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Lamellar Body Count and Fetal Lamellar Body Count and Fetal Respiratory Distress SyndromeRespiratory Distress SyndromeRespiratory Distress SyndromeRespiratory Distress Syndrome
Leo Serrano, FACHE, DLM(ASCP)Leo Serrano, FACHE, DLM(ASCP)cmcm
A M K H it l dA M K H it l dAvera McKennan Hospital and Avera McKennan Hospital and University Health CenterUniversity Health Center
Sioux Falls, S.D.Sioux Falls, S.D.
Conflict of InterestsConflict of Interests
There are no conflicts of interest to declare.There are no conflicts of interest to declare.
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Resources and CitationsResources and CitationsThere are several great resources for There are several great resources for information on Fetal Lung Maturity Testinginformation on Fetal Lung Maturity Testing David Grenache PhDDavid Grenache PhD ARUPARUP Salt Lake CitySalt Lake City David Grenache, PhD David Grenache, PhD –– ARUP ARUP –– Salt Lake CitySalt Lake City FLMoptions.comFLMoptions.com Anne Gronowski, PhD Anne Gronowski, PhD –– Washington Washington
University School of Medicine University School of Medicine –– St. LouisSt. Louis AACC Pediatric and MaternalAACC Pediatric and Maternal--Fetal DivisionFetal Division Neerhoff et alNeerhoff et al Obstet GynecolObstet Gynecol 2001;97:3182001;97:318-- Neerhoff, et.al. Neerhoff, et.al. Obstet Gynecol.Obstet Gynecol. 2001;97:3182001;97:318--
320.320.All of these sources were used in the preparation of this program. All of these sources were used in the preparation of this program.
Their contributions are gratefully acknowledged.Their contributions are gratefully acknowledged.
Fetal Respiratory Distress Fetal Respiratory Distress SyndromeSyndrome
AKA AKA –– Hyaline Membrane DiseaseHyaline Membrane DiseaseCli i l M if t ti f FRDSCli i l M if t ti f FRDS Clinical Manifestations of FRDSClinical Manifestations of FRDS Tachypnea Tachypnea –– rapid shallow breathingrapid shallow breathing Prominent, audible gruntingProminent, audible grunting Nasal FlaringNasal Flaring SubSub--costal and intercostal and inter--costal retractionscostal retractions Cyanosis unresponsive to O2 administrationCyanosis unresponsive to O2 administration Progressive worsening of cyanosis and Progressive worsening of cyanosis and
dyspnea.dyspnea. Apnea and irregular respirationsApnea and irregular respirations
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FRDS Risk FactorsFRDS Risk Factors
Prematurity (<37 weeks )Prematurity (<37 weeks ) Prematurity (<37 weeks )Prematurity (<37 weeks ) Male genderMale gender Caucasian raceCaucasian race 22ndnd born infant of twinsborn infant of twins
More frequent in multiple birthsMore frequent in multiple births More frequent in multiple birthsMore frequent in multiple births
History of FRDS in siblingHistory of FRDS in sibling
RDS Causes and Surfactant RDS Causes and Surfactant CompositionComposition
Caused by a deficiency in pulmonary surfactantCaused by a deficiency in pulmonary surfactant Pulmonary surfactants decrease surface tension Pulmonary surfactants decrease surface tension u o a y su acta ts dec ease su ace te s ou o a y su acta ts dec ease su ace te s o
of waterof water Composition of surfactantsComposition of surfactants
85% phospholipids85% phospholipids PhosphatidylcholinePhosphatidylcholine --76%76% PhosphatidylglycerolPhosphatidylglycerol --13%13% Phosphatidylinositol Phosphatidylinositol –– 4%4% PhosphatidylethanolaminePhosphatidylethanolamine --3%3% SphingomyelinSphingomyelin 2% and other phospholipids 2%2% and other phospholipids 2%
10 % protein10 % protein 5% neutral lipids5% neutral lipids
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FRDS IncidenceFRDS Incidence
Most common cause of respiratory failure in Most common cause of respiratory failure in neonatesneonates
Incidence is Incidence is indirectlyindirectly proportional to proportional to gestational age at deliverygestational age at delivery 6060--80% of births at <28 weeks have FRDS80% of births at <28 weeks have FRDS 1515--30% of births at 3230% of births at 32--36 weeks have FRDS36 weeks have FRDS 55--6% of births at 37 weeks have FRDS6% of births at 37 weeks have FRDS FRDS of Full Term deliveries is rareFRDS of Full Term deliveries is rareFRDS of Full Term deliveries is rareFRDS of Full Term deliveries is rare
Ref: Grenache, DavidRef: Grenache, David
Tests for FLMTests for FLMFor detail, refer to “Contemporary Issues in FLM Testing” For detail, refer to “Contemporary Issues in FLM Testing”
presentation by David Grenache, PhD.presentation by David Grenache, PhD.
