Sequential G-Banding and Fluorescent In SituHybridization on Peripheral Blood, Bone Marrow,and Amniotic Fluid Samples

Anne Wiktor, Regina Puskorius, Kristina Zubrickas, and Daniel L. Van Dyke*Department of Medical Genetics, Henry Ford Health System, Detroit, Michigan

We describe a method for fluorescent in situhybridization (FISH) on previously G-banded slides and mounted coverslip prepa-rations. The technique is successful on pre-viously G-banded slides from peripheralblood and bone marrow specimens and onamniotic fluid samples from in situ har-vests. Satellite sequence probes, wholechromosome paints, and unique sequenceprobes have all produced reliable results.Am. J. Med. Genet. 79:38–41, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: FISH; fluorescent in situ hy-bridization; sequential band-ing; G-banding

INTRODUCTION

Fluorescent in situ hybridization (FISH) is widelyused as an adjunct to conventional cytogenetic analy-sis. Depending on the types of probes used, FISH canprovide useful information in the study of mosaicism,the identification of microdeletions, and the evaluationof complex rearrangements. While the use of FISHtechniques on freshly prepared slides is common prac-tice in most cytogenetics laboratories, previously G-banded preparations may be the only source of mate-rial for retrospective analysis or when sample volumeis small. We have developed a technique for sequentialFISH using previously G-banded peripheral blood andbone marrow preparations and on in situ harvestedG-banded amniotic fluid samples.

MATERIALS AND METHODSCulture and Staining Procedures

Chromosome preparations from blood and bone mar-row specimens were prepared for G-banding following

standard cytogenetic procedures. Slides were air driedand aged at 90°C for 1.25 hr before G-banding. Amnio-cytes were cultured in situ and harvested using a ro-botic harvester (Tecan U.S., Chapel Hill, NC). Afterharvest, coverslips were flame dried and aged at 90°Cfor 60 min. Each slide or coverslip was treated withtrypsin and stained with Leishman stain. Coverslipswere mounted (cell side down) on microscope slides us-ing Flo-Tex (Lerner Laboratories) mounting medium.Blood and marrow specimen slides were not mounted.

Sequential Staining

Following analysis of G-banded microscope slides,the residue oil was removed by soaking the slide inxylene and allowed to air dry. The slide was destainedin methanol for 10 min, rinsed in distilled water, andair dried. Coverslipped slides were wiped clean to re-move excess oil and demounted by soaking in freshxylene and allowed to air dry (the length of time neededto soak off coverslips varies with the age of the slide).Coverslips were then destained in methanol for 10 min,pretreated in glacial acetic acid for 8 min, rinsed indistilled water, and air dried.

Fluoroescent In Situ Hybridization

The FISH procedure used is a modification of themanufacturer’s protocol (Oncor, Inc., Gaithersburg,MD) and all probes used were from Oncor, Inc. Slidesand coverslips were incubated in 2 × SSC for 30 min at37°C and dehydrated in 70%, 95%, and 100% ethanolfor 2 min each at room temperature. (The initial incu-bation in 2 × SSC is optional because they are agedprior to banding. No difference had been noted if thisstep was omitted.) Before hybridization, probes wereprepared as follows: satellite sequence probes, 1.5 ml ofprobe and 20 ml Hybrisol VI (Oncor, Inc.) denatured for5 min at 72°C; unique sequence probes, 20 ml pre-warmed for 10 min at 37°C; paints, 20 ml denatured for10 min at 72°C and preannealed 30 min at 37°C. (Probevolumes are for hybridization of a standard microscopeslide but can be decreased by half for a 22 mm2 cover-slip area.) Both biotin- and digoxigenin-labeled probeswere used. Slides or coverslips were denatured in 70%formamide/2 × SSC for 2 min (3–4 min for marrows) at72°C, dehydrated in 70%, 95%, and 100% ethanol for 2

*Correpondence to: Daniel L. Van Dyke, Ph.D., Henry FordHospital, Cytogenetics Laboratory, 2799 W. Grand Blvd., CFP461, Detroit, MI 48202.

