704

ClinicalChemistiy 42:5

704-710 (1996)

Automated HPLC screening of newborns forsickle cell anemia and other hemoglobinopathies

JOHN W. EASTMAN,* RUTH WONG, CATHERINE L. Lao, and DANIEL R. MORALES

Automated HPLC is used to test dried blood-spot speci-

mens from newborns for hemoglobms (ITh) F, A, S, C, E,and D. We present the method and report on its perfor-

mance determined during >4 years of testing 2.5 x 106

newborns. The method features automated derivation ofpresumptive phenotypes; quantitative quality control and

proficiency testing; throughput of one specimen per

minute; small sample volume; hemoglobin concentrationsquantified with an mterlaboratory CV of 14-18%; retention

times with interlaboratory CV of <2% and matching,

within ± 0.03 miii, of laboratories and reagent lots; control

of peak resolution; 0.5% detection limit for Hb S and C, and

1.0% for Hb F, A, E, and D; few interferences; and negli-gible background and carryover. Shortcomings of themethod are the absence of microplate barcode identifica-

tion and the need for manually pipetting the sample eluate

into the microplate.

INDEXING mie1s: dried blood-spot specimens #{149}Guthrie cards.

chromatography, cation-exchange #{149}phenotypes

In keeping with national recommendations, California legisla-

tion mandates screening all newborns for sickle cell disease

[1-3].This legislation was implemented for the State of Cali-

fornia by the Department of Health Services’ Genetic DiseaseLaboratory (GDL) and Genetic Disease Branch, Berkeley, CA

[4]. Because of its potential as a quantitative method of analysis

amenable to automation, GDL chose cation-exchange HPLC

[5-12] as the screening method. Cation-exchange HPLC has

been used to screen cord blood for hemoglobinopathies [13].

Here, we report on the application of this technique to screen

newborns by assaying their dried blood-spot (DBS) specimens.

The method reported here is designed to resolve hemoglo-

bins (Hb) F, A, 5, C, E, and D. The California program requires

California Department of Health Services, Genetic Disease Laboratory, 700

Heinz St., Suite 100, Berkeley, CA 94710.*AUthOr for correspondence. Fax 510-540-2228.

‘Nonstandard abbreviations: GDL, Genetic Disease Laboratory; DBS, dried

blood spot; NB, newborn; Hb, hemoglobin(s); and AU, hemoglobin concentration

in area units, the area of the chromatographic peak.

Received October 4, 1995; accepted January 25, 1996.

that newborns with hemoglobinopathy patterns (e.g., FS, FSC,

FSA, F only) be recalled for mandatory follow-up. Also, new-

borns with FE patterns are recalled to differentiate EE andE//3-thalassemia. For trait patterns of FAS, FAC, and FAD,

the families of the affected newborns are offered voluntary

counseling.

Materialsand MethodsEQUIPMENT/REAGENT SYSTEM

Modular instrumentation was adapted for the California pro-gram by Bio-Rad Labs., Hercules, CA. A similar integrated

instrument, ASCeNT#{174},has been marketed [14]. Each of the

modular instruments used in this work consists of one to three

HPLC column systems run by one Model 700 chromatography

workstation. Each of the column systems consists of a Model

AS-I00 HRLC automated sampling system, a 20-g.tL-loop

injection valve, two Model 1350 gradient elution pumps, thecation-exchange column in a Model 1250425 35 #{176}Cheater, and

a dual-wavelength (415/690 nm) filter photometer (cat. no.

1961042). The 6 X 40 mm columns are packed with a nonpo-rous 7-.tm-diameter Bio-Rad MA 7 polymeric cation-exchange

material. Each column can be used for as many as 500 injections.

Two sodium phosphate buffers are used as eluents, run as a

gradient from -4 g/L (buffer A) to 14 g/L (buffer B) at pH 6.4.

