BACKGROUND: Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays.
METHODS: After overnight incubation (16–20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC–tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid–liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate.
RESULTS: Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory.
CONCLUSIONS: Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.
In recent years various approaches for high-throughput tandem mass spectrometry (MS/MS)7 screening of lysosomal storage disorders (LSDs) have been developed. The first assays were developed for the screening of Pompe, Gaucher, Niemann-Pick A/B, Fabry, and Krabbe disease in 2004 (1). The protocols have continuously evolved, and have been refined and optimized for high-throughput analysis (2, 3). The screening panel for LSDs was recently expanded and currently includes MS/MS assays for mucopolysaccharidosis (MPS) I, II, IVA, and VI (4,–,7).
However, newborn screening for LSDs is still a technological challenge. One of the major technical challenges in implementing MS/MS-based multiplex enzyme assays by using the currently available substrates is the complexity of sample preparation. Specimens collected from newborns are incubated with enzyme-specific buffers containing substrates for each lysosomal enzyme, followed by MS/MS detection of all respective enzymatic products and internal standards. The technology for screening several enzyme activities at once from more or less 1 single blood punch is complicated, time-consuming, and laborious, and includes the handling of hazardous reagents such as ethyl acetate (1, 3).
Currently, routine newborn screening for LSDs has been introduced for Pompe disease in Taiwan (8) and by the state of New York for Krabbe disease (9). Other US states such as Washington (10) and countries such as Austria started the first pilot studies for multiplex MS/MS screening, in which a variety of LSDs are screened within a single assay (11,–,13). For future implementation of high-throughput LSD assays in routine clinical diagnostics, sample handling and MS analysis must be simplified; specifically, sample pretreatment, separation, and finally detection must become more integrated (14). The implementation of online multidimensional chromatography combining sample preparation with analysis in 1 protocol facilitates ease-of-use sample introduction and increases speed of analysis (15).
Turbulent flow chromatography (Turboflow) involves the use of large-diameter particles (approximately 30 μm) with a high surface area packed into narrow-bore columns (0.5–1 mm i.d.) and available in a variety of stationary phases. Relatively crude samples such as dilute serum, cerebrospinal fluid, plasma, urine, blood-spot extracts, and whole blood can be injected directly into these columns under high mobile-phase linear velocities, inducing turbulent-flow conditions. TurboFlow simultaneously eliminates matrix interferences from salts and proteins, the most common suppressors of MS/MS performance, and eliminates the need to perform liquid–liquid extraction (LLE) and solid-phase extraction (SPE) cleanup steps of previous methods (3, 16). The Turboflow cleanup is accomplished online in a matter of seconds and the analytes of interest, after cleanup of potential matrix interferences, are subsequently transferred to an analytical column for ultra-HPLC (UHPLC) separation before MS/MS analysis (17).
We recently introduced a method for screening for LSDs that uses Turboflow chromatography technology (18). In the current study, we modified and optimized the system for high-throughput screening of 6 different LSDs in a single assay. To demonstrate the technical feasibility and robustness of this novel method, we applied it within a comprehensive pilot screening program that runs up to several thousand samples in a routine newborn screening laboratory, including samples from affected patients. Moreover, the protocol was expanded for the screening of MPS I, and parallel measurements were performed in collaboration with the newborn screening (NBS) laboratory in New York state, New York State Department of Health, Albany, NY, for evaluation of its use with their Krabbe assay (9).
Material and Methods
Deionized water (18 MΩ) produced by a Millipore Milli-Q Reference A+ System. Methanol (#106035) and isopropanol (#109634) were purchased from Merck Chemicals. Acetone (#650501), formic acid (#94318), and trifluoroacetic acid (#T62200) were purchased from Sigma Aldrich. Acetonitrile (#A/0627/17), Cyclone P 0.5 × 50–mm Turbo Flow HTLC columns, and Hypersil Gold C8 2.1 × 50–mm, 1.9-μm particle size columns were purchased from Thermo Fisher Scientific. Microplates 96/U (#0030 601.203), microplates 96/F (#0030 601.106) and deep well plates (#0030 505.301) were purchased from Eppendorf.
