45427532 wostenholme-cytogenetics-in-70-80s-prenat-diagn-2010
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PRENATAL DIAGNOSIS Prenat Diagn 2010; 30: 605–607. Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pd.2478 30 th Anniversary Issue of Prenatal Diagnosis HISTORICAL PERSPECTIVES Cytogenetics in the 1970s and 1980s J. Wolstenholme 1 * and D. E. Rooney 2 1 Cytogenetics Laboratory, Institute for Human Genetics, Newcastle upon Tyne, UK 2 Regional Cytogenetics Laboratory, NE Thames Regional Genetics Service, Great Ormond Street Hospital, London, UK KEY WORDS: prenatal diagnosis; cytogenetics; history By the mid to late 1970s, much of the groundwork for the prenatal diagnosis of cytogenetic disorders was already in place; although compared with today, there was relatively little in the garden. Fledgling cytogenetics laboratories in the UK had already established postna- tal services using blood lymphocyte cultures (although often without the benefit of G-banding) and were slowly introducing karyotyping of amniocyte cultures. How- ever, with no effective screening for high-risk pregnan- cies, other than maternal age, referrals were predomi- nantly from women with a previous history of trisomy 21, or because of a familial translocation; indeed, in the early days samples were as likely to be for diagno- sis of biochemical disorders, fetal sexing or neural tube defects, as for chromosomal errors. Due to the limited resolution or lack of availability of ultrasound, the risk of the amniocentesis procedure itself was such that it was not something to be performed lightly. With experience in tissue culture, but none what- soever in genetics, JW’s initial training in Dundee in 1978 consisted of a 1-day trip to Glasgow to meet ‘the Ferguson-Smiths’ and to be told how to grow and har- vest amniocytes. This was followed by analyzing blood chromosomes for 3 weeks, before the first cultures were ready. A year later, a rather more genetically qualified DER arrived in Pendlebury (Manchester) equipped with a cut-out G-banded karyotype kept from an undergrad- uate practical. Facilities were highly variable: Dundee had the advan- tage of a modern laboratory, whereas in contrast, the Oxford laboratory was based in some prefabs (temporary buildings of high asbestos content, erected after WWII), which had previously housed an MRC Research Unit in the grounds of an ex-US military hospital. The Newcas- tle laboratories were in the upstairs bedrooms of a late Victorian terraced house, complete with boarded-up fire- places, ornamental plaster work ceilings and most of the electrics nailed round the walls. Tissue culture medium *Correspondence to: J. Wolstenholme, Cytogenetics Laboratory, Northern Genetics Service, Institute of Human Genetics, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. E-mail: [email protected] was an inconsistent entity, made up from a selection of concentrated stock solutions (some home-made) and human or fetal calf serum, often of unreliable prove- nance. Tissue culture plastics were new and equally variable in quality, so the use of glass culture vessels was still widespread, including Carrel flasks first described in 1912. Batch testing of everything was routine. The early gassed and humidified incubators were as likely to grow Aspergillus as amniocycytes, which led to two camps of cytogeneticists: those who loved them and scrubbed them meticulously clean on a regular basis and those who avoided them like the plague. G-banding was still a new technique and somewhat unreliable; solid stain- ing, using unpleasant concoctions such as aceto-orcein, made up by boiling up glacial acetic acid and powdered stain in a fume cupboard, was considered to give supe- rior chromosome morphology (but unlike Giemsa stain, it could not be removed from fingers using bleach). Many happy hours were spent washing coverslips for in situ cultures, and washing, siliconizing, plugging and sterilizing glass pipettes. Health and Safety considerations were somewhat sec- ondary. Latex gloves were for wimps. Recirculating ster- ile cabinets were new, expensive and not very effective. It was far more likely that you had a sterile cabinet that blew straight at you or that you worked on the open bench. Flaming everything in a Bunsen burner, bacteri- ologist style, was essential, particularly if you worked on the open bench. Leaving the Bunsen burner on in a ster- ile cabinet after you turned off the airflow was an excel- lent way to start a fire, especially if you left items such as metal tweezers and coverslips sitting in pots of methanol inside them. These old sterile cabinets also welded your contact lenses to your eyes. Nobody worried about Hep- atitis B, or even knew about HIV, but nobody died. Cytogenetics in the 1970s also involved a lot of photography, hours in the darkroom developing and printing, and even longer cutting out and sticking down chromosomes on bits of card (Figure 1). The going rate was three cut out, glued down and laminated karyotypes an hour, although four was achievable. Finding a number 5 chromosome stuck to your sleeve when you got home was an occupational hazard. Copyright 2010 John Wiley & Sons, Ltd. Received: 14 December 2009 Accepted: 9 January 2010
