Comparison of Two Newborn Screening Systems for Hemoglobinopathy

Identification

Christine D. McKeever, MSP, MT(ASCP)

de t cat o

Tennessee Dept. of Health, Division of Laboratory Services, Nashville, TN

ObjectiveObjective To compare the overall performance of HPLC (Bio-rad

VNBS)to capillary electrophoresis (Sebia )in the identification of Hb Bart’s , S, C, D, E, G and A2 from newborn dried blood spotsnewborn dried blood spots.

InstrumentationInstrumentationBio-Rad VNBS Cation exchange

Column for separation

Gradient/increasing ionic strength

Dual wavelength photometer( 415 & 690 nm)

Chromatogram

Retention times

ID f k Hb ID of unknown Hbs

Pattern assignment

P i t d t Printed report

Qualitative assay

InstrumentationInstrumentationSebia Capillarys

Electrophoretic separation by charge, pH and electroosmoticflow

Silica capillaries

Anode to cathode

Wavelength detection at 415 nm

Pattern adjustment based on F

R l di l d i Results displayed in zones N1-N13

Qualitative determination Qualitative determination

MethodologyMethodology 54 samples chosen based on phenoytpe pattern then de-identified

Sample DistributionReported Phenotype pattern # of samples included

FA or AF (Normal hemoglobin) 16( g )

S trait or Disease 12

D/G trait 4

Bart’s Hemoglobin 9Bart s Hemoglobin 9

C trait 7

E/A2 trait or Disease 6

Simultaneous analysis on VNBS and CE

Two FAG-Philadelphia samples analyzed later by CE to see

Total 54

Two FAG Philadelphia samples analyzed later by CE to see distinction of Hb D from G

ResultsResults

VNBS (normal Hb) Capillarys (normal Hb)VNBS (normal Hb) Capillarys (normal Hb)

ResultsResults

VNBS Hb Bart’s Capillarys Hb Bart’sVNBS – Hb Bart s Capillarys – Hb Bart s

ResultsResults

VNBS FAC Capillarys FACVNBS -FAC Capillarys - FAC

ResultsResults

VNBS FAS Capillarys FASVNBS – FAS Capillarys – FAS

ResultsResults

VNBS FAD/G Capillarys FAVVNBS – FAD/G Capillarys - FAV

ResultsResults

VNBS FAE/A2 Capillarys FAEVNBS – FAE/A2 Capillarys - FAE

ResultsResults

Demonstration of A2

ResultsResults

VNBS FAD/G Capillarys FADVNBS – FAD/G Capillarys - FAD

ResultsResults

G-Philadelpia trait

ResultsResults Punch size VNBS – Standard punch 3.2 mm

or 1/8” Capillarys - 3 8 mm punch or Capillarys 3.8 mm punch or

15/100”

Testing Format VNBS – Standard Microtiter

plates C ill Dil ti Capillarys – Dilution

segments 96 wells

ResultsResults Daily maintenance

QC

Throughput

ConclusionConclusion CE distinguishes Hb D from Hb G Hb E from A2

VNBS i i t VNBS requires more maintenance

CE’s controls have only 2 variants Hb S and Hb C Hb S and Hb C

Throughput

Both are reliable for neonatal Hb screeningBoth are reliable for neonatal Hb screening

ReferencesReferences Bio-Rad Variant nbs Sickle Cell Program Instruction Manual,

L50031605 O b 2008L50031605, October 2008

Capillary S Neonat Hb Fast Training Manual, Sebia Inc., October 2009

AcknowledgementsAcknowledgements TDH newborn screening lab

Dr. Pilar Aguinaga of Meharry Medical College

Bonny Champagne of Sebia