Nominal conditions of analysis are a flow rate of 2 mL/min

at a pressure of 25 kg/cm2 and the following gradient (time frominjection in minutes/percent of eluent that is buffer B): 0.0/0%;

0.3/10%; 0.5/24%; 1.0/52%; 1.8/100%; 1.9/100%; and 2.0/0%.

Including the 1-mm wash between specimens, each chromato-

gram takes 3 mm. When all three columns are used, the rate of

analysis is one specimen pen minute.

Operation of the instrument is automated through a menu-

driven software program that generates the worklist, injects the

sample, controls the gradient for hemoglobin separation, mea-

sures and integrates the peaks, derives the hemoglobin pattern,

stores the data on electronic media, and telecommunicates the

data to a remote central site.The reagent kits include whole-blood primer, wash solution,

three linearity calibrators containing Hb F and A, and two

lyophilized controls, one containing Hb F, A, E, and S and oneHb F, A, D, and C. To maintain the separation of hemoglobmns

among all lots of the cation-exchange resin, the software that

Clinical Chemistry 42, No. 5, 1996 705

controls the gradient is modified when needed to accommodate

any differences in the performance of the different lots.

A key feature of the test system is its quantification of the

concentration of the hemoglobin variants. Chromatographic

peaks are reported with heights in microvolts and with areas in

relative response units (AU, area units). With the integration

settings used for this screening method, 1 AU is approximately

three times the area in p.V-min. The three linearity calibrators

are used to monitor the dose-response curve for photometer

readings vs hemoglobin concentration. The reagent-instrument

system must maintain the photometer readings for the linearity

calibrators (-0.2, 0.4, and 0.6 g/L Hb F) within ±20% of a

stated nominal value.

HPLC SCREENING METHOD

Specimen collection and preparation.Blood from a subject is

absorbed into S&S 903 specimen collection paper (Schleicher &

Schuell, Keene, N}-l). A disc 0.95 cm (3/8 in.) in diameter is

punched from the DBS specimen, and the blood is eluted into

1.00 mL of water for 30 mm with periodic shaking (equivalent

to a 36-fold dilution of the whole-blood specimen). The eluate

is further diluted 1:6 with water and dispensed into a 96-well

microplate, which is loaded onto the autosampler for injection.

Worklistgenerationand reports.A worklist is generated automat-

ically when the analyst sets up the run file information. At the

end of each run, a Neonatal Hemoglobin Summary Report is

printed out. The summary lists the identification number of

each specimen and the percentages of all the hemoglobin

variants found in the specimen. A Pattern Report is also printed

out, listing the hemoglobin pattern (presumptive phenotype) for

each specimen. This report lists, in order of decreasing concen-

tration, the letter designation of each of the hemoglobins found

in the specimen.

Quality control. For each column system, the FAES and FADC

liquid controls are run at the beginning and end of each run. At

the beginning and end of each tray (microplate) of newborn

specimens, eluates of blood spots containing Hb S are run as tray

controls. The mean and SD values for retention time are fixed as

quality-control action limits in the controller software by spec-

ification. Mean and SD values for hemoglobin concentration are

established by replicate analysis of the controls at GDL before

they are put into use. Trays of newborn results are flagged for

review by a quality-control officer when (a) the retention time of

Hb S in the tray control exceeds the ±3SD limit; (b) the

concentration of Hb S in the tray control exceeds the ± 3 SD

limit; or (c) the blank water injections give a chromatographic

peak height >5000 j.tV.

Because the microplates are not barcoded for identification,

a positional control is also included on each tray. This is an

eluate of a blank disc of blood-collection paper placed in a

unique position such that it documents each tray’s number in the

sequence of analysis (e.g., tray 2 must have a blank result at

position 2). The positional control also serves as a water blank

on each tray.

DERIVATION OF PATTERNS

For each newborn tested, a hemoglobin pattern presents the

observed hemoglobins in order of relative concentration from

the highest to lowest. GDL developed algorithms (see Appendix)

for use on the chromatography workstation software so that

chromatographic peaks from noise and minor hemoglobins are

not included in the hemoglobin patterns.