After we received informed consent from the parents, we collected dried blood spots (DBS) consecutively from 8586 newborns during the national routine Austrian NBS program. In Austria, NBS is centralized and conducted by one single laboratory located at the Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, for the entire population (approximately 78 000 births/year). In addition, NBS samples and 51 samples from patients with known LSDs were analyzed anonymously for 6 different lysosomal enzyme activities beyond the current screening panel for endocrine and metabolic disorders, including cystic fibrosis (19). The study was approved by the local ethics committee (EK 478/2009) and conducted in accordance with the institutional guidelines. The expanded screening for LSDs included the analysis of acid β-glucocerebrosidase (ABG), α-galactosidase (GLA), α-glucosidase (GAA), acid sphingomyelinase (ASM), galactocerebrosidase (GALC), and iduronidase (IDUA) enzyme activities in the anonymized DBS samples.
Sample preparation was performed according to a 2-day working protocol. On day 1, the protocol for the incubation of DBS with enzyme reagent cocktails was adapted from Zhang et al. (3) for ABG, ASM, GAA, and GLA and from Blanchard et al. (20) for IDUA. The GALC protocol was modified from Orsini and his coworkers (9). All protocols for day 1 and 2 are described in detail in the Supplementary Materials 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol57/issue9. In brief, on day 1 aliquots of a single blood spot were incubated with enzyme-specific buffers containing substrates and internal standard for each lysosomal enzyme. On day 2, after an overnight incubation of 20–22 h, the enzymatic reactions for ABG, ASM, GAA, GLA, and IDUA were quenched with 100 μL stop solution (80 mL acetonitrile/19.8 mL water/0.2 mL formic acid). After an overnight incubation of 19.5–21.5 h (30-min shorter than the previous incubation), the GALC enzyme reaction was quenched with 100 μL stop solution, sealed with silicone covers, and incubated again (30 min, 37 °C, 750 rpm) to maximize the product outcome. Finally, all 6 assays were transferred into 1 single deep well plate, sealed with aluminum foil, centrifuged for 15 min at 3320g, and measured with the TLX2 system by using online sample cleanup. Table 1 provides a short overview of the Zhang et al. protocol with the use of LLE and SPE compared with the new simplified protocol.
Transcend UHPLC systems (part of the TLX-2) with Allegro quaternary pumps were used (ThermoFisher Scientific). The mobile phases were A: 0.1 mL formic acid/0.01 mL trifluoroacetic acid/99.89 mL water; B: 0.1 mL formic acid/0.01 mL trifluoroacetic acid/99.89 mL acetonitrile; C: 45 mL isopropanol/45 mL acetonitrile/10 mL acetone; D: 80 mL acetonitrile/19.8 mL water/0.2 mL formic acid. Mobile phase C was used as a wash solvent and mobile phase D was used to prevent air bubbles in the pump system. The injection volume was 10 μL. The Transcend system employs 2 HPLCs and two 6-port valves per channel, which were configured in the standard focus mode (17). The online sample cleanup and separation performed by use of the TLX-Turboflow System was recently described by Kasper et al. (18). The cleanup and chromatographic sequence are described in detail in the online Supplementary Materials 1.
MULTIPLEXED SAMPLE INTRODUCTION
The total time for the multidimensional TurboFlow UHPLC experiment was 4 min. The analytes of interest emerged from the analytical column at around 2.15 min in a window that was 1.30-min wide. MS/MS data acquisition started 2.15 min after injection and continued for 1.3 min until all analyte signals were recorded (see Fig. 1). In the current experimental setup, a dual-channel TLX-2 system was used (2 TurboFlow and 2 analytical columns). We used staggered injections, so that while one sample was being cleaned up on the Turboflow column another was being chromatographed into the MS/MS analyzer. Hence, the effective analysis time for each sample was <2 min instead of the 4 min required if samples were run serially.
MS was performed on a Thermo Scientific TSQ Quantum Ultra with an HESI-2 heated electrospray probe. We monitored single-reaction–monitoring transitions for the 6 products and their respective internal standards. A chromatogram that includes the retention and peaks for substrates, products, and internal standards for all 6 lysosomal enzymes is shown in Fig. 1. The MS settings for each compound of interest are provided in Table 2. In the final step, the amount of product was calculated from the ion abundance ratio of the product to the internal standard for a sample multiplied by the amount of added internal standard, time of incubation, and blood spot volume, according to the method reported by Li et al. (16). Blank values (obtained with extracts of 3.2-mm blood-free filter disks) were subtracted to give the final values of lysosomal enzyme activity.
