Clinical Genetics 199 1 : 40: 190-1 93

Enhancement of amniotic fluid cell growth for genetic amniocentesis

THOMAS MATHEWS AND RAM S. VERMA Division of Genetics, The Long Island College Hospital, SUNY Health Science Center,

at Brooklyn, New York, USA

The last two decades have witnessed a logarithmic growth in demand for prenatal diagnosis of human disease through amniocentesis. Consequently, culture turn-around-time has become the major concern of all those who are primary care providers for patients secking help. We report a rapid method for obtaining cytogenctic resuits from amniocytes as early as 5 days. Early tapping and reducing the turn-around-time during cytogenetic analysis may provide an alternative to chorionic villi sampling.

Received 17 September 1990. revised and accepted for publication 18 February 1991

Key words: amniocentesis; amniotic fluid; chromosome; prenatal diagnosis.

Almost a quarter of a century ago, it was first demonstrated that amniotic fluid cells could be cultured to reveal the fetal karyotype (Steel & Breg 1966). Techniques for cell cul- ture have developed slowly and a number of prerequisites have been established to obtain successful growth (Van Dyke et al. 1980, Ro- driguez et al. 1986, Verma & Babu 1989). There are a number of factors responsible for cell growth, and cytogeneticists continue to face these problems. A major concern in any prenatal diagnosis laboratory is time. Conse- quently, physicians have tried to tap the am- niotic fluid around the 14th week of preg- nancy, rather than the usual 16 weeks. Alter- natively, fetal tissues have been obtained by CVS methods, at a gestation age of 9 to 12 weeks, reducing further delays (Elejalde 1990). However, this technology is only available in a limited number of centers. Thus, tissues obtained by amniocentesis re- main the universal approach for prenatal di- agnosis. Some cytogenetic laboratories, in-

cluding our own, claim that they can provide cytogenetic reports within 7 days. We have continued to improve the technique and state that with the present approach, results can now be obtained in about 5 days. We wish to report a detailed protocol for those labora- tories who wish to expedite their turn- around-time.

Material and Methods

The cultures were set up using standard pro- tocol (Verma & Babu 1989). Two systems were used as follows:

Culture Initiation and Maintenance 1. Transfer the amniotic fluid sample (10

cc) into two centrifuge tubes (A&B) and centrifuge at 800 rpm for 10 min.

2. Remove the supernatant and save in two new sterile tubes (one tube for analysis of alpha-fetal-protein and the other as a back-up).

A M N I O T I C F L U I D C E L L G R O W T H 191

3.

4.

5.

6.

7.

Resuspend the pellets (tubes A&B) in about 2.5 mI of Chang’s compIete me- dium (Chang 90 ml plus basal medium A 10 ml; L-glutamine 1 ml (200 mM); penicillin and streptomycin solution 1 ml (10 000 pglml and 10 000 ng/ml, respec- tively) or MEM (complete MEM 75 ml. Plus complete Chang 25 ml)). Place 0.5 ml of suspension gently onto each coverslip (CSlK Plasteka) in a Mat- Tek 35-mm petri dish (MatTekE Corp. Ashland, MA), preventing any overflow beyond the coverslip. Number the top and bottom halves of the petri dishes before initiating the culture. Incubate the dishes in a 5% C02 incu- bator at 37’C for 48 h. After 48 h, add an additional 2 ml of fresh medium to each petri dish and con- tinue incubation. Examine the cultures daily under an in-

verted microscope and, on the 4th day, growth medium in the culture should be renewed.

8. Harvest the cultures on the 5th day, if the growth is adequate.

Harvesting In Situ Cultures 1.

2. 3.

4.

5 .

6.

Add 2 drops of colcemid solution (10 pg/ml) to each 2 ml of culture medium using 22 1 /2 G needle. Incubate for 1 h and 15 min. Decant the medium as completely as possible by slightly tilting the dish to one side and pipetting from the bottom without disturbing the coverslips. Gently add 2 ml of hypotonic solution (prewarmed at 37’C) to each culture. Allow the culture to stand at room tem- perature for 20 min. Add 1 ml of freshly prepared 3:1 meth-

Fig. 1. Metaphase spread [46,XX] obtained from amniocytes harvested after 5 days using in situ technique.

192 M A T H E W S AND V E R M A

anol-acetic acid fixative directly to the hypotonic solution for 5 min.

7. Decant the solution and add 2 ml of fresh fixative for 20 min.

8. Repeat step 7 twice for an interval of 10-15 min.

9. Following the last change of fixative, prepare the coverslip as follows: Drying the PlasteP coverslip: Drying conditions, which include ambient tem- perature and relative humidity, are ex- tremely crucial in obtaining proper spreading of chromosomes for in siru harvests. Optimum conditions should be standardized according to available facilities. A relative humidity of 50 to 60% at an ambient temperature of 24°C

to 27°C has proven to be the most satis- factory. Further spreading of chromo- somes is achieved by blowing air gently over the coverslip.

10. The coverslips should be carefully re- moved from the petri dish with a blunt forceps (facilitated by gently pressing the middle of the bottom half of the petri dish with a finger).

1 1. The coverslips should then be mounted on standard glass microscope slides using MatTek's mounting medium (Cat no. MM 125). The mounted coverslips should then be kept at room tempera- ture for 15 min or until dry. The use of the correct mounting medium and adequate drying time are critical to suc-

Table 1

Culturing duration for amniotic fluids using two types of coverclass

Coverslips Coverslips Gestat. Gestat.

