Fetal Laboratory Medicine: On the Frontier of - Clinical Chemistry

Fetal Laboratory Medicine:On the Frontier of Maternal–Fetal Medicine

Sharon M. Geaghan1*

BACKGROUND: Emerging antenatal interventions andcare delivery to the fetus require diagnostic support,including laboratory technologies, appropriate meth-odologies, establishment of special algorithms, and in-terpretative guidelines for clinical decision-making.

CONTENT: Fetal diagnostic and therapeutic interven-tions vary in invasiveness and are associated with aspectrum of risks and benefits. Fetal laboratory assess-ments are well served by miniaturized diagnostic meth-ods for blood analysis. Expedited turnaround times aremandatory to support invasive interventions such ascordocentesis and intrauterine transfusions. Health-associated reference intervals are required for fetal testinterpretation. Fetal blood sampling by cordocentesiscarries substantial risk and is therefore performed onlywhen fetal health is impaired, or at risk. When the sus-pected pathology is not confirmed, however, norma-tive fetal data can be collected. Strategies for assuranceof sample integrity from cordocenteses and confirma-tion of fetal origin are described. After birth, definitiveassessment of prenatal environmental and/or drug ex-posures to the fetus can be retrospectively assessed byanalysis of meconium, hair, and other alternative ma-trices. A rapidly advancing technology for fetal assess-ment is the use of fetal laboratory diagnostic tech-niques that use cell-free fetal DNA collected frommaternal plasma, and genetic analysis based on molec-ular counting techniques.

SUMMARY: Developmental changes in fetal biochemicaland hematologic parameters in health and disease arecontinually delineated by analysis of our collectiveoutcome-based experience. Noninvasive technologiesfor fetal evaluation are realizing the promise of lowerrisk yet robust diagnostics; examples include samplingand analysis of free fetal DNA from maternal blood,and analysis of fetal products accessible at maternalsites. Application of diagnostic technologies for non-

medical purposes (e.g., sex selection) underscores theimportance of ethical guidelines for new technologyimplementation.© 2011 American Association for Clinical Chemistry

Fetal laboratory medicine offers a pioneer opportunityfor diagnostic testing, forging the frontier of maternal–fetal medicine. Clinical trends in antenatal technolo-gies demand laboratory support and expertise,beginning with diagnostic sample procurement, estab-lishment of healthy fetal reference values, strategies forconfirmation of fetal specimen identity, fetal health as-sessment in the context of biochemical and hemato-logic development, alternative matrices for analysis,and maternal sampling for identification of fetal secre-tory products, and extending to guidance for futuredirections in noninvasive prenatal molecular diagnos-tics for fetal health assessment. The objective of thisreview is to evaluate the current scope and contribu-tions of fetal laboratory testing. A systematic literaturereview was performed. Extensive electronic searcheswere carried out in the PubMed database for fetal lab-oratory testing. Articles were selected on the basis ofstudy characteristics, quality, and results. Referencelists of articles obtained were searched for any furtherarticles. There were no language restrictions.

Fetal Therapeutic Interventions

The spectrum of fetal therapeutic interventions rangesin degree of invasiveness from open fetal surgeries per-formed in utero to minimally invasive fetoscopic sur-gery, which allows for small incisions under sono-graphic guidance (Table 1). The field of fetal surgerywas first developed on animal models approximately 3decades ago at the University of California, San Fran-cisco, by Dr. Michael Harrison. The first human openfetal surgery was completed by Dr. Harrison on a fetuswith congenital hydronephrosis due to congenital uri-nary tract blockage. Obstruction to urinary flow cancause permanent renal damage and other sequelae,such as poor lung development, if untreated. A vesicos-tomy (placement of a catheter into the bladder) wasperformed, allowing urine to pass until definitive revi-sion of the obstruction postnatally (1 ).

1 Department of Pathology, Stanford University School of Medicine, Palo Alto,California.

* Address correspondence to this author at: Stanford Hospital and Clinics ClinicalLaboratory, 300 Pasteur Dr., Palo Alto, CA 94304. Fax 650-736-1734-; [email protected].

Received May 4, 2011; accepted November 21, 2011.Previously published online at DOI: 10.1373/clinchem.2011.166991

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Open fetal surgery begins with a hysterotomy, oruterine incision, performed under general anesthesia.Maternal safety is prioritized above surgical success andpreterm birth avoidance. Fetal size and fragility prohibitssurgery before 18 weeks gestation. The fetus is exposedand the surgery performed (Fig. 1). The fetus remainsdependent on placental support and is returned to theuterus following surgery. Before uterine closure, amnioticfluid is replaced, and the abdominal wall closed. Antenatalsurgical interventions may be an interim procedure, toallow further development and maturation in utero untila definitive postnatal surgery can be performed.

Following open fetal surgery, preterm birth iscommon and cesarean section is mandatory. Thesesurgeries are high risk, and are still considered experi-mental(2 ). For prenatal diagnosis and management ofbilateral hydronephrosis (the indication for the firstfetal surgery), for example, very few fetuses will un-dergo prenatal surgery. Laboratory diagnostic testswere employed for case selection, including fetal mea-surements: (a) urine Na �100 mmol/L; (b) urine Cl�90 mmol/L; (c) urine osmolarity �210 mosmol; (d)renal sonographic evaluation; (e) amniotic fluid status;and (f) urine output at fetal bladder catheterization.More recently, other metrics have been shown to beperhaps more predictive of poor renal function (seelater discussion). Other indications for fetal interven-tions include: thoracic space-occupying lesions; amni-otic bands; chorangioma; cardiac malformations;

congenital diaphragmatic hernia; sacrococcygeal tera-toma; and neural tube defect closure (3 ).

The field has expanded to include a range of ante-natal surgical techniques: Fetendo, or minimally inva-sive fetoscopic surgery, is performed in utero underreal-time ultrasonographic guidance, through minuteuterine incisions or even without incisions, by usingintrauterine ultrasonographic and endoscopic views.Clinical indications include: fetoscopic closure of spinabifida; atrial septostomy; aortic or pulmonary valvulo-plasty; treatment of fetal bladder obstructions; and bal-loon treatments of tracheal occlusions associated withcongenital diaphragmatic hernia (3 ).

Most compelling was a recent randomized trial ofprenatal vs postnatal repair of myelomeningocele, inwhich the trial was stopped for substantially greaterefficacy of prenatal surgery based on results at 12months of age. Shunt placement rates were 40% in theprenatal surgery group, compared with 82% in thepostnatal surgery group (P � 0.001). Significant im-provement for composite mental development andmotor function scores at 30 months and secondaryoutcomes of ambulation by 30 months and hindbrainherniation by 12 months were manifest (4 ). Maternaland fetal risks were increased (uterine dehiscence atdelivery and preterm delivery rate).

This randomized trial represents the most defini-tive study to date demonstrating the value of prenatalsurgery for a clinical indication. The results of this trial

Table 1. Fetal therapeutic interventions (in order of increasing invasiveness) and clinical indications.a.b

Antenatal intervention Clinical indication(s)

Delivery of therapeutic drugs, antibiotics, or steroidsto fetus by maternal administration

Thyroid hormone in cases of maternal thyroid dysfunction; antibiotics forintrauterine infection; and steroid administration for accelerated lungmaturation and to reduce the incidence of respiratory distress andneonatal mortality in the setting of premature births, or for confirmedcongenital adrenal hyperplasia

Intrauterine transfusion Fetal anemia (e.g., Rh isoimmunization)

Surgery on fetal membranes Amniotic band syndrome (release)

Placental surgery For placental chorangioma, to prevent cardiac failure and hydrops

Laser ablation or photocoagulation of placentalvasculature

IUGR or twin–twin transfusion syndrome due to placental vascularanastamoses between monochorionic twins

Urinary obstruction decompression byvesicoamniotic catheter placement/shunting

Congenital hydronephrosis (urinary obstruction), with adequate renalfunction and pulmonary immaturity such that delivery must be delayed

Fetoscopy (direct endoscopic visualization) Percutaneous tracheal occlusion procedure for lung growth promotion incongenital diaphragmatic hernia; tissue biopsies; vascular access;diagnosis and treatment of urinary tract obstruction (see below);surgeries on placenta, membranes, cord

Open fetal surgery Meningomyelocele repair; congenital diaphragmatic hernia repair;thoracic space-occupying lesion removal; cardiac malformation repair;lower urinary tract obstruction decompression; sacrococcygealteratoma resection

a Harrison et al. (1 ), Crombleholme et al. (2 ), Deprest et al. (3 ), Adzick et al. (4 ), Quintero et al. (5 ), Leviton et al. (6 ).b Adapted with permission from Geaghan (95 ).