DEVELOPMENT OF A HIGH-THROUGHPUT HEXAPLEX SCREENING ASSAY FOR LSDs
The multiplexed, multidimensional UHPLC MS/MS screening method was optimized (Table 2) without the need for time-consuming offline sample preparation such as LLE and SPE (see online Supplementary Material 1). The assays for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and MPS I disease were less laborious, which led to time savings of 4–5 h per 400 samples on day 2 (Table 1). In a first pilot study, all 6 lysosomal enzyme activities for 8586 newborns and 51 patients with known LSDs were analyzed (Table 3). The cutoff values were chosen as ≤3.2 μmol · L−1 · h−1 for Gaucher, ≤0.9 μmol · L−1 · h−1 for Niemann Pick A/B, ≤3.3 μmol · L−1 · h−1 for Pompe, ≤2.9 μmol · L−1 · h−1 for Fabry, and ≤0.7 μmol · L−1 · h−1 for MPS I disease, respectively, according to the 0.1 percentile of the normal population. The cutoff for Krabbe disease was below 20% of the daily mean activity according to Orsini et al. (9). We did not observe any statistical difference in lysosomal enzyme activities between single and multiplex analysis with the use of the combined online sample cleanup and the separation of substrates and products with an analytical column (see online Supplemental Fig. 1).
DETECTION OF AFFECTED PATIENTS WITH LSDs
For method evaluation, we analyzed a total of 51 patients with known LSDs (12 patients with Pompe, 13 with Gaucher, 22 with Fabry, 1 with Niemann-Pick A/B, 1 with Krabbe, and 2 with MPS I disease). All disease control patients had diminished enzyme activities that showed little or no overlap with enzyme activity values obtained from the normal reference population (Table 3; also see online Supplemental Table 3).
METHOD COMPARISON FOR THE KRABBE DISEASE ASSAY
The GALC enzyme activities for 176 newborns were measured in parallel by the New York screening laboratory to evaluate the Turboflow assay (Table 4). The coefficient of determination (R2) between both methods was 0.79. The mean activity measured with the Turboflow assay was lower, but SDs and CVs were similar (Table 4). Both assays clearly differentiated the affected and nonaffected newborns.
LSDs result in the accumulation of macromolecular substrates that would normally be degraded by lysosomal enzymes. The combined incidence has been estimated at 1 per 7700 live births for whites (21, 22). The relatively high combined incidence, availability of effective therapies for some of these diseases, and dire clinical outcomes for lack of timely treatment justify implementation of LSDs into routine NBS protocols. Substantial medical challenges still remain for the development of successful therapies for many of the other LSDs, as well as challenges regarding the availability of those therapies that are successful (23).
The screening for several LSDs simultaneously in a single sample can be time-consuming, expensive, and work-intensive without the use of highly automated online systems. Current limitations include the requirement to perform LLE and SPE cleanup, and the use of hazardous reagents such as ethyl acetate (1,–,3). The introduction of online sample cleanup that uses multidimensional chromatography essentially eliminates the use of such organic compounds from the screening of LSDs by MS/MS (Table 1), and reduces the need for large amounts of consumables (see online Supplemental Material 2).
The use of an analytical column in conjunction with an online cleanup column improves a multiplex approach by separating several enzymatic products from residual substrates. The first such protocol was described in a report by la Marca et al. (24), in which they suggested that online multidimensional HPLC sample preparation eliminated the need for laborious preanalytical steps with the use of a homebuilt multidimensional LC method in which a POROS cleanup column (Applied Biosystems) and an analytical C18 column were used.
A similar protocol that included the use of a Turboflow cleanup column was recently reported by our group (18); this protocol was expanded to a hexaplex assay for the screening of MPS I in the present work. A serious problem with some published LSD assays is the potential for interference of the enzyme product signal from excess substrate due to insource fragmentation (14, 15). In these previous studies interference was minimized by either detuning the ion source, i.e., purposefully making the instrument less sensitive, or adding off-line cleanup steps such as SPE. We found that the use of an online analytical column in conjunction with the Turboflow online sample cleanup column, and the higher resolving powers of UHPLC, completely eliminated such interference while keeping the total analysis time to <2 min per sample and facilitated the expansion of the screening panel. This expansion has practical value because new substrates for the screening for MPS II, IVA, and VI are on the horizon (4,–,7). To illustrate this point, we integrated the screening of GALC activity with our published pentaplex assay (18) and evaluated the resulting GALC activities in collaboration with the New York screening laboratory by performing parallel measurements. Although our GALC enzyme activities were lower than those measured by the New York laboratory, we could clearly differentiate the affected Krabbe patient and healthy newborns [patient's GALC activity was below the cutoff for retesting of <12% according to Orsini et al. (9)]. The lower GALC activity might be due to slightly different sample workup. These preliminary results demonstrated that both assays are comparable, but further studies with more positive controls are required.
