of colonies needed for accurate analysis, Prenat. Diagn., 14;

1009- 1017

Hsu L.Y.F., Kaffe S., Jenkins E.C., Alonso L., Benn P.A., David K., Hirschhorn K., Lieber E., Shanske A.,

Shapiro L.R., Schutta E., Warburton D.(1992). Proposed guidelines for diagnosis of chromosome mosaicism

in amniocytes based on data derived from chromosome mosaicism and pseudomosaicism studies, Prenat.

Diagn., 12, 555- 573

Richkind K.E. and Risch N.J. (1990). Sensivity of chromosomal mosaicism detection by different tissue

culture methods, Prenat. Diagn., 10; 519- 527

Chapter 4


120

Appendix

The question is: what is the probability that M colonies are represented by at least one

cell in the sample?

Solution (assuming an equal contribution of the different colonies)

If one cell is sampled, one colony is present. We represent the probability that M

colonies are present in a sample of K cells, assuming N colonies by P(M,K,N). M may

vary from 1 (at least one colony is represented in the sample) to the lower of the

numbers K and N, denoted min (K,N) (there may not be more colonies than there are

samples and there may not be more colonies than there are colonies).

The computation of P(M,K,N) is recursive. Assume that we want to compute

P(M,K,N). This probability is related to the probability that in K-1 samples, M and M-1

colonies are present:

P(M,K,N)= (N - M + 1)/

N x P(M - 1,K - 1,N) + (M)/

N x P(M,K -1,N) if K≤N,

assuming that P(M,K,N)=0 if M > min(K,N) or M < 1.

The recursion stops at P(1,1,N)=1.0.

The factor (N - M + 1)/N relates to the probability that a cell is observed from the

not yet observed pool of cells, while the factor (M)/N is the probability of observing a

cell from an already observed pool.

Summary,future prospects, and suggested guidelines


121

Chapter 5

Summary, future prospects, andsuggested guidelines

Chapter 5


122

5.1 Summary

The scope of the thesis is to establish guidelines for quality controlled processing

of amniotic fluid cells and chorionic villi obtained for purposes of prenatal

cytogenetic diagnosis. We, therefore, have investigated which factors are of critical

importance and we have quantified their influence. We have introduced controlled

experimental variations in the workflow in order to find main and possibly

interactive effects. The results will be discussed with respect to the three parts that

can be distinguished in the cytogenetic process, namely cell culture, slide

preparation and chromosomal analysis.

Factors influencing the quality of cell culture

Amniotic fluid as obtained by amniocentesis following the guidelines of the Dutch

Society of Obstetrics and Gynaecology (1997) will be in general a good source for

culturing amniocytes for cytogenetic diagnosis (Chapter 2.1). The volume of the

admitted material, the reason for amniocentesis, gestational age at amniocentesis

as well as the chromosomal diagnosis, appeared as no major critical factors where

small variations would lead to unacceptable variation in outcome. We found that

only the variation in appearance of the amniotic fluid has a relatively major impact.

However, the effect of bloody or brown amniotic fluid was an extended culture time

rather than culture failure (Chapter 2.1). The minimal amount of amniotic fluid

required for culturing leading to a reliable cytogenetic result appeared to be 3.2 ml

(Chapter 2.2). Commercial hormone-supplemented media are generally of

sufficient quality for prenatal cytogenetic purposes. However, because of the

inclusion of in-vivo produced serum, intrinsic variations in quality may occur that

make acceptance testing necessary. To address this issue, we have developed an

acceptance test based on the principle of stress testing (Chapter 2.3). We

established the validity and sensitivity of this test by investigating the influence of

decreased oxygen tension during culturing and by comparison with standard

growth curve determination. A reduced oxygen tension during culturing caused a



123

2.5-fold increase in the number of AF cells within a clone. The number of clones,

however, is only slightly increased (Chapter 2.3).

Factors involved in culturing chorionic villi have not yet been systematically

investigated. To shorten the culture time and to increase the number of

metaphases per preparation, a systematic investigation of factors involved in this

process, similar to what we have done for amniocytes, is recommended.

Factors influencing the quality of slide preparation

In preparations from primary in-situ cultures of AF cells and chorionic villi we found

that metaphase quality is only to a limited degree affected by the composition of

fixative, the relative humidity, the ambient temperature and the air flow at

harvesting (Chapter 3). Variations in the conditions commonly used in prenatal

cytogenetic diagnosis have a limited influence and cannot be considered as critical

for the quality of slide preparation. Thus, expensive measures for fine control of

these conditions are not necessary at all. Within this limited influence it is the

composition of fixative which mostly affects chromosome spreading. The presence

or absence of cytoplasm over the metaphase is for amniotic fluid cells most

affected by the relative humidity and for chorionic villi by the air flow (Chapter 3).

We have not been able to discover factors that could lead to less variation in

quality.

We recommend in-situ harvesting rather than suspension type of harvesting,

since the turn-around time is much shorter and less chromosomal analyses have to

be made for a reliable diagnosis of in-situ cultures.

Factors influencing the quality of chromosomal analysis

All previously mentioned factors lead to the final quality of the chromosome

preparation; an appropriate number of metaphases on the slide, a sufficient length

of the chromosomes and within a short turn-around time. The required conditions

have already been well-defined in existing guidelines (Hsu et al., 1992; Hsu and

Chapter 5


124

Benn, 1999; ACC Working Party, 2001; Mellink et al., 2003). Following these

guidelines will result in an accurate and reliable detection or exclusion of structural

and numerical chromosomal abnormalities. However, an additional guideline for

the exclusion of chromosomal mosaicism in AF cell cultures would be desirable.

We, therefore, developed a probability table which may help laboratories to

standardise the confidence levels of prenatal diagnosis regarding mosaicism when,

despite routinely using the in-situ method, trypsinised cultures need to be used to

complete the analysis (Chapter 4.3). This probability table can of course also be

applied when using solely the flask method.

For chorionic villi, chromosomal analysis from both a direct preparation and a

culture is recommended. Provided that maternal contamination is limited, the

culture method alone is sufficiently reliable for prenatal diagnosis to be used as the

sole method (Chapter 4.2). The risk of a false negative result when using a direct

preparation has been calculated as 1-3 percent per 1000 and is negligible when

analysing preparations from cultures (Chapter 4.1). The risk of a false positive

result (positive predictive value) is in general higher in analysis from direct

preparations (Hahnemann and Vejerslev, 1997) and depends on the type of

abnormality (Chapter 4.2).