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demonstrate that certain fetal surgeries can be per-formed safely without laboratory support. Only theposterior aspect of the fetus is exposed in these repairs.Intraoperatively, maternal status is stable and placentalcirculation continues to support the fetus.

Other technologies include fetoscopically directedselective laser (photocoagulation) ablations of placen-tal vascular communications for intrauterine growthretardation (IUGR)2 due to vascular anastomoses(communications between twin circulations) inmonochorionic twin pregnancies. If successful, thepregnancy is thereafter functionally dichorionic (withindependent circulations), and risk of serious sequelaeof twin–twin transfusion syndrome (neurologic dam-age, morbidity, and mortality) is reduced. Improvedneurological outcomes have been reported for twinstreated at �26 weeks gestational age (5 ). Proceduralrisks include IUGR, twin deterioration, or even demise.

Minimally invasive modalities include therapeuticpharmaceuticals delivery via maternal administration

and placental transport to the fetus, such as antibioticsfor infectious diseases, steroid administration for ac-celeration of lung maturity to reduce respiratory dis-tress syndrome and neonatal mortality in cases ofthreatened preterm birth (6 ), or thyroid medicationsin the setting of maternal thyroid disease. In manytypes of fetal intervention, the elegant mechanisms ofplacental circulation allow for compensation andmaintenance of fetal homeostasis.

The spectrum of diagnostic laboratory support re-quired for these interventions ranges from no testing(myelomeningocele repair) to pathologic tissue analy-sis (sacrococcygeal teratoma resection), serial moni-toring by urinalysis (lower urinary tract obstruction),and life-saving transfusion when required (e.g., laserphotocoagulation of placental anastomoses in twin–twin transfusion syndrome).

Fetal Diagnostic Technologies

Fetal diagnostic modalities vary widely in invasivenessand associated risk to mother and fetus (Table 2). Um-bilical cordocentesis, or ultrasound-guided percutane-ous umbilical cord blood sampling (PUBS), is per-formed for fetal health assessment and for fetal diseasemanagement. Despite the growing number of applica-tions for this procedure, cordocentesis is not without

2 Nonstandard abbreviations: IUGR, intrauterine growth retardation; PUBS, per-cutaneous umbilical cord blood sampling; Hb F, hemoglobin F or fetal hemo-globin; MCV, mean corpuscular volume; 21-OH, 21-hydroxylase; WBC, whiteblood cell; MCA-PSV, fetal middle cerebral artery peak systolic velocity; AFP,�-fetoprotein; fFN, fetal-specific fibronectin; IL-6, interleukin-6; CRP, C-reactiveprotein; CVS, chorionic villus sampling.

Fig. 1. Fetal neck dissection at 25 weeks.Courtesy of Dr. Michael R. Harrison.

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risk. The amount of blood sampled depends on theindication, but can be 1–3.5 mL (7 ), a substantialamount for a fetus. In a large series of 341 cases re-ported complications included: bradycardia (9.38%)related to repeated and prolonged punctures; cord he-matomas (1.47%) associated with puncture attemptstargeting a free loop of cord; and fetal deaths (5.87%), 3of which were directly related to the procedure(0.88%). Literature review and metanalysis of 4922cases revealed similar rates (bradycardia 3.1%–11%;total fetal deaths 3.84%; fetal deaths directly related toprocedure 0.98%) (8 ). Other complications, reported

in the largest published series of 606 consecutive cases,include spontaneous abortion (0.8%), growth retarda-tion (8%), in utero death (1.1%), and premature birth(5%) (9 ). Because the complication rate is muchhigher if the procedure exceeds 10 min or if more than3 punctures are attempted, practice guidelines recom-mend that attempts should not exceed 10 min, and amaximum of 2 punctures be performed at 1 session.Overall, the procedure failure rate (no sample pro-cured) reported in the literature is 1.68%– 8.71% (8 ). Asingle umbilical artery (rather than the expected2-artery, 1-vein cord) precludes this procedure, be-

Table 2. Antenatal diagnostic laboratory technologies: clinical applications, associated risks and benefits.a,b

Diagnostic Test Clinical Application Risks Benefits

Amniocentesis Detection of aneuploidy oramniocytes for moleculargenetic analysis on fetalcells; determination ofamniotic fluid OD 450 in Rhincompatibility (infrequentdue to use of MCA-PSA);lamellar body count

Amniotic fluid leakage 1%–2%;spontaneous loss rate 0.7% ifperformed at 14 weeks orlater

Reassurance or allows for geneticcounseling, OD 450 guidesinterventions; lamellar body countindicates fetal lung maturity

Chorionic villus sampling Detection of aneuploidy ormolecular genetic analysison fetal cells

If performed at an experiencedcenter after 9 weeks, lossrate approaches that ofmidtrimester amniocentesis

Reassurance if normal, if abnormal,allows for genetic counseling

Cordocentesis: fetalhormones

Testing for congenital adrenalhyperplasia by CYP21genotyping and assessmentof antenatal steroid therapyby following marker 17-hydroxyprogesterone

�2% Fetal loss related toprocedure; 9.38% fetalbradycardia

Reduce risk of gender misassignment;possible reduction of genderconfusion; may prevent need forcorrective genital surgery forambiguous genitalia

Cordocentesis: blood gases Fetal acidemia Same as above Correlates with reduced developmentquotient; indicator for emergentdelivery

Cordocentesis: coagulationtesting and plateletcounts

Diagnosis and management ofhereditary and acquiredimmune bleeding disorders

Same as above Planning for minimizing birth trauma(caesarean section); in uterotransfusions

Maternal site sampling forfetal products

Detection of fetal fibronectinfor risk of premature labor;IL-6 and CRP incervicovaginal fluid; use of�-microglobulin-1 test incervicovaginal fluid

None fFN: negative predictive value forlabor, may reduce admissions; IL-6and CRP proposed for detection ofchorioamnionitis; �-microglobulin-1for detection of premature ruptureof membranes

Meconium analysis; fetal orsegmental maternal hairanalysis

Detection of drugs of abuseand alcohol (fatty acid ethylesters) exposures;environmental exposures

Maternal sanctions, legal andsocial

Allows for child protection, maternalrehabilitation; allows for removalof environmental risk and ongoingrisk surveillance

Fetal urine analysis bypercutaneous catheter

Assess need to decompressurinary obstruction: if Na�100 mEq/L Cl �90 mEq/L;osmolarity �210 mosmol

Shunt can dislodge, obstruct,migrate

Avert renal damage; possibly promotelung growth

a Harrison et al. (1 ), Crombleholme et al. (2 ), Deprest et al. (3 ), Leviton et al. (6 ), Soothill et al. (30 ), Ghizzoni et al. (37 ), Sulcova et al. (38 ), Daffos et al. (43 ),Bevis et al. (46 ), Liley (47 ), Queenan et al. (48 ), Mari et al. (49 ), Gareri et al. (51 ), Gourley et al. (52 ), Verma et al. (53 ), Koren et al (54 ), Chan et al. (55 ), Cernichiariet al. (56 ), Loew et al. (61 ), Swamy et al. (62 ), Wei et al. (63 ), Ashwood et al. (64 ), Cousins et al. (65 ), American College of Obstetricians and Gynecologists (68 ),Borgida et al. (69 ), Botto et al. (70 ), Brambati and Tului (71 ).b Adapted with permission from Geaghan (95 ).