The retention time of Hb A, is similar to that of Hb E.

However, Hb A2 is never expressed in a hemoglobin pattern

derived for a newborn specimen because its concentration is

low, and the interpretation algorithms remove such low peaks

from the pattern (Appendix). Satellite hemoglobins (e.g., E1, 5,,

C,) produce small chromatographic peaks that elute -0.17 mm

faster than the peak of the corresponding major hemoglobin

[5,7]. GDL introduced a 1:4 rule to eliminate the satellite

hemoglobmns from the reported patterns. Also, based on pub-

lished information on the relative percentage concentrations of

Hb A in thalassemia cases [8, iS], GDL developed a 1:2 rule to

differentiate patterns FAS (sickle cell trait) from FSA (S/f3-

thalassemia) (see Appendix).

Hb (1), an unidentified hemoglobin eluting between Hb F

and Hb A, appears in the HPLC chromatograms for the

linearity calibrators, the liquid controls, surrogate in-house DBS

samples, and in most DBS specimens from newborns. The

retention time for Hb (1) corresponds to that expected for Hb

A,d [16]. The program used to derive the hemoglobin pattern

adds the concentration of Hb (1) to the concentration of Hb A

and reports the total concentration as Hb A (Appendix).

SPECIMENS ANALYZED

GDL collected data on three types of samples: the liquid

Bio-Rad FAES and FADC controls, which are not processed

through the punching, elution, and dilution steps of the method;

surrogate DBS specimens prepared in house (GDL-DBS sam-

ples); and newborns’ specimens collected on paper (NB-DBS

specimens). The liquid controls contain Hb F, A, 5, C, E, D inconcentrations similar to those found in actual newborn speci-

mens, i.e., mostly Hb F with 10-20% Hb A and 3-10% of the

other variants. The GDL-DBS samples, which contain Hb F, A,

and 5, were prepared by mixing commercial bulk AS blood or

cord blood with adult outdated bank blood and spotting the

mixture onto specimen collection paper. These DBS sample

pools contain various concentrations of the hemoglobin variants

for use as controls and proficiency-test samples. For GDL-DBS

pools containing Hb S, the concentration of Hb A is large

(-75%), and the ratio of [Hb A]/[Hb S] does not match the ratio

found in newborn specimens. Nevertheless, these samples yield

satisfactory chromatograms.

EVALUATION OF PERFORMANCE CHARACTERISTICS

The precision and accuracy of retention times, as well as the

precision of the variant quantification, were determined from

data collected with 23 column systems at nine laboratories using

one lot of cation-exchange resin. We also evaluated the accuracy

and precision of data collected with four different lots of resin by

one laboratory using three column systems. The other perfor-

‘20OEa)(I)

0

100

0

400

a)Cl)

0a-C’)a)

0

0 2

Li0 2

706 Eastman et al.: Screening newborns for hemoglobinopathies by HPLC of dried blood spots

mance characteristics were determined for the aggregate of 23

column systems and four lots of resin.

ResultsPERFORMANCE ASSESSMENT

Chromatograms. Fig. 1 is a chromatogram obtained from a liquid

sample made by combining controls with Hb F, A, 5, C, E, and

D. Fig. 2 is a chromatogram of an eluate of a DBS specimen

obtained from a newborn with sickle cell disease.

Precision of retention times.Table 1 compares our results with the

instrument specifications. The SD specification is one-sixth the

range of the retention time identification window (window

� ± 3SD). The observed SD for Hb S exceeds the limit some-

what; nonetheless, at this precision, all cases of sickle cell disease

have been correctly identified.

Accuracy of retention times. The observed mean retention time

(Table 1) for each hemoglobin is compared with the retention

time for the center of the hemoglobin identification window in

the integration software. In most cases the observed mean values

are within 0.01 mm of the specified value, and in no case does

the bias exceed 0.03 mm.

