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Clinical

Chemistiy 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- that newborns with hemoglobinopathy patterns (e.g., FS, FSC,
mens from newborns for hemoglobms (ITh) F, A, S, C, E, FSA, F only) be recalled for mandatory follow-up. Also, new-
and D. We present the method and report on its perfor- borns with FE patterns are recalled to differentiate EE and
mance determined during >4 years of testing 2.5 x 106 E//3-thalassemia. For trait patterns of FAS, FAC, and FAD,
newborns. The method features automated derivation of the families of the affected newborns are offered voluntary
presumptive phenotypes; quantitative quality control and counseling.
proficiency testing; throughput of one specimen per
minute; small sample volume; hemoglobin concentrations Materials and Methods
quantified with an mterlaboratory CV of 14-18%; retention EQUIPMENT/REAGENT SYSTEM
times with interlaboratory CV of <2% and matching, Modular instrumentation was adapted for the California pro-
within ± 0.03 miii, of laboratories and reagent lots; control gram by Bio-Rad Labs., Hercules, CA. A similar integrated
of peak resolution; 0.5% detection limit for Hb S and C, and instrument, ASCeNT#{174},has been marketed [14]. Each of the
1.0% for Hb F, A, E, and D; few interferences; and negli- modular instruments used in this work consists of one to three
gible background and carryover. Shortcomings of the HPLC column systems run by one Model 700 chromatography
method are the absence of microplate barcode identifica- workstation. Each of the column systems consists of a Model
tion and the need for manually pipetting the sample eluate AS-I00 HRLC automated sampling system, a 20-g.tL-loop
into the microplate. injection valve, two Model 1350 gradient elution pumps, the
cation-exchange column in a Model 1250425 35 #{176}C heater, and
INDEXING mie1s: dried blood-spot specimens Guthrie
#{149} cards. a dual-wavelength (415/690 nm) filter photometer (cat. no.
chromatography, cation-exchange phenotypes
#{149} 1961042). The 6 X 40 mm columns are packed with a nonpo-
rous 7-.tm-diameter Bio-Rad MA 7 polymeric cation-exchange
In keeping with national recommendations, California legisla- material. Each column can be used for as many as 500 injections.
tion mandates screening all newborns for sickle cell disease Two sodium phosphate buffers are used as eluents, run as a
[1-3].This legislation was implemented for the State of Cali- gradient from -4 g/L (buffer A) to 14 g/L (buffer B) at pH 6.4.
fornia by the Department of Health Services’ Genetic Disease Nominal conditions of analysis are a flow rate of 2 mL/min
Laboratory (GDL) and Genetic Disease Branch, Berkeley, CA at a pressure of 25 kg/cm2 and the following gradient (time from
[4]. Because of its potential as a quantitative method of analysis injection in minutes/percent of eluent that is buffer B): 0.0/0%;
amenable to automation, GDL chose cation-exchange HPLC 0.3/10%; 0.5/24%; 1.0/52%; 1.8/100%; 1.9/100%; and 2.0/0%.
[5-12] as the screening method. Cation-exchange HPLC has Including the 1-mm wash between specimens, each chromato-
been used to screen cord blood for hemoglobinopathies [13]. gram takes 3 mm. When all three columns are used, the rate of
Here, we report on the application of this technique to screen analysis is one specimen pen minute.
newborns by assaying their dried blood-spot (DBS) specimens. Operation of the instrument is automated through a menu-
The method reported here is designed to resolve hemoglo- driven software program that generates the worklist, injects the
bins (Hb) F, A, 5, C, E, and D. The California program requires 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
California Department of Health Services, Genetic Disease Laboratory, 700
data to a remote central site.
Heinz St., Suite 100, Berkeley, CA 94710. The reagent kits include whole-blood primer, wash solution,
*AUthOr for correspondence. Fax 510-540-2228.
three linearity calibrators containing Hb F and A, and two
‘Nonstandard abbreviations: GDL, Genetic Disease Laboratory; DBS, dried
lyophilized controls, one containing Hb F, A, E, and S and one
blood spot; NB, newborn; Hb, hemoglobin(s); and AU, hemoglobin concentration
in area units, the area of the chromatographic peak. Hb F, A, D, and C. To maintain the separation of hemoglobmns
Received October 4, 1995; accepted January 25, 1996. among all lots of the cation-exchange resin, the software that