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cause there is less opportunity for collateral circulationin the event of spasm or damage.

Therapeutic PUBS procedures, such as in uterotransfusions, have reportedly higher complicationrates (8 ). Intrauterine fetal transfusions require provi-sion of blood counts in real time, at the point of care, toguide treatment. Our institution has a dedicated smallfootprint instrument mounted on a wheeled, stablecart for easy transport to the maternal bedside (Fig. 2).The instrument must be optimized for small samplevolume, ideally 25 �L or less to conserve fetal bloodvolume. Intravascular fetal blood transfusion by cor-docentesis represents a significant therapeutic advancefor red blood cell isoimmunization, or hemolytic dis-ease of the newborn. Goals of limiting the duration oftransfusion and reducing the risk of repeated proce-dures call for a brisk injection rate with maternallycompatible transfused blood. Packed red cells are highhematocrit (ranging from 60% to over 80%), and rep-resent significant transfused blood volumes (60 –160mL) (10, 11 ). In one study of 12 fetuses expansion offetoplacental blood volumes ranged from 36.34% to106.37%, and the rate of increase from 2.41% to16.47% per minute. Substantial expansion of the feto-

placental volume impacts cardiac dynamics; however,alterations are resolved within 2 h (12 ).

Following intrauterine transfusion, diagnosticblood testing reflects donor cells and plasma. Testing ofposttransfusion samples can produce false-positiveand false-negative test results, and cannot be reliedupon for newborn screening or other definitive labora-tory diagnostics. The role of the laboratory is to sup-port cordocentesis procedures by offering technologiesthat minimize sample volumes and offer expeditedturnaround times (most often, but not limited to, he-moglobin and hematocrit measurements).

Healthy Fetal Reference Intervals

Intrauterine biochemical assessments are evaluatedwith reference to gestational age and programmed de-velopment. Postnatal umbilical cord blood samples areroutinely available but cannot be assumed to reflectfetal values, even at term. Colossal changes in the fetalphysiology and anatomy occur during the transitionfrom the in utero to the postnatal environment; forexample, massive fluid shifts take place in the firsthours of postnatal life, altering the distribution of bodywater (13 ). In the first postnatal week, physiologic con-traction of the extracellular space accounts for an ex-pected weight loss (attributable to water loss) of 5%–10% for term neonates (14 ) and 10%–20% for lowbirth weight/premature infants (15 ). Before birth, ma-ternal and placental homeostatic mechanisms managefetal water and electrolyte balance. After delivery, theneonate is subject to a more labile environment andmust manage fluid and electrolyte changes that charac-teristically occur during the first days of life (16 ).

Values obtained from fetoscopic sampling fromearly mid- through late gestation have been used toestablish reference intervals. These samples better re-flect healthy fetal physiology than did earlier studiesbased on spontaneous or therapeutic abortuses. To es-tablish fetal reference values for many analytes, the useof leftover blood from cordocenteses may be the onlyavailable option. Because cordocenteses carry substan-tial risks and are performed when fetal health is typi-cally at risk or impaired, these blood samples do notordinarily provide an ideal healthy reference popula-tion. With the use of strict exclusion criteria, however,robust data can be produced. Data collected from neg-ative cordocenteses can provide reference values, ide-ally confirmed by physical examination and attestationof health at birth.

Small population sizes are associated with statisti-cal uncertainty and wider confidence limits for theendpoints of each reference interval. Published con-sensus guidelines provide guidance on the use of cen-tiles to describe sample sizes �10 (17 ).

Fig. 2. Have instrument will travel.Laboratory support cart for cordocentesis at the point of care.



The most extensive body of work that establisheshealthy fetal reference intervals is found outside theclinical laboratory, in ultrasonographic measurementof fetal physical parameters of all types. Like laboratorymeasurements, these data are highly dependent onchoice of method, are related to gestational age, and arecritically important to inform the obstetrician aboutthe health and development of the fetus. Recognizeddifficulties relative to accurate pregnancy dating andestablishment of gestational age adds imprecision todata sets. It is recommended in the radiology literatureto record measurements by gestational age in days(18 ). Recording of normative data by age in completedweeks allows for inclusion of fetuses that are up to 6days older than the reference group age (as expressed inweeks), and contributes error. The recommendation touse age in days merits consideration in the laboratorycommunity. The role of the laboratory is to provide thebest available fetal reference data to support clinicaldecision-making in these specialized clinical settings.

Confirmation of Sample Integrity and Fetal Origin

Fetal laboratory diagnostics require that specimens canbe definitively identified as fetal in origin (Table 3).

Fetal red blood cells obtained by umbilical cordocente-sis are distinguished from maternal cells by theKleihauer–Betke test, or by flow cytometry that usesantibodies such as anti-hemoglobin F (Hb F), and/orby differences between maternal and fetal mean cor-puscular volume (MCV). The Kleihuaer–Betke usesthe different physical properties of Hb F, the predom-inant hemoglobin in fetal red cells, and Hb A, the pre-dominant hemoglobin in adult cells, to determinesource. A thin smear of maternal blood is treated withacid, rinsed, and counterstained. Hb F in fetal cells isresistant to the acid elution treatment, so that fetal cellswill stain bright pink with hemoglobin; in contrast,maternal cells with Hb A– containing cells will appearas colorless “ghosts” following acid elution. The sub-jectivity of the test (microscopic categorization of cellsas fetal or adult on the basis of staining characteristics)contributes to poor reproducibility. Flow cytometricanalysis using a monoclonal anti-Hb F antibody toquantify Hb F– containing fetal cells is far superior inprecision and objectivity in large multiyear multicentertrials, and in the aggregate experience of the nationalCollege of American Pathologists proficiency surveys(19 ). However, flow cytometry preparation and analy-sis requires more time, and is therefore not suited for

Table 3. Laboratory technologies for confirmation of fetal sample origin.a,b

Test Method Principle Performance characteristics

Red cell MCV Hematology analyzer, impedancemeasurement

Fetal MCV higher thanmaternal MCV

MCV is an average; Overlapcan occur in pathologicalstates (e.g. maternalmacrocytic anemia, or fetalmicrocytic anemia)

Kleihauer–Betke, manual Acid elution test followed bymanual microscopy, counting offetal cells which stain brightlyowing to Hb F resistance to acidelution

Semiquantitative estimation offetal hemoglobin containingcells in maternal circulationrepresents size offetomaternal hemorrhage

Manual microscopysubjectivity, counting ofsmall numbers of cells, highCVs: low sensitivity andpoor reproducibility

Fetomaternal hemorrhagedetection by flow cytometry

Flow cytometry of red cells labeledwith monoclonal antibody, e.g.,anti-Hb F

Quantification of fetal cells inmaternal circulation toassess size of fetomaternalhemorrhage

Counting of large numbers ofcells, low CVs: precise andreproducible

Molecular analysis of fetalnucleated cells frommaternal blood

Enrichment and isolation of rarefetal cells for analysis

Fetal nucleated cells present inmaternal blood are used fordiagnostics

Low throughput: rare cellsrequire enrichment andisolation: time-consumingand implementationtechnically difficult

Molecular analysis of cell-freefetal DNA and RNA frommaternal blood

Varies: real-time quantitative PCR;high-throughput shotgunsequencing; electrophoretictechniques or smaller PCRamplicons to extract (shorter)fetal DNA

Fetal DNA present in maternalblood are used fordiagnostics

Higher throughput, �20� theamount of cellular-free fetalDNA present as comparedwith nucleated, cellularfraction

a Chen et al. (19 ), College of American Pathologists (20 ), Raeihae (21 ), Fan et al. (73 ), Chiu et al. (74 ), Chiu et al. (75 ), Lo et al. (76 ), Li et al. (77 ), Chan et al.(78 ), Fan et al. (79 ), Lun et al. (80 ), Hahn et al. (81 ), Jorgez et al. (82 ), Lo et al. (83 ), Fan et al. (84 ), Chui et al. (85 ), Liao et al. (86 ), Avent (87 ), Tynan et al.(88 ), Wright et al. (89 ).b Adapted with permission from Geaghan (95 ).