Received 2 February 1998; Accepted 10 April 1998

American Journal of Medical Genetics 79:38–41 (1998)

© 1998 Wiley-Liss, Inc.

min each at room temperature, and air dried. Theprobe mixture was applied to the microscope slides,and sealed under a coverslip with rubber cement. Forhybridization of coverslips, the probe mixture was ap-plied to a clean microscope slide, and the denaturedcoverslip was placed cell side down on the slide andsealed with rubber cement. Hybridization was over-night in a moist chamber at 37°C.

Following hybridization, the rubber cement was re-moved and slides or coverslips were immersed in pre-warmed solutions for 5 min at 70°C. Satellite probeswere washed in 0.4 × SSC; unique sequence probes in 2× SSC, and paints in 1 × SSC. Preparations were thenrinsed in BN Buffer (0.1M NaBicarbonate/0.05% Noni-det P-40)[Pinkel et al., 1986] for 2 min at room tem-perature. The hybridization signal was detected usingeither FITC-Digoxigenin/Texas red-Biotin for dualprobe detection or Rhodamine-Digoxigenin for singleprobe detection. Detection was performed using themanufacturer’s protocol except for using BN buffer (in-

stead of 1 × PBD (Oncor, Inc.)) as the wash solution.Excess wash solution was allowed to drain and slideswere counterstained with 10 ml of DAPI and cover-slipped. For coverslips, DAPI was applied to a cleanmicroscope slide, the coverslip was placed cell sidedown on the slide, and the edges sealed with clear nailpolish.

Metaphase and interphase cells were analyzed usinga Zeiss Axioskop epifluorescence microscope with a50W fluorescent bulb. Cells were imaged using a Cyto-vision Imaging system (Applied Imaging, Pittsburgh,PA) equipped with filters specific for each fluorochromeand dual and triple band pass filters.

RESULTS

We have used this technique in eight cases and findthe procedure simple to perform and the results accu-rate and reproducible.

A peripheral blood specimen was analyzed sequen-

Fig. 1. A: G-banded metaphase from a bone marrow specimen indicating normal chromosome 8 homolog (large arrows) and normal chromosome 12homologs (small arrows). B: Metaphase following FISH with 8cen (red signal) and 12cen (green signal) probes.

Fig. 2. A: G-banded metaphase from a bone marrow specimen with arrows indicating chromosomes 9, 12, 22, and Ph1. B: Using a chromosome 22paint, a complex t(9;12;22) with ins(12;22) identified.

Sequential G-Banding and FISH Analysis 39

tially using the Y heterochromatic region probe (DYZ1)to confirm a Yqh+ polymorphism and rule out an un-balanced translocation.

Two blood samples were tested for deletion of theN25 region associated with velocardiofacial syndromeusing the D22S75 probe. One of the samples analyzedhad been G-banded 15 months earlier.

Sequential analysis of bone marrow samples using acombination of chromosome 8 and chromosome 12 cen-tromere probes was used to validate probe specificity(Fig. 1).

A complex t(9;12;22) translocation was confirmed us-ing a chromosome 22 painting probe. By FISH analy-sis, it was observed that the complex rearrangementinvolved an insertion of a chromosome 22 segment intochromosome 11 (Fig. 2).

To distinguish an inv(16)(p13q22) from adel(16)(q22) in a bone marrow specimen, two separatehybridization experiments were performed. A 16q telo-mere probe confirmed the presence of the 16q telomeresequence on the distal end of the q-arm of both chro-mosome 16s. A second hybridization was performed onthe same slide using the inv(16) probe. Signals werepresent on the p-arm of both 16 homologs and the ex-pected cross-hybridization on 18p. This analysis con-firmed deletion of chromosome 16 rather than an in-version (Fig. 3).