Precision of quantification of hemoglobins. According to the speci-

fications (Table 2, last column), with a one-column system (with

one photometer) the interrun CV for Hb F in the middle-

concentration linearity calibrator should be no more than 5%.Also, among all systems the mean should always lie between

80 000 and 120 000 AU (±20% range for matching the 23

systems). The results are in acceptable agreement with the

specifications. The CV of 10.9% over all 23 column systems is

equivalent to a ±2SD range of 21.8%, which is close to the±20% range limit.

On multiple column systems the observed CVs for liquid

controls and DBS samples span a range from 14% at 30 000

300

Time (mm)

Fig. 1. Chromatogram of a 1:1 mixture of two Bio-Rad controlscontaining Hb FAES and Hb FADC.Peaks (from left to right): Hb FAST, Fl, F, (1), A, E, D, S, and C. (See Appendixfor hemoglobin nomenclature.)

AU to 18% at 5000 AU. The 5000 AU (18% CV) is equivalentto a NIB-DBS specimen containing a hemoglobin variant at

2.5% relative concentration (in a total area of 200 000 AU). The

30 000 AU (14% CV) is equivalent to a NB-DBS specimen

containing a hemoglobin variant at 15% relative concentration.

Detection limits. HPLC peak criteria in the integration parame-ters are set so that Hb S and C are expressed in the hemoglobin

pattern when the relative concentration of each exceeds 0.5%.

For Fib F, A, E, or D the detection limit is 1.0%. To achieve

these detection limits requires that the height threshold used toeliminate background noise be reduced to 500 jV. To test thepracticality of this threshold, GDL submitted two GDL-DBS

samples containing 1.1% and 0.8% Hb S for analysis at the

satellite laboratories. In 12 weekly shipments (one sample perweek) to nine laboratories (n = 106 analyses after excluding 2

results that were invalid for other reasons), there were no missed

Hb S peaks. By now, >450 000 newborns have been tested with

use of the 500-.tV height threshold, and there have been no

known missed cases of Hb S or other clinically significant

variant exceeding 0.5% in the newborns’ specimens.

That a 500 .tV threshold is required is shown by results

obtained during the first 3.5 years of HPLC screening, during

which time we used a threshold of either 3000 or 2000 j.tV. At

3000 j.V not all satellite laboratories reported the presence of

Hb 5, even though it made up 1.0% of the total hemoglobin in

the GDL-DBS proficiency-test samples. However, retrospective

examination of the chromatograms, which are stored on mag-

netic tape, and reprocessing at a lower threshold detected all of

the Fib S peaks. Also, during that period, -2.1 million newborns

were screened. In rare instances Hb S, A, E, and D that were not

reported in the newborn screening pattern were found inchildren at an older age (Table 3). In all cases, when the

chromatographic raw data were electronically reprocessed at alower threshold (e.g., 500 .tV), we found a well-resolved bell-

shaped peak at the retention time of the missing variant.

bOO

Time (mm)

FIg. 2. Chromatogram of a newborn dried blood-spot specimen withpattern ES.Peaks (from left to right):void volume and Hb FAST, Fl, F, (1), and S.

n

Table 1. PrecisIon and accuracy of retention times.Retention time, mm x 100

MeanMean

specification

FAEDSC

BIas

422

422211211211211

SD

64.0883.2998.14

107.93118.25

172.60

Hb

Liquid samples

Dried blood spots

Tables 1 and 2: Data collected from nine sites using resin lot 4 on 23 column systems.

SDspecification

F

61.0083.00

98.00107.00118.00172.00

CV, %

AS

+ 3.08+ 0.29+0.14+0.93+0.25+0.60

90

0.971.001.26

1.391.391.59

97204

63.3784.29

117.57

2.331.671.331.671.332.00

61.0083.00

118.00

1.51.2

1.31.41.2

0.9

-2.37+ 1.29

-0.43

1.17

1.02

1.41

2.331.671.33

1.81.2

1.2

The concentrations determined after reprocessing are given in

Table 3.Theoretically, the detection limit of the method might be

decreased by a further reduction in the threshold setting.