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real-time guidance during an invasive procedure. Con-firmation of fetal identity also uses MCV measure-ments: maternal MCV is expected to be markedly lowerthan the developmentally large fetal MCV, without ap-preciable overlap in size ranges. A disease state, such asa macrocytic anemia, could certainly confound this de-termination, for example, by increasing maternal cellsize, or MCV, into the fetal range. Likewise, increases infetal hemoglobin percentage in adults with disease statessuch as thalassemias are fairly common, and hereditarystates of persistence of fetal hemoglobin in adult life aredescribed, which could also (rarely) confound a Kleihauer–Betke test.For these reasons, a flow cytometric test offersa more reliable, reproducible, and superior metric, butis not yet widely employed and not available at thepoint of care. The assays currently available are primar-ily designed for another purpose: detection of fetal redcells in the maternal circulation, for prevention andtreatment of maternal alloimmunization, or immunesensitization from exposure to paternal (foreign) anti-gens expressed by the fetus. In the College of AmericanPathologists Survey HFB 2011-A, principal methodsfor identification of fetal hemoglobin across the USlaboratories enrolled in proficiency testing programsincluded modified Kleihauer–Betke method (n � 854);immunoassays (n � 1730); and flow cytometric meth-ods (n � 42), primarily anti-Hb F assays (20 ).

Amniotic fluid contamination is also a small riskin PUBS; in one series, contamination was reported inapproximately 2.5% of umbilical blood samples (15/606) (7 ). Amniotic fluid has a lower pH (pH � 7.1) andother important chemical differences (higher lactate)compared with healthy fetal blood (21 ). The laborato-ry’s role is to confirm specimen integrity and fetal spec-imen identity for each fetal sampling by one of thesetechnologies to assure quality and patient safety.

Biochemical Assessments of the Fetus

Knowledge of developmental biochemistry as well asestablishment of healthy fetal reference values is requi-site for laboratory data interpretation. Certain analyteschange appreciably as a function of gestational age andothers do not. From a series of 171 fetuses that under-went PUBS between 18 and 40 weeks gestation, a subsetof 72 healthy fetuses for biochemical analyses was iden-tified after suspected pathologies were not confirmed.Physical examination and laboratory screening at birthconfirmed these infants to be healthy. Testing for 18biochemical analytes was performed, and the followingtrends noted: total protein and albumin plasma con-centrations increase with advancing age, but remainlower than those in adults (22 ); this finding was repli-cated in several studies (23, 24 ). Creatine kinase in-creases with gestational age, then falls at term to neo-

natal concentrations. Alkaline phosphatase peaks at21–25 weeks at concentrations higher than those inadults, and then falls to concentrations lower than inadults; this pattern has also been confirmed in severalstudies (24, 25 ). Concentrations of sodium, potas-sium, urea nitrogen, creatinine, lactate dehydrogenase,uric acid, and magnesium are not substantially differ-ent in fetuses than in term newborns. Cholesterol andtriglyceride concentrations are much lower than adultconcentrations (25, 26 ). Fetal total bilirubin concen-trations are lower than in neonates, but higher thanin adults. Also notable are phosphorus and�-glutamyltransferase enzymatic activity levels, whichare higher in fetuses compared with both newbornsand adults. In the fetus, activity levels of enzymes suchas aspartate aminotransferase and alanine aminotrans-ferase appear to be independent of maternal levels, andfar lower than those of other adults (22, 23 ). Valuesfrom multiple studies are generally consistent, but dif-ferences exist owing to differences in instrumentationand small sample size.

The acquisition of passive immunity from mater-nal source immunoglobulin is evident in the increase infetal IgG from week 20 to 34, from a mean of 256 to amean of 566 mg/dL (P � 0.001). Fetal immunoglobu-lin A and M, representing solely fetal production, alsoappeared to increase with gestational age, but the val-ues did not reach statistical significance (27 ). The ma-jority of the maternal IgG is transferred to the fetusduring the last 4 weeks of pregnancy, rendering prema-ture infants relatively deficient in maternal antibodies(28 ). These trends are important manifestations of theontogeny of the fetal immune response.

Could fetal blood biochemical analysis be helpfulfor timing of delivery? Fetal cord sampling for oxygen-ation and acid– base balance demonstrates fetal pH tobe slightly higher than maternal pH, but close to 7.40;fetal PO2 ranges in the 60s and 70s for the majority, andfetal percentage saturations from the high 70s to thehigh 80s. Human fetal umbilical vein pH and PO2 arehigher, and PCO2 much lower than those observed atdelivery (27 ). In growth-retarded human fetuses, cordsampling for blood gases demonstrated that for fetusesin whom clinical assessment (independent and blindedto laboratory values) warranted immediate delivery,pH was lower (P � 0.001), and PCO2 higher (P � 0.002)compared with fetuses with more favorable clinical as-sessments, indicating that the pregnancies should beallowed to continue (29 ). Fetal lactate concentrationwas inversely correlated with pH (P � 0.0009). An-other study showed an association between chronic fe-tal acidemia and subsequent impaired neurodevelop-ment, as measured in 36 fetuses who had cordocentesesfor growth retardation. Those who had acidemia as fe-tuses had significantly lower developmental quotients



(30 ). These data suggested that fetal biochemical anal-yses could be helpful for clinical decision-making re-garding timing of delivery. Fetal blood sampling for pHand lactate has been employed when further informa-tion about the fetal status is desired, in the presence ofan abnormal or nonreassuring fetal heart rate pattern.One review of the literature found a statistically signif-icant higher sample yield rate for lactate compared withpH sampling, but no reported difference in correlationwith neonatal or maternal outcomes (31 ). Fetal scalppH and lactate sampling are technologies that were in-troduced into practice, as was fetal electric monitoringby use of cardiac tocography (32 ), before a clear evi-dence base in terms of neonatal/child/maternal out-come was established, and benefit remains unproven.

Fetal laboratory measurements may potentially offerprognostic information regarding postnatal organ func-tion. A prospective noninterventional study measuredcystatin C and �2-microglobulins (low molecular weightproteins that are markers of kidney function) for theprediction of renal function impairment in excess serumfrom 129 cordocenteses in 84 fetuses. Maternal and fetalserum concentrations did not correlate for either cystatinC or �-microglobulin, but correlated closely for serumcreatinine. For the prediction of renal dysfunction,creatinine from the mother confounds fetal assessment.Overall, mean plus 1 SD for cystatin C was 1.66 (0.202)mg/L (upper limit of reference interval at mean plus1.96 SD � 2.06 mg/L), independent of gestational age.�2-microglobulin, in contrast, decreased with gestationalage; the upper limit was established as a dynamic rangedetermined to be 7.19 mg/L � 0.052 mg/L, multipliedby gestational age in weeks. Cystatin C had a higherspecificity for detection of renal dysfunction (91.8%)compared to �2-microglobulin (85.5%), whereas �2-microglobulin had a higher sensitivity than cystatin C(90.0% vs 63.6%), and diagnostic efficiency for the 2 testswas equivalent. Cystatin C is produced by nucleated cells;�2-microglobulin is produced by lymphocytes and morelikely to vary with infections and other factors (33). How-ever, a large systematic review and metaanalysis con-cluded an overall poor predictive accuracy for fetal urinal-ysis (34). Instead, percutaneous fetal cystoscopy (directvisualization) may determine those urinary tract lesionsthat are appropriate for in utero therapy: a recent system-atic review found that fetal cystoscopy revised the ultra-sound pathologic diagnosis in 25%–36.4% of fetuses(35). This evidence was limited to 2 series. At present,measurement of amniotic fluid volume and the appear-ance of the renal (parenchymal) cortex appear to be themost predictive features of poor postnatal renal function(36). However, this disappointing review of urinalysis islikely attributable to confounding factors, including: lackof use of gestational age specific reference intervals in

studies, different assay methodologies, and lack of defin-itive cutoff values.

