Cover Story

The ABCs of
PRE-, NEO-, and
POST-NATAL
TESTING

Scientists seeking answers for fetuses and infants

Edited by Carren Bersch, Editor

MLO invited David G. Grenache, PhD, D(ABCC), and Brad S. Karon, MD, PhD, to respond to questions regarding the topics of the presentations they made at AACC 2009 in Chicago recently. Those presentations are available on DVD through AACC at http://aacc.eventmediastore.com. Dr. Grenache is assistant professor of Pathology at the University of Utah School of Medicine, and medical director of Special Chemistry at ARUP Laboratories, both in Salt Lake City, UT. Dr. Karon, is assistant professor of Laboratory Medicine and Pathology, and director of the Hospital Clinical Laboratories, Point-of-Care Testing, and Phlebotomy Services at Mayo Clinic in Rochester, MN.

MLO: Briefly explain how you became interested in studying fetal lung-maturity testing.

David G. Grenache, PhD: When I was a post-doctoral fellow in clinical chemistry the laboratory called to tell me they had accidently centrifuged an amniotic fluid specimen that had been submitted to the lab for a fetal lung-maturity (FLM) test. As these specimens are not to be centrifuged prior to analysis, the specimen was clearly compromised but, being that it was amniotic fluid, it was not easily re-collected. I began to wonder what effect centrifugation and resuspension had on the result of the FLM test, so I set about doing a series of investigations that examined not only the centrifugation effect but also other preanalytical factors that might affect the test. The results of this study were published in Clinical Chemistry.

MLO: How often do changes occur in fetal lung-maturity testing? Based on your experience in fetal lung-maturity testing, what changes in equipment or tests do you believe might be implemented in the near future that would affect laboratory testing or testing interpretation?

Grenache: The first test of fetal lung maturity was the L/S (lecithin/sphingomyelin) ratio that was introduced in 1971. It was an important test because, until then, there were no tests to detect lung maturity.

Numerous tests for fetal lung maturity have been introduced since 1971 when the first test, the L/S ratio, was described; but there have really been no changes since 1988 when the LBC (lamellar body count) was first introduced. The most widely used FLM test is the S/A (surfactant/albumin) ratio that is commercially available from Abbott Laboratories; and the company has recently announced that it is retiring its "legacy systems," which include the TDx and TDxFLx instruments that perform the S/A ratio. Although no formal announcement has been made about the retirement of the S/A ratio, it would appear as though the writing is on the wall.

The loss of this test to the laboratory community would have profound effects for FLM testing. Labs that currently offer the test may need to find a replacement for it or begin sending FLM test requests to a referral lab. Since physicians expect FLM tests results to be returned quickly (<12 hours), sending specimens out is not an ideal solution. When considering a replacement test, there are a few options. If laboratories do not currently do the L/S ratio — and only ~15% of labs do — they might consider doing so, but the test is technically difficult to perform, requires considerable expertise, and is imprecise. The rapid detection of PG (phosphatidylglycerol) is an option, but PG is a late marker of lung maturity and the result is qualitative, so it is not an appropriate replacement for the quantitative S/A ratio. That really only leaves the LBC test and, in my opinion, that is probably the most suitable and practical replacement test.

MLO:What do you believe is the most important single thing a laboratory scientist should know about fetal lung-maturity testing in terms of the most up-to-date information you have derived from your studies — and why? How does this impact the medical laboratory in light of its current tests and procedures?

Grenache: As the lamellar body count is the most appropriate replacement test for the S/A ratio, as I just mentioned, then laboratorians need to be aware of its strengths and limitations. Consensus guidelines for the LBC have been published (Obstet Gynecol. 2001;97:318), which suggested a maturity cutoff of 50,000 LBC/µL, but Ann M. Gronowski, PhD, an associate professor at the Department of Pathology and Immunology Washington University School of Medicine in St. Louis, MO, and her colleagues have shown that this cutoff is not appropriate for all brands of cell counters (Clin Chem. 2003;49:995-997).

Labs wishing to offer the LBC will obviously need to do a thorough validation, because it is a lab-developed test, and the issue of a maturity cutoff is very important aspect of that validation. Fortunately, the cutoff recommended in the guidelines was derived from a Beckman-Coulter (BC) cell counter, and studies indicate that concordance among all BC-brand counters, regardless of model, are quite good. If a lab is using a BC-type counter, then the 50K maturity cutoff is likely appropriate.

