May 1, 2009 / Vol. 34, No. 9 / OPTICS LETTERS 1483

Light-scattering spectroscopy differentiates fetalfrom adult nucleated red blood cells: may

lead to noninvasive prenatal diagnosis

Kee-Hak Lim,1 Saira Salahuddin,1 Le Qiu,1 Hui Fang,1 Edward Vitkin,1 Ionita C. Ghiran,1

Mark D. Modell,1 Tamara Takoudes,1 Irving Itzkan,1 Eugene B. Hanlon,1,2

Benjamin P. Sachs,1 and Lev T. Perelman1,*1Biomedical Imaging and Spectroscopy Laboratory, Department of ObGyn and Reproductive Biology, Beth Israel

Deaconess Medical Center, Harvard University, Boston, Massachusetts 02215, USA2Department of Veterans Affairs, Medical Research Service and Geriatric Research Education and Clinical Center,

Bedford, Massachusetts 01730, USA*Corresponding author: ltperel@bidmc.harvard.edu

Received February 23, 2009; revised April 7, 2009; accepted April 10, 2009;posted April 13, 2009 (Doc. ID 107845); published April 30, 2009

Present techniques for prenatal diagnosis are invasive and present significant risks of fetal loss. Noninva-sive prenatal diagnosis utilizing fetal nucleated red blood cells (fNRBC) circulating in maternal peripheralblood has received attention, since it poses no risk to the fetus. However, because of the failure to findbroadly applicable identifiers that can differentiate fetal from adult NRBC, reliable detection of viablefNRBC in amounts sufficient for clinical use remains a challenge. In this Letter we show that fNRBC light-scattering spectroscopic signatures may lead to a clinically useful method of minimally invasive prenatalgenetic testing. © 2009 Optical Society of America

OCIS codes: 170.6510, 170.1530, 280.1350.

The incidence of birth defects in the United Statesdue to chromosomal abnormalities is estimated to be34,000 per year[1]. Of these, approximately 0.65%cause significant morbidity, and another 0.2% even-tually will affect reproduction [1]. Fetuses with aneu-ploidy, an abnormal number of chromosomes, accountfor 6% to 11% of all stillbirths and neonatal deaths[2]. The most common form of aneuploidy at birth(1:800–1:1000 live births) is Down syndrome or tri-somy 21 (three copies of chromosome 21). Aside fromDown syndrome, other autosomal trisomies, sex chro-mosome abnormalities, and structural balanced andunbalanced abnormalities can be associated withhigh neonatal mortality and both short- and long-term morbidity. The risk of having a child with aneu-ploidy increases with increasing maternal age. Pre-natal screening or diagnostic testing for chromosomalabnormality in the fetus or embryo carried out earlyin pregnancy, combined with appropriate geneticcounseling, can help parents make informed deci-sions about the pregnancy and neonatal care. Cur-rently, prenatal diagnosis of aneuploidy relies on fe-tal cells obtained by amniocentesis, chorionic villussampling, or percutaneous umbilical blood sampling.It is assumed approximately 9% of all deliveries ev-ery year in the United States involve midtrimesteramniocentesis [3]. These are invasive procedures pre-senting significant risk to the fetus. Alternatively,noninvasive methods to obtain fetal cells or free fetalDNA circulating in maternal peripheral blood duringpregnancy have received much attention [4,5].

Fetal nucleated red blood cells (fNRBC) have gen-erated the greatest interest for prenatal diagnosis[6–9]. They present a full complement of genes, pos-sess morphologic characteristics that distinguishthem from other nucleated cells [10], and with a lim-

ited life span of about 25 to 35 days, it is unlikely

0146-9592/09/091483-3/$15.00 ©

that fNRBC could persist from one pregnancy to thenext [11]. While NRBC are extremely rare in normaladult circulation, fNRBC do make up a small fraction(perhaps 1/109) of circulating cells during pregnancy.Some success in isolating fNRBC from the maternalcirculation has been realized (Bianchi et al. [12] iso-lated approximately one cell per milliliter of mater-nal blood when the karyotype is normal), but currentmethods to identify, isolate, and analyze fNRBC gen-erally have been found to be clinically unsuitable forprenatal diagnosis [5].