704
Clinical Chemistry 42, No. 5, 1996 705

controls the gradient is modified when needed to accommodate DERIVATION OF PATTERNS


any differences in the performance of the different lots. For each newborn tested, a hemoglobin pattern presents the
A key feature of the test system is its quantification of the observed hemoglobins in order of relative concentration from
concentration of the hemoglobin variants. Chromatographic the highest to lowest. GDL developed algorithms (see Appendix)
peaks are reported with heights in microvolts and with areas in for use on the chromatography workstation software so that
relative response units (AU, area units). With the integration chromatographic peaks from noise and minor hemoglobins are
settings used for this screening method, 1 AU is approximately not included in the hemoglobin patterns.
three times the area in p.V-min. The three linearity calibrators The retention time of Hb A, is similar to that of Hb E.
are used to monitor the dose-response curve for photometer However, Hb A2 is never expressed in a hemoglobin pattern
readings vs hemoglobin concentration. The reagent-instrument derived for a newborn specimen because its concentration is
system must maintain the photometer readings for the linearity low, and the interpretation algorithms remove such low peaks
calibrators (-0.2, 0.4, and 0.6 g/L Hb F) within ±20% of a from the pattern (Appendix). Satellite hemoglobins (e.g., E1, 5,,
stated nominal value. C,) produce small chromatographic peaks that elute -0.17 mm
faster than the peak of the corresponding major hemoglobin
HPLC SCREENING METHOD [5,7]. GDL introduced a 1:4 rule to eliminate the satellite
Specimen collection and preparation.Blood from a subject is hemoglobmns from the reported patterns. Also, based on pub-
absorbed into S&S 903 specimen collection paper (Schleicher & lished information on the relative percentage concentrations of
Schuell, Keene, N}-l). A disc 0.95 cm (3/8 in.) in diameter is Hb A in thalassemia cases [8, iS], GDL developed a 1:2 rule to
punched from the DBS specimen, and the blood is eluted into differentiate patterns FAS (sickle cell trait) from FSA (S/f3-
1.00 mL of water for 30 mm with periodic shaking (equivalent thalassemia) (see Appendix).
to a 36-fold dilution of the whole-blood specimen). The eluate Hb (1), an unidentified hemoglobin eluting between Hb F
is further diluted 1:6 with water and dispensed into a 96-well and Hb A, appears in the HPLC chromatograms for the
microplate, which is loaded onto the autosampler for injection. linearity calibrators, the liquid controls, surrogate in-house DBS
samples, and in most DBS specimens from newborns. The
A worklist
Worklistgenerationand reports. is generated automat- retention time for Hb (1) corresponds to that expected for Hb
ically when the analyst sets up the run file information. At the A,d [16]. The program used to derive the hemoglobin pattern
end of each run, a Neonatal Hemoglobin Summary Report is adds the concentration of Hb (1) to the concentration of Hb A
printed out. The summary lists the identification number of and reports the total concentration as Hb A (Appendix).
each specimen and the percentages of all the hemoglobin
variants found in the specimen. A Pattern Report is also printed SPECIMENS ANALYZED
out, listing the hemoglobin pattern (presumptive phenotype) for GDL collected data on three types of samples: the liquid
each specimen. This report lists, in order of decreasing concen- Bio-Rad FAES and FADC controls, which are not processed
tration, the letter designation of each of the hemoglobins found through the punching, elution, and dilution steps of the method;
in the specimen. surrogate DBS specimens prepared in house (GDL-DBS sam-
ples); and newborns’ specimens collected on paper (NB-DBS
Quality control. For each column system, the FAES and FADC specimens). The liquid controls contain Hb F, A, 5, C, E, D in
liquid controls are run at the beginning and end of each run. At concentrations similar to those found in actual newborn speci-
the beginning and end of each tray (microplate) of newborn mens, i.e., mostly Hb F with 10-20% Hb A and 3-10% of the
specimens, eluates of blood spots containing Hb S are run as tray other variants. The GDL-DBS samples, which contain Hb F, A,
controls. The mean and SD values for retention time are fixed as and 5, were prepared by mixing commercial bulk AS blood or
quality-control action limits in the controller software by spec- cord blood with adult outdated bank blood and spotting the
ification. Mean and SD values for hemoglobin concentration are mixture onto specimen collection paper. These DBS sample
established by replicate analysis of the controls at GDL before pools contain various concentrations of the hemoglobin variants
they are put into use. Trays of newborn results are flagged for for use as controls and proficiency-test samples. For GDL-DBS
review by a quality-control officer when (a) the retention time of pools containing Hb S, the concentration of Hb A is large
Hb S in the tray control exceeds the ±3SD limit; (b) the (-75%), and the ratio of [Hb A]/[Hb S] does not match the ratio
concentration of Hb S in the tray control exceeds the ± 3 SD found in newborn specimens. Nevertheless, these samples yield
limit; or (c) the blank water injections give a chromatographic satisfactory chromatograms.
peak height >5000 j.tV.
Because the microplates are not barcoded for identification, EVALUATION OF PERFORMANCE CHARACTERISTICS
a positional control is also included on each tray. This is an The precision and accuracy of retention times, as well as the
eluate of a blank disc of blood-collection paper placed in a precision of the variant quantification, were determined from
unique position such that it documents each tray’s number in the data collected with 23 column systems at nine laboratories using
sequence of analysis (e.g., tray 2 must have a blank result at one lot of cation-exchange resin. We also evaluated the accuracy
position 2). The positional control also serves as a water blank and precision of data collected with four different lots of resin by
on each tray. one laboratory using three column systems. The other perfor-
706 Eastman et al.: Screening newborns for hemoglobinopathies by HPLC of dried blood spots

