1. 704
ClinicalChemistiy 42:5
704-710 (1996)
Automated HPLC screening of newborns for
sickle 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 of
presumptive phenotypes; quantitative quality control and
proficiency testing; throughput of one specimen per
minute; small sample volume; hemoglobin concentrations
quantified 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 the
method 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 Disease
Laboratory (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 and
E//3-thalassemia. For trait patterns of FAS, FAC, and FAD,
the families of the affected newborns are offered voluntary
counseling.
Materialsand Methods
EQUIPMENT/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, the
cation-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 from
injection 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 one
Hb F, A, D, and C. To maintain the separation of hemoglobmns
among all lots of the cation-exchange resin, the software that

2. 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 ± 3SD
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 in
concentrations 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-

3. ‘20O
E
a)
(I)
0
100
0
400
a)
Cl)
0
a-
C’)
a)
0
0 2
Li
0 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.
Results
PERFORMANCE 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 controls
containing Hb FAES and Hb FADC.
Peaks (from left to right): Hb FAST, Fl, F, (1), A, E, D, S, and C. (See Appendix
for hemoglobin nomenclature.)
AU to 18% at 5000 AU. The 5000 AU (18% CV) is equivalent
to 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 to
eliminate background noise be reduced to 500 jV. To test the
practicality 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 per
week) 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 in
children at an older age (Table 3). In all cases, when the
chromatographic raw data were electronically reprocessed at a
lower 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 with
pattern ES.
Peaks (from left to right):void volume and Hb FAST, Fl, F, (1), and S.

4. n
Table 1. PrecisIon and accuracy of retention times.
Retention time, mm x 100
Mean
Mean
specification
F
A
E
D
S
C
BIas
422
422
211
211
211
211
SD
64.08
83.29
98.14
107.93
118.25
172.60
Hb
Liquid samples
Dried blood spots
Tables 1 and 2: Datacollectedfrom nine sites using resin lot 4 on 23 columnsystems.
SD
specification
F
61.00
83.00
98.00
107.00
118.00
172.00
CV, %
A
S
+ 3.08
+ 0.29
+0.14
+0.93
+0.25
+0.60
90
0.97
1.00
1.26
1.39
1.39
1.59
97
204
63.37
84.29
117.57
2.33
1.67
1.33
1.67
1.33
2.00
61.00
83.00
118.00
1.5
1.2
1.3
1.4
1.2
0.9
-2.37
+ 1.29
-0.43
1.17
1.02
1.41
2.33
1.67
1.33
1.8
1.2
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 which
the 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 an
atypical 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 the
Appendix 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. of
column
Hb systems
F
AU
No. of
samples Mean SO
F
Liquid samples
Dried blood spots
A
S
CV, %
18 40
23b 105
23 213
23 213
103 834
101 679
29 057
7 338
F
4621
11 112
4 295
1 002
Table 3. ComparIson of the newborn screening result with
the result obtaIned at an older age.a
Pattern
Resuft after reprocessing
Case no. Newborn Older data, % of total Hb
1 FS FSaC HbA, 0.9
2 FS FSa HbA, <0.5
3 ES FSa HbA, <0.5
4 ES FSAC HbA, 0.8
5 ES FAS HbA, <1.0
6 FAd FAS HbS, 0.5
7 FA#{176} FAS FIbS, 0.88
8 F only FAS HbS, 1.08
9 FE FEa HbA, <0.5
10 FA’ FAE HbE, 0.8
11 F only HbA, 0.9, HbD, 0.8
8 Differencesattributableto 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 specimen gave 1.1% HD E and a patternofFAE.
g v represents an unidentifiedvariant.
4.4
10.9
14.8
13.6
A
S
23 27 37838
23 97 5284
23 206 5 113
5 147
918
894
13.6
17.4
17.5
ClinicalChemistry 42, No. 5, 1996 707
8CV <5% specified.
Rangeof ±20% specified.

5. 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 tImes
for Hb S, E, and D.
Screening pattern Follow-up pattern Follow.upmethodb
FAV IEF
FA IEF
FAS
1 FAE
2 FAE
3 FAE
4 FAE
5 FAE
6 FAE
7 FAE
8 FDA
9 FDE
10 FAS
11 FAS
FA
FAV
FAV
FAC
FAD/G
FAD/G
FAG
FETak
FAV
IEF
IEF
IEF
Mother
CAE
CAE
Mother
MS
IEF
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
Comment
Hb 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 on
HPLC 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 on
F$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%.
Discussion
The 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

6. 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 the
cation-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 relatively
few and do not compromise the detection of newborns with
sickle cell disease. Also, degradation (if any) of hemoglobmns in a
DBS 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 with
HPLC 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 and
proficiency 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 from
the 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’s
Hospital Oakland Research Institute, directed by F. Shafer, B.
Lubin, and E. Vichinsky.
References
1. Office of Medical Applications of Research. Newborn screening for
sickle cell disease and other hemoglobinopathies. National Insti-
tutes of Health consensus development conference statement,
Vol. 6, No. 9. Bethesda, MD: National Institutes of Health, Public
Health Service, US Department of Health and Human Services,
1987.
2. Sickle Cell Disease Guideline Panel. Sickle cell disease: screen-
ing, diagnosis, management, and counseling in newborns and
infants. Clinical Practice Guideline No. 6. AHCPR Pub. No. 93-
0562. Rockville, MD: Agency for Health Care Policy and Research,
Public Health Service, US Department of Health and Human
Services, 1993.
3. California Health and Safety Code §309.5 (West 1990 & Suppl
1995).
4. Lorey F, Cunningham GC, Shafer F, Lubin B, Vichinsky E. Universal
screening for hemoglobinopathies using high performance liquid
chromatography: clinical results of 2.2 million screens. Eur J Hum
Genet 1994;2:262-71.
5. Wilson JB, Headlee ME, Huisman THJ. A new high-performance
liquid chromatographic procedure for the separation and quanti-
tation of various hemoglobin variants in adults and newborn
babies. J Lab Clin Med 1983:102:174-86.
6. Ou C-N, Buffone GJ, Reimer GL. High-performance liquid chroma-
tography of human hemoglobins on a new cation exchanger. J
Chromatogr 1983:266:197-205.
7. Huisman THJ. Percentages of abnormal hemoglobins in adults
with a heterozygosity for an a-chain and/or a chain variant. Am
J Hematol 1983;14:393-404.
8. Kutlar A, Kutlar F, Wilson JB, Headlee MG, Huisman THJ. Quanti-
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Appendix: Rules for Derivation of Hemoglobin Pattenis
A 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 Hb
A 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 Hb
A. The computer program adds the concentration of Hb (1) to

7. Peak name
FAST
Fl
F
A
E
0
S
C
(1)
(2)
(3)
(4)
(5)
0.18
0.45
0.61
0.83
0.98
1.07
1.18
1.72
0.73
0.91
1.13
1.33
1.55
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 concentration
of 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, the
pattern 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 FAST
plus 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 designated
inadequate 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 excluded
Always added to F, so the total of [Fl] + [F] is reported in the pattern as F
Always included
Included, 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 is
deleted from the pattern
Included, unless S is present and [E] <[S]/4, in which case E is deleted from
the pattern
Included,unless S is present and [Dl <[S]/4, in which case D is deleted from
the pattern
Always included
Always included
If 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 deleted
from the pattern
Always included
Always included
Included, unless C is present and [(5)] <[C]/4, in which case (5) is deleted
from the pattern
Always included