Established methods can be used to separateNRBC from other cells in maternal circulation. How-ever, owing to very low concentration in the maternalblood (1 per 109 cells) and interference by maternal,adult nucleated red blood cells (aNRBC) [13] presentat a similar concentration (1 fNRBC to 1–100aNRBC), reliable detection of fNRBC [14] and isola-tion from aNRBC for clinical use remains a chal-lenge. For example, chromatographic methods riskcell damage and loss of genetic material, and currentcell sorting methods require tagging, which inter-feres with and/or limits the applicability of availablediagnostic analyses. In this Letter we use light-scattering spectroscopy (LSS) to address the un-solved problem of differentiating fNRBC fromaNRBC.

Recently we developed biomedical LSS and showedits unique ability to optically probe nuclear morphol-ogy without damaging cells [15]. Since fNRBC differfrom aNRBC in nuclear morphology [10], this ap-proach may lead to a robust optical technique to en-able prenatal genetic testing based on enrichmentand recovery of intact fNRBC from maternal periph-eral blood.

We performed experiments with fNRBC from fetal

umbilical cord blood and aNRBC from adult female

2009 Optical Society of America

1484 OPTICS LETTERS / Vol. 34, No. 9 / May 1, 2009

bone marrow. The protocol was reviewed by the Insti-tutional Review Board of Beth Israel DeaconessMedical Center, and the requisite approvals were ob-tained. Standard immunofluorescence techniqueswere used to distinguish NRBC in each of the follow-ing specimens.

To identify fNRBC in umbilical cord blood, we usedtwo different monoclonal antibody techniques, as noavailable technique is 100% specific. First, we usedmouse monoclonal antibody (MAB 3432, Chemicon,Temecula, Calif.) directed against glycophorin A, anerythrocyte-specific cell surface antigen. As the sec-ondary antibody we used a red fluorescence marker,phycoerythrin (PE) conjugated goat antimouse IgG(Jackson ImmunoResearch Laboratories, Inc., WestGrove, Pa.). These cells were then double stained byincubating with green fluorescence DNA specific dyeSyto 16 (Molecular Probes, Inc. Carlsbad, Calif.),which stains the nucleus. Using this technique, thenucleated red blood cells were double stained withboth green and red fluorescers [Fig 1(a)].

Second, we used a mouse monoclonal antibody di-rected against human erythroid cell surface antigenHAE9 (MAB 2115, Chemicon, Temecula, Calif.),which recognized only NRBC. Green fluorescentisothiocyanate (FITC) conjugated antimouse IgG(Jackson ImmunoResearch Laboratories, Inc., WestGrove, Pa.) was used as the secondary antibody [Fig.1(b)].

To obtain aNRBC from bone marrow specimensfrom adult females, we used monoclonal antibodyHAE9 as the primary antibody with Alexa Fluor 488(Molecular Probes, Inc., Carlsbad, Calif.) conjugatedgoat antimouse IgM as the secondary antibody.

Cells successfully identified by the above tech-niques were studied with our recently developed Con-focal Light Absorption and Scattering Spectroscopic(CLASS) microscopy system [16], which combinesconfocal microscopy with LSS, to obtain the light-scattering spectra of the immunostained cells.

The CLASS image of aNRBC and spectra of theirvarious subcellular compartments are presented inFig. 2. Each compartment of the aNRBC has an in-formative spectrum. For example, Fig. 2(a) exhibitsan oscillatory structure of approximately 30 nm pe-riod, characteristic of light scattering from a largeparticle such as the nucleus. The spectrum in Fig.2(b), which is taken close to the nucleus, in additionto the high-frequency oscillations, also exhibits a low-

Fig. 1. (Color online) Individual cells in an umbilical cordblood sample. (a) MAB-PE-Syto 16 stained cells, (b) MAB-

FITC stained cells.

frequency oscillation characteristic of smaller scat-terers, such as submicron organelles (about 300 nmperiod). In Fig. 2(c), the scattering signatures are al-most gone, but the hemoglobin absorption bands at540 nm and 580 nm, present in all three compart-ments, are clear.