We minimized a potential source of ion suppression and residual substrate interference due to insource fragmentation by using the analytical column to separate the substrate from the product. Other sources of ion suppression such as salt, proteins, and phospholipids were eliminated by using Turboflow chromatography (18).
The purchase of a commercial 2-channel online sample cleanup system (TLX-2) is approximately €150 000, and screening for LSDs requires one additional mass spectrometer for the high-throughput analysis of approximately 100 000 samples per year. A detailed overview of cost calculations for both LLE and SPE screening and a Turboflow assay is provided in the online Supplementary Materials 2. The latter protocol has lower run-time costs (<€0.80) and fewer personnel requirements, and does not require the purchase of liquid-handling stations for automated processing of SPE.
One limitation of the current protocol is the separate incubations on day 1, which require several additional microtiter plates and sample handling. However, Gelb et al. recently demonstrated that these preanalytical steps can be simplified by combining incubation buffers for Pompe, Fabry, and MPS I diseases; hence additional time and expenses for consumables can be saved (2).
With a biochemical assay it is not possible to discriminate between pseudodeficiency genes that result in low enzyme activities and genes that lead to a severe phenotype. Prospective screening for LSDs has to be accompanied by genetic confirmatory testing [e.g., Krabbe screening in New York state (9)]. Results of this and other studies have demonstrated that enzymatic assays that use MS are robust and accurate for the detection of known symptomatic LSD patients (2, 11, 12). The rarity of some of these diseases will, however, necessitate more extensive population-based studies to accurately evaluate the true frequency of both false-negative and false-positive results that occur with the use of this biochemical screening approach.
Quality of screening is an important issue, and hence certified substrates and QCs must be produced under good manufacturing practices with regulatory approvals. Quality assurance programs, e.g., the Newborn Screening Quality Assurance Program at the CDC (Atlanta, GA) provides QC standards that are essential to maintain a high quality of screening (25). Worldwide collaborative projects such as the Region 4 Stork website (http://www.region4genetics.org) are necessary to ensure safety and consistency of screening between states and even within countries (26).
The availability of suitable treatments for some of these disorders has resulted in increased efforts to develop new, reliable, and robust methods that can be used to perform high-throughput population screening through established NBS programs worldwide. New protocols and technologies are now available that permit higher efficiency (2, 27, 28). Nonetheless, the current experience of nationwide screening for LSDs is limited. Several small pilot projects have been started, but whole-population LSD NBS has been implemented on a routine basis in only 2 jurisdictions. Screening for Pompe disease was introduced in Taiwan because of the high population prevalence of this disease (8), and screening for Krabbe disease was started in 2006 in New York state (29) because hematopoietic stem cell transplantation during the newborn stage demonstrated improved outcomes (30).
We are faced with rapidly advancing technical possibilities and industry-driven development of enzyme replacement therapies that are still limited to just a small number of LSDs, a situation that is raising controversial discussions worldwide. These disorders were low-priority targets in the American College of Medical Genetics expert panel, in part because detection was not considered feasible, cost-effective, and simple (31). This viewpoint has changed owing to recent developments regarding high-throughput screening technologies (3, 27, 28) that allow simplified, less laborious, and more cost-effective (<€1 per sample) multiplex screening of several LSDs from a single blood spot.
We are grateful to Joseph DiBussolo and Jeffrey Zonderman of Thermo Scientific for support and technical assistance with mass spectrometry, Hui Zhui and Victor de Jesus of the CDC, and Joan Keutzer of Genzyme.
↵† Thomas F. Metz and Thomas P. Mechtler contributed equally to the work, and both should be considered as first authors.