5.2 Future prospects

Amniocentesis and chorion villus biopsy will in the foreseeable future remain the

methods of choice in prenatal diagnosis. It is to be expected that the cytogenetic

diagnosis of AF cells and chorionic villi will increasingly be supplemented by

molecular techniques. For a more rapid test and a reduction of maternal anxiety,

detection of the most common aneuploidies, such as trisomies 13; 18; 21 and sex

chromosomal aneuploidies, interphase fluorescence in situ hybridisation (FISH)

has been introduced (Klinger et al.,1992; Bryndorf et al., 1997; D’Alton et al., 1997;

Pergament et al., 2000). In this technique, a set of chromosome-specific

fluorescence-labelled probes is hybridised to interphase nuclei of uncultured

prenatally obtained cells. The number of fluorescent signals in each nucleus



125

represents the copy number of the chromosome. A result is ultimately available

after two days, however, due to the significantly higher costs and labour

requirements this testing is offered only for a subset of prenatal referrals, mainly for

the indication ‘foetal structural abnormalities found upon ultrasound examination’.

More recently, quantitative molecular tests, such as quantitative fluorescent

(QF)-PCR or multiplex ligation-dependent probe amplification (MLPA), have

become available for high-throughput, reliable, fast and robust aneuploidy

detection (Mann et al., 2004; Slater et al., 2003). MLPA is a PCR-based method for

relative quantification of up to 40 DNA sequences by means of a simple single-tube

assay (Schouten et al., 2002). During the procedure, several sequence-specific

probe sets are hybridised to genomic DNA, covalently ligated into single probes

after successful hybridisation, and only than amplified using just one primer set.

Afterwards, the resulting PCR-products are analysed semi-quantitatively using a

capillary sequencer. In practice, a first study on MLPA for detection of aneuploidy

of chromosomes 13; 18; 21; X or Y in almost 600 AF samples, leading to no false

negative or false positive results, has been successfully conducted (Slater et al.,

2003).

QF-PCR involves the relative quantification of microsatellite alleles to determine

sequence copy number. Multiplex amplification using fluorescence labelled primers

is followed by size separation and allele peak measurement on a semi-automated

genetic analyser. In testing more than 7000 prenatal samples for trisomy 13; 18

and 21 no false positive nor false negative results were detected for non-mosaic

trisomies (Mann et al., 2004).

DNA-microarray analysis is another molecular technique which allows a

simultaneous quantitative analysis of genomic copy numbers, using many probes

consisting of mapped DNA fragments that are spotted on a glass slide (Pinkel et

al., 1998; Snijder et al., 2001). Microarray technology was initially conceived to

detect the expression of thousands of genes simultaneously, but quantification of

genomic copy numbers is also possible. The resolution of an array depends solely

on its contents. Presently, subtelomere-specific arrays (Veltman et al., 2002),

Chapter 5


126

chromosome-specific arrays (Veltman et al., 2004) and whole genome arrays, with

resolutions varying from 2Mb to tiling path density, are being used in diagnostic

practice. The comparative nature of the technique implies that only net gains and

losses can be found. Balanced rearrangements will remain undetected. In prenatal

diagnosis, routine microarray analysis of AF cells or chorionic villi is too labour-

intensive and too expensive to date. However, this technique can be used for

further investigation of small chromosomal unbalances at a higher resolution when

an apparently balanced karyotype is found in the group of referrals with

‘abnormalities at ultrasound’, or it can be used to determine whether a de-novo

structural abnormality is balanced or not.

Cytogenetic analysis of AF cells and chorionic villi is associated with

invasive techniques to obtain the material. These techniques carry a risk for the

mother and the foetus. Therefore, they need to be restricted to women whose

increased risk of having a foetus with an abnormality outweighs the risk of the

procedure. Introduction of a better risk estimator than maternal age will reduce the

number of invasive tests, especially in the group of women with advanced maternal

age (Beekhuis et al., 1994; Kornman et al., 1995). At the same time the detection

rate for Down syndrome for each procedure as well as the absolute number has

increased (Cornel et al., 1993). One approach is biochemical screening of maternal

serum possibly combined with a measurement of thickening of the nuchal fold at

ultrasound (nuchal translucency). This procedure for prenatal detection of Down

syndrome is estimated to result in a detection rate of 93% with 5.9% false positive

rate (Steenhouse et al., 2004). Only if this screening indicates an increased risk for

Down syndrome, subsequent cytogenetic diagnosis by use of AF cells or chorionic

villi should be performed. Recently, a model for contingent screening already in the

first trimester was recommended which avoids further testing in up to 75% of

unaffected pregnancies (Wright et al., 2004). In most European countries

screening for Down syndrome is offered to all pregnant women. In the Netherlands

the advice of the Health Council (2004) was in this line. However, the last

population screening act (Ministry of Health, 2004), passed after this advice,

allowed health care providers only to offer screening for Down syndrome to



127

pregnant woman older than 36 years of age. Pregnant women younger than 36

years of age have to be informed about screening, but the use is at their own

expense. Since most Down syndrome children are born to younger mothers, this

policy means a highly ineffective way of screening for Down syndrome.

Alternative non-invasive techniques for prenatal diagnosis that can be

offered to all pregnant women are actively explored. Isolation of intact foetal cells

from the maternal circulation has been extensively studied (Adinolfi, 1995; Bianchi

et al., 1999; Simpson and Elias, 1993). The major limitation in applying this

technique clinically, however, is the in general low number of foetal cells found in

the maternal circulation; approximately one foetal cell per ml of maternal blood

(Bianchi et al.; 1997, Hamada et al.; 1993). Furthermore, foetal cells can persist in

the circulation, sometimes many years after delivery (Bianchi et al.; 1996). As an

alternative, Lo et al. (1997) was the first to describe the presence of cell-free foetal

DNA in maternal plasma and serum. Cell-free foetal DNA is cleared from the

circulation in less than an hour (Lo et al., 1999). Several clinical applications for

cell-free foetal DNA have been described, such as foetal sex determination

(Sekizawa et al., 2001) and foetal Rhesus- D genotyping (Faas et al., 1998).

Maternal plasma/ serum analysis has the advantage of being rapid, reliable,

reproducible, and easily carried out for a large number of samples. The main

limitation at present appears to be the availability of uniquely foetal gene

sequences that identify and/or measure the presence of foetal DNA in both male

and female foetuses (Bianchi, 2004; Wataganara et al., 2004).

