The Pregnancy Lab

Diligence recommended during lamellar body count validation

Today's post is by a guest author, Patrick Kyle, Ph.D.  Dr. Kyle is the Director of Analytical Toxicology and an Associate Director of Clinical Chemistry at the University of Mississippi Medical Center in Jackson, MS.  He discovered a curious phenomenon when the lamellar body count is performed on a specific cell counter and he shares his observations here.  A report of this phenomenon has been published Clinical Chemistry and Laboratory Medicine.

The lamellar body count (LBC) is a relatively recent assay used for determination of fetal lung maturity (FLM) that has been discussed in previous posts (here and here) on this blog.

As with any laboratory-developed test, laboratorians should use the utmost care during LBC validation.  As reported here, during recent validation protocols in my laboratory, striking imprecision was noted in LBC values acquired from human amniotic fluid using a Beckman Coulter UniCel DxH 800.  This was unexpected because the manufacturer’s previous model (LH 750) had always yielded good precision (<10.0 CV%) with LBC.  In hopes that the problem was confined to a single instrument, the LBC results of amniotic fluid acquired from three DxH 800 platforms, a Beckman Coulter 750, and a Sysmex XE-5000 were compared.  Each of the five instruments was used to analyze two pools (low and high concentrations) of human amniotic fluid twice per day for ten non-consecutive days.  During the course of the experiment, samples were stored at 4^oC and were never centrifuged or frozen.  Each day of analysis, samples were allowed to come to room temperature then inverted 5-10x immediately prior to analysis.  Each instrument aspirated sample from the same tubes during the same days.

Aberrantly low counts were randomly produced with each DxH 800 instrument, whereas the XE-5000 and LH 750 produced consistent counts.  The aberrant counts were consistently 25-50% lower than target values obtained on the Coulter LH750 and Sysmex XE-5000.  The coefficients of variability (CV%) ranged from 28.1-45.3% for the three DxH 800 instruments and were considerably higher than those of the Beckman LH750 (6.1-7.0%) and Sysmex XE-5000 (4.4-8.0%).

Interestingly, a review of the daily quality control values obtained with each instrument using three concentrations of manufacturer-specific controls revealed less than 10 CV% with each instrument.  This seemed to indicate that the DxH 800 instruments were performing as designed.  Therefore, LBC proficiency test (PT) data was examined in order to compare the results of these DxH 800 platforms to those of other laboratories.  My laboratory’s College of American Pathologists 2011 LBC-B survey results were all acceptable.  As a whole, the results of the DxH 800 group were comparable to those of other Coulter instrument groups.  Most importantly, the standard deviations of the DxH 800 group results were comparable to that of other instruments and exhibited <2.0 CV%.  Because this was inconsistent with the imprecision described above, the matrix of the PT samples was questioned.  When asked, the College of American Pathologists indicated that the PT samples were composed of synthetic amniotic fluid to which porcine platelets had been added.  In other words, the PT samples had tested the instruments’ ability to count porcine platelets, not their ability to count lamellar bodies.   

The manufacturer’s instrument literature was reviewed in order to investigate the source of the issue.  The DxH 800 incorporates Beckman Coulter’s new “Data Fusion” software, which allows intercommunication between flow cells to automatically correct values when specific morphologies are detected.  For example, lymphocyte counts are automatically corrected upon detection of giant platelets.  The DxH 800 includes particles from 2-25 fL in the platelet counts.  Particles less than 2 fL are categorized as debris, whereas particles >20 fL are categorized as giant platelets.  The DxH 800 histograms of EDTA blood (A) and amniotic fluid (B) below, reveal that the volumes of many lamellar bodies are smaller than those of platelets with many less than 2 fL.  Therefore, the aberrant values may have been caused by the limitations in platelet inclusion criteria (2 fL cutoff) and/or the algorithms applied by the Data Fusion technology.

This issue emphasizes a very important fact: the LBC test should be validated using actual amniotic fluid
samples.  In recent communications with two laboratories in separate states that are using the DxH 800 for LBC testing, I learned that they had not used human amniotic fluid for validation nor were they using it for quality control purposes.  One lab chose to use commercial hematology controls for validation due to the lack of commercial controls, and to avoid issues with sample stability.  Given this information, many laboratories may not be aware of this issue and its potential problems.

In summary, all laboratory-developed assays, such as the LBC test, should be rigorously validated.  Matrix appropriate materials should be used whenever possible.  Beckman Coulter representatives are aware of the LBC issue on the new DxH 800.  However, imprecise LBC test results may preclude use of the DxH 800 for this assay.

CLSI publishes guideline on the assessment of fetal lung maturity by the lamellar body count

I’ve blogged about fetal lung maturity (FLM) tests before but this is exciting news!

The Clinical and Laboratory Standards Institute (CLSI) has just published a document that provides guidance to labs that wish to perform the lamellar body count as a test for fetal lung maturity. Disclaimer: I participated in creating this guideline.