mance characteristics were determined for the aggregate of 23 AU to 18% at 5000 AU. The 5000 AU (18% CV) is equivalent
column systems and four lots of resin. to a NIB-DBS specimen containing a hemoglobin variant at
2.5% relative concentration (in a total area of 200 000 AU). The
Results 30 000 AU (14% CV) is equivalent to a NB-DBS specimen
PERFORMANCE ASSESSMENT containing a hemoglobin variant at 15% relative concentration.
Chromatograms. Fig. 1 is a chromatogram obtained from a liquid
sample made by combining controls with Hb F, A, 5, C, E, and Detection limits. HPLC peak criteria in the integration parame-
D. Fig. 2 is a chromatogram of an eluate of a DBS specimen ters are set so that Hb S and C are expressed in the hemoglobin
obtained from a newborn with sickle cell disease. 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
Precision of retention times.Table 1 compares our results with the these detection limits requires that the height threshold used to
instrument specifications. The SD specification is one-sixth the eliminate background noise be reduced to 500 jV. To test the
range of the retention time identification window (window practicality of this threshold, GDL submitted two GDL-DBS
± 3SD). The observed SD for Hb S exceeds the limit some- samples containing 1.1% and 0.8% Hb S for analysis at the
what; nonetheless, at this precision, all cases of sickle cell disease satellite laboratories. In 12 weekly shipments (one sample per
have been correctly identified. week) to nine laboratories (n = 106 analyses after excluding 2
results that were invalid for other reasons), there were no missed
Accuracy of retention times. The observed mean retention time Hb S peaks. By now, >450 000 newborns have been tested with
(Table 1) for each hemoglobin is compared with the retention use of the 500-.tV height threshold, and there have been no
time for the center of the hemoglobin identification window in known missed cases of Hb S or other clinically significant
the integration software. In most cases the observed mean values variant exceeding 0.5% in the newborns’ specimens.
are within 0.01 mm of the specified value, and in no case does That a 500 .tV threshold is required is shown by results
the bias exceed 0.03 mm. 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
Precision of quantification of hemoglobins. According to the speci- 3000 j.V not all satellite laboratories reported the presence of
fications (Table 2, last column), with a one-column system (with Hb 5, even though it made up 1.0% of the total hemoglobin in
one photometer) the interrun CV for Hb F in the middle- the GDL-DBS proficiency-test samples. However, retrospective
concentration linearity calibrator should be no more than 5%. examination of the chromatograms, which are stored on mag-
Also, among all systems the mean should always lie between netic tape, and reprocessing at a lower threshold detected all of
80 000 and 120 000 AU (±20% range for matching the 23 the Fib S peaks. Also, during that period, -2.1 million newborns
systems). The results are in acceptable agreement with the were screened. In rare instances Hb S, A, E, and D that were not
specifications. The CV of 10.9% over all 23 column systems is reported in the newborn screening pattern were found in
equivalent to a ±2SD range of 21.8%, which is close to the children at an older age (Table 3). In all cases, when the
±20% range limit. chromatographic raw data were electronically reprocessed at a
On multiple column systems the observed CVs for liquid lower threshold (e.g., 500 .tV), we found a well-resolved bell-
controls and DBS samples span a range from 14% at 30 000 shaped peak at the retention time of the missing variant.