Two amniotic fluid samples were hybridized follow-ing G-banded analysis. A specimen that was two weeksold was tested using the Williams syndrome probe andresults were normal. Another specimen was hybridizedwith the 21q22 region specific probe to confirm i(21q)mosaicism that had been identified in 2 of 37 coloniesby G-band analysis. Interphase cells demonstratedthree signals, confirming the presence of three copies ofthe 21q22.2 region in a colony with only one metaphase(Fig. 4). Normal interphase cells (two signals) were ob-served in other colonies (both with and without meta-phases) on both coverslips with the i(21q) colonies.

DISCUSSIONThe benefits of G-banding and FISH analysis are op-

timized when analysis can be performed sequentially

on both slides and in situ harvested preparations. Incases of complex chromosome karyotypes, G-bandingalone does not always permit complete characteriza-tion of rearrangements and FISH analysis may provideinformation to better characterize abnormalities. Thisis especially true in leukemia studies where sample

Fig. 3. A: G-banded metaphase from a bone marrow specimen with del(16). Arrows indicate chromosome 16 homologs. B: FISH with 16q telomereprobe. C: Rehybridized metaphase with inv(16) probe demonstrating normal hybridization pattern on chromosome 16 homologs and the usual cross-hybridization on chromosome 18s.

Fig. 4. A: Partial G-banded metaphase from in situ harvested amnio-cytes. Arrows indicate normal 21 homolog and i(21q). B: FISH using a21q22.2 probe confirms i(21q) in both metaphase and interphase cells.

40 Wiktor et al.

size may be limited or where mosaicism is questionedand analysis of previously G-banded cells would provethe most informative.

Other techniques combining G-banding and FISHanalysis have been described [Babu and Wiktor, 1991;Speleman et al., 1991; Jalal et al., 1993; Epstein et al.,1995; El-Rifai et al., 1996; Gerdes et al., 1997]. Theseprotocols required formaldehyde-acetone pretreat-ment, increased probe concentration, a double denatur-ation step when rehybridizing slides with a secondprobe, or described the use of only one type of probe.The present technique is a modification and a muchless time-consuming method than was first describedby our laboratory.

The method described is reliable for hybridizationand rehybridization of G-banded slides and mountedchromosome preparations. The procedure has provensuccessful using satellite sequence, unique sequenceand chromosome painting probes and works well onperipheral blood, bone marrow, and amniotic fluidsamples.

REFERENCES

Babu VR, Wiktor A (1991): A fluorescence in situ hybridization techniquefor retrospective cytogenetic analysis. Cytogenet Cell Genet 57:16–17.

El-Rifai W, Knuutila S (1996): Fluorescent in situ hybridization on archivalG-banded slides. Cytogenet Cell Genet 73:322–324.

Epstein L, DeVries S, Waldman FM (1995): Reutilization of previouslyhybridized slides for fluorescence in situ hybridization. Cytometry 21:378–381.

Gerdes A, Pandis N, Bomme L, Dietrich CU, Teixeira MR, Bardi G, HeimS (1997): Fluorescence in situ hybridization of old G-banded andmounted chromosome preparations. Cancer Genet Cytogenet 98:9–15.

Jalal SM, Law ME, Christensen ER, Spurbeck JL, Dewald GW (1993):Method for sequential staining of GTL-banded metaphases with fluo-rescent-labeled chromosome-specific paint probes. Am J Med Genet46:98–103.

Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quanti-tative, high-sensitivity, fluorescence hybridization. Proc Natl Acad SciUSA 83:2934–2938.

Speleman F, Leroy JG, Van Roy N, de Paepe A, Suijkerbuijkr, Brunner H,Looijenga L, Verschraegen-Spae M-R, Orye E (1991): Pallister-Killiansyndrome: Characterization of the isochromosome 12p by fluorescent insitu hybridization. Am J Med Genet 41:381–387.

Sequential G-Banding and FISH Analysis 41