For the further future combining the isolation of foetal DNA from maternal

serum/ plasma with molecular techniques such as MPLA or whole genome

microarrays, would certainly be a desirable way to go, in order to perform prenatal

diagnosis without any risk to mother and foetus.

As an alternative to prenatal diagnosis, preimplantation genetic diagnosis

(PGD) was introduced at the beginning of the 1990s to prevent termination of

pregnancy in couples with a high risk for offspring affected by a sex-linked genetic

Chapter 5


128

disease (Sermon et al., 2004). It is a kind of early prenatal diagnosis, in which at

least one cell of an 8-cells in-vitro embryo is analysed for well defined genetic

defects. Only embryos free of this defect are replaced in utero. Recently, PGD for

aneuploidy screening has been applied to improve a low success rate of in-vitro

fertilisation (IVF) attributed to chromosomal aneuploidies in the embryos, as is

sometimes the case in woman older than age 36 –40 years (Wilton et al., 2002). In

the Netherlands, a blind randomised study has been started to answer this

question (Mastenbroek et al., 2004). However, PGD will be restricted to women

undergoing IVF and to a few couples with a high risk of an affected foetus for well-

defined diseases.

Reducing the costs of health care is an important issue since the budget is

not increasing at the same rate as the demands. From a medical and financial

point of view, a screening program is the most cost-effective policy (Wortelboer et

al., 2000). It leads to a better use of cytogenetic diagnosis, with more benefit and

less harm to the pregnant women and their babies. Furthermore the question rises

whether a complete cytogenetic diagnosis is essential for the indication ‘advanced

risk at screening’ (Hulten et al.,2003). A diagnosis for just the common

aneuploidies, such as 13; 18; 21; X and Y by a molecular technique, e.g. MLPA,

might be sufficient. In a retrospective study among 65,189 cases in the advanced

maternal age group 0.2% false negative or ‘clinically relevant’ abnormalities would

have been missed if women would have had the MLPA test for chromosomes 13;

18; 21, X and Y instead of karyotyping (Niewint et al., 2004). The false negative

rate could be further reduced by the use of an MLPA test with probes for the

subtelomeric regions of all chromosomes (Suijkerbuijk et al., 2004).

Although (prenatal) cytogenetics is moving into the direction of molecular

approaches, these developments by no means imply that ‘conventional’

cytogenetic diagnosis will disappear from the laboratories in the near future. Only

by regular karyotyping, an overview of the complete human genome is obtained

and balanced aberrations, such as translocations, inversions or insertions can be

visualised, whereas DNA techniques can only detect unbalanced aberrations.



129

In summary, possibilities for prenatal diagnosis will further increase in the

future. The choice of the method will remain difficult. On the one hand there is the

wish of pregnant women to get maximal certainty of a healthy baby, on the other

hand there is a limited health care budget. Moreover, with every introduction of a

new technique the question should be discussed whether the test should be

available to all pregnant women. Professionals in the field of prenatal diagnosis

should inform and advice the government, health care providers and pregnant

women on the implications of the different tests. Eventually, it is up to the society to

determine whether all questions in prenatal diagnosis should be answered, which

tests should be offered to whom and at which costs. The answer to these

questions might be different for different countries.

5.3. Suggested guidelines for handling amniotic fluid and chorionic villi

In general, our prenatal cytogenetic practice has proven to be robust and reliable.

Yet, based on our findings a few additional guidelines are formulated.

Guidelines for culturing AF cells:

It can be concluded that AF can be stored, if necessary, for at least two days either

at room temperature or in the refrigerator without any decrease of growth potential

(Chapter 2.3). The desired amount of AF is 6 ml. According to our standard

procedure for culturing, four in-situ cultures will be initiated with 1.5 ml AF per dish

(Chapter 2.2). However, 3.2 ml AF, 0.8 ml AF per in-situ dish, are sufficient for a

reliable diagnosis, while culture time is extended with two days. We suggest to

register a bloody or brown appearance of AF and AF sample volumes less than 3.2

ml for explaining a possible delay in culture time or culture failure (Chapter 2.1). A

new lot or type of culture medium needs to be subjected to stress testing before

introducing it in the routine practice (Chapter 2.3). The growth potential of AF cells,

as assessed by an increase of the number of cells within a clone, can be enhanced

2.5-fold under a decreased oxygen tension (Chapter 2.3).

Chapter 5


130

Guidelines for harvesting and slide preparation of in-situ cultures:

As demonstrated in Chapter 3, metaphase quality is only to a limited degree

affected by the composition of fixative, the relative humidity, the ambient

temperature and the air flow at harvesting. Within this limited influence, the

following recommendations can be given. (1) The higher the concentration of

acetic acid before slide preparation the better the spreading of chromosomes. (2)

The presence of cytoplasm in in-situ preparations is slightly influenced by relative

humidity and is minimal between 30% and 40%. (3) Extra air flow of 0.6 m/s further

decreases the presence of cytoplasm for chorionic villi preparations. Expensive

instruments in climate control during harvesting and slide preparation are not

necessary.

Guidelines for chromosomal analysis:

Guidelines for analysing preparations from AF cells and chorionic villi have already

been well defined. Therefore, only two additional suggestions are given. (1) The

table for exclusion of mosaicism, especially developed for preparations from

trypsinised in-situ AF cell cultures, should be used to complete analysis from in-situ

culture dishes in case an insufficient number of clones is obtained in the regularly

harvested dishes. (2) Metaphase preparations from cultured mesenchymal cells

from chorionic villi alone can be analysed for cytogenetic diagnosis provided that

maternal cell contamination is limited.



131

5.4 Literature

Adinolfi M. Non- or minimally invasive prenatal diagnostic tests on maternal blood samples or

transcervical cells. Prenat Diagn. 1995; 15: 889-96.

Association of Clinical Cytogeneticists (ACC) Working Party. Professional guidelines for clinical

cytogenetics. BMJ Publishing Group 2001. http://www.cytogenetics.org.uk/information/Guidelines.pdf

Beekhuis JR, de Wolf BTMH, Mantingh A, Heringa MP. The influence of serumscreening on the

amniocentesis rate in women of advanced maternal age. Prenat Diagn. 1994; 14: 199-202.

Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, De Maria MA. Male foetal progenitor cells persist in

maternal blood for long as 27 years postpartum. Proc Natl Acad Sci USA1996; 93: 705-8.

Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP. PCR quantitation of foetal

cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet. 1997; 61: 822- 9.

Bianchi DW. Foetal cells in the maternal circulation: feasibility for prenatal diagnosis. Br J Haematol.

1999 ; 105: 574-83.

Bianchi DW. Circulating foetal DNA: its origin and diagnostic potentials; A review. Placenta 2004; 25:

S93-S101.

Bryndorf T, Christensen B, Vad M, Parner J, Brocks V, Philip J. Prenatal detection of chromosome

aneuploidies by fluorescence in situ hybridisation: experience with 2000 uncultured amniotic fluid

samples in a prospective preclinical trial. Prenat Diagn. 1997; 17(4): 333-41.

Cornel MC, Breed ASPM, Beekhuis JR, Meerman te GJ, Kate ten, LP. Down syndrome: effects of

demographic factors and prenatal diagnosis on the future livebirth prevalence. Hum Genet. 1993; 92:

163-68.

D’Alton ME, Malone FD, Chelmow D, Ward BE, Bianchi DW. Defining the role of fluorescence in situ

hybridisation on uncultured amniocytes for prenatal diagnosis of aneuploidies. Am J Obstet Gynecol.

1997; 176: 769-76.

Dutch Society of Obstetrics and Gynaecology. 1997. Guidelines for invasive prenatal diagnosis.

http://www.nvog.nl

Faas BH, Beuling EA, Christiaens GC, dem Borne AE, van der Schoot CE. Detection of foetal RHD-

specific sequences in maternal plasma. Lancet 1998; 352 : 1196.

Hahnemann JM, Vejerslev LO. European collaborative research on mosaicism in CVS (EUCROMIC) –

foetal and extrafoetal cell lineages in 192 gestations with CVS mosaicism involving single autosomal

trisomy. Am J Med Genet. 1997; 70: 170-87.

Hamada H, Arinami T, Kubo T, Hamaguchi H, Iwasaki H. Foetal nucleated cells in maternal peripheral

blood: frequency and relationship to gestational age. Hum Genet. 1993; 91: 427-32.

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132

Health Council of the Netherlands. Prenatal screening (2); Down’s syndrome, neural tube defects. The

Hague: Health Council of the Netherlands, 2004; publication no. 2004/06.

Hulten MA, Dhanjal S, Pertel B. Rapid and simple prenatal diagnosis of common chromosome

disorders: advantages and disadvantages of the molecular methods FISH and QF-PCR. Reproduction

2003; 126(3): 279-97.

Hsu LY, Kaffe S, Jenkins EC, Alonso L, Benn PA, David K, Hirschhorn K, Lieber E, Shanske A, Shapiro

LR, et al. Proposed guidelines for diagnosis of chromosome mosaicism in amniocytes based on dataderived from chromosome mosaicism and pseudomosaicism studies. Prenat Diagn. 1992;12(7):555-73

Hsu LYF, Benn PA. Revised guidelines for the diagnosis of mosaicism in amniocytes. Prenat Diagn.

1999; 19: 1081-82.

Kornman LH, Beekhuis JR, Mantingh A, Morssink LP. Serumscreening for foetal Down syndrome in

pregnant women at advanced maternal age: less amniocentesis. Ned Tijdschr Geneeskd. 1995; 139:

1840-4.

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Ongeveer 1 op de 200 baby's wordt geboren met een chromosoomafwijking. De

gevolgen daarvan kunnen sterk uiteenlopen, van verwaarloosbaar (het kind zal er

in zijn of haar ontwikkeling geen last van ondervinden) tot aan 'niet levensvatbaar'.

Veel chromosomale afwijkingen blijken gepaard te gaan met hartafwijkingen,

ontwikkelingsachterstand en uiterlijke bijzonderheden.

Chromosomale afwijkingen kunnen in twee categorieën worden ingedeeld:

numerieke en structurele. Bij een numerieke afwijking is een chromosoom teveel of

te weinig aanwezig. Bij een structurele afwijking mist er materiaal van een

chromosoom of heeft een chromosoom juist extra materiaal. Ook kan het zijn dat

bepaalde stukken van chromosomen zijn uitgewisseld. Alles is wel aanwezig maar

zit niet op de juiste plaats. De ernst van de aandoening wordt over het algemeen

bepaald door de mate waarin chromosomaal materiaal is toe- dan wel afgenomen.

Door middel van prenatale cytogenetische diagnostiek is het mogelijk om

het overgrote deel van de chromosoomafwijkingen uit te sluiten en zwangeren op

dat punt gerust te stellen. Indien bij dergelijk onderzoek wel een

chromosoomafwijking wordt aangetoond, kunnen de toekomstige ouders na

grondige voorlichting over wat hen mogelijk te wachten staat, eventueel kiezen

voor afbreking van de zwangerschap. Ook al wordt er d.m.v. prenataal

chromosomenonderzoek geen afwijking gevonden, dan blijft er nog een algemeen

risico bestaan van ca. 2% - 3% op een kind met een erfelijke ziekte of aangeboren

afwijking berustend op een gen-afwijking, die niet aan de chromosomen te zien is.

De meest gebruikte methodes om uit foetaal materiaal cytogenetisch

onderzoek te verrichten zijn de vruchtwaterpunctie en de vlokkentest (Figuur 1).

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Figuur 1: Schematische weergave van een vroege zwangerschap tijdens de

afname van chorionvlokken (uit Genetics: Human Aspects; Mange & Mange,

Second Edition, Sinauer Associates, INC. Publishers Sunderland, pg. 504)

Bij de vruchtwaterpunctie wordt normaliter tussen de 16e en 18e week van de

zwangerschap 15- 20 ml vruchtwater afgenomen. Foetale cellen die in het

vruchtwater zijn terecht gekomen worden onder bepaalde omstandigheden in

kweek gebracht. Door deling groeien de vitale cellen uit tot een samenhangend

groepje cellen, een zogenaamde celkloon. Als er redelijk wat klonen van

voldoende grootte zijn, dit is na circa acht dagen, wordt een mitose-remmer aan de

kweken toegevoegd, zodat de cellen in het delingsstadium blijven steken. Om tot

een chromosoompreparaat te komen worden de cellen met een hypotone vloeistof

behandeld, bedoeld om de cellen te laten opzwellen en de chromosomen beter te

spreiden. Vervolgens worden de cellen met een mengsel van methanol en

azijnzuur gefixeerd. Na het verwijderen van het fixatief worden de cellen gedroogd.