300
bOO

‘20O
E 400
a)
(I) a)
Cl)
0
0
100 a-
C’)
a)

0 2
0 Li
0 2
Time (mm)
Time (mm)
Fig. 1. Chromatogram of a 1:1 mixture of two Bio-Rad controls
containing Hb FAES and Hb FADC. FIg. 2. Chromatogram of a newborn dried blood-spot specimen with
Peaks (from left to right): Hb FAST, Fl, F, (1), A, E, D, S, and C. (See Appendix pattern ES.
for hemoglobin nomenclature.) Peaks (from left to right): void volume and Hb FAST, Fl, F, (1), and S.
Clinical
Chemistry 42, No. 5, 1996 707

Table 1. PrecisIon and accuracy of retention times.


Retention time, mm x 100

Mean SD
Hb n Mean specification BIas SD specification CV, %
Liquid samples
F 422 64.08 61.00 +3.08 0.97 2.33 1.5
A 422 83.29 83.00 +0.29 1.00 1.67 1.2
E 211 98.14 98.00 +0.14 1.26 1.33 1.3
D 211 107.93 107.00 +0.93 1.39 1.67 1.4
S 211 118.25 118.00 +0.25 1.39 1.33 1.2
C 211 172.60 172.00 +0.60 1.59 2.00 0.9
Dried blood spots
F 90 63.37 61.00 -2.37 1.17 2.33 1.8
A 97 84.29 83.00 + 1.29 1.02 1.67 1.2
S 204 117.57 118.00 -0.43 1.41 1.33 1.2
Tables 1 and 2: Data collected from nine sites using resin lot 4 on 23 column systems.