↵7 Nonstandard abbreviations:
- tandem mass spectrometry;
- lysosomal storage disorder;
- liquid-liquid extraction;
- solid-phase extraction;
- turbulent flow chromatography;
- dried blood spots;
- newborn screening;
- acid β-glucocerebrosidase;
- acid sphingomyelinase;
Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest:Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: B. Shushan, Thermo Scientific.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Grant from the Austrian Ministry of Health (GZ 20.501/40-IV/A/2007).
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
- Received for publication March 1, 2011.
- Accepted for publication June 30, 2011.
- © 2011 The American Association for Clinical Chemistry
There where 3 questions wrong due to missing answers for all that apply. Here they are:
Infant Care at Birth
The nursery RN places the infant under a radiant warmer and starts to dry her quickly.
What is the rationale for these actions?
A) Heat production is increased through stimulation.
Drying the infant may increase heat production, but this is not the best method to correct the newborn's body temperature.
B) Convective heat loss from evaporation is reduced.
Drying the infant quickly and placing her under a radiant warmer reduces heat loss through evaporation and radiation.
C) Newborns in an incubator are more difficult to access than those in a radiant warmer.
Although a radiant warmer allows healthcare personnel easy access to the infant, that is not the reason for its use in this situation.
D) Bonding is promoted by enhancing the infant's appearance.
Drying and warming do not necessarily enhance the infant's appearance or promote bonding.
Which action should the nurse take prior to drying the infant's back?
A) Note if the infant has passed any meconium stool.
Although this observation is important, it is not related to drying the back.
B) Observe the sacral area for possible Mongolian spots.
Mongolian spots are normal variations in the pigment of the skin, and they do not hinder drying the back.
C) Assess the amount and location of vernix caseosa.
The amount of vernix caseosa is related to gestation age, but is not related to drying the back.
D) Inspect the back for possible neurological defects.
To prevent harm while drying the newborn, the back should always be inspected for possible neurological defects, like spinal bifida.
At 1 minute of age, the infant is crying and has a heart rate of 160 and a respiratory rate of 58. Both of the infant's arms and legs are flexed, and her hands and feet are cyanotic.
Which APGAR score should the nurse assign?
Review the findings again.
One point is deducted for acrocyanosis.
Review the findings again.
Review the findings again.
The nurse conducts a physical assessment of the infant looking for normal as well as abnormal findings.
Upon inspection of the umbilical cord, which finding should the nurse report to the healthcare provider?
A) The cord is covered with Wharton's jelly.
This is a normal finding.
B) Pulsations are felt at the base of the cord.
This is a normal finding.
C) One artery and one vein are present.
Two arteries and one vein should be present.
D) The cord is glistening with a pearl-like coloring.
This is a normal finding.
The Carson baby's head is molded from the vaginal delivery. Upon seeing the baby, Ms. Carson says, "Oh, she is so beautiful, but something is wrong with her head."
How should the nurse respond?
A) "No nothing is wrong with her head. She really is a beautiful baby."
This response does not fully address the mother's concern.
B) "'Yes, it is misshaped, but we will show you how to change it over time."
Parents can be taught to change an infant's sleeping positions to correct a misshaped head, but this is not the best response.
C) "Her head has been molded from delivery through the birth canal, which is normal."
Molding commonly occurs in babies delivered vaginally, and the head will become more symmetrical over time.
D) "I know you are concerned. Would you like to talk further with the midwife?"
Acknowledging Ms. Carson's feelings is a thoughtful response, but referral to the midwife is not necessary.
Ms. Carson is offered the opportunity to breastfeed. After securing a comfortable position for herself and the baby, Ms. Carson puts the infant to her breast. The baby latches onto the nipple, and with some encouragement, she begins to nurse. After a time of family interaction, Ms. Carson is taken to the postpartum unit, and the infant is transferred to the transition care nursery.
The nurse checks the identification bands for both the baby and the mother upon admission to the nursery. One ID number is incorrect.
Which action should the nurse take to solve this problem?
A) Document the presence of the incorrect number on the charts for the baby and the mother.
Although the nurse should probably document the discrepancy, it does not solve the problem.
B) Explain to the mother that there is an incorrect number on one of the bands.
Although an explanation should be given to the mother, that will not solve the problem.
C) Redo the identification bands with another nurse witnessing the process.
Identification bands must be correct to ensure the safety and security of all hospitalized clients, especially newborns.