So why is this exciting news? Currently, the most widely used FLM test is one made by Abbott Diagnostics called the “TDx Fetal Lung Maturity II” test. It’s popular because it’s commercially available, it can be performed quickly, it’s precise, and it’s an excellent predictor of fetal lung maturity. Abbott is the only in vitro diagnostic company that makes this test and a couple of years ago they announced that they would stop doing so at the end of 2011. Labs that perform this test have been left wondering what test they would replace it with. While the lamellar body count is the most logical option, it’s not a well-known test and there are some issues that have to be considered.

One of the biggest hurdles facing labs that wish to offer the lamellar body count test is the fact that it’s a laboratory developed test. The test is performed on FDA-approved automated blood cell counters but the manufacturers of those cell counters have never sought FDA approval for using them to count lamellar bodies in amniotic fluid. Lack of FDA approval doesn’t mean that the test can’t be performed because FDA doesn’t regulate clinical laboratories. In the U.S., The Centers for Medicare & Medicaid Services regulates lab testing performed on humans through the Clinical Laboratory Improvement Amendments (CLIA). CLIA requires that all clinical tests be validated before they are used but the requirements for a laboratory developed test are more stringent than they are for FDA-approved tests.

Many labs are not accustomed to validating laboratory developed tests because they only perform those that are FDA-approved. After Abbott announced the retirement of their FLM test it became clear that labs would need some sort of guidance if they wanted to offer the lamellar body count tests as a replacement. In 2009 I proposed to CLSI that a guideline document on this topic be created. The proposal was approved and several well-qualified volunteers stepped up to help write it. Writing began at the end of 2010 and the final version was approved by CLSI earlier this month.

The new CLSI guidelines will help educate people about the lamellar body count test and it provides a framework that labs can use to validate the test for clinical use. According to a press release, the guideline 1) describes the use of automated cell counting to perform the lamellar body count test, 2) describes methods to assist in test verification and validation, and 3) describes methods to select an appropriate maturity cutoff.

Diabetes, fetal lung maturity, and the lamellar body count

I've written a few posts on diabetes during pregnancy lately so thought I'd stick with it a bit longer. This time, however, the focus is on the effect that maternal diabetes has on fetal lung maturity.

It's generally believed that fetal lung maturity is delayed in women who have diabetes (gestational or otherwise). The reasons for this problem are not well understood, however high concentrations of glucose and insulin in the fetal blood have been postulated as a possible cause. Despite this, respiratory distress syndrome rarely occurs in term infants born to mothers with diabetes. Still, the issue of delayed fetal lung maturity and diabetes remains controversial.

I'll go on record for saying that there is no valid reason to perform amniocentesis solely to determine fetal lung maturity if maternal diabetes is well-controlled. However, if mom's diabetes is poorly controlled, then there may be value in performing fetal lung maturity tests. I'd like to emphasis the "may" in that last statement. As I've written before, it seems like all tests of fetal lung maturity should become obsolete. Realistically, however, they probably aren't going to go away any time soon.

Because the most widely used test of fetal lung maturity is about to disappear forever, many clinical laboratorians are planning to offer the lamellar body count test as a replacement. I've been asked several times what, if any, effect that maternal diabetes has on the results of this test.

Not surprisingly, that issue has been addressed only by a few published studies. In one study from 2002, a mature result from the LBC result was believable regardless of diabetes status (note, however, that this study did not include any neonates with RDS so a true assessment of the performance of the LBC could not be made). Another study from 2009 determined that the results of an LBC test were not influenced by maternal diabetes although it did suggest that the test was still useful in preterm pregnancies and in poorly controlled maternal diabetes.

What the take-home message? Granted, the data are limited but it's probably no more necessary to perform the LBC test in women with diabetes than it is in women without the disorder.

What the loss of a widely used fetal lung maturity test will mean to doctors and labs

The test most widely used to assess fetal lung maturity is going away.  Earlier this year I wrote about the different laboratory tests that doctors use to determine if a fetus' lungs were mature.  One of those tests, the surfactant-to-albumin ratio, is marketed and sold by Abbott Laboratories as the TDx FLM II test.  Last year, they announced that they were discontinuing the manufacture of that test.

The main reason this test will no longer be made is that the instrument that performs the test is several decades old.  The TDx line of instruments is aging and the technology it uses is becoming outdated.  The decision to stop supporting the TDx makes sense but it does leave labs that offer the FLM II assay in a bit of a quandary.  The Abbott test performs well and is rapid and simple to perform.  Doctors are familiar with it and know how to interpret the results.  The test is an excellent predictor of fetal lung maturity and the risk of respiratory distress syndrome as a function of both gestational age and TDx FLM II result is known.  Labs that currently offer the TDx FLM II test and that wish to continue offering a test for fetal lung maturity have to figure out what to do.  Doctors will also have to learn what the loss of this test will have on their clinical practice.