The concentrations determined after reprocessing are given in Background and carryover. Water blanks are included on the
Table 3. calibration tray and on each tray of newborns’ samples for
Theoretically, the detection limit of the method might be analyses. GDL staff review all chromatograms that show a peak
decreased by a further reduction in the threshold setting. with height >5000 .tV. These situations occur infrequently (9
However, all cases of sickle cell disease are detected at the times per 100 000 newborns) and do not affect newborns’
current value. hemoglobin patterns. In some cases small spikes are observed,
usually at the void volume (retention time 0.2 mm). However,
Specificity. In the past 4 years, GDL has been notified of 12 the retention times are too fast, the peak widths too narrow, and
instances in which diagnostic follow-up results did not agree the heights too low for these spikes to affect the hemoglobin
with the newborn screening hemoglobin patterns, and for which patterns generated by the method algorithms (Appendix).
the discrepancy could be attributed to the specificity of the
HPLC method (Table 4). Also, we have found that -1% of the Peak shape. Laboratory staff monitor the Hb F peak shapes for
chromatographic peaks at the retention time for Hb S have an the controls and for 5% of all newborns’ specimens. By remov-
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
Table 3. ComparIson of the newborn screening result with
chromatographic peaks, is readily identified by visual inspection
the result obtaIned at an older age.a
of the chromatogram. According to the rules given in the
Pattern
Appendix for derivation of patterns, and depending on the Resuft after reprocessing
particular lot of cation-exchange resin in use, Hb G has been Case no. Newborn Older data, % of total Hb
reported in newborn screening as Hb E, Hb D, or combinations 1 FS FSaC HbA, 0.9
of Hb E and D with Hb (2). 2 FS FSa HbA, <0.5
3 ES FSa HbA, <0.5
4 ES FSAC HbA, 0.8
5 ES FAS HbA, <1.0
Table 2. PrecIsion of quantification.
6 FAd FAS HbS, 0.5
No. of AU
column No. of 7 FA#{176} FAS FIbS, 0.88
Hb systems samples Mean SO CV, % 8 F only FAS HbS, 1.08
Liquid samples 9 FE FEa HbA, <0.5
F 18 40 103 834 4621 4.4 10 FA’ FAE HbE, 0.8
F 23b 105 101 679 11 112 10.9 11 F only HbA, 0.9, HbD, 0.8
A 23 213 29 057 4 295 14.8 8 Differencesattributable to prior use of higherthreshold (>500 MV).
S 23 213 7 338 1 002 13.6 b Original newborn screening data reprocessed to reflect current 500 V
Dried blood spots threshold.
r FSApatterns are reported as trait
unless(Hb SJ >2 lHb Al,inwhichcase the
F 23 27 37838 5 147 13.6
pattern isreported as FSa forS/p.thalassernia
(see Appendix).
A 23 97 5284 918 17.4 d Follow-up initiated
from familystudies.
S 23 206 5 113 894 17.5 8 HbS >0.5%.

8CV <5% specified. ‘Reanalysis ofNB-DBS specimen gave 1.1% HD E and a patternofFAE.
Rangeof ±20% specified. g v represents an unidentifiedvariant.
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.
Case no. Screening pattern Follow-up pattern Follow.up methodb Comment
See comment FAS FAV IEF Hb variant elutes on HPLC as FIb S. Incidence
1/10 000 newborns (1% of FAS patterns).
[FIb A1/[Hb V] - 6/1
1 FAE FA IEF Hb variant,not resolved by IEF, elutes on
HPLC as Hb E.
2 FAE FA IEF Same as case 1.
3 FAE FAV IEF Hb variant (not Hb C) elutes on HPLC as
Hb E.
4 FAE FAV IEF Same as case 3.
5 FAE FAC Mother Same as case 3.
6 FAE FAD/G CAE Hb G elutes on HPLC as Hb E. (Hb D ruled
out by the HPLC method.)
7 FAE FAD/G CAE Same as case 6.
8 FDA FAG Mother FIb G elutes on I-IPLC as FIb D.
9 FDE FETak MS Hb Tak elutes on HPLC as Hb D.
10 FAS FAV IEF Hb variant elutes on HPLC as Kb S. [Hb Al/
[Hb V] - 1.
11 FAS FA IEF Kb variant, not resolved by IEF, elutes on
F$PLCas HbS. [Hb A]/[Hb V] - 1.
V represents an unidentified variant.
8 Not the same as the cases in Table 3.

IEF,isoelectric
focusing; CAE,cellulose acetate electrophoresis;MS, mass spectrometry;Mother, the newborn’smother was tested for hemoglobinopathies.