MLO: If not in fetal lung-maturity testing, in what other area(s) of pre-natal testing do you foresee improvements in equipment or changes in tests that would affect laboratory testing or testing interpretation?

Grenache: Various issues surrounding hCG (human chorionic gonadotropin) tests, both quantitative and qualitative, have been getting a lot of attention lately. Like most immunoassays for heterogeneous molecules, hCG assays are not standardized. Recent work by Sturgeon and colleagues has demonstrated the variability of results between hCG assays (Clin Chem. 2009;55:1484) — and also in an editorial I co-authored with Dr. Gronowski (Clin Chem. 2009;55:1447) — by identifying the analytical specificity of hCG assays. Similar studies in my lab have confirmed Sturgeon’s report, and Dr. Gronowki and my report is currently under review.

I foresee that there will be improvements in hCG assay standardization, and this will certainly improve the clinical utility of hCG assays, not so much for pregnancy diagnosis but for oncology applications (which, in the United States, are off-label uses) where serial measurements of hCG are common.

As a side note, work on hCG assay standardization was begun in 1994 when the International Federation of Clinical Chemistry and Laboratory Medicine established a group to work on that issue. If successful, it would serve as a model for other protein hormones as well.

The qualitative detection of hCG in urine-using rapid point-of-care (POC) tests are also in need of improvement. A few years ago, I reported that qualitative hCG tests could detect hCG variants that were unexpected (Clin Chem. 2007;53:989). By design, these tests should only detect the dimeric hormone; but, surprisingly, some of them can detect non-intact variants. A recent report from Dr. Gronowski and colleagues has now shown that false-negative results can occur when the urine sample contains high concentrations of an hCG variant called the "beta core fragment," which is the major hCG variant in urine from the fifth week of gestation to term. False-negative hCG results are particularly dangerous because they suggest the absence of pregnancy, and the mother may undergo a procedure or treatment that is potentially harmful to the fetus. Manufacturers of POC hCG tests need to address this issue to prevent this type of interference from occurring.

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MLO: Briefly explain how you became interested in neo-natal studies, particularly hyperbilirubinemia.

Brad Karon, MD, PhD: I received an e-mail from my colleague, Walter J. Cook, MD, in Pediatrics who was interested in the idea of transcutaneous bilirubin-screening but had no idea how to conduct a device or method validation. Our initial communication has now led to two clinical studies and a clinical protocol in our nursery for transcutaneous bilirubin screening. It has been an enjoyable and rewarding collaboration with Dr. Cook. In addition, it is satisfying to see a new clinical protocol in our large practice being developed directly from data that we obtained together.

MLO: What do you believe is the most important single thing a laboratory scientist should know about that in terms of the most up-to-date information you have — and why?

Karon: We need to continue to raise the level of awareness about the risk of kernicterus in neonates if we do not employ systematic risk-assessment protocols for hyperbilirubinemia in our hospitals and nurseries. For laboratorians, the most important message is probably to find a way to communicate bilirubin values, whether serum/plasma or transcutaneous, in ways that allow clinicians to interpret them with regard to the infant’s post-natal age in hours. Age-specific nomograms or protocols exist or can be developed for both laboratory and transcutaneous bilirubin — and we need to collaborate with our clinician colleagues to make sure we give them enough information to do this.

MLO: How often do changes occur in this area of neo-natal testing? Based on your experience, what changes in equipment or tests do you believe might be implemented in the near future that would affect laboratory testing or testing interpretation?

Karon: The biggest challenge I see for implementation of transcutaneous-screening protocols is the variability in the relationship between laboratory (serum/plasma) and transcutaneous bilirubin. This is clearly due to variability in laboratory methods and procedures and may be due to variability in the performance of transcutaneous devices, though there is no good way to obtain data on this currently. We need to move forward with better standardization of laboratory bilirubin assays and come up with a simple way to perform quality-control monitoring for transcutaneous devices. Right now hospitals or healthcare networks that would like to implement a transcutaneous bilirubin-screening protocol cannot predict how these values will relate to the laboratory measurement of bilirubin already being performed. Every study published seems to obtain a different relationship between transcutaneous and serum bilirubin, so changes occur often. I would say the most recent change has been the gradual acceptance of the concept that this relationship will likely vary by institution.