Prenatal endocrine analysis is another area of fetaldiagnostic impact. Congenital adrenal hyperplasias are agroup of autosomal recessive endocrine disorders attrib-utable to enzyme deficiency in steroid synthesis. The mostcommon etiology is 21-hydroxylase (21-OH) deficiency.The goal of prenatal diagnosis and treatment and of21-OH deficiency is prevention of prenatal virilization inaffected female fetuses, and avoidance of consequencessuch as risk of gender misassignment, gender confusion,and indications for possible corrective genital surgery(37). Early diagnosis (before the 15th gestational week) isdesirable. Genotyping for the responsible CYP21 (cyto-chrome P450, family 21, subfamily A, polypeptide 2)3

gene can be performed from chorionic villi sampled at 10–11 weeks gestation, although technical factors can lead to di-agnostic mishaps (37). Amniotic 17-hydroxyprogesteroneis the diagnostic biochemical marker for 21-hydroxylasedeficiency, is unaffected by sex, and variation with gesta-tional age is not significant (38). Guidance for fetal treat-ment of congenital adrenal hyperplasia is therefore pos-sible by monitoring 17-hydroxyprogesterone duringantenatal dexamethasone treatment. Although outcomestudies of patients after antenatal exposure to dexameth-asone have shown no significant adverse effects, confir-mation of the safety of this treatment (e.g., on brain de-velopment) awaits longer-term randomized controlledstudies of treated vs untreated pregnancies (38).

For other endocrine disorders, fetal studies are notavailable, and only data from preterm births are avail-able. For example, luteinizing and follicle-stimulatinghormone concentrations have been reported for pre-mature infants at 24 –29 weeks (39 ).

Ideally, collaboration among laboratories in-volved in fetal sampling at maternal fetal medicine cen-ters could lead to the creation of multiinstitutional datasets (for example, by investigations on excess samples,when available) to expand the limited literature in fetalbiochemistry, and better characterize health and dis-ease states in the antepartum setting.

Hematologic Assessments of the Fetus

Fetal hematologic measurements inform our under-standing of developmental hematopoiesis and help guidetransfusion therapy (Table 4) (40). Fetal red cell count,hemoglobin, and hematocrit sequentially rise over thecourse of gestational weeks 15 through term. Simultane-ously, the red cell MCV gradually and progressively falls toterm. Total white blood cell (WBC) count also rises from

3 Human genes: CYP21, cytochrome P450, family 21, subfamily A, polypeptide 2;RHD, Rh blood group, D-antigen.

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the late second trimester to term. Total WBC count mustbe manually corrected in the presence of fetal nucleatedred blood cells. Without correction, total WBC countoverestimation occurs because of impedance count inter-ference from the similar-sized nucleated red blood cells inthe WBC channel. Quantitative WBC counts in thesecond-trimester fetus are a fraction of the numbers ob-served in the term neonate. Platelet counts, on the otherhand, reach levels considered to be adequate for olderchildren and adults during the second and third trimes-ters, and continue to rise in number to term (41, 42).

Prenatal diagnosis and management of hereditaryand acquired immune bleeding disorders by fetal bloodsampling has been described in 103 cases, allowing forin utero transfusions of coagulation factor concen-trates and platelets; assessment of success of certainmaternal therapies for fetal conditions; and effectiveplanning for minimizing trauma by the caesareanmode of birth (43 ).

This area of fetal hematology and coagulation test-ing would also benefit from multiinstitutional consor-tiums; collective experience could expand the currentlimited fund of knowledge on fetal hematologic ontog-eny and also serve to better characterize fetal health anddisease states.

Alternative Sample Matrices for Fetal HealthAssessment

Several alternative matrices are clinically useful for fetalhealth assessment. Fetal urine production increasesbriskly with gestational age, from 5 mL/h at 20 weeksgestation to 51 mL/h at 40 weeks gestation (44 ). Fetalurine production is the primary component of amni-

otic fluid. Amniotic fluid, obtained by invasive amnio-centesis, is one of the best-studied matrices for fetalanalysis. The composition varies throughout gesta-tion: in the first trimester, it is essentially a plasmaultrafiltrate. Later, in the second and third trimes-ters, the developing renal, urinary, gastrointestinal,and pulmonary organ systems contribute their se-cretory products to amniotic fluid to yield a morecomplex matrix (45 ).

The first widespread application of amniotic fluidanalysis was for prenatal assessment for Rh-group im-munized pregnancies. In these pregnancies, the fetus isRh-positive and mother Rh-negative, causing animmune-mediated maternal destruction of fetal redcells leading to severe anemia and even intrauterinedeath. The field advanced in 1953 when Bevis et al.demonstrated that amniotic bilrubin pigment concen-trations correlate with clinical hemolytic disease of thenewborn (46 ). The Liley system for prediction of fetalcondition was based on amniotic fluid absorbance,spectophotometrically measured at wavelength 450nm (� OD 450). This system, introduced in the 1960s,was clinically useful in categorization of pregnancies 27weeks to term into mild/absent and severe disease, with amidzone requiring repeat testing (47). However, the in-ability of this system to be extrapolated to earlier gesta-tional ages was a limitation better addressed by the gener-ation of the Queenan system. This dataset, also based onamniotic fluid � OD 450, spanned 14–40 weeks gesta-tion, and offered a new management protocol based on 4zones of risk (unaffected, indeterminate, affected, and in-trauterine death zone). Excellent clinical correlation andoutcomes were demonstrated on the basis of a dataset of789 pregnancies (48).

Table 4. Hematologic parameters in healthy fetuses by gestational age.a,b

Gestationalage

Sampleno.

Hemoglobin,g/dL

Redblood cells,

�106/�LHematocrit,

%MCV,

fL

TotalWBC count,

�103/�L

CorrectedWBC count,

�103/�LPlatelets,�103/�L

15c 6 10.9 (0.7) 2.43 (0.26) 34.6 (3.6) 143 (8) 1.6 (0.7) — 190 (31)

16c 5 12.5 (0.8) 2.68 (0.21) 38.1 (0.21) 143 (12) 2.4 (1.7) — 208 (57)

17c 16 12.4 (0.9) 2.74 (0.23) 37.4 (0.28) 137 (8) 2.0 (0.8) — 202 (25)

18–21d 760 11.69 (1.27) 2.85 (0.36) 37.3 (4.32) 131.1 (11.0) 4.68 (2.96) 2.57 (0.42) 234 (57)

22–25d 1200 12.20 (1.6) 3.09 (0.34) 38.59 (3.94) 125.1 (7.8) 4.72 (2.82) 3.73 (2.17) 247 (59)

26–29d 460 12.91 (1.38) 3.46 (0.41) 40.88 (4.4) 118.5 (8.0) 5.16 (2.53) 4.08 (0.84) 242 (69)

�30d 440 13.64 (2.21) 3.82 (0.64) 43.55 (7.2) 114.4 (9.3) 7.71 (4.99) 6.4 (2.99) 232 (87)

a Hematologic measurements by a Coulter S-plus II instrument on 2860 fetal blood samplings for prenatal diagnostic purposes. Data expressed as mean (SD). TotalWBC count includes NRBCs. Corrected WBC count includes WBCs only, after subtracting NRBCs, based on a 100-cell count manual differential.b Adapted with permission from Geaghan (95 ).c Data from Millar et al. (41 ).d Data from Forestier et al. (42 ).