Hierna worden de chromosomen bewerkt en gekleurd om een bepaald

bandenpatroon te verkrijgen. Aan de hand van dit bandenpatroon kunnen de


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chromosomen in paren worden gerangschikt (karyotype)1, zodat eventuele

numerieke of structurele afwijkingen kunnen worden gedetecteerd. Het gehele

proces van afname van het vruchtwater tot diagnose duurt twee tot drie weken.

Bij een vlokkentest worden 5- 50 mg chorionvlokken (embryonaal weefsel

dat deel uitmaakt van de placenta) afgenomen, normaliter tussen de 10e en de

12e week van de zwangerschap. Na afname worden de chorionvlokken eerst van

aanhangend moederlijk weefsel ontdaan. De vlokken bestaan uit verschillende

celtypes. Van één daarvan, de cytotrofoblast cellen, kunnen via de zogenaamde

directe afwerking zonder kweek chromosoompreparaten gemaakt worden,

aangezien meestal voldoende cellen in deling zijn. Een cytogenetische diagnose is

dan reeds na enkele uren mogelijk, echter de kwaliteit van deze

chromosoompreparaten is slecht. Zodoende kunnen kleine structurele afwijkingen

over het hoofd worden gezien.

Als alternatief kan van een ander celtype, de mesenchymale cellen, een kweek

worden ingezet. Meestal worden hierbij eerst de cytotrofoblast cellen verwijderd en

de mesenchymale cellen met behulp van een enzym tot een suspensie (cellen

bevattende vloeistof) verteerd. Deze celsuspensie wordt in kweek gebracht. Het

proces om tot een chromosoompreparaat te komen is dan hetzelfde als bij

vruchtwatercellen. Een cytogenetische diagnose is na acht tot veertien dagen

mogelijk en de kwaliteit is dusdanig dat ook kleine structurele afwijkingen kunnen

worden gedetecteerd. Een nadeel van deze methode is de mogelijke bijmenging

van moederlijke cellen, waardoor in plaats van foetale cellen moederlijke cellen

1 Een mens heeft normaliter in elke cel 46 chromosomen; 22 paren van autosomen en één paar

geslachtschromosomen. Bij een vrouw zijn beide geslachtschromosomen X- chromosomen, bij de man

bestaan ze uit één X- en één Y-chromosoom. Chromosomen zijn tijdens een celdeling (mitose) het

meest gecondenseerd in de metafase. Zij zijn dan lineair van gestalte en kunnen als paar naar lengte,

positie van het centromeer en bandenpatroon gerangschikt worden. Deze ordening wordt karyotype

genoemd.

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zouden kunnen worden geanalyseerd. Vanwege de kans op discrepanties tussen

het chromosomenpatroon van de foetus en weefsel van de placenta wordt een

diagnose gebaseerd op de resultaten van cytotrofoblast en mesenchymale cellen

dan ook het betrouwbaarst geacht.

De belangrijkste kwaliteitscriteria ten behoeve van de diagnostiek voor het

genoemde foetale materiaal zijn de sensitiviteit, of wel de mate waarin het lukt een

in de foetale cellen aanwezige afwijking ook inderdaad aan te tonen; en de

specificiteit, die aangeeft in hoeverre kan worden vastgesteld dat een afwijking ook

daadwerkelijk voorkomt in de foetale cellen en niet stamt uit moederlijke cellen of

berust op een pas in de kweek ontstane afwijking. Het doel van prenatale

cytogenetische diagnostiek is immers om een mogelijke chromosomale afwijking

bij de foetus vast te stellen. Er is sprake van een optimale diagnostiek wanneer

sensitiviteit en specificiteit zo goed als 100% zijn. Bovendien dient een diagnose zo

snel mogelijk tegen zo laag mogelijke kosten tot stand te komen. Om dit te kunnen

realiseren is kennis nodig van alle factoren die hierbij een rol spelen en van de

mate van hun invloed. Gebaseerd op deze kennis kunnen richtlijnen worden

opgesteld met betrekking tot de wijze waarop vruchtwatercellen en chorionvlokken

bewerkt moeten worden om uiteindelijk tot betrouwbare prenatale diagnostiek te

komen.

Het cytogenetisch proces kan in de volgende de stappen gesplitst worden;

afname van materiaal, celkweek, preparaatbereiding en analyse. Voor de analyse

van chorionvlokken en vruchtwatercellen bestaan reeds richtlijnen, opgesteld door

de Werkgroep van de Nederlandse Vereniging voor Klinische Cytogenetica. Deze

richtlijnen zijn van voldoende kwaliteit om een betrouwbare diagnose te

waarborgen. Voor de andere stappen in dit proces zijn echter vrijwel geen

richtlijnen voorhanden. Deze aan de analyse voorafgaande stappen zijn wel van

groot belang voor de uiteindelijke kwaliteit van het chromosoompreparaat inzake

de hoeveelheid klonen en delende cellen en de lengte en structuur van de

chromosomen. Bovendien is de invloed van factoren die hierbij een rol zouden


140

kunnen spelen nog nooit systematisch onderzocht. Dergelijk onderzoek is wel van

belang, met name voor prenatale diagnostiek, omdat het verkrijgen van

vruchtwater of chorionvlokken gepaard gaat met een zeker risico voor zowel de

aanstaande moeder alsook voor de foetus en dus een gewaarborgde kwaliteit

gewenst is. Mislukkingen die herhaling van de procedure noodzakelijk zouden

maken, dienen daarom zo veel mogelijk te worden vermeden dan wel beperkt.

Het doel van dit proefschrift is om te trachten richtlijnen op te stellen voorhet hele proces dat vruchtwatercellen en chorionvlokken ondergaan om toteen juiste cytogenetische diagnose te komen.

In ons onderzoek hebben wij de invloed van mogelijk belangrijke factoren binnen

dit proces onderzocht om, al naar gelang de mate van hun invloed, te kunnen

bepalen welke factoren kritisch bewaakt dienen te worden en voor welke er

richtlijnen moeten worden opgesteld.

In een algemene introductie in hoofdstuk 1 wordt het proces beschreven

dat vruchtwatercellen en chorionvlokken ondergaan om tot een cytogenetische

diagnose te komen. Tevens worden bestaande richtlijnen voor dit proces

beschreven en wordt aangegeven wat reeds bekend is over de invloed van

verschillende factoren op de kwaliteit van het cytogenetisch proces.