MLO: In what other area(s) of neo-natal testing do you foresee improvements in equipment or changes in tests that would affect laboratory testing or testing interpretation?

Karon: There are several groups interested in looking at genetic determinants of bilirubin rise after birth, something that could add to the tools already available to predict which infants will need close monitoring or treatment for hyperbilirubinemia. Right now, it is not clear whether better tests or better systems are necessary to prevent kernicterus; though, if I had to guess, I would say better systems are needed.

MLO: In the "history" or "timeline" of neo-natal testing, when did the particular subject matter that commands your attention initially arise; and where, in terms of importance in that timeline, does your particular interest lie?

Karon: Kernicterus has been around for centuries, though interest in this waned significantly in the 1970s and 1980s after the routine introduction of RhoGAM to prevent alloimmunization of Rh-negative mothers. Interest in kernicterus re-emerged in 2004 with the publication of new guidelines by the American Association of Pediatrics (AAP). The AAP guidelines basically pointed out that with shortened maternal and, thus, infant, admissions, healthcare systems had not produced effective ways to evaluate infants for the risk of dangerously high bilirubin levels that might develop after discharge from the nursery. My interest developed in 2004 and beyond with the new recommendations.

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Exsanguinating infants: laboratorians seeking limits

By Dennis Ernst, MT(ASCP)

Concern over excessive volumes of blood withdrawn from newborns and infants is prompting facilities to adopt policies to monitor total blood volumes that reduce the chance of phlebotomy-induced (i.e., iatrogenic) anemia. Removing as little as 10 mL of blood from a newborn can result in up to a 10% depletion of the infant’s total blood volume.¹ Because newborns have between 80 mL and 110 mL of blood per kilogram of body weight, those subject to multiple sampling during the first week of birth (e.g., severely jaundiced newborns) are especially vulnerable to complications.¹

In one study, researchers concluded that phlebotomy overdraws were responsible for up to 15% of the packed-cell transfusions given to very low birth rate infants.² An article published in Clinical Leadership & Management Review reports of a study conducted in Denmark that showed blood losses from diagnostic sampling constitute up to 45% of the total blood volume of infants studied.³

This lack of clear-cut standards on maximum sampling volumes, especially for premature infants and neonates, continues to frustrate phlebotomists and their managers who want to minimize the impact of the procedure on those most susceptible to phlebotomy-induced anemia.

Further, a study reported in Laboratory Medicine shows that patients lose 4 mg of iron for every 10-mL tube of blood drawn.⁴ Therefore, not only do newborns and infants suffer from red blood-cell depletion due to multiple sampling, but [also] the risk of developing iron deficiency over time increases dramatically, [thus] increasing the impact of blood-volume depletion.

According to the Clinical and Laboratory Standards Institute’s document H3-A4, Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture, "A mechanism should be in place to monitor the amount of blood drawn for pediatric and critically ill patients, in order to avoid phlebotomy-induced anemia."⁵

The College of American Pathologists prompts volume considerations in its accreditation checklist by asking a question about the lab’s review of "phlebotomy practices to minimize unnecessarily large blood draw volumes."³

Two publications suggest limitations on the volumes of blood to be collected from pediatric patients. Phlebotomy Handbook contains a chart that places limits on quantities withdrawn both per collection and per admission on pediatric patients from six to 100 pounds (2.7 kg to 45.5 kg). According to the authors, the recommended limit for an infant who weighs between six and eight pounds (2.7kg to 3.6 kg) is 2.5 mL per draw and 23 mL per hospital stay, up to one month.

A chart published in the CLSI Document H4-A6 shows that 10 mL of blood constitutes 7.9% of the total blood volume in a 1.1-pound premature infant.⁶ But guidance in the literature stops there, leaving it up to phlebotomists and their managers to devise their own tracking system and develop protocols when their limits are met.

More guidance is clearly needed from pediatricians and researchers so that laboratories can continue to provide critical test results without threatening the already-fragile physiologies of neonates and geriatrics.