ing defective columns from use in testing specimens, the staff FREQUENCY DISTRIBUTION OF TOTAL HEMOGLOBINS
maintain the height/area ratio for Hb F in liquid controls within When applied to the California newborn population, the HPLC
a specification of 3.5 MV/AU. During a recent 12-month screening method gave the following distribution for the total
period when two lots of resin were in use, GDL rejected 102 area for all hemoglobins: n = 151 000; mean = 208 000 AU; SD
(6%) of 1769 columns because of broad Hb F peaks. = 43 400 AU; skewness, 0.283; 1st percentile, 110 000 AU; 50th
percentile, 207 000 AU; 99th percentile, 320 000 AU. Within
Hb F, A, E, D, 5, and C are well resolved
Resolution. (Fig. 1). the overall CV of 21% for the frequency distribution, the
variance (SD)2 is estimated to be distributed among its compo-
Effects ofchangingthereagentlot. In 4 years we have used four lots nents as follows: physiological 14%, DBS sample collection
of resin. The CV for retention times on the different lots was 14%, and HPLC methodology 72%.
comparable with that given in Table 1. The accuracy of
retention times for four lots of resin was comparable with that Discussion
reported in Table 1 for lot 4. In two-thirds of the measurements, The HPLC screening method quantifies the relative concentra-
the observed mean values for retention times were within 0.01 tions of hemoglobin variants and has good reproducibility with
mm of the specified center of the hemoglobin identification singleton determination. Quantitative ratio rules are invoked to
window. The maximum observed bias was 0.03 mm. The derive automatically the presumptive phenotype for each new-
observed mean values for all hemoglobins were the same for born. Setting quantitative limits allows application of routine
liquid controls and eluates from DBS samples (no matrix effect). quality-control rules. Proficiency tests are scored with the use of
The precision of quantification was determined by measur- quantitative acceptability limits.
ing Hb F in the linearity calibrators with three column systems Analyte contents measured in newborns’ DBS specimens are
at one laboratory; the CVs were <6% for each of the four lots dependent on the adequacy of the specimen. A hemoglobinop-
of resin. Photometer readings for the three linearity calibrators athies screening test result of an extremely low or high concen-
were within acceptable limits (nominal value ±20%). CVs for tration of hemoglobin reveals specimens that are not suitable for
measuring Hb F, A, and S in DBS samples were <16% for all determinations of any of the newborn screening analytes (phe-
lots, which is similar to the results obtained on one lot (Table 2). nylalanine, thyroxine, thyrotropin, uridyl transferase, etc.). In
such cases a second blood specimen must be obtained from the
Linearity. For four lots of resin and the corresponding lots of newborn.
linearity calibrators, the dose-response curves for the three The HPLC screening method requires only a small sample.
relative concentrations gave the following multiple R2 values: lot One punch of a 0.95-cm-diameter disc from a blood-collection
1, 0.977; lot 2, 0.975; lot 3, 0.976; and lot 4, 0.985. card is eluted in water to separate the hemoglobmns. This same
Clinical
Chemistry 42, No. 5, 1996 709