D) Mark the incorrect numbers in red to denote the correction made to the bands.
This is not the proper action for the nurse to take to solve this problem.
Upon admission to the transition care nursery, the Carson baby's axillary temperature is 97.4° F.
Which action should the nurse take?
A) Continue monitoring and document this finding in the record.
Another action should be taken in this situation.
B) Place the infant in a radiant warmer and monitor her temperature.
The baby's temperature is not within normal range (97.5°-99° F). The infant should remain in the radiant heat warmer until her temperature has stabilized.
C) Remove a blanket from the infant and check the temperature again.
This action will make the situation worse, not improve it.
D) Notify the healthcare provider immediately about the temperature.
The nurse is capable of providing care to remedy this situation. It is not necessary to notify the healthcare provider.
While examining the infant's head, the nurse notes soft swelling of the scalp that extends across the suture lines of the fetal skull.
Which action should the nurse take in response to this finding?
A) Document the finding in the record.
This finding indicates caput succedaneum, which commonly occurs after a vaginal birth.
B) Monitor the tension of the anterior fontanel.
Although the anterior fontanel should be monitored, it is not related to this finding.
C) Report the finding to the healthcare provider.
It is not necessary to report this finding to the healthcare provider.
D) Apply cool compresses to prevent more swelling.
Applying cool compresses is not the proper action for the nurse to take.
The nurse notes a bluish discoloration of the skin across the infant's sacral area.
Which should the nurse do in response to this finding?
A) Assess the infant for cold stress.
An overall mottled appearance is usually more indicative of cold stress.
B) Refer the parent to the care of a pediatric specialist.
A referral to a pediatric specialist is not necessary as a result of this assessment.
C) Document this finding in the record.
This bluish discoloration of the skin is a birthmark, commonly referred to as Mongolian spots. They are merely a dense collection of normal skin cells deep in the skin. This is a common finding, which should simply be noted in the baby's record.
D) Evaluate the infant's neurological status.
It is not necessary to further evaluate the infant's neurological status based on this finding. However, tufts of hair or dimples in the sacral area might indicate a need for a more in-depth neurological evaluation.
Which physical finding, if present, should the nurse report to the healthcare provider?
A) Presence of unopened sebaceous glands.
These pinhead-size whiteheads on the newborn are referred to as milia, and they usually disappear without treatment. Their presence does not need to be reported.
B) Loose natal teeth that are not covered by the gums.
Natal teeth present at birth is an unusual occurrence that should be reported to the healthcare provider. Loose natal teeth are frequently removed to prevent aspiration.
C) White, cream cheese-like substance on skin.
This substance is vernix caseosa, which covers and protects the fetus from the amniotic fluid in utero. Because its presence on the infant at birth is normal, this finding does not need to be reported.
D) Enlarged breasts secreting a thin, watery discharge.
This temporary condition in the newborn is caused by the influence of the mother's hormones on the fetus prior to birth. The secretion is often referred to as witch's milk. This is a normal finding that does not need to be reported.
When examining the baby's extremities, which finding would warrant additional assessment by the nurse?
A) Toenails blanch with pressure and quickly refill.
This is a normal response that does not require additional assessment.
B) Feet that turn in, but can be manipulated to midline.
This is a normal finding that does not require additional assessment.
C) Hands are plump and clenched into fists.
This is a normal finding and does not require additional assessment.
D) Limited hip abduction in the supine position.
Because this finding could indicate developmental dysplasia of the hip, formerly known as congenital hip dislocation, additional assessment is warranted.
Which finding by the nurse is consistent with an infant born at 39 weeks gestation?
A) Presence of abundant lanugo hair across face and back.
A baby born at 39 weeks gestation has minimal lanugo hair, which is the soft prenatal hair that is shed during the last few weeks of pregnancy.
B) Plantar creases covering the entire sole of foot.
This finding is consistent with a baby born at 39 weeks gestation.
C) Head and Neck 25% of body surface
D) Slightly soft, curved pinna with slow recoil.
The ear of a baby born at 39 weeks gestation should be well formed and firm with instant recall.
E) Skin is smooth and pink with visible veins.
This finding is more consistent for an infant with an earlier gestational age.
Continued Transition Care
A nursing student is assisting the RN in caring for the infants in the nursery. The RN questions the student about vitamin K (Aqua MEPHYTON) as preparations are made for administration.