However, all cases of sickle cell disease are detected at the

current value.

Specificity.In the past 4 years, GDL has been notified of 12

instances in which diagnostic follow-up results did not agree

with the newborn screening hemoglobin patterns, and for whichthe discrepancy could be attributed to the specificity of the

HPLC method (Table 4). Also, we have found that -1% of the

chromatographic peaks at the retention time for Hb S have anatypical concentration ratio for [Hb A}:[Hb S] of -6:1 (usually

the ratio is -1:1). The screening patterns for these newborns are

FAS (also in Table 4). Hb G, an a-chain variant with four

chromatographic peaks, is readily identified by visual inspection

of the chromatogram. According to the rules given in theAppendix for derivation of patterns, and depending on the

particular lot of cation-exchange resin in use, Hb G has been

reported in newborn screening as Hb E, Hb D, or combinations

of Hb E and D with Hb (2).

Background and carryover. Water blanks are included on the

calibration tray and on each tray of newborns’ samples for

analyses. GDL staff review all chromatograms that show a peak

with height >5000 .tV. These situations occur infrequently (9

times per 100 000 newborns) and do not affect newborns’

hemoglobin patterns. In some cases small spikes are observed,

usually at the void volume (retention time 0.2 mm). However,

the retention times are too fast, the peak widths too narrow, and

the heights too low for these spikes to affect the hemoglobin

patterns generated by the method algorithms (Appendix).

Peak shape. Laboratory staff monitor the Hb F peak shapes for

the controls and for 5% of all newborns’ specimens. By remov-

Table 2. PrecIsion of quantification.No. ofcolumn

Hb systems

F

AUNo. of

samples Mean SO

F

Liquid samples

Dried blood spots

AS

CV, %

18 4023b 10523 21323 213

103 834101 679

29 0577 338

F

4621

11 1124 295

1 002

Table 3. ComparIson of the newborn screening result withthe result obtaIned at an older age.a

PatternResuft after reprocessing

Case no. Newborn Older data, % of total Hb

1 FS FSaC HbA, 0.92 FS FSa HbA, <0.5

3 ES FSa HbA, <0.54 ES FSAC HbA, 0.85 ES FAS HbA, <1.06 FAd FAS HbS, 0.5

7 FA#{176} FAS FIbS, 0.888 F only FAS HbS, 1.089 FE FEa HbA, <0.5

10 FA’ FAE HbE, 0.8

11 F only HbA, 0.9, HbD, 0.88 Differencesattributable to prior use of higherthreshold (>500 MV).b Original newborn screening data reprocessed to reflect current 500 V

threshold.r FSApatterns are reported as traitunless(Hb SJ>2 lHbAl,inwhichcase the

pattern isreported as FSa forS/p.thalassernia(seeAppendix).d Follow-up initiatedfromfamilystudies.

8 HbS >0.5%.

‘Reanalysis ofNB-DBS specimengave 1.1% HD E and a patternofFAE.

g v represents an unidentifiedvariant.

4.4

10.914.813.6

AS

23 27 37838

23 97 5284

23 206 5 113

5 147918894

13.617.417.5

ClinicalChemistry 42, No. 5, 1996 707

8CV <5% specified.Rangeof ±20% specified.

708 Eastman et al.: Screening newborns for hemoglobmnopathies by HPLC of dried blood spots

Table 4. Newborn screening results dIfferIng from clinIcal foilow-up results because other Variants eluted at retention tImesfor Hb S, E, and D.

Screening pattern Follow-up pattern Follow.upmethodb

FAV IEF

FA IEF

FAS

1 FAE

2 FAE3 FAE

4 FAE5 FAE6 FAE

7 FAE8 FDA

9 FDE10 FAS

11 FAS

FAFAV

FAVFACFAD/G

FAD/GFAGFETak

FAV

IEFIEF

IEFMotherCAE

CAEMotherMSIEF

FA IEF

V represents an unidentified variant.8 Notthesame as the cases in Table3.IEF,isoelectricfocusing;CAE,cellulose acetate electrophoresis;MS, mass spectrometry;Mother, the newborn’smotherwas tested for hemoglobinopathies.