Typical CLASS spectra of fetal and adult NRBCare presented in Fig. 3. For a given cell, spectra wereacquired by scanning the entire cell point by pointand summing the spectra together. These summedspectra were averaged for three to four cells of eachtype, and the mean spectra were plotted in Fig. 3. Er-ror bars show the standard deviation. Light scatter-ing spectra clearly differentiate fetal from adultNRBC in the spectral region from 600 to 780 nm,where the standard deviations do not overlap. The pvalue for a Student’s t test using the intensities at780 nm is p=0.0004, which is statistically significant.This is the same as using the slopes of the spectra.

Spectra of NRBC of both fetal and adult bone mar-row origin are dominated by oscillatory behavior

Fig. 2. (Color online) CLASS image and spectra of the in-dividual subcellular compartments of adult bone marrowNRBC: (a) nucleus, (b) smaller organelle near nucleus(dashed curve, oscillating with low frequency, is provided toguide the eye), (c) compartment containing onlyhemoglobin.

Fig. 3. (Color online) Fetal and adult NRBC have dis-tinctly different spectra. The upper curve is the mean LSSspectrum of fNRBC, and the lower curve is the mean LSSspectrum of aNRBC. Bars are the standard deviation of

measurements taken on multiple cells.

May 1, 2009 / Vol. 34, No. 9 / OPTICS LETTERS 1485

characteristic of light scattering by the nucleus [16].Overlap of the light-scattering spectra of the cellnucleus and other cell compartments also contributesto the oscillatory features seen in Fig. 3. While the re-ported spectra were acquired with CLASS, it is im-portant to note that the summed, whole-cell spectradifferentiate fNRBC from aNRBC, so confocal, intra-cellular spatial resolution, which might slow poten-tial screening and harvesting protocols, is not re-quired.

We note that the presence of the immunofluores-cence markers should not interfere significantly withthe light-scattering spectra of the NRBC. First, thefluorescence emission produced under the conditionsof the light-scattering measurement is very weakrelative to the elastically scattered light. Second,the fluorescence band structure is narrow �25 nm to50 nm� compared with the scattering bandwidth��400 nm� and broad compared with the higher fre-quency oscillations ��10 nm� of the light-scatteringspectra. Finally, the dimensions of the markers areon the order of 10 nm, well below the light-scatteringwavelengths (500 nm to 900 nm). Therefore, alter-ation of the inherent light-scattering properties ofthe labeled cells compared with native cells is negli-gible and does not affect the conclusion drawn here.Of course, a working clinical system will not requirefluorescence markers.

The light-scattering spectra of fNRBC and aNRBCare clearly distinguishable, and the standard devia-tions do not overlap at wavelengths longer than600 nm. The distinctions between the spectra ofthese two cell types arise from differences in mor-phology and biochemistry. While morphological dif-ferences are not readily apparent in visual imagesobtained with conventional microscopy techniques,light-scattering spectra respond sensitively to smalldifferences in nuclear size, nuclear volume to cell vol-ume ratio, and organelle size and number densitydistributions [15,16], resulting in the reported differ-ences in the light-scattering spectra of fNRBC andaNRBC. Identification of fNRBC in maternal circula-tion using LSS has the potential to yield fNRBC withminimal damage, thereby providing useful fetal ge-netic material for prenatal diagnosis.

The results we report here indicate that CLASS iscapable of reliably distinguishing fetal NRBC fromadult NRBC without tagging, fixation, or risk of celldamage. In addition, because sample preparation isminimal for CLASS or LSS measurements, multi-stage preparation procedures that result in cumula-tive cell loss are eliminated. There may be a wealth ofinformative cell biology and chemistry revealed byanalysis of the detailed structure of fNRBC light-scattering spectra, some of which may itself be ofvalue for prenatal diagnosis. Nonetheless, the quali-tative differences in the overall spectra of fNRBC and

aNRBC presented above indicate LSS may provide auseful technique for developing a clinical method ca-pable of recovering sufficient fNRBC from peripheralmaternal blood for minimally invasive prenatal ge-netic testing.

This study was supported by the National Insti-tutes of Health (NIH) grant RR017361 and the Na-tional Science Foundation (NSF) grant BES0116833and in part by the Office of Research and Develop-ment, Department of Veterans Affairs. Adult marrowcells were generously provided by G. Pihan and P.Bhargava, Department of Pathology, Beth IsraelDeaconess Medical Center.References

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