Which response by the student indicates an understanding of the purpose for administering this drug?
A) "The purpose of this drug is to prevent hyperbilirubinemia in the newborn."
Further teaching is needed because vitamin K does not prevent hyperbilirubinemia.
B) "Vitamin K is a fat-soluble vitamin and promotes a positive nutritional status."
This is not the primary reason for giving vitamin K.
C) "This drug is given to the newborn to prevent and/or treat hemorrhagic disease."
Because this vitamin does not cross the placenta and there is very little in breast milk, supplemental vitamin K should be given to newborns at birth to help clot the blood. Therefore, this is an accurate response by the student and no further client teaching is needed.
D) "Vitamin K is produced and stored in the liver, which is immature in the infant."
Vitamin K is produced in the gut, but stored in the liver.
The nurse is preparing to give the baby her first bath.
Which assessment data indicates that it is safe for the baby to be given her bath at this time?
A) Respiratory rate of 46.
This respiratory rate is high-normal and will rise with the activity of bathing.
B) Axillary temperature of 98° F.
A bath may potentially lower the temperature, which will not be harmful because the core temperature is near 99° F.
C) Apical heart rate of 160.
This heart rate is high-normal and will rise further with the activity of bathing.
D) Pulse oximeter of 90%.
This value is below normal and could become lower with the activity of bathing.
At 2400 hours the infant is crying, her skin is mottled, and her hands are shaking.
Which action should the nurse take first?
A) Assess the infant's respiratory efforts.
Assessing the respiratory efforts of the infant is unnecessary since the infant is crying, and crying is a good indicator of respiratory effort.
B) Monitor the blood glucose level.
Since it has been 2 hours since delivery, the infant may be experiencing hypoglycemia.
C) Give the infant some formula.
Because this infant is breastfeeding, an attempt should be made to let her nurse before offering the formula.
D) Evaluate for possible seizures.
The infant is not exhibiting any signs associated with seizures.
The baby's vital signs have stabilized by 0100 hours. Upon completion of assessment and documentation, the nurse takes the baby to Ms. Carson who wants to breastfeed and 'room-in' with the baby. After checking the ID bands, the infant is positioned for breastfeeding.
The nurse checks on Ms. Carson and the baby at 0200 hours. Both are asleep in the bed, with the baby lying beside Ms. Carson.
What should the nurse do next?
A) Pick up the baby and return her to the crib while letting Ms. Carson sleep.
Although returning the baby to her crib is the proper response, this action is incomplete.
B) Wake Ms. Carson and remind her that keeping the baby in the bed is unsafe.
Although waking Ms. Carson is the proper response, this action is incomplete.
C) Tell Ms. Carson that the baby must be returned to the nursery for safety reasons.
Although the baby can be returned to the nursery if Ms. Carson is too tired to care for her, there is a better response in this situation.
D) Remind Ms. Carson about infant safety and assist her to place the infant in the crib.
This action protects the baby while reinforcing teaching to the mother.
When returning the baby to the crib, the nurse notices that the blanket covering the baby is loose, and the cap is off her head. The nurse takes the baby's temperature, which is 97.6° F.
Which should the nurse do next?
A) Bundle the baby and place the cap on her head.
Although this would help the baby, another action is more effective.
B) Cover the baby with a blanket, but leave the cap off.
This action is incomplete.
C) Show Ms. Carson how to wrap the baby for warmth and apply the cap to her head.
This action not only protects the baby, but also involves and teaches the mother.
D) Immediately take the baby and place her under a heat source.
Other actions should be done first.
The nurse checks on Ms. Carson and her baby every 2 hours throughout the night. The baby is breastfed at 0300 and 0600 hours without difficulty. After the change of shift report at 0700 hours, the day nurse assesses the mother and baby.
Ms. Carson states that the baby had a bowel movement after breastfeeding. She tells the nurse that she attempted to change the diaper, but had difficulty doing so.
What action should the nurse implement?
A) Reassure Ms. Carson that she will get plenty of practice.
Ms. Carson's statement implied a need that should be addressed by the nurse.
B) Observe Ms. Carson as she performs a diaper change.
This approach helps the nurse evaluate the problems Ms. Carson is experiencing so the most effective teaching can be provided.
C) Place the baby on the bed and demonstrate how to change a diaper.