Case no.

See comment

CommentHb variant elutes on HPLC as FIb S. Incidence

1/10 000 newborns (1% of FAS patterns).[FIb A1/[Hb V] - 6/1

Hb variant,not resolved by IEF, elutes onHPLC as Hb E.

Same as case 1.Hb variant (not Hb C) elutes on HPLC as

Hb E.Same as case 3.Same as case 3.Hb G elutes on HPLC as Hb E. (Hb D ruled

out by the HPLC method.)

Same as case 6.

FIb G elutes on I-IPLC as FIb D.

Hb Tak elutes on HPLC as Hb D.

Hb variant elutes on HPLC as Kb S. [Hb Al/[Hb V] - 1.

Kb variant, not resolved by IEF, elutes onF$PLCas HbS. [Hb A]/[Hb V] - 1.

ing defective columns from use in testing specimens, the staff

maintain the height/area ratio for Hb F in liquid controls within

a specification of �3.5 MV/AU. During a recent 12-month

period when two lots of resin were in use, GDL rejected 102(6%) of 1769 columns because of broad Hb F peaks.

Resolution.Hb F, A, E, D, 5, and C are well resolved (Fig. 1).

Effectsofchangingthereagentlot.In 4 years we have used four lots

of resin. The CV for retention times on the different lots was

comparable with that given in Table 1. The accuracy of

retention times for four lots of resin was comparable with that

reported in Table 1 for lot 4. In two-thirds of the measurements,

the observed mean values for retention times were within 0.01

mm of the specified center of the hemoglobin identification

window. The maximum observed bias was 0.03 mm. The

observed mean values for all hemoglobins were the same for

liquid controls and eluates from DBS samples (no matrix effect).

The precision of quantification was determined by measur-

ing Hb F in the linearity calibrators with three column systems

at one laboratory; the CVs were <6% for each of the four lots

of resin. Photometer readings for the three linearity calibrators

were within acceptable limits (nominal value ±20%). CVs for

measuring Hb F, A, and S in DBS samples were <16% for all

lots, which is similar to the results obtained on one lot (Table 2).

Linearity. For four lots of resin and the corresponding lots of

linearity calibrators, the dose-response curves for the three

relative concentrations gave the following multiple R2 values: lot

1, 0.977; lot 2, 0.975; lot 3, 0.976; and lot 4, 0.985.

FREQUENCY DISTRIBUTION OF TOTAL HEMOGLOBINS

When applied to the California newborn population, the HPLC

screening method gave the following distribution for the total

area for all hemoglobins: n = 151 000; mean = 208 000 AU; SD

= 43 400 AU; skewness, 0.283; 1st percentile, 110 000 AU; 50th

percentile, 207 000 AU; 99th percentile, 320 000 AU. Within

the overall CV of 21% for the frequency distribution, the

variance (SD)2 is estimated to be distributed among its compo-

nents as follows: physiological 14%, DBS sample collection

14%, and HPLC methodology 72%.

DiscussionThe HPLC screening method quantifies the relative concentra-

tions of hemoglobin variants and has good reproducibility with

singleton determination. Quantitative ratio rules are invoked to

derive automatically the presumptive phenotype for each new-

born. Setting quantitative limits allows application of routine

quality-control rules. Proficiency tests are scored with the use of

quantitative acceptability limits.

Analyte contents measured in newborns’ DBS specimens are

dependent on the adequacy of the specimen. A hemoglobinop-athies screening test result of an extremely low or high concen-

tration of hemoglobin reveals specimens that are not suitable for

determinations of any of the newborn screening analytes (phe-

nylalanine, thyroxine, thyrotropin, uridyl transferase, etc.). In

such cases a second blood specimen must be obtained from the

newborn.