Several sources provide assistance in establishing blood-sampling limits based on body weight and on estimating circulating erythrocyte volumes. For adults, blood volume can be calculated by applying the general assumption that a patient’s average blood volume is equal to 70 mL per kilogram of weight.³ Full-term neonates have a blood volume of 80mL/kg to 110 mL/kg, while the volume for premature infants is 115 mL/kg.¹

Using these statistics, a patient’s circulating erythrocyte volume can be calculated by multiplying the blood volume with the hematocrit. For example, a full-term infant weighing 2.7kg (six pounds) has an estimated total blood volume of 229 mL. If the infant’s hematocrit is 55%, the erythrocyte volume is 126 mL (229 mL x .55).

If the facility caring for the infant adopts a policy not to withdraw more than 7% of any patient’s total erythrocyte volume in any 24-hour period (arbitrary), then a calculation of the maximum allowable whole blood volume that can be removed from the infant without extracting more than 7% of the RBCs can be made.

Monitoring the volume of blood withdrawn as CLSI recommends can be accomplished with a log sheet attached to the infant’s chart upon which phlebotomists and nurses can record volumes withdrawn or by recording volumes in the laboratory records. Regardless of the approach, all mechanisms have the potential to breakdown if all who collect specimens in the facility do not comply.

Even with full compliance, however, tracking serves no purpose unless limits are established on volumes sampled, and a protocol is adopted and implemented when those limits are met or exceeded. Because no current guideline exists in the literature, such a protocol takes a coordinated effort between the laboratory, nursing, and medical staff, and must begin with thorough research on the current thinking about newborn and premature-infant blood volumes and the degree of blood loss the facility is willing to establish as a limit.

This lack of clear-cut standards on maximum sampling volumes, especially for premature infants and neonates, continues to frustrate phlebotomists and their managers who want to minimize the impact of the procedure on those most susceptible to phlebotomy-induced anemia.

More guidance is clearly needed from pediatricians and researchers so that laboratories can continue to provide critical test results without threatening the already-fragile physiologies of neonates and geriatrics. Until standards emerge, facilities will continue to face the challenge of setting their own limits on sampling volumes.

Reprinted with kind permission from Dennis J. Ernst, MT(ASCP), the director of the Center for Phlebotomy Education in Corydon, IN, and MLO editorial advisory board member.

References

1. Becan-McBride K, Garza D, Phlebotomy Handbook. Upper Saddle River, NJ: Pearson; 2010.
2. Lin J, Strauss R, Kulhavy J, Johnson K, Zimmerman M, et al. Phlebotomy overdraw in the neonatal intensive care nursery. Pediatrics. 2000;106(2).
3. McPherson R. Blood sample volumes: emerging trends in clinical practice and laboratory medicine. Clin Leadership Mgmt Rev. 2001;Jan/Feb:3-10.
4. Q&A. Blood volumes needed for common tests. Lab Med. 2001;4(2):187.
5. Clinical and Laboratory Standards Institute. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—Sixth Edition. Wayne, PA: CLSI; 2007. CLSI Document H3-A6.

Clinical and Laboratory Standards Institute. Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard—Sixth Edition. Wayne, PA: CLSI; 2008. CLSI Document H04-A6.

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Trends in low-weight and premature-infant nutrition

Creamatocrit testing

Mother’s milk is the preferred feeding for low-weight, premature infants because of its unmatched nutrition, immunological, and anti-inflammatory properties. Because the caloric density of mother’s milk varies greatly, neo-natal intensive care units are performing creamatocrit testing in order to measure the milk’s fat content and estimate the calories. Not only does this test provide a baseline of what the infant is getting, certain processes can be employed to raise the fat and calorie content, if desired. These processes include determining the time a mother must pump in order to express the calorie-dense hindmilk, capturing the lower-volume, higher-calorie milk that generally occurs late in the day, correcting storage techniques, and so forth.

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Non-invasive prenatal blood test

A non-invasive pre-natal blood test could detect whether a fetus will have genetic disorders such as cystic fibrosis or sickle cell anemia, according to a study published online in the Proceedings of the National Academy of Science, the Wall Street Journal reports. Pre-natal diagnoses of such disorders currently are possible through amniocentesis, a procedure that involves inserting a needle into the uterus and carries a small risk of miscarriage, according to the Journal.