In 2000, the practice of fetal medicine changed withthe demonstration that noninvasive Doppler ultrasoundof the fetal middle cerebral artery peak systolic velocity(MCA-PSV) could be used to better assess and guidetreatment of hemolytic disease of the newborn, as firstreported by Mari et al. (49). The study included 111 fe-tuses at risk for hemolytic disease of the newborn due tored cell isoimmunization, and 265 healthy fetuses. A no-mogram was created using a reference interval for gesta-tional age–specific hemoglobin concentration developedfrom healthy fetuses at 18–40 weeks gestation, and gesta-tional age–specific reference values developed for theMCA-PSV. It was demonstrated that with the use of thisnomogram and measurement of MCA-PSV, 70% of in-vasive cordocenteses could have been avoided. In thesecases, hemoglobin sampling by cordocentesis revealedthat the fetuses were only mildly or not at all anemic. Byusing the decision limit of a multiple of the mean of 1.5 orgreater for MCA-PSV, all significant anemias would havebeen identified and none missed, with a false-positive rateof 12%. Additionally, in the remaining 30% of affectedfetuses, the fetuses were already hydropic (indicating ad-vanced disease) in 40% of cases, but could have been iden-tified earlier without risk of invasive procedure, by serialDoppler MCA assessments. Doppler MCA velocimetryoffers utility in noninvasive diagnosis and management ofother types of fetal anemia, and is now also pivotal inassessment of intrauterine growth retardation.

The study of enzymes in amniotic fluid between 14and 35 weeks gestation demonstrates that timely and re-liable amniotic enzyme quantification is possible on aconventional analyzer, with minor adaptation. In cases ofsonographically diagnosed bowel disorders, assays of am-ylase, � glutamyl transferase, 5�-nucleotidase, and totalalkaline phosphatase were found to be of diagnostic valueas confirmatory tests. For example, � glutamyl transferaseand 5�nucleotidase have high activities in bile, and aretherefore low (�5th percentile) activities in biliary atresiadue to restriction of bile flow. Increases in all 5 enzymesare associated with gastroschisis, a congenital defect in theanterior abdominal wall through which the abdominalorgans freely protrude. In these cases, enzyme activitiesare usually at �95th percentile, due to the open commu-nication between amniotic fluid and the bowel in thatdisorder (45).

Glycosaminoglycan accumulation from fetal urinehas also been analyzed in amniotic fluid. Chondroitin sul-fate, the dominant human glycosaminoglycan, togetherwith hyaluronic acid comprise the majority found by am-niotic fluid analysis; heparin sulfate and dermatan sulfateare present in small amounts and undetectable in healthypregnancies, respectively. Relative changes in the propor-tions of these components have been noted in several con-genital metabolic disorders, and may have diagnosticvalue (50).

A matrix entirely unique to the fetus is meconium,the fetal waste product accumulated over the second andthird trimesters of gestation. In term infants, 99% passtheir first formed stool by 48 h. Meconium is completelyevacuated within the first 125 h after birth, and for ana-lytic purposes, collection is recommended within the first72 hours of postnatal life (thereafter the matrix is a tran-sitional one, from meconium to feces). In preterm, ex-tremely low birth weight infants, the evacuation of meco-nium is comparatively delayed: median age at first stool is3 days, with 90% of infants evacuating meconium by 12postnatal days (51–53). Meconium analysis is therefore aretrospective evaluation of fetal exposures in utero, butnonetheless a highly valuable tool for assessment. Specificutilities include testing for environmental toxin exposuresand maternal drugs of abuse. According to current under-standing, once meconium is deposited, this matrix repre-sents a stable record of antenatal exposures.

Exposures to alcohol, drugs of abuse, and smokingpresent variable degrees of risk to the fetus, mother,and family. The use of segmental hair analysis is helpfulin maternal–fetal pharmacology and toxicology, facili-tating identification of chronological patterns of drugand alcohol abuse in pregnant women. This matrix isunique, because detection of patterns of illicit drugs ofabuse and prenatal alcohol use over time is possible,including confirmation of abstinence or changes indrug abuse for child protection (54 ). Fatty acid ethylesters are a marker of prenatal alcohol exposure avail-able from segmental hair analysis, and nicotine con-centrations are available as a marker for maternalsmoking (55 ). Maternal hair analysis is also recom-mended for monitoring prenatal exposure to methyl-mercury. Methylmercury poses an environmental riskto which the fetus is exquisitely sensitive owing to neu-rotoxic effects on the developing central nervous sys-tem. As a stable, historical record of concentrations oftransportable species in plasma, hair is analyzed as asurrogate for fetal tissue (brain) concentrations (56 ).Noninvasive collection, easy and inexpensive transportand storage, and minimal issues of sample integritymake hair an ideal matrix for analysis.

The spectrum of bioanalytic matrices and meth-ods to monitor in utero drug exposures also includesvernix caseosa (thick lipid and cellular fetal covering),oral fluid, amniotic fluid, urine, sweat, blood, hair,nails, and cord tissue (57 ). Although these measure-ments actually take place at or after birth, they are ret-rospective assessments of the exposures and environ-ment of fetal life. The clinical laboratory’s role is toexpand the repertoire of available matrices for testingin the future, or to make such options available on areferral basis, as clinically appropriate.

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Fetal Secretory Products Accessible by MaternalSampling

The identification or quantification of fetal-specific se-cretory products (in relation to gestational age) in ac-cessible maternal sites can offer valuable antenatal di-agnostics and aid decision-making for preterm laborand delivery. Maternal serum �-fetoprotein (AFP)screening is offered between 14 and 22 weeks gestation,as part of evolving algorithms and often in combina-tion with additional tests, as a marker for neural tubedefects and a variety of common congenital abnormal-ities. AFP is a fetal glycoprotein that is synthesized inthe yolk sac early in gestation, and is later formed inthe fetal liver and gastrointestinal tract. Though AFPconstitutes the major serum protein in the fetus, itsfunction is unknown. AFP circulates in extraordinarilyhigh concentrations in fetal serum, passing into thefetal urine, which is the major contributor to amnioticfluid. The concentration of AFP in fetal serum and inamniotic fluid peaks at 13 weeks and decreases there-after (58 ). After 12-weeks gestational age, this proteinis found in increasing concentrations in maternal se-rum (59 ), because it diffuses across fetal membranesand is also transported by diffusion into the maternalplacental circulation (60 ). Measurement of maternalserum AFP is expressed as a multiple of the mean of ahealthy population. This reporting mechanism is usedto normalize AFP values to a statistical distribution,and facilitate comparison of screening program resultsacross different populations and laboratories. Thesematernal measurements of a ubiquitous fetal proteinform the basis of widely adopted prenatal screeningprograms, and combined with 2 or 3 additional tests(known as a “triple” or “quad” screen, the details ofwhich are outside the scope of this review) can helpidentify a wide variety of fetal conditions.