Hoofdstuk 2 richt zich op het kweken van vruchtwatercellen volgens de in-

situ procedure (in-situ = ter plaatse 2). Hierbij werd middels retrospectief

onderzoek bekeken in welke mate de gynaecoloog, de hoeveelheid vruchtwater,

de samenstelling van het vruchtwater (met name al dan niet ook bloed bevattend),

de grootte van het neerslag van cellen na centrifugatie, de zwangerschapsduur bij

afname, de reden voor diagnostiek, de chromosomale diagnose en het type of de

2 In dit geval wil dit zeggen, dat vruchtwater in een kweekschaaltje wordt gebracht waarin op de bodemeen glaasje is geplaatst en dat de cellen van het vruchtwater uitzakken naar de bodem en zich terplaatse aan het glasoppervlak hechten. Ze kunnen daar dan eventueel tot klonen uitgroeien.

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geleverde partij van het kweekmedium van invloed zijn op de uiteindelijke kwaliteit

van de celkweken. Kwaliteit wordt in dit verband gemeten aan de totale

hoeveelheid klonen welke aanwezig zijn op de eerste dag van de beoordeling van

de celkweken en aan de tijdsduur van het in kweek brengen van de

vruchtwatercellen tot aan de preparaatbereiding (hoofdstuk 2.1). Uit ons onderzoek

is gebleken dat vruchtwater afgenomen volgens de richtlijnen van de Nederlandse

Vereniging voor Gynaecologie en Obstetrie over het algemeen van voldoende

kwaliteit is voor cytogenetisch onderzoek. Verder kwam naar voren dat de

hoeveelheid vruchtwater, de reden voor afname, de zwangerschapsduur bij

afname, alsmede de chromosomale diagnose niet van invloed zijn op de kwaliteit

van vruchtwater celkweken. Kleine veranderingen binnen deze factoren leiden niet

tot een onacceptabel resultaat. Daarentegen werd duidelijk, dat bloederig of bruin

vruchtwater een grote invloed heeft op de kwaliteit van de kweken. Hoewel de

kweken hierdoor weliswaar niet mislukken, wordt de kweekduur er met maar liefst

drie dagen door verlengd.

Verder hebben wij de minimale hoeveelheid vruchtwater bepaald die voor

een betrouwbare diagnose nodig is (hoofdstuk 2.2). Dit bleek 3,2 ml te zijn.

Commercieel medium voor vruchtwatercellen dat is aangevuld met hormonen, is

over het algemeen van voldoende kwaliteit voor het kweken. Omdat medium

tevens in-vivo geproduceerd serum bevat, kan de kwaliteit tussen verschillende

geleverde partijen uiteenlopen. Om deze reden hebben wij een acceptatietest voor

medium ontwikkeld, gebaseerd op het principe van een stresstest (hoofdstuk 2.3).

Om de validiteit en sensiviteit van deze test te kunnen bepalen, hebben wij als

voorbeeld de invloed van een verlaagde zuurstofspanning tijdens het kweken

onderzocht. De groei van vruchtwatercellen is m.b.v. de stresstest en groeicurven3

vergeleken onder gereduceerde zuurstofspanningen van 10%, 5% en 2,5% en bij

de 20% zuurstof in de normale atmosfeer. Het bleek dat een gereduceerde

3 Vanuit samengevoegde vruchtwatermonsters zijn verschillende kweken opgezet. De groei van dezekweken wordt op verschillende dagen gestopt om de hoeveelheid cellen te tellen en zo een groeicurveover die dagen op te stellen.


142

zuurstofspanning resulteerde in een 2,5 voudige toename in het aantal cellen per

kloon. Het aantal klonen nam slechts licht toe.

Hoofdstuk 3 richt zich op de preparaatbereiding. Tijdens de

preparaatbereiding van in-situ kweken4 van vruchtwatercellen en chorionvlokken is

de invloed bepaald van de samenstelling van het fixatief, de relatieve vochtigheid,

de temperatuur en de luchtstroom op de kwaliteit van chromosomen. Kwaliteit

wordt in dit verband gemeten als de mate van spreiding van de chromosomen en

de mate van het (niet) aanwezig zijn van cytoplasma. Hierbij heeft de

samenstelling van het fixatief de grootste invloed op de spreiding van de

chromosomen. Het beperken van de aanwezigheid van cytoplasma wordt bij

vruchtwatercellen gerealiseerd door het beïnvloeden van de luchtvochtigheid en bij

chorionvlokken door het beïnvloeden van de luchtstroom. We hebben echter

vastgesteld, dat de genoemde factoren maar in beperkte mate van invloed zijn op

de kwaliteit van de chromosomen. Variatie in deze condities leidt nauwelijks tot

kwaliteitsveranderingen. Desalniettemin verdient de in-situ preparaatbereiding

onze voorkeur boven de preparaatbereiding van celsuspensies5, omdat de in-situ

procedure veel sneller is en daarbij minder delende cellen nodig zijn voor een

betrouwbare diagnose.

Hoofdstuk 4 richt zich op de analyse van preparaten van vruchtwatercellen

en chorionvlokken. Voor de analyse bestaan reeds richtlijnen met betrekking tot de

hoeveelheid cellen die geanalyseerd moeten worden per indicatie en per gevonden

afwijking en inzake de benodigde bandenresolutie van de chromosomen. In

aanvulling hierop hebben wij in een tabel gedefinieerd hoeveel cellen geanalyseerd

moeten worden voor het kunnen uitsluiten van een chromosomaal

4 Dat wil zeggen dat de in-situ celkweken tijdens de preparaatbereiding op het glaasje blijven en na hetdrogen op een objectglaasje geplakt worden.

5 Dat wil zeggen dat de cellen meestal in een plastic flesje worden gebracht en op de bodem groeien.Wanneer bijna het gehele oppervlak met cellen is bedekt, worden de cellen met behulp van een enzymvan de bodem losgehaald (getrypsiniseerd). De zo onstaande celsuspensie wordt tot chromosoompreparaat verwerkt.

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mozaïekpatroon6 in getrypsiniseerde in-situ preparaten van vruchtwatercelkweken.