Another action is more effective to meet the client's needs.
D) Tell Ms. Carson that the nurses can change the diapers until they go home.
Although the nurses may assist with diaper changing, the mother's involvement in the care of her baby is essential.
E) Advise Ms. Carson that classes to teach infant care are provided on the unit
When Ms. Carson removes the diaper, the nurse notices that the baby has caked powder in the inguinal leg folds and vulva areas.
What action should the nurse take?
A) Show Ms. Carson how to remove the caked-on powder.
This should be done, but further instruction is needed.
B) Explore with Ms. Carson why powder was used.
This may provide some information, but does not correct the problem.
C) Praise Ms. Carson for wanting to keep her baby dry.
This response does not teach Ms. Carson about the proper use of powder on her baby.
D) Instruct Ms. Carson to use plain water instead of powder.
Until the baby is 4 days old, only plain warm water is recommended (after the initial bath) because soaps, ointments, powders, lotions, and baby wipes can disrupt the acid mantle on the skin and provide a medium for bacterial growth. Ointments are prescribed only if a rash develops in the first few days of life. Use of powder also places the infant at risk for fine particle aspiration.
While changing the diaper, Ms. Carson notices blood-tinged mucous in the vulva area and asks the nurse what is causing this with her baby.
Which explanation should the nurse provide?
A) "Your baby probably has the beginning of a urinary tract infection."
This finding is not consistent with a urinary tract infection.
B) "Apparently your baby had some trauma at birth to cause this."
There is usually a much more reasonable explanation for this finding. This response could cause the mother unnecessary anxiety.
C) "Withdrawal of maternal hormones is the usual cause of this occurrence."
This is called pseudomenstruation, which is due to the effects of maternal hormones.
D) "This is unusual, and I will notify the pediatrician about the mucous."
This is not an unusual occurrence.
Preparation for Discharge
At two days post birth, Ms. Carson and her baby are doing well and preparing for discharge. The baby's weight at birth was 7 lb 15 oz (3600 gms), and today she weighs 7 lb 3 oz (3300 gms).
Ms. Carson expresses her concern to the nurse when she realizes that her baby has lost almost a pound since birth.
How should the nurse respond?
A) "I can tell you are concerned. Would you like to talk with the pediatrician?"
She can certainly talk with the pediatrician, but the nurse can and should respond to this mother's concern.
B) "Yes, this is a concern. The pediatrician may want to keep the baby here for another day."
Based on the data regarding the baby's weight loss, it would not be necessary to keep the baby another day.
C) "Don't worry. Your baby will gain weight in a few days when your milk comes in."
This response offers false reassurance. In addition, it may lead the mother to believe that her breast milk is not adequate at this time, which is incorrect.
D) "Don't be concerned. Your baby's weight loss is in the typical range for all babies."
Babies may lose up to approximately 10% of their birth weight.
Ms. Carson is told that a neonatal screening test needs to be done before they are discharged.
When asked the reason for including the PKU test in the screening, which information should the nurse provide?
A) An error in metabolism of the amino acids leucine, isoleucine, and valine can cause death if not detected and treated early.
This describes another error in metabolism.
B) A problem converting the protein, phenylalanine, may be present, which can lead to mental retardation if not found and treated early.
PKU testing is done to detect the level of phenylalanine in the baby's blood.
C) Screening for an error in metabolism of the sugars galactose and lactose can prevent liver and brain damage in the newborn.
This describes a different error in metabolism.
D) This test detects the level of thyroxin produced by the thyroid. If too little is produced or if treatment is not started early, mental retardation can result.
This describes another metabolic disorder, not PKU.
How should the nurse collect the blood needed for PKU screening?
A) Clean the heel with alcohol swap, dry with gauze, and collect blood in a capillary tube.
A capillary tube is used to collect blood for hemoglobin, not PKU screening.
B) Puncture the lateral heel after warming and collect blood samples on the designated lab form.
The heel should be warmed, cleaned with alcohol, and dried with gauze. After puncturing the heel with a microlancet, blood is collected on a special neonatal screening form.
C) Collect heel blood using a transfer pipette and place a drop of blood on a reflectance meter.
This is the usual technique to collect blood for glucose analysis, not PKU screening.
D) After grasping the baby's lower leg and foot, use a microlancet to puncture the middle portion of the heel.
The middle portion of the heel should not be used.