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Transcript of 45427532 wostenholme-cytogenetics-in-70-80s-prenat-diagn-2010
PRENATAL DIAGNOSISPrenat Diagn 2010; 30: 605–607.Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/pd.2478
30th Anniversary Issue of Prenatal Diagnosis
HISTORICAL PERSPECTIVES
Cytogenetics in the 1970s and 1980s
J. Wolstenholme1* and D. E. Rooney2
1Cytogenetics Laboratory, Institute for Human Genetics, Newcastle upon Tyne, UK2Regional Cytogenetics Laboratory, NE Thames Regional Genetics Service, Great Ormond Street Hospital, London, UK
KEY WORDS: prenatal diagnosis; cytogenetics; history
By the mid to late 1970s, much of the groundworkfor the prenatal diagnosis of cytogenetic disorders wasalready in place; although compared with today, therewas relatively little in the garden. Fledgling cytogeneticslaboratories in the UK had already established postna-tal services using blood lymphocyte cultures (althoughoften without the benefit of G-banding) and were slowlyintroducing karyotyping of amniocyte cultures. How-ever, with no effective screening for high-risk pregnan-cies, other than maternal age, referrals were predomi-nantly from women with a previous history of trisomy21, or because of a familial translocation; indeed, inthe early days samples were as likely to be for diagno-sis of biochemical disorders, fetal sexing or neural tubedefects, as for chromosomal errors. Due to the limitedresolution or lack of availability of ultrasound, the riskof the amniocentesis procedure itself was such that itwas not something to be performed lightly.
With experience in tissue culture, but none what-soever in genetics, JW’s initial training in Dundee in1978 consisted of a 1-day trip to Glasgow to meet ‘theFerguson-Smiths’ and to be told how to grow and har-vest amniocytes. This was followed by analyzing bloodchromosomes for 3 weeks, before the first cultures wereready. A year later, a rather more genetically qualifiedDER arrived in Pendlebury (Manchester) equipped witha cut-out G-banded karyotype kept from an undergrad-uate practical.
Facilities were highly variable: Dundee had the advan-tage of a modern laboratory, whereas in contrast, theOxford laboratory was based in some prefabs (temporarybuildings of high asbestos content, erected after WWII),which had previously housed an MRC Research Unit inthe grounds of an ex-US military hospital. The Newcas-tle laboratories were in the upstairs bedrooms of a lateVictorian terraced house, complete with boarded-up fire-places, ornamental plaster work ceilings and most of theelectrics nailed round the walls. Tissue culture medium
*Correspondence to: J. Wolstenholme, Cytogenetics Laboratory,Northern Genetics Service, Institute of Human Genetics, CentralParkway, Newcastle upon Tyne, NE1 3BZ, UK.E-mail: [email protected]
was an inconsistent entity, made up from a selectionof concentrated stock solutions (some home-made) andhuman or fetal calf serum, often of unreliable prove-nance. Tissue culture plastics were new and equallyvariable in quality, so the use of glass culture vessels wasstill widespread, including Carrel flasks first described in1912. Batch testing of everything was routine. The earlygassed and humidified incubators were as likely to growAspergillus as amniocycytes, which led to two campsof cytogeneticists: those who loved them and scrubbedthem meticulously clean on a regular basis and thosewho avoided them like the plague. G-banding was stilla new technique and somewhat unreliable; solid stain-ing, using unpleasant concoctions such as aceto-orcein,made up by boiling up glacial acetic acid and powderedstain in a fume cupboard, was considered to give supe-rior chromosome morphology (but unlike Giemsa stain,it could not be removed from fingers using bleach).Many happy hours were spent washing coverslips forin situ cultures, and washing, siliconizing, plugging andsterilizing glass pipettes.
Health and Safety considerations were somewhat sec-ondary. Latex gloves were for wimps. Recirculating ster-ile cabinets were new, expensive and not very effective.It was far more likely that you had a sterile cabinet thatblew straight at you or that you worked on the openbench. Flaming everything in a Bunsen burner, bacteri-ologist style, was essential, particularly if you worked onthe open bench. Leaving the Bunsen burner on in a ster-ile cabinet after you turned off the airflow was an excel-lent way to start a fire, especially if you left items such asmetal tweezers and coverslips sitting in pots of methanolinside them. These old sterile cabinets also welded yourcontact lenses to your eyes. Nobody worried about Hep-atitis B, or even knew about HIV, but nobody died.
Cytogenetics in the 1970s also involved a lot ofphotography, hours in the darkroom developing andprinting, and even longer cutting out and sticking downchromosomes on bits of card (Figure 1). The going ratewas three cut out, glued down and laminated karyotypesan hour, although four was achievable. Finding a number5 chromosome stuck to your sleeve when you got homewas an occupational hazard.