Criteria for FLM Test Selection:Criteria for FLM Test Selection: Criteria for FLM Test Selection:Criteria for FLM Test Selection: Rapid performanceRapid performance Must have high sensitivity for immaturity and Must have high sensitivity for immaturity and
high predictive value for maturityhigh predictive value for maturity Used for clinical decision makingUsed for clinical decision making Performed on Amniotic Fluid at 32 to 38 Performed on Amniotic Fluid at 32 to 38
weeks of gestation (ACOG weeks of gestation (ACOG –– 2008)2008)
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Tests for FLMTests for FLM Precursor Tests Precursor Tests
L/S Ratio was the “gold standard” but of L/S Ratio was the “gold standard” but of questionable reproducibility and accuracy.questionable reproducibility and accuracy.questionable reproducibility and accuracy. questionable reproducibility and accuracy. Was developed in the early 1970’s.Was developed in the early 1970’s.
PG by TLC was slow and tedious.PG by TLC was slow and tedious. PG by agglutination technically easy, but not PG by agglutination technically easy, but not
very sensitive very sensitive –– developed early 1980’sdeveloped early 1980’s S/A ratio S/A ratio –– (Abbott FLM) (Abbott FLM) –– automated test automated test ( )( )
developed in the late 1980’s and will no developed in the late 1980’s and will no longer be available. Sensitive, quantitativelonger be available. Sensitive, quantitative
LBC LBC –– first described in late 1980’s but not first described in late 1980’s but not commonly performed. Sensitive, quantitativecommonly performed. Sensitive, quantitative
Lamellar Body CountLamellar Body Count
Lamellar bodies are similar in size to blood Lamellar bodies are similar in size to blood platelets (1platelets (1--5 5 uMuM or 2 or 2 --10 10 fLfL).).
They can be accurately quantified using They can be accurately quantified using routine hematology analyzers.routine hematology analyzers.
They are formed by the Type II They are formed by the Type II pneumocytespneumocytes through a “packaging” through a “packaging” process and stored as granular materialprocess and stored as granular materialprocess and stored as granular material.process and stored as granular material.
They are excreted from the fetal lung into They are excreted from the fetal lung into the amniotic fluid.the amniotic fluid.
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Lamellar BodiesLamellar Bodies
Human PlateletsHuman Platelets
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Lamellar Body CountsLamellar Body Counts
AdvantagesAdvantages High Sensitivity for immaturityHigh Sensitivity for immaturity Quick and simple to performQuick and simple to perform Require low sample volumeRequire low sample volume Instruments are readily available in labsInstruments are readily available in labs
DisadvantagesDisadvantagesAffected by blood and meconiumAffected by blood and meconium Affected by blood and meconiumAffected by blood and meconium
Lab developed test Lab developed test -- not FDA approvednot FDA approved Instrument specific cutoffs requiredInstrument specific cutoffs required
Varies by model within manufacturersVaries by model within manufacturers
Lab Consensus GuidelinesLab Consensus Guidelines(Neerhoff, et. al.)(Neerhoff, et. al.)
Amniotic Fluid should NOT be centrifugedAmniotic Fluid should NOT be centrifuged Amniotic Fluid should NOT be centrifugedAmniotic Fluid should NOT be centrifuged Grossly bloody or contaminated Amniotic Grossly bloody or contaminated Amniotic
Fluid should NOT be usedFluid should NOT be used Cutoffs are Instrument and Model Cutoffs are Instrument and Model
dependentdependent regardless of manufacturerregardless of manufacturerFF tt l il i For For mostmost analyzers, range is:analyzers, range is: Mature = >50,000/Mature = >50,000/uLuL Intermediate/transitional = 15,000 to 50,000/Intermediate/transitional = 15,000 to 50,000/uLuL Immature = <15,000/uImmature = <15,000/u
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Consensus Range CautionsConsensus Range Cautions
Consensus range was established without Consensus range was established without o u a g a ab d ouo u a g a ab d ouregard of the hematology analyzer used.regard of the hematology analyzer used.
Not all analyzers use the same parameters Not all analyzers use the same parameters for counting platelets; thus, it is for counting platelets; thus, it is IMPERATIVEIMPERATIVE that analyzer specific cutthat analyzer specific cut--off off
l b d t i d b th l b tl b d t i d b th l b tvalues be determined by the laboratory.values be determined by the laboratory.
PrePre--Analytic ConsiderationsAnalytic Considerations Amniotic Fluid (AF) is a heterogeneous Amniotic Fluid (AF) is a heterogeneous
mixture containing sloughed cells, hair and mixture containing sloughed cells, hair and other fetal debrisother fetal debrisother fetal debris. other fetal debris.