Deze tabel draagt bij aan de verdere standaardisering van de

betrouwbaarheidsgrenzen om een mozaïekpatroon uit te sluiten wanneer, de in-

situ kweek ten spijt, getrypsiniseerde kweken nodig zijn om de diagnose te

completeren (hoofdstuk 4.3). Deze tabel kan ook gebruikt worden wanneer in

flessen gekweekt wordt en vanuit een suspensie het preparaat bereid wordt van

waaruit cellen geanalyseerd worden.

In de literatuur wordt voor chorionvlokken de chromosomale analyse van

cytotrofoblast cellen (directe afwerking) en mesenchymale cellen (kweek)

aangeraden. Wij hebben aangetoond dat analyse uit kweekpreparaten alleen

voldoende betrouwbaar is mits bijmenging van moederlijke cellen beperkt is

(hoofdstuk 4.2). De kans op een fout-negatieve diagnose (foetus is chromosomal

afwijkend maar de geanalyseerde cellen zijn normaal) is bepaald op 1-3 procent

per 1000 wanneer slechts cytotrofoblast cellen geanalyseerd worden en is te

verwaarlozen wanneer mesenchymale cellen worden gebruikt (hoofdstuk 4.1). De

kans op een fout- positieve diagnose (foetus is chromosomal normaal maar de

geanalyseerde cellen zijn afwijkend) is over het algemeen hoger bij analyse van

cytotrofoblast cellen dan bij analyse van mesenchymale cellen en hangt af van de

gevonden afwijking (hoofdstuk 4.2).

Samengevat kan geconcludeerd worden dat onze cytogenetische

diagnostiek in vruchtwatercellen en chorionvlokken robuust en betrouwbaar is. De

volgende aanvullende conclusies en richtlijnen worden door ons in hoofdstuk 5

gegeven.

Vruchtwater kan zonder verlies van groeipotentiaal van de cellen twee

dagen in de koelkast of bij kamertemperatuur bewaard worden. De gewenste

hoeveelheid vruchtwater per patiënt is 6 ml. Volgens onze standaard procedure

voor in-situ kweken zouden vier kweken met 1,5 ml vruchtwater opgezet moeten

6 Mozaïekpatroon wil zeggen dat naast elkaar verschillende cellen met verschillende chromosomalesamenstelling voorkomen.


144

worden. Met 3,2 ml vruchtwater (0,8 ml per kweekschaaltje) is echter reeds (met

een twee dagen langere kweekduur) een betrouwbare diagnose te stellen. Het in

kweek brengen van minder dan 3,2 ml vruchtwater zou geregistreerd moeten

worden, evenals een bloederige of bruine samenstelling van het vruchtwater. Dit

om een mogelijke groeivertraging of mislukking te kunnen verklaren. Een nieuw

geleverde partij of type medium dient eerst op groeipotentiaal getest te worden

d.m.v. een stresstest voordat het in de diagnostiek wordt gebruikt. De

groeisnelheid van vruchtwatercellen binnen een kloon kan 2,5 keer vergroot

worden door een verlaagde zuurstofspanning tijdens het kweken.

Tijdens de preparaatbereiding is de chromosoomkwaliteit van in-situ

kweken van vruchtwatercellen en chorionvlokken slechts beperkt afhankelijk van

de samenstelling van het fixatief, de relatieve vochtigheid, de temperatuur en de

luchtstroom. Binnen deze beperkte invloed kan het volgende worden vastgesteld:

(1) hoe hoger de concentratie van azijnzuur in het fixatief voor het drogen van de

preparaten, hoe beter de spreiding van de chromosomen, (2) het minste

cytoplasma is zichtbaar wanneer de relatieve vochtigheid tijdens het drogen tussen

30% en 40% is, (3) een extra luchtstroom van 0,6 m/s kan de aanwezigheid van

cytoplasma in chorionvlokken preparaten verder beperken.

Analyse uit mesenchymale cellen van chorionvlokken (kweek) is voldoende

voor een betrouwbare diagnostiek mits bijmenging van moederlijke cellen beperkt

is. Het aantal te analyseren cellen uit getrypsiniseerde vruchtwater celkweken zou

middels de hiervoor ontwikkelde tabel bepaald moeten worden om zo nodig de

diagnostiek uit in-situ preparaten aan te vullen.


145

Curriculum Vitae

Birgit Sikkema-Raddatz was born the 28th of February 1965 in Güstrow (Germany).

In 1989 she graduated in Biology (specialisation Genetics) at the Martin Luther

University in Halle.

In 1990 she married Jan Sikkema, a Dutchman, which made her decide to move to

the Netherlands. That same year she started her career in the Department of

Medical Genetics (chair: Prof.dr. C.H.C.M. Buys) at the University of Groningen,

first as a technician and since 1991 as a cytogeneticist. In 1992 she became

manager of the laboratory for prenatal cytogenetics. In 2000 she switched to the

molecular cytogenetic laboratory where she now works as head of this group.

Birgit Sikkema-Raddatz is registrated as clinical cytogeneticist with the Dutch

Association of Clinical Cytogeneticists.


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147

List of publications

Sijmons RH, Sikkema-Raddatz B, Kloosterman MD, Briet JW, de Jong, Leschot NJ.

46,XY,dup(10q) in direct CVS preparation and mosaic 48,XXXY,dup(10q) in CVS long-term

culture and fetal tissue. Prenat Diagn.1995; 15 (3): 285-90.

Sikkema-Raddatz B, Sijmons RH, Tan-Sindunata MB, van der Veen AY, Brunsting R, de

Vries B, Beekhuis JR, Bekedam DJ, van Aken, de Jong B. Prenatal diagnosis in two cases

of de novo complex balanced chromosomal rearrangements. Three-year follow-up in one

case. Prenat Diagn. 1995; 15 (5): 467-73.

Morssink LP, Sikkema-Raddatz B, Beekhuis JR, de Wolf BT, Mantingh A. Placental

mosaicism is associated with unexplained second-trimester elevation of MShCG levels, but

not with elevation of MSAFP levels. Prenat Diagn. 1996; 16 (9): 845-51.

Morssink LP, de Wolf BT , Kornman LH, Beekhuis JR, van der Hall TP, Sikkema-Raddatz B,

Mantingh A. The significance of placental pathology in pregnancies with unexplained

abnormal concentrations of MSAFP or MShCG. Early Hum Dev. 1996; 30; 47 Suppl: S63-5.

