Category Archives: AmnioStat-FLM


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 4oC 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.

Image 3

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.

Tests of Fetal Lung Maturity

This month the National Academy of Clinical Biochemistry (NACB) launched its blog, appropriately Slide1 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.