Fetal-specific fibronectin (fFN) is an extracellularglycoprotein uniquely produced by fetal placental tis-sue, also known as “trophoblast glue” for its role insecuring placental trophoblasts to maternal decidua.fFN detection in cervicovaginal secretions can be usedas a prognosticator for preterm labor. fFN is measuredon samples free of blood and amniotic fluid by enzyme-linked immunoassay; a positive value exceeds 50 �g/L.In a randomized trial, a negative fFN in women pre-senting with threatened preterm labor was linked tofewer hospital admissions and decreased length of hos-pital stay (61 ). A robust negative predictive value of98% for delivery within 1 week of an assay performed at22–34 weeks is useful; positive predictive values aremuch lower (62 ). The poor positive predictive valuehas discouraged use of this test; however, a negativepredictive value can potentially be leveraged in an ap-

propriate clinical algorithm. Inflammatory cytokineinterleukin-6 (IL-6) in cervicovaginal fluid, and bothIL-6 and C-reactive protein (CRP) in amniotic fluid arestrongly associated with spontaneous preterm birth inasymptomatic women. This strong association is notfound for IL-6 and CRP in maternal plasma, suggestingthat inflammation occurring at the maternal–fetal in-terface, rather than systemic inflammation, could bethe key factor in spontaneous preterm births (63 ).Clinical application awaits development of standard-ized assays for detection of increased IL-6 and CRP incervicovaginal fluid to assess the utility in obstetriccare.

The quantification of lamellar body counts fromthird trimester amniotic fluid provides evidence oflung maturity, and is predictive for respiratory statuspostdelivery (64 ). The lamellar bodies are fetal inorigin and are produced by type II alveolar cells ofthe lung. Lamellar bodies are whorled aggregates ofsphingomyelin, found in the amniotic fluid owing tocontinuity of fetal respiratory secretions (includingthe lamellar bodies) with the surrounding amnioticfluid, and by fetal swallowing of respiratory secre-tions and passage via fetal urination into the amni-otic fluid. Lamellar body counts are handily countedby impedance counters on conventional hematologyanalyzers, and are gaining popularity due to relativeease of use and favorable cost per test (no need forpurchase of a proprietary test kit). Over an estab-lished quantitative threshold, lamellar body countprovides evidence of adequate surfactant to reducealveolar surface tension sufficiently to prevent alve-olar collapse and ensure adequate postnatal pulmo-nary function.

Another example of fetal health assessment bymaternal sampling of products that are of fetal originis a recently introduced method for the diagnosis ofrupture of fetal membranes (ROM). A fetal product,placental �-microglobulin-1, is present at muchhigher concentrations in amniotic fluid (2000 –25 000 �g/L) compared with cervicovaginal fluid(0.05–2.0 �g/L). When high concentrations of thisprotein are detected in cervicovaginal fluid, a diag-nosis of rupture of fetal membranes is made with ahigh degree of accuracy; a sensitivity of 98.0%, spec-ificity of 100%, positive predictive value of 100%,and negative predictive value of 99.1% have beenreported (65 ).

The role of laboratories that support obstetric pa-tients is to offer rapid turnaround times on these ma-ternal samples of fetal products (lamellar body counts,placental �-microglobulin-1, and specific markers ofpreterm labor as the evidence base develops) for expe-dited clinical decision-making.



Fetal Laboratory Testing Contributes toUnderstanding of Disease

Fetal studies can elucidate, challenge, and revise ourunderstanding of the natural history of fetal diseases inimportant ways. Twin–twin transfusion syndrome,manifested in 4%–25% of monochorionic multiplepregnancies, is responsible for 17% of twin mortality(66 ). The syndrome is posited to be due to vascularanastomoses (connections) between twin circulations,and inadequate compensatory vascular communica-tions. Unbalanced unidirectional flow becomes evi-dent early in the second trimester, with discordant fetalgrowth and unequal amniotic sac fluid volumes. The“donor” twin is growth retarded and oliguric, with oli-gohydramnios (insufficient amniotic fluid volume);the “recipient” twin is larger and polyuric, with poly-hydramnios (excess amniotic fluid volume). Studies offetal iron metabolism in monochorionic twin pregnan-cies with twin–twin transfusion syndrome have dem-onstrated higher ferritin concentrations in recipienttwins than in donor twins in utero; however, these con-centrations, as well as stainable postnatal liver iron, ap-pear to be comparable to twins without this syndrome.These data fail to support a theory of iron overload inrecipient twins, and iron depletion in donor twins. Infact, recipient ferritin concentrations were far belowthose associated with iron overload (which are �1000ug/L), and donor ferritin concentrations were withinreference intervals expected for fetuses from singletonpregnancies, sampled for intrauterine growth retardation(67). Such studies challenge our theories and augmentour knowledge of fetal health and disease in the high-riskgroup of monochorionic twins. Again, use of discarded orleftover fetal blood samples from maternal–fetal medicinecenters is a largely untapped resource for potential inves-tigation of fetal parameters.

Prenatal Testing for Aneuploidy and FutureDirections in Fetal Laboratory DiagnosticEvaluation

Invasive prenatal testing for fetal aneuploidy includesconventional amniocentesis offered at 15–20 weeksgestation and chorionic villus sampling (CVS), offeredafter 9 weeks completed gestation, respectively. Cyto-genetic accuracy is �99% on these samples. Theprocedure-related fetal loss rate for CVS appears to ap-proach that of midtrimester amniocentesis, about 1 in300 –500, or lower depending on the center (68 ). Foramniocentesis the rate of cell culture failure is 0.1% ofsamples. Complications such as amniotic fluid leakageand vaginal spotting occur in 1%–2% of all amniocen-teses; chorioamnionitis occurs in �1 in 1000 cases(69 ). For CVS, complications include vaginal spotting

or bleeding in up to 32.2% of patients after transcervi-cal CVS, less if the procedure is performed transab-dominally. Initial reports of limb reduction anomalieswere linked to procedures before 9 weeks of age (70 ).The overall complication rate, including amniotic fluidleakage infection and culture failure, is �0.5% (71 ).

Most recently, the American College of Obstetri-cians and Gynecologists Committee on Genetics rec-ommended that targeted (rather than genome-wide)array comparative genomic hybridization be offeredfor fetal abnormal sonographic anatomic findings anda normal karyotype, or for fetal demise cases with un-determined karyotype. Despite improved resolutionover conventional karyotype for detecting chromo-somal abnormalities �3 Mb, limitations of the targetedarray include inability to detect balanced chromosomalrearrangements, uncertain significance of copy num-ber variations, and higher costs, and give caution towidespread screening application (72 ).

The discovery of fetal cell-free nucleic acids inplasma of expectant mothers has led to the develop-ment of noninvasive technologies for antenatal diag-nosis of fetal aneuploidy. Fetal DNA is detectable asearly as day 18 after embryo transfer in cases of assistedreproduction. Advantages of very early detection, with-out the possibility of procedure-related loss of an un-affected fetus, hold great promise. The first report offetal aneuploidy detection by high-throughput shot-gun sequencing of cell-free DNA, and mapping of shortsequences to the chromosome of origin, was reportedin 2008 (73 ). Small changes in numerical representa-tion of chromosomes, detected by counting millions ofDNA sequences, are indicative of an aneuploid fetus.The overrepresentation of chromosomes 13, 18, and 21in trisomies and underrepresentation of chromosomeX in male pregnancies was demonstrated, and findingshave been independently reproduced (74, 75 ). Earlystudies in this area often lacked a fully blinded andprospective design. Progress on several challengingtechnical areas follows.

First, the portion of the DNA sample derived fromthe fetus is comparatively small. The high background ofmaternal DNA limits assay sensitivity. The magnitude ofthe fetal DNA fraction varies between pregnancies andthroughout gestation (76). Many investigators have re-ported �10% fetal DNA from sequencing studies (77).Using high-throughput paired-end sequencing, size dis-tributions of maternal and fetal cell-free DNA have con-firmed that cell-free DNA has a peak size of 160–180 bp,and originates primarily from apoptotic cells. Fetal DNAis shorter (usually �500 bp) than maternal DNA (78, 79)and techniques to enrich for shorter (�150 bp) DNA toincrease sensitivity are a focus of study. The sensitivity forfetal aneuploidy detection is dependent on the originalamount of fetal DNA, the relative enrichment by size se-

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lection techniques, and the residual number of moleculesfor analysis after size selection. Enrichment techniquescan reduce the number of molecules available to becounted (80). Techniques to increase assay sensitivity forfetal genotyping or detection of fetal point mutations byfetal DNA enrichment also include use of smaller PCRamplicons (81), microchips (77), and techniques(74, 75, 82, 83) to extract lower molecular weight DNAfractions. These technologies offer a radically shortenedturnaround time for aneuploidy detection. Microfluidicdigital PCR, for example, uses uncultured amniocytes andchorionic villus tissue, and analysis for chromosomes X,Y, 18, 13, and 21 is complete in �6 h (84).