The HPLC screening method requires only a small sample.

One punch of a 0.95-cm-diameter disc from a blood-collection

card is eluted in water to separate the hemoglobmns. This same

ClinicalChemistry 42, No. 5, 1996 709

eluate is used for the determinations of two other newborn

screening analytes, phenylalanmne and uridyl transferase.

According to Bio-Rad, the rapid separation of hemoglobins is

possible because proteins do not penetrate the resin. Also, any

degraded hemoglobins and other proteins are removed from thecation-exchange resin before the hemoglobmns of interest are

eluted.The interferences from variant hemoglobmns that have reten-

tion times similar to Hb S, C, E, and D (Table 4) are relativelyfew and do not compromise the detection of newborns with

sickle cell disease. Also, degradation (if any) of hemoglobmns in aDBS sample does not interfere with the reporting of an accurate

phenotype.

Disadvantages of the method include the requirement for

manual aliquoting and dilution of the specimen eluate into the

microplate, which is subject to specimen identification error,

given that a specimen may be pipetted into the wrong well of the

microplate. Also, because the microplates have no barcode

identification, a positional control is needed to maintain the

sequence of analysis.

Although the California program does not screen for Hb

Barts, this variant is measurable with the HPLC screening

method. The chromatographic peak for Hb Barts elutes with a

retention time close to that of the void volume and can be seen,

for example, in some DBS specimens from newborns with Hb E,

giving a chromatogram characteristic of EJa-thalassemia.

Other methods used to screen newborns for hemoglobinop-

athies are cellulose acetate (basic) and citrate agar (acidic)

electrophoresis and isoelectric focusing. In a large-scale screen-

ing program, these methods do not compare favorably withHPLC screening, because they are not automated and quanti-

tative. When electrophoresis is used, the presumptive pheno-

types are derived by visual inspection, consensus decision-making, and manual data entry, all of which are subject to

human error and judgment. With HPLC screening, presump-

tive phenotypes are derived automatically. Quality control andproficiency testing are quantitative. Compared with other

HPLC techniques such as anion-exchange chromatography, the

cation-exchange chromatography used here has the advantage

that hemoglobin degradation products are eluted rapidly fromthe column and do not interfere with quantification of the

principal hemoglobmns.

Results of the laboratory analyses in clinical follow-up presented

in Tables 3 and 4 were determined under contract to the

California Department of Health Services by the Children’sHospital Oakland Research Institute, directed by F. Shafer, B.

Lubin, and E. Vichinsky.

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14. Loomis SJ, Go M, Kupeli L, Bartling DJ, Binder SR. An automatedsystem for sickle cell screening. Am Clin Lab 1990;Oct.:33-4O.

15. Weatherall DJ. The thalassaemia syndromes. Oxford, UK: Black-well Scientific Publications, 1965:268 pp.

16. Bisse E, Wieland H. High-performance liquid chromatographicseparation of human haemoglobins, simultaneous quantitation offoetal and glycated haemoglobins. J Chromatogr 1988;434:95-110.

Appendix: Rules for Derivation of Hemoglobin PattenisA list of the hemoglobmns with their retention times is given

below. Numbers in parentheses are used for unidentified spe-cies. For example, Hb (1) elutes between Hb F and Hb A.

Noise and minor hemoglobins. When present, Hb A is used as an

internal standard. Any chromatographic peak with an area <0.1

the area of the Hb A peak is not included in the pattern. If HbA is not present, the other adult hemoglobin (e.g., Hb S in sickle

cell disease) is used as the internal standard.

Hb (1).Hb (1) (possibly Hb Aid) elutes between Hb F and HbA. The computer program adds the concentration of Hb (1) to

Peak name

FAST

Fl

FA

E

0

S

C(1)(2)

(3)(4)(5)

0.180.450.610.83

0.98

1.07

1.181.72

0.730.91

1.131.331.55

710 Eastman et al.: Screening newborns for hemoglobinopathies by HPLC of dried blood spots

(6) 1.85

the concentration of Hb A. The total concentration is expressed

in the pattern as Hb A.