eluate is used for the determinations of two other newborn 2. Sickle Cell Disease Guideline Panel. Sickle cell disease: screen-
screening analytes, phenylalanmne and uridyl transferase. ing, diagnosis, management, and counseling in newborns and
According to Bio-Rad, the rapid separation of hemoglobins is infants. Clinical Practice Guideline No. 6. AHCPR Pub. No. 93-
0562. Rockville, MD: Agency for Health Care Policy and Research,
possible because proteins do not penetrate the resin. Also, any
Public Health Service, US Department of Health and Human
degraded hemoglobins and other proteins are removed from the Services, 1993.
cation-exchange resin before the hemoglobmns of interest are 3. California Health and Safety Code §309.5 (West 1990 & Suppl
eluted. 1995).
The interferences from variant hemoglobmns that have reten- 4. Lorey F, Cunningham GC, Shafer F, Lubin B, Vichinsky E. Universal
tion times similar to Hb S, C, E, and D (Table 4) are relatively screening for hemoglobinopathies using high performance liquid
few and do not compromise the detection of newborns with chromatography: clinical results of 2.2 million screens. Eur J Hum
Genet 1994;2:262-71.
sickle cell disease. Also, degradation (if any) of hemoglobmns in a
5. Wilson JB, Headlee ME, Huisman THJ. A new high-performance
DBS sample does not interfere with the reporting of an accurate
liquid chromatographic procedure for the separation and quanti-
phenotype. tation of various hemoglobin variants in adults and newborn
Disadvantages of the method include the requirement for babies. J Lab Clin Med 1983:102:174-86.
manual aliquoting and dilution of the specimen eluate into the 6. Ou C-N, Buffone GJ, Reimer GL. High-performance liquid chroma-
microplate, which is subject to specimen identification error, tography of human hemoglobins on a new cation exchanger. J
given that a specimen may be pipetted into the wrong well of the Chromatogr 1983:266:197-205.
7. Huisman THJ. Percentages of abnormal hemoglobins in adults
microplate. Also, because the microplates have no barcode
with a heterozygosity for an a-chain and/or a chain variant. Am
identification, a positional control is needed to maintain the J Hematol 1983;14:393-404.
sequence of analysis. 8. Kutlar A, Kutlar F, Wilson JB, Headlee MG, Huisman THJ. Quanti-
Although the California program does not screen for Hb tation of hemoglobin components by high-performance cation-
Barts, this variant is measurable with the HPLC screening exchange liquid chromatography. Am J Hematol 1984;17:39-53.
method. The chromatographic peak for Hb Barts elutes with a 9. Rogers BB, Wessels RA, Ou C-N, Buffone GJ. High performance
retention time close to that of the void volume and can be seen, liquid chromatography in the diagnosis of hemoglobinopathies and
thalassemias; report of three cases. Am J Clin Pathol 1985;84:
for example, in some DBS specimens from newborns with Hb E,
67 1-4.
giving a chromatogram characteristic of EJa-thalassemia.
10. Wilson JB, Wrightstone RN, Huisman THJ. Rapid cation-exchange
Other methods used to screen newborns for hemoglobinop- high-performance liquid chromatographic procedure for the sepa-
athies are cellulose acetate (basic) and citrate agar (acidic) ration and quantitation of hemoglobins S, C, and 0 Arab in cord
electrophoresis and isoelectric focusing. In a large-scale screen- blood samples. J Lab Clin Med 1986:108:138-41.
ing program, these methods do not compare favorably with 11. Huisman THJ. Separation of hemoglobins and hemoglobin chains
HPLC screening, because they are not automated and quanti- by high-performance liquid chromatography. J Chromatogr 1987;
418:277-304.
tative. When electrophoresis is used, the presumptive pheno-
12. Ou C-N, Rognerud CL. Rapid analysis of hemoglobin variants by
types are derived by visual inspection, consensus decision-
cation-exchange HPLC. Clin Chem 1993;39:820-4.
making, and manual data entry, all of which are subject to 13. van der Dijs FPL, van den Berg GA, Schermer JG, Muskiet FD,
human error and judgment. With HPLC screening, presump- Landman H, Muskiet FAJ. Screening cord blood for hernogmobinop-
tive phenotypes are derived automatically. Quality control and athies and thalassernia by HPLC. Clin Chem 1992;38:1864-9.
proficiency testing are quantitative. Compared with other 14. Loomis SJ, Go M, Kupeli L, Bartling DJ, Binder SR. An automated
HPLC techniques such as anion-exchange chromatography, the system for sickle cell screening. Am Clin Lab 1990;Oct.:33-4O.
cation-exchange chromatography used here has the advantage 15. Weatherall DJ. The thalassaemia syndromes. Oxford, UK: Black-
well Scientific Publications, 1965:268 pp.
that hemoglobin degradation products are eluted rapidly from
16. Bisse E, Wieland H. High-performance liquid chromatographic
the column and do not interfere with quantification of the
separation of human haemoglobins, simultaneous quantitation of
principal hemoglobmns. foetal and glycated haemoglobins. J Chromatogr 1988;434:95-
110.

Results of the laboratory analyses in clinical follow-up presented Appendix: Rules for Derivation of Hemoglobin Pattenis
in Tables 3 and 4 were determined under contract to the A list of the hemoglobmns with their retention times is given
California Department of Health Services by the Children’s below. Numbers in parentheses are used for unidentified spe-
Hospital Oakland Research Institute, directed by F. Shafer, B. cies. For example, Hb (1) elutes between Hb F and Hb A.
Lubin, and E. Vichinsky.
Noise and minor hemoglobins. When present, Hb A is used as an
internal standard. Any chromatographic peak with an area <0.1
References the area of the Hb A peak is not included in the pattern. If Hb
1. Office of Medical Applications of Research. Newborn screening for
A is not present, the other adult hemoglobin (e.g., Hb S in sickle
sickle cell disease and other hemoglobinopathies. National Insti-
tutes of Health consensus development conference statement, cell disease) is used as the internal standard.
Vol. 6, No. 9. Bethesda, MD: National Institutes of Health, Public
Health Service, US Department of Health and Human Services, Hb (1).Hb (1) (possibly Hb Aid) elutes between Hb F and Hb
1987. A. The computer program adds the concentration of Hb (1) to
710 Eastman et al.: Screening newborns for hemoglobinopathies by HPLC of dried blood spots