Culture age Glass Plaatek Culture age Glass Plastek

AFW-1 17.5 13 6 AF90-26 21.0 12 5 AFW-2 19.0 9 5 AF90-27 21 .o 10 5 AF90-3 16.0 11 6 AF90-28 16.2 9 5 AFSW 21.0 10 5 AF90-29 17.0 10 6 AF90-5 17.5 11 5 AFW-30 19.5 12 6 AF90-6 19.0 12 5 AF9031 18.0 13 5 AFW-7 23.0 9 5 AF90-32 18.0 8 5 AF90-8 16.0 10 6 AFSC-33 16.0 13 5 AF90-9 16.5 10 5 A F W 17.0 11 5 AF90-10 16.5 9 5 AF90-35 17.0 12 5 AFW-11 21 .o 9 5 AFW-36 17.5 12 6 AF90-12 17.0 10 6 AF90-37 20.0 12 5 AF90-13 17.5 9 5 AFSC-38 17.5 9 5 AFW14 15.5 9 5 AFW-39 16.0 13 6 AFSO-15 16.0 8 5 A F W O 17.0 12 5 AFW-16 22.5 12 6 A F W 1 19.0 6 6 AF90-17 16.0 13 6 AF90-42 17.5 9 5 AF90-16 16.0 10 5 AF90-43 18.5 8 5 AFSO-19 18.5 13 5 A F W 19.0 13 5 AFgO-20 21.5 9 6 A F M 18.5 9 5 AFW-21 18.0 12 7 AF90-46 21.5 9 5 AF90-22 17.0 11 5 AF90-47 17.0 11 6 AF90-23 22.5 13 6 AF90-46 17.0 13 5 AF90-24 17.0 13 6 AF90-49 19.5 12 5 AF90-25 17.0 8 5 AFW-50 18.0 8 5

~~~ -~

Average culturing time of 50 cases

'Gestational age was in weeks while culturing time waa in days.

10.62 f 1.75 5.34 f 0.52

A M N I O T I C F L U I D C E L L G R O W T H 193

cessful focusing at high magnification. Once the coverslips have dried, they should be heated at 60°C for 2 h.

12. Slides can then be processed for band- ing chromosomes. Cells can be photo- graphed using a lOOx plan-Apochromat oil immersion lens (Fig. 1).

Discusslon

Recent advances in technology have helped to enhance the culture growth of fetal cells primarily through the use of hormone sup- plemented medium (Chang et al. 1982). However, the most important factor is the initiation of cell attachment to the coverslip using in situ technique. In the present inves- tigation, we used PlastekB coverslip CSl (MatTeka Corporation, Ashland MA) which is both oxygen rich and has a nega- tively charged surface that enhanced the cell attachment and growth. Fifty amniotic fluids were cultured; half the amniotic fluid using the standard glass coverslips and the other half using Pfustek coverslips. Faster cell growth was observed with the plastek coverslip enabling earlier harvesting of 5-6 days (5.34f0.52) as compared to ordinary glass coverslips, which takes 8-13 days (1 0.62 f 1.75) (Table 1). Increased colony formation was also noted, allowing a culture to be harvested as early as within 5 days. With increased fetal risk and the decreased time period during which a retap is possible, minimizing any culturing time is pertinent for providing a rapid diagnosis. This is a major concern of all those who are primary care providers to patients seeking such help (Crane et al. 1988). Elejalde and hs col- leagues (1990) have clearly demonstrated that tapping can be performed as early as the 9th week of gestation. Therefore early tapping and reducing the turn-around-time during cytogenetic analysis may provide an

alternative to chorionic villi sampling pro- cedure.

Acknowledgements

The financial support from MatTek@ Cor- poration, Ashland, MA is gratefully ac- knowledged. Presented at the 41st Annual Meeting of the American Society of Human Genetics, Cincinnati, Ohio, Oct. 1990.

References

Chang, H. C., 0. W. Jones & H. Masui (1982). Human amniotic fluid cell grown in a hormone supplemented medium: suitability for prenatal diagnosis. Proc. Natl. Acad. Sci. [USA). 79, 47954199.

Crane, J. P., H. A. Beaver & S. W. Cheung (1988) First trimester chorionic villus sampling versus mid trimester genetic amniocentesis. Prelimi- nary results of a controlled propective trial. Prenat. Diag. 8, 355-366.

Elejalde, B. R., M. M. deElejalde, J. M. Acuna, D. Thelen C. Trujillo & M. Karrmann (1990). Prospective study of amniocentesis performed between weeks 9 and 16 of gestation. Its feasi- bility, risk, complications and use in early gen- etic prenatal diagnosis. Am. J. Med. Genet. 35, 188-196.

Rodriguez, J. G., A. Babu & R. S. Verma (1986). A rapid method of culturing amniotic fluid for chromosome analysis. Am. J. Obstet. Gynecol. 154,969-970.

Steel, M. W. & W. R. Breg (1966). Chromosome analysis of human amniotic fluid cells. Lancer I, 383-385.

Van Dyke, D. L., L. Weiss, J. Clark, M. Worsh- am & K. Beisel (1980). Culture and karyotyp- ing of amniotic fluid cells. J. Histotechnol. 3,

Verma, R. S. & A. Babu (1989). Humun Chromo- some: Manual of Basic Technique. New York, Pergamon Press, pp. 13-19.

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Address: Dr. Ram S. Verma Division of Genetics Long Island College Hospital 340 Henry Street Brooklyn, N . L I1201