Hb F1 The concentration of Hb F1 (acetylated Hb F) is added

to the concentration of Hb F and the sum is expressed in the

pattern as Hb F.

Satellite hemoglobins. Consider Hb X as the major hemoglobin

and Hb Z as a potential satellite hemoglobin [5, 7]: If the

concentration of Hb Z is <0.25 the concentration of Hb X, Hb

Z is deleted from the pattern. In this work the combinations of

major and satellite hemoglobmns are (X/Z): E/A, D/(2), D/A,

SIE, S, and Cl(s).

Thalassemia flag. If Hb A and Hb Y (any variant) are both

present, divide the concentration of Hb Y by the concentrationof Hb A. If the quotient is >2, then change the representation

of Hb A in the pattern from “A” to “a”. For example, once it has

been determined that Hb A and Hb S are both present, thepattern report code of Hb A is changed to Hb a if the

concentration of Hb S is more than twice that of Hb A. Thus

S/-thalassemia is reported as FSa. For follow-up, FAS and FSA

are both reported as trait, unless [Hb S] >2[Hb A], in which case

the pattern is reported as S1f3-thalassemia [8, 15].

Inadequate specimens. When the total area is <60 000 AU, the

pattern report is “not determined (low area).” Similarly, when

the total area exceeds 420 000 AU, the pattern report is “not

determined (high area).” When a repeat analysis confirms ei-

ther low area or high area, the specimen is declared made-

quate for all the newborn screening analytes, and a new speci-

men is requested. In the California program, the number of

inadequate specimens so detected is -16 per 100 000 new-

borns tested.

Sample degradation. A flag is used to identify specimens with

excessive concentrations of hemoglobin degradation products.

In the chromatography system used, these compounds are

eluted before Hb F and appear in the two identification windows

defined as FAST and Fl. (In most specimens, window Fl holds

Hb F1, the acetylated form of Hb F, and no degradation

products.) When the total relative concentrations of Hb FASTplus Fl exceed 50%, the hemoglobin pattern is reported as “not

determined (FAST exceeds 50%).” In practice, after review of

the chromatogram by a quality-control officer, valid Hb pat-

terns can be reported with a total [FAST + Fl] as high as 75%.In the California program the incidence of chromatograms with

[FAST + Fl] >50% is <1 per 100 000 newborns tested. Many

of those found are the result of improper collection of the blood

sample from an umbilical line. Such specimens are designatedinadequate for determining all of the newborn screening

analytes.

F only. If the pattern is F only, the result is printed out as “not

determined (F only).” This result, which is expected only in

cases of f3-thalassemia major, must be confirmed by repeat

injection of the DBS eluate.

Peak criteria. The peak criteria for inclusion of a species in the

hemoglobin pattern are summarized in Table 5.

Table 5. Peak criteria for Inclusion of a species In the hemoglobin pattern report.Retention time, mm Peak criterion for Inclusion In pattern report

Always excludedAlways added to F, so the total of [Fl] + [F] is reported in the pattern as FAlways includedIncluded, unless: (a) E is present and [A] <[E]/4, in which case A is deleted

from the pattern, or (B) D is present and [Al <[D]/4, in which case A isdeleted from the pattern

Included, unless S is present and [E] <[S]/4, in which case E is deleted fromthe pattern

Included,unless S is present and [Dl <[S]/4, in which case D is deleted fromthe pattern

Always includedAlways includedIf F is present, the total of [(1)1 + [A] is reported in the pattern as A

Included, unless D is present and [(2)] <[D]/4, in which case (2) is deletedfrom the pattern

Always includedAlways includedIncluded, unless C is present and [(5)] <[C]/4, in which case (5) is deleted

from the patternAlways included