Copyright 2010 John Wiley & Sons, Ltd. Received: 14 December 2009Accepted: 9 January 2010
606 J. WOLSTENHOLME AND D. E. ROONEY
Figure 1—Case A1 from Newcastle, for ‘maternal age 42, screening for mongolism’. Sample taken 5th April 1973, reported 15th May 1973
The early 1980s saw the arrival of Prenatal Diagno-sis (the Journal) and CVS. In 1981, Bob Williamson’sgroup at St Mary’s Hospital, London showed that itwas possible to use DNA from chorionic villi for thediagnosis of hemoglobinopathies, kick-starting a con-certed effort to make CVS for early prenatal diagnosisof chromosome abnormalities a reality. The St Mary’sObstetric and Cytogenetic teams, eventually in collabo-ration with other London centres, explored various sam-pling and culturing techniques. During these early days,Guiseppe Simoni, a cytogeneticist from Milan, visitedthe St Mary’s group to observe first-hand Meena Niazi’smethod of separating the different trophoblastic layersfor culture and chromosome analysis of mesenchymecells. It was during this visit that the idea of usingspontaneous mitotic cells in the cytotrophoblast layer,first described in 1975 by Yamamoto, occurred to him(Simoni, personal communication to DER, 1983) andin 1983 he published his ‘direct’ method of obtain-ing metaphase spreads from chorionic villi aspiratedfrom first trimester pregnancies immediately prior totermination.
In 1984, the Milan group hosted the 1st InternationalSymposium on First Trimester Fetal Diagnosis in theItalian resort of Rapallo. By then, many laboratories allover the world were in the process of building up theirown expertise, and the ‘Rapallo Conference’ was thefirst opportunity for many of us (including the authors)to meet, exchange experiences, enthuse and drink Chi-anti with our international counterparts. The proverbial‘buzz’ was in the air, and passionate, though somewhatchaotic Italy was a fitting choice of venue.
From these early days, opinions on ‘directs’ versus‘cultures’ were already highly polarized. Although theadvantages of the direct method were numerous, some
groups, particularly in the UK, found the sole use ofthe comparatively inferior direct chromosome prepara-tions unacceptable, preferring to use them for a prelim-inary result to be followed by a more detailed analysisfrom cultured cells. The Italians, Dutch and some otherEuropeans used only the direct method and, for a time,debates regarding their respective merits could becomequite heated. But evidence quickly mounted that theresults obtained from the two approaches (representingcells of two different origins) could be discordant, notonly from each other but also from the fetus itself, lead-ing to interpretive dilemmas and false positive/negativeoutcomes in some cases. This phenomenon, termed ‘con-fined placental mosaicism’ (CPM), had in fact alreadybeen described by Dagmar Kalousek in 1983, based onher work with pregnancy losses and intrauterine growthrestriction, so perhaps we should not have been too sur-prised when CPM (and collection of data about it) tookover our lives.
One of the early collectors of CVS data was ‘Uncle’Laird Jackson of Philadelphia who, in 1983, startedsending out a newsletter to a small group of knownactive investigators, mostly in the United States, to col-lect and circulate data on all aspects of CVS in diag-nostic practice. In the UK, Kim Smith sent a shortquestionnaire on CVS and CPM to the British labs tocanvass their experience. These and other similar collec-tions of CPM data grew over the years into the massiveinternational databases that still facilitate the interpre-tation of ambiguous results and have led to a greaterunderstanding of cell distribution in the early embryo.
As young clinical cytogeneticists during those years,we were both fortunate to be directly involved in theintegration of amniocentesis and CVS into diagnosticpractice. With many opportunities for publication and
Copyright 2010 John Wiley & Sons, Ltd. Prenat Diagn 2010; 30: 605–607.DOI: 10.1002/pd
CYTOGENETICS IN THE 1970S AND 1980S 607
presentation, at home and overseas, we soon madecontacts in laboratories across the world. This meantplenty of visitors from and visits to other centres todemonstrate/watch each other’s techniques, leading tofruitful collaborative studies and enduring professionalrelationships, many of which last to this day.
ACKNOWLEDGEMENTS
JW worked in the laboratories in Dundee and Oxfordand is now based in Newcastle. He feels that he is
getting over his initial lack of experience in genetics, andthanks to all those who have helped with this. DER spenther formative years at Royal Manchester Childrens’Hospital, Pendlebury, and St Mary’s Hospital, London.She fondly remembers a summer afternoon in 1979when Harold Ward (Laboratory Manager at Pendlebury)sent all the Cytogenetics staff home to ‘enjoy thesunshine while you are still young’ staying himself tocomplete the day’s work. It was to be his last summer.
Copyright 2010 John Wiley & Sons, Ltd. Prenat Diagn 2010; 30: 605–607.DOI: 10.1002/pd