Sikkema-Raddatz B, Castedo S, te Meerman GJ. Probability tables for exclusion of

mosaicism in prenatal diagnosis. Prenat Diagn. 1997; 17 (2): 115-8.

Sikkema-Raddatz B, Verschuuren-Bemelmans CC, Kloosterman M, de Jong B. A

46,XX,der(13;14)(q10;q10),+21 child born after a 45,XX,der(13;14)(q10;q10) chromosomal

finding in CVS. Prenat Diagn. 1997; 17 (11): 1086-8.


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Sikkema-Raddatz B and Sijmons RH. De novo balanced complex chromosomal

rearrangements in prenatal diagnosis. From: Chorionic villus sampling; Eds. Rao KA and

Nicolaides K; Jaypee Brothers, Medical Publishers (P) LTD, New Delhi1998.

Leegte B, Sikkema-Raddatz B, Hordijk R, Bouman K, van Essen T, Castedo S, de Jong.

Three cases of mosaicism for balanced reciprocal translocations. Am J Med Genet 1998;

79(5): 362-5.

Sikkema-Raddatz B, Bouman K, Verschuuren-Bemelmans CC, de Jong B. Trisomy 12

mosaicism in CVS culture confirmed in the fetus. Prenat Diagn. 1999; 19 (12): 1176-7.

Sikkema-Raddatz B, Bouman K, Verschuuren-Bemelmans CC, Stoepker M, Mantingh A,

Beekhuis JR, de Jong B. Four years' experience with the culture of chorionic villi. Prenat

Diagn. 2000; 20 (12):950-5.

Leegte B, Sikkema-Raddatz B, Hordijk R, Davelaar I, van der Veen, Cobben JM. Two

unbalanced segregation products due to a maternal t(7;16)inv(16). Prenat Diagn. 2001;

21(7): 550-2.

Sikkema-Raddatz B, van Echten J, van der Vlag J, Buys CHCM, te Meerman GJ. Minimal

volume of amniotic fluid for reliable prenatal cytogenetic diagnosis. Prenat Diagn. 2002; 22

(2): 164-5.

Westra JL, Boven LG, van der Vlies P, Faber H, Sikkema B, Schaapveld M, Dijkhuizen T,

Hollema H, Buys CHCM, Plukker JTM, Kok K, Hofstra RMW. A substantial proportion of

microsatellite-instable colon tumours carries TP53 mutations while not showing

chromosomal instability. Genes Chromosomes and Cancer 2004; accepted for publication

Mastenbroek S, Engel C, van Echten-Arends J, Sikkema-Raddatz B, van Wassenaer, AG,

de Vries WA, Heineman MJ, Repping S, van der Veen F. Preimplantatie Genetische

screening naar numerieke chromosoomafwijkingen bij embryo’s van vrouwen van 35 jaar en

ouder: de eerste resultaten in Nederland. Ned Tijdschr Geneeskd. 2004; 148(50): 2486-90.

proefdruk1.doc

149

Dankwoord

Allereerst wil ik mijn copromotor Dr. Gerard te Meerman bedanken daar hij aan de

wieg stond van deze promotie. Gerard, jouw enthousiasme voor dit onderzoek is

van begin tot eind onverminderd groot geweest en was voor mij een belangrijke

bron voor inspiratie. Hartelijk dank voor al jouw berekeningen, alle uitvoerbare en

niet uitvoerbare ideeën en jouw support op het praktisch vlak.

Veel dank ben ik ook verschuldigd aan mijn promotor Prof. Charles Buys.

Charles, bedankt voor jouw kritische blik ten aanzien van de resultaten van onze

experimenten waardoor vaak nieuwe invalshoeken konden ontstaan. Daarnaast

ben ik je zeer erkentelijk voor het lezen en redigeren van mijn manuscripten

waardoor ze aan helderheid toenamen.

Mijn direct leidinggevende Klasien Gerssen-Schoorl wil ik bedanken voor het

feit dat zij mij in mijn promotieplannen heeft gesteund waardoor ik in de

gelegenheid werd gesteld één en ander te realiseren. Anneke van der Veen wil ik

bedanken voor het feit dat zij zorg droeg voor de dagelijkse werkzaamheden

waardoor ik de handen vrij had voor mijn onderzoek.

Ron, Katelijne, Trijnie en Marga, bedankt voor jullie hulp in de vorm van ‘even

meedenken, even een manuscript lezen’ en ‘even een computerprobleem

oplossen’.

Tevens wil ik op deze plek Prof. Bauke de Jong bedanken. Bauke, dankzij het

vertrouwen dat jij in mij stelde door mij in 1991 aan te nemen en mij op te leiden tot

cytogeneticus heb jij de basis gelegd voor dit proefschrift.


150

Verder wil ik mijn collega’s van de FISH-groep bedanken; Gineke, Hanny, Harke,

Hendrika, Inge, Marjan en Yolanthe, met name het laatste half jaar hebben jullie

door je hoge inzet er voor gezorgd dat de ‘tent bleef draaien’. Yolanthe wil ik

tevens bedanken voor het ontwerpen van de cover van mijn proefschrift.

Uiteraard gaat ook een woord van dank naar de prenatale groep voor het

verrichten van alle proeven ten behoeve van mijn onderzoek. Annet, Anke, Eddy,

Fia, Janet, Jolanda, Jurjen, Lennart, Madeleine en Reina, jullie moesten een stapje

harder werken om mijn paranimfen Jakob en Marian gelegenheid te bieden om

zich aan mijn onderzoek te wijden.

Jakob en Marian; zonder jullie niet aflatend enthousiasme en inzet had ik het

onderzoek nimmer kunnen voltooien. Heel veel dank daarvoor.

I would like to thank the members of the reading committee for their willingness

and quick review of the manuscript.

Auch möchte ich mich bei allen Raddatz’en bedanken. Schliesslich habt Ihr auf

direkte und indirekte Weise dafür gesorgt, dass ich Biologie studiert habe und dass

alles was ich mir beruflich vorgenommen habe, auch gelungen ist.

Mijn echtgenoot Jan wil ik bedanken voor diens aandeel in het redigeren en voor

zijn morele ondersteuning.

Tot slot zijn er nog veel mensen die een bijdrage hebben geleverd aan het tot

stand komen van dit proefschrift zonder dat zij hier met hun naam zijn genoemd.

Jullie allen ook hartstikke bedankt.

Birgit

university of groningen quality assessment of prenatal ...chapter 2.2 minimal volume of amniotic...

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