Chiu et al. compared massively parallel sequencingof maternal plasma DNA against full karyotyping on 753pregnancies at high risk for trisomy 21, using prospec-tively collected and archived maternal samples (85). Noprior validation of the diagnostic accuracy and feasibilityanalysis of multiplexed maternal plasma DNA sequenc-ing for trisomy 21 detection had been conducted on alarge scale. Two protocols with different sample through-puts were tested: a 2-plex and an 8-plex sequencing pro-tocol. The 2-plex protocol demonstrated superior results,available for 314 pregnancies: 100% sensitivity and 97.9%specificity, and a positive predictive value of 96.6% andnegative predictive value of 100%. The efficacy of thenoninvasive sequencing test suggests that a reduction inthe number of high-risk pregnancies requiring an amnio-centesis or a CVS, both of which carry procedure-associated risk, is possible (85). Investigators have dem-onstrated that targeted sequencing of genomic regionsusing enrichment kits allows for increased coverage offetus-specific alleles within targeted regions; this may in-crease throughput and reduce costs compared with non-selectively sequencing the genome. Protocols that com-bine bioinformatics and sequencing can noninvasivelysurvey the entire genome of a fetus for mutations andgenetic loci (86).

The minimum amount of requisite fetal DNAfraction (i.e., the number of DNA molecules largeenough to be statistically meaningful) and depth of se-quencing for different diagnostic targets is in the pro-cess of definition through additional clinical trials.

In the immunohematology arena, an important lim-itation for fetal blood-type genotyping was an immuno-logic phenomenon: fetal–maternal ABO or Rh incompat-ibility can limit the lifespan and survival of fetal red cells inthe maternal circulation (87). Free fetal DNA analysis al-lows for such genotyping despite immunological destruc-tion of fetal cells; a novel multiplex assay to detect the fetalRh blood group, D-antigen gene (RHD) loci is one exam-ple. This RHD genotyping assay includes: exons 4, 5, 7,and 10; the RHD� (pseudogene) of the RHD gene; thesex-determining region, or Y chromosome–specific assay;and a generic PCR amplification control. Plasma samples

from 150 randomly selected pregnant women were as-sayed for fetal RHD genotype using the MassARRAY sys-tem. The fetal RHD status of 148 of 150 samples (98.7%)was correctly classified; 86 (57.3%) and 62 (41.3%) werepositive and negative, respectively. Routine fetal RHDgenotyping using a multiplex assay on cell-free fetal DNAcollected from maternal plasma holds several advantages,as described in editorial commentary: neither mother norfetus is exposed to the risks associated with invasive pro-cedures such as amniocentesis or chorionic villus sam-pling, including the risk of immunological sensitizationassociated with invasive procedures. Furthermore, rou-tine testing of nonimmunized RhD-negative pregnantwomen may be justified to avoid unnecessary cost and useof RhD immune globulin. When compared with real-time PCR, the multiplexing of the MassARRAY systemallows for many loci to be analyzed simultaneously, sothat only a single reaction is required. Lastly, RHD vari-ants that yield negative antigen presentation but do notrepresent an entire gene deletion (e.g., the � pseudogeneor various exon conversions) can be correctly typed froma single small sample of cell-free fetal DNA in a singlereaction (88). A commercial assay is offered for noninva-sive assessment of fetal RhD status (SEQureDx™ tech-nology, LENETIX� Medical Screening Laboratory) usingcell-free DNA extracted from 2 separate aliquots ofmaternal blood, each performed in triplicate. The assay isapproved by the New York Department of Health to per-form noninvasive RHD genotyping in pregnancies of 15-weeks gestational age or greater (www.lenetix.com/html/rhd___sry_genotyping.html). The fetal Y chromosomesequences and PSI (�) allele are also included. If findingsare that of a female Rh-negative fetus, the entire protocolis repeated to confirm the diagnosis. Comparison of thisassay with current serologic testing in broad-based clinicaltrials will more fully characterize the clinical utility.

Ethical issues raised by noninvasive prenatal test-ing include nonclinical applications of the technologyfor early determination of fetal sex and paternity test-ing as well as performing such testing without adequateinformed consent (89, 90 ). The potential for prenatalscreening without procedural risks may allow for wide-spread accessibility and usage. Commercial tests foraneuploidy detection have recently been approved inthe US; by early 2012, 2 are likely to be available (Dr.Steven Quake, personal communication, December 6,2011). This technology may partially replace invasiveprenatal diagnostic sampling procedures such as am-niocentesis for cytogenetic analysis, as Doppler MCA-PSV measurements replaced amniocentesis for amni-otic fluid OD 450 analysis for hemolytic disease of thenewborn. Risk reduction for patients would be real-ized, accompanied by changes for healthcare deliverysystems, such as fewer billable procedures for obstet-rics, and a dramatic decrement in test workload, and



revenues for conventional cytogenetic laboratories.Concerns include additional cost for each pregnancy,and to the healthcare system(s) if the risk reduction ofnoninvasive prenatal diagnosis leads to widespread de-mand. Without adequate genetic counseling resources,the test results may not lead to a well-informed choice.In both India and China, unbalanced sex ratios have ledto prohibition of prenatal sex selection for social rea-sons (91, 92 ). Internet advertisement and regulation ofconsumer access are also areas of controversy. Thewider impact of, and controversies raised by, genomictechnologies on preconception, preimplantation, andpostconception genetic screening and testing are re-viewed in detail (93).

Fetal interventions in the future are also likely toinclude reconstruction of congenital fetal defects inutero from multipotent mesenchymal stem cells ob-tained by amniocentesis and use of biologic matrices,an active and promising area of investigation in tissueengineering (94 ). The clinical laboratory’s role will beone of support and guidance, and will evolve alongwith this therapeutic frontier.

Summary

As the field of fetal surgery advances, new techniqueswill allow additional congenital defects to be treated,and an expanded portfolio of minimally invasive tech-nologies will be developed. Laboratory support of thesesurgical interventions, when required, mandates: min-iaturized diagnostic blood methodologies, the abilityto provide blood counts in an expedited fashion to as-

sess for intrauterine transfusion requirements, and ap-propriate technologies for biochemical assessment andcontinuous monitoring of the fetus (95 ). The labora-tory community can contribute to improved fetalhealth by studies of developmental changes in fetal bio-chemical and hematologic parameters in health anddisease on discard samples, and by analysis of our in-stitutional and collective outcome-based monitoringexperience. Although opportunities for noninvasiveantenatal diagnosis and fetal therapeutics are limitedonly by the imagination, the application of such tech-nologies for nonmedical purposes (sex selection) un-derscore the need to develop ethical approaches for im-plementation, in concert with the technologies.

Author Contributions: All authors confirmed they have contributed tothe intellectual content of this paper and have met the following 3 re-quirements: (a) significant contributions to the conception and design,acquisition of data, or analysis and interpretation of data; (b) draftingor revising the article for intellectual content; and (c) final approval ofthe published article.

Authors’ Disclosures or Potential Conflicts of Interest: Upon man-uscript submission, all authors completed the Disclosures of PotentialConflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.Consultant or Advisory Role: S.M. Geaghan, Abbott Corp andARUP.Stock Ownership: None declared.Honoraria: S.M. Geaghan, Abbott Corp.Research Funding: None declared.Expert Testimony: None declared.

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