New tests could be developed because of a discovery by researchers at the Chinese University of Hong Kong who found fetal DNA circulates in maternal blood, Reuters reports. Digital blood-testing technology was used to count abnormal DNA sequences in the pregnant woman’s plasma to determine the number of abnormal genes inherited by the fetus and the fetus’ probability of developing a genetic disorder. Also notes was that the accuracy of the test depends on the concentration of fetal DNA in maternal blood. The test currently is expensive and inefficient, but is offered as "proof" that the technique can be used to diagnose fetal disorders.

Reprinted with kind permission from www.nationalpartnership.org. The National Partnership for Women and Families, published by The Advisory Board Company, offers the Daily Women’s Health Policy Report as a free service that includes archives and e-mail updates.

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The future of post-natal testing and newborn screening

Uniform panels in the making

MLO approached Piero Rinaldo, MD, PhD, professor of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, MN, for information about newborn screening. All 50 states screen newborns for various core conditions like hearing, congenital hypothyroidism, sickle-cell anemia, and a host of others.

According to Dr. Rinaldo, since the 2006 recommendations of the American College of Medical Genetics (Toward a Uniform Screening Panel and System written by Rinaldo with Michael S. Watson, PhD; Marie Y. Mann, MD, MPH; Michele A. Lloyd-Puryear, MD, PhD; and R. Rodney Howell, MD, editors), "the degree of implementation of a uniform panel of 29 conditions is practically complete."

“In fact, more than 98% of U.S. births are screened for that panel, and most are also screened for the 25 secondary targets. It will be 100% before the end of the year,” Rinaldo notes.

"In fact, more than 98% of U.S. births are screened for that panel, and most are also screened for the 25 secondary targets. It will be 100% before the end of the year," Rinaldo notes.

His interests and training in pediatrics and human genetics combine to support his ongoing studies of what he terms "the next generation of newborn conditions, including SCID (severe combined immunodeficiency), LSD (lysosomal storage diseases, e.g., Pompe disease and Krabbe disease), and hyperbilirubinemia."

Much of Dr. Rinaldo’s expertise is contained in his writings. Included among his publications are 39 items listed on PubMed, a service of the National Library of medicine.

The listing of these articles can be found online at: www.ncbi.nlm.nih.gov/sites/entrez?cmd=PureSearch&db=pubmed&term=%28rinaldo%20p%5BAuthor%5D%20AND%20mayo%5BAll%20Fields%5D%29. Among them are:

• Newborn Screening of Metabolic Disorders: Recent Progress and Future Developments, written by Piero Rinaldo with James S. Lim, Silvia Tortorelli, Dimitar Gavrilove, and Dietrich Matern: Nestle Nutr Workshop Ser Pediatr Program. 2008; 62:81-93; discussion 93-96.

According to the description of this article, with the development of tandem mass spectrometry (MS/MS), more than 40 conditions could be detected by a single test. Thus, the evolution of newborn screening has grown until the panel of conditions recommended by the American College of Medical Genetics included, at the time of the article, 20 primary conditions and 22 secondary targets, detectable by MS/MS.

• Making the case for objective performance metrics in newborn screening by tandem mass spectrometry, written by Piero Rinaldo with Saba Zafari, Silvia Tortorelli, and Dietrich Matern: Ment Retard Dev Disabil Res Rev. 2006;12(4):255-261.

This article focuses on the expansion of newborn-screening programs which might include multiplex testing by MS/MS. The authors propose particular targets as evidence of adequate analytical and post-analytical performance, based on their experience with performance metrics in a program limited to MS/MS testing.

• Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots by Piero Rinaldo with Coleman Turgeon, Mark J. Magera, Pierre Allard, Silvia Tortorelli, Dimitar Gavrilow, Devin Oglesbee, Kimlo Raymond, and Dietrich Matern: Clin Chem. 2008 Apr;54(4):657-664. Epub 2008 Feb 15.

This article focuses on tyrosinemia type 1, a disorder that can cause early death if not treated. Because screening newborns for TYR1’s diagnostic marker is problematic, the authors developed a new assay using dried blood spots in concert with flow-injection MS/MS as a solution.

See page 18 for the National Newborn Screening and Genetics Resource Center’s Suggested Uniform Panels for the United States.

NOTE: CE Test questions include all articles of this month’s Cover Story section, except the product announcement.

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