It should be tested promptly (< 4 hours) It should be tested promptly (< 4 hours) after collection for best results.after collection for best results.
It should not be centrifuged prior to It should not be centrifuged prior to analysis It should be gently mixed wellanalysis It should be gently mixed wellanalysis. It should be gently mixed well, analysis. It should be gently mixed well, not vortexed.not vortexed.
Grossly bloody or contaminated AF should Grossly bloody or contaminated AF should be rejected.be rejected.
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Comparison of Various AnalyzersComparison of Various Analyzers
Most early studies were performed using Most early studies were performed using different models of Beckman Coulter different models of Beckman Coulter
llanalyzers.analyzers. These analyzers use electrical impedance These analyzers use electrical impedance
differences through a 50 differences through a 50 uMuM aperture.aperture. Multiple studies have shown this technology Multiple studies have shown this technology
to compare favorably with previous FLM tests to compare favorably with previous FLM tests (L/S ratio, PG, FLM/SA).(L/S ratio, PG, FLM/SA).
LBC is faster and easier to perform than other LBC is faster and easier to perform than other quantitative methods.quantitative methods.
Comparison of Various AnalyzersComparison of Various Analyzers When compared to the Beckman Coulter Gen S When compared to the Beckman Coulter Gen S
counter, various studies found:counter, various studies found:Concordance Rates as follow:Concordance Rates as follow:Concordance Rates as follow:Concordance Rates as follow: Sysmex XESysmex XE--2100 = 86%2100 = 86% Siemens Siemens AdviaAdvia 120 = 78%120 = 78% Abbott CellAbbott Cell--DynDyn 3500 = 66%3500 = 66%
Manufacturers use different apertures and Manufacturers use different apertures and various impedance mechanismsvarious impedance mechanismsvarious impedance mechanismsvarious impedance mechanisms..
Ref: Lu, Ref: Lu, GronowskiGronowski, et.al., et.al.
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Various Analyzer Maturity CutVarious Analyzer Maturity Cut--OffsOffs(suggested from literature)(suggested from literature)
Beckman Coulter Beckman Coulter ((GenSGenS, STKR), STKR) 50 000/50 000/uLuL 50,000/50,000/uLuL
Sysmex Sysmex (XE(XE--2100, K2100, K--800)800) 50,000/50,000/uLuL except XEexcept XE--50005000
Siemens Siemens ((AdviaAdvia 120)120) 50,000/50,000/uLuL
Abbott Abbott (Cell (Cell DynDyn 3500)3500) 80,000/80,000/uLuL
Ref: Lu, Ref: Lu, GronowskiGronowski, et.al. , et.al.
Analyzer DifferencesAnalyzer Differences SysmexSysmex
Uses electrical and radio Uses electrical and radio m,frequencym,frequency impedance plus impedance plus an 80 an 80 uMuM aperture in addition to forward and side aperture in addition to forward and side scatter and hydroscatter and hydro--dynamic focusing.dynamic focusing.
For all except XEFor all except XE--5000, they compare very well with 5000, they compare very well with the consensus ranges. the consensus ranges.
SiemensSiemens Uses low angle (volume) and high angle (refractive Uses low angle (volume) and high angle (refractive
index) light scatter from a laser. Any particle with a index) light scatter from a laser. Any particle with a volume of <60 volume of <60 fLfL and a high refractive index. They and a high refractive index. They compare well with the consensus ranges.compare well with the consensus ranges.p gp g
AbbottAbbott Combine optical scatter and electrical impedance. Combine optical scatter and electrical impedance.
They have a positive slope compared to the other They have a positive slope compared to the other analyzers and thus use a higher cutoff value for analyzers and thus use a higher cutoff value for maturity than the consensus ranges.maturity than the consensus ranges.
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Sysmex XESysmex XE--5000 5000 Uses different software parameters for the Uses different software parameters for the
platelet count.platelet count. Where most analyzers size the platelet Where most analyzers size the platelet
parameter down to 1 parameter down to 1 fLfL or less, the XEor less, the XE--5000 5000 sizes platelets using a 2 sizes platelets using a 2 fLfL low discriminator. low discriminator.
Thus, LBCs on the XEThus, LBCs on the XE--5000 will tend to be as 5000 will tend to be as much as 50% lower in some cases.much as 50% lower in some cases.
This led to our study comparing LBC and L/S This led to our study comparing LBC and L/S to FRDS at delivery.to FRDS at delivery.