I recently published the results of a survey that provide some insight as to what might be expected. Here is a summary of those data:

• Across the US, labs are performing fewer fetal lung maturity tests each year and the most widely used test is Abbott's TDx FLM II test.
• Although 25% of physicians indicated that their use of fetal lung maturity tests is decreasing, 90% of them said that they could not provide their current level of care without access to a test of fetal lung maturity.
• Without the TDx FLM II test, 65% of physicians would order the lecithin-to-sphingomyelin (L/S) ratio and 40% would order the lamellar body count (LBC).
• Fewer than 20% of labs offer the L/S ratio and even fewer offer the LBC.
• Laboratories could simply refer fetal lung maturity test requests to a lab that performs other lung maturity tests but doctors want results within 12 hours of ordering the test.  That expectation probably can't be realized if the test is not performed in the lab.

Of all the available tests that could potentially replace the TDx FLM II test, the LBC test is the most logical:

• The instrumentation required to perform the test is an automated cell counter and these are widely available in clinical labs.
• The test is performed rapidly and with high precision.
• Several outcome-based studies have demonstrated the diagnostic utility of the LBC and it performs as well as the TDx FLM II test.

When I'm asked by laboratorians what they should do when the TDx FLM II test goes away I tell them that they should consider the LBC test.  Of all the other tests of fetal lung maturity, the LBC, for the reasons outlined above, is the most logical replacement test.  I also caution them that time is running out and that NOW is the time to communicate with their physicians about the need for the change and to get their input.

It's worth re-stating that perhaps, just maybe, we could consider stop performing fetal lung maturity tests altogether.  Time will tell.

Tests of Fetal Lung Maturity

This month the National Academy of Clinical Biochemistry (NACB) launched its blog, appropriately called the NACBlog.  The first post is on the topic of fetal lung maturity tests and how the medical community might respond to the upcoming loss of a popular test of fetal lung maturity that is being discontinued by its manufacturer (disclaimer: I am the author of that post).

Seems like a good reason to talk about FLM tests here!  So, how can a lab test evaluate a fetus' lungs?

First the basics:

1. The lungs are one of the last organs to mature in a fetus.  In order for them to work properly after birth the alveoli (i.e. air sacs) have to open up and stay open once the baby takes its first few breaths.  This is not as easy as it seems because the inside lining of the alveoli has a thin coating of water and the surface tension of this water promotes their collapse.  A collapsed alveoli doesn't work very well!  Fortunately, our lungs secrete chemicals called surfactants that lower the surface tension of the water that coats the alveoli thus preventing the alveoli from collapsing.
2. Having enough of these surfactants in the lungs at birth is extremely important because that's
   
   when the baby has to make a transition from getting its oxygen from mom to getting it from the air.  If there's not enough surfactant then the alveoli may collapse and the baby may have a difficult time breathing.  When this does happen it's called respiratory distress syndrome of the newborn, or just RDS.
3. Lung surfactants begin to be made around the 25th week of pregnancy but there's usually not enough of it present to prevent RDS until the 37th week.  That means babies born prematurely are at greater risk of developing RDS than those born at term.
4. Because the fetus essentially "breathes" amniotic fluid in and out of its lungs, the amount of surfactant in the lungs can be determined by measuring surfactants in the amniotic fluid.  In the lab, there are a few ways we can do just that using a sample of amniotic fluid.

Several fetal lung maturity tests have been developed since the 1970's but only a few are still in use today:

• AmnioStat-FLM
  • This test looks for the presence of a lung surfactant called phosphatidylglycerol (usually just called PG).
  • This is an agglutination  test that uses antibodies to detect PG in amniotic fluid.  If PG is present then visible agglutinates (clumps of particles) can be seen and the fetal lungs are considered mature.
• Lamellar Body Count
  • In certain cells of the lungs, surfactants are packaged into granules called lamellar bodies and secreted from the cells into the alveoli.  This test actually counts the number of lamellar bodies in amniotic fluid.
  • The higher the lamellar body count, the more likely it is that the fetal lungs are mature.
• Lecithin/Sphingomyelin Ratio
  • This was the first test of fetal lung maturity ever developed and is more commonly known as the L/S ratio.  It's a measure of the ratio of two lung surfactants, lecithin and sphingomyeli, that's determined using a technique known as thin-layer chromatography.
  • Lecithin is the most important lung surfactant and provides the greatest surface tension-lowering properties of all the surfactants.  It increases dramatically in the last few weeks of pregnancy.  Sphingomyelin is a minor lung surfactant and that amount of it in the lungs stays about the same throughout pregnancy so it serves as a good baseline against which the increasing amount of lecithin can be compared.  A ratio that is 2.5 or greater is usually used to indicate lung maturity.
  • Many doctors consider this to be the "best" fetal lung maturity test but that is not true.
• TDx FLM II
  • This test measures the ratio of surfactant to albumin and so is sometimes called the S/A ratio.
  • The test relies on a technique known as fluorescence polarization and is the most widely used fetal lung maturity test; unfortunately it will no longer be available to clinical labs at the end of this year because the manufacturer has decided to stop making it.
  • The effect that the loss of this test will have on patients, doctors, and labs remains to be seen!

There a whole lot more to say about fetal lung maturity tests but those will have to wait for future posts.