NOT FDA Approved NOT FDA Approved –– Lab Developed TestLab Developed Test
McKennan XEMcKennan XE--5000 FRDS study5000 FRDS study
Study performed on 68 patientsStudy performed on 68 patients Compared LBC, L/S and RDS at deliveryCompared LBC, L/S and RDS at delivery There was 1 still born, 1 twin delivery and There was 1 still born, 1 twin delivery and
2 cases with 2 specimens submitted.2 cases with 2 specimens submitted. Summary of specimen results: Summary of specimen results:
LBC L/SLBC L/S RDSRDSMature 52 Mature 52 2525 56 none56 noneIntermedIntermed 44 18 (m/18 (m/cautcaut) ) --Immature 13Immature 13 55 12 RDS12 RDS
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McKennan CommentsMcKennan Comments
Both of the twins had respiratory distressBoth of the twins had respiratory distress All of the newborns with RD admitted to All of the newborns with RD admitted to
NICUNICU The still born fetus had meconium The still born fetus had meconium
contaminated AF so could not be tested.contaminated AF so could not be tested.Several immature by LBC were calledSeveral immature by LBC were called Several immature by LBC were called Several immature by LBC were called “mature w/caution” by L/S ratio.“mature w/caution” by L/S ratio.
McKennan CutMcKennan Cut--offs for XEoffs for XE--50005000
CutCut--offs validated for XEoffs validated for XE--5000 at AMcK5000 at AMcK CutCut--offs validated for XEoffs validated for XE--5000 at AMcK.5000 at AMcK.
Mature = Mature = >>25,000/uL25,000/uL Indeterminate/transitional = 21 to 24,000/uLIndeterminate/transitional = 21 to 24,000/uL Immature = < 21,000/uLImmature = < 21,000/uL, /, /
Validated for our analyzer(s) in our labValidated for our analyzer(s) in our lab
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Proficiency SurveyProficiency Survey
CAP SurveyCAP Survey LBCLBC ––A (May 2011)A (May 2011) CAP Survey CAP Survey LBC LBC ––A (May 2011)A (May 2011) Ungraded Educational ChallengeUngraded Educational Challenge Demonstrated that different analyzers give Demonstrated that different analyzers give
different results on the same sampledifferent results on the same sample Lumps instrument by manufacturer without Lumps instrument by manufacturer without
d f d l ( f S )d f d l ( f S )consideration of model. (Issue for Sysmex)consideration of model. (Issue for Sysmex) XEXE--5000 lumped in w/ other Sysmex analyzers5000 lumped in w/ other Sysmex analyzers Affects the Mean, SD and CVAffects the Mean, SD and CV
LBCLBC--AA## mean SD CV Medianmean SD CV Median
LBCLBC--0101 Coulter 133 6.9 1.1 15.9 7.0Coulter 133 6.9 1.1 15.9 7.0 Siemens 11 9.2 1.7 18.7 9.0Siemens 11 9.2 1.7 18.7 9.0 Sysmex 77 4.9 0.8 16.6 5.0Sysmex 77 4.9 0.8 16.6 5.0
LBCLBC--0202 Coulter 134 65.8 2.1 3.3 66Coulter 134 65.8 2.1 3.3 66 Siemens 11 82.5 4.2 5.1 81Siemens 11 82.5 4.2 5.1 81 Sysmex 78 58 8 3 0 5 0 59Sysmex 78 58 8 3 0 5 0 59 Sysmex 78 58.8 3.0 5.0 59Sysmex 78 58.8 3.0 5.0 59
LBCLBC--0303 CoulterCoulter 136 20.8 1.5 7.1 21136 20.8 1.5 7.1 21 Siemens 11 27.1 2.1 7.6 27Siemens 11 27.1 2.1 7.6 27 Sysmex 79 16.7 1.6 9.3 17Sysmex 79 16.7 1.6 9.3 17
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SummarySummary
FLM II assay by Abbott will no longer be FLM II assay by Abbott will no longer be available.available.
L/S ratio is labor intensive, uses very toxic L/S ratio is labor intensive, uses very toxic reagents and requires expertise. It is not reagents and requires expertise. It is not standardized.standardized.
Fetal Lung Maturity Testing is not Fetal Lung Maturity Testing is not i di d i h j i f ii di d i h j i f iindicated in the majority of pregnancies indicated in the majority of pregnancies ––only in premature deliveries <38 weeks only in premature deliveries <38 weeks with a few exceptions.with a few exceptions.
SummarySummary LBC is an excellent predictor of fetal lung LBC is an excellent predictor of fetal lung
maturity and FRDS.maturity and FRDS. Can be performed on most hematology Can be performed on most hematology
analyzers.analyzers. Test is NOT FDA approved Test is NOT FDA approved –– must be must be
validated in your lab.validated in your lab. CutCut--off values vary by manufacturer as off values vary by manufacturer as
well as by instrument model.well as by instrument model.