the concentration of Hb A. The total concentration is expressed quate for all the newborn screening analytes, and a new speci-
in the pattern as Hb A. men is requested. In the California program, the number of
inadequate specimens so detected is -16 per 100 000 new-
Hb F1 The concentration of Hb F1 (acetylated Hb F) is added borns tested.
to the concentration of Hb F and the sum is expressed in the
pattern as Hb F. Sample degradation. A flag is used to identify specimens with
excessive concentrations of hemoglobin degradation products.
Satellite hemoglobins. Consider Hb X as the major hemoglobin In the chromatography system used, these compounds are
and Hb Z as a potential satellite hemoglobin [5, 7]: If the eluted before Hb F and appear in the two identification windows
concentration of Hb Z is <0.25 the concentration of Hb X, Hb defined as FAST and Fl. (In most specimens, window Fl holds
Z is deleted from the pattern. In this work the combinations of Hb F1, the acetylated form of Hb F, and no degradation
major and satellite hemoglobmns are (X/Z): E/A, D/(2), D/A, products.) When the total relative concentrations of Hb FAST
SIE, S, and Cl(s). plus Fl exceed 50%, the hemoglobin pattern is reported as “not
determined (FAST exceeds 50%).” In practice, after review of
Thalassemia flag. If Hb A and Hb Y (any variant) are both the chromatogram by a quality-control officer, valid Hb pat-
present, divide the concentration of Hb Y by the concentration terns can be reported with a total [FAST + Fl] as high as 75%.
of Hb A. If the quotient is >2, then change the representation In the California program the incidence of chromatograms with
of Hb A in the pattern from “A” to “a”. For example, once it has [FAST + Fl] >50% is <1 per 100 000 newborns tested. Many
been determined that Hb A and Hb S are both present, the of those found are the result of improper collection of the blood
pattern report code of Hb A is changed to Hb a if the sample from an umbilical line. Such specimens are designated
concentration of Hb S is more than twice that of Hb A. Thus inadequate for determining all of the newborn screening
S/-thalassemia is reported as FSa. For follow-up, FAS and FSA analytes.
are both reported as trait, unless [Hb S] >2[Hb A], in which case
the pattern is reported as S1f3-thalassemia [8, 15]. 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
Inadequate specimens. When the total area is <60 000 AU, the cases of f3-thalassemia major, must be confirmed by repeat
pattern report is “not determined (low area).” Similarly, when injection of the DBS eluate.
the total area exceeds 420 000 AU, the pattern report is “not
determined (high area).” When a repeat analysis confirms ei- Peak criteria. The peak criteria for inclusion of a species in the
ther low area or high area, the specimen is declared made- hemoglobin pattern are summarized in Table 5.

Table 5. Peak criteria for Inclusion of a species In the hemoglobin pattern report.
Peak name Retention time, mm Peak criterion for Inclusion In pattern report
FAST 0.18 Always excluded
Fl 0.45 Always added to F, so the total of [Fl] + [F] is reported in the pattern as F
F 0.61 Always included
A 0.83 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
E 0.98 Included, unless S is present and [E] <[S]/4, in which case E is deleted from
the pattern
0 1.07 Included,unless S is present and [Dl <[S]/4, in which case D is deleted from
the pattern
S 1.18 Always included
C 1.72 Always included
(1) 0.73 If F is present, the total of [(1)1 + [A] is reported in the pattern as A
(2) 0.91 Included, unless D is present and [(2)] <[D]/4, in which case (2) is deleted
from the pattern
(3) 1.13 Always included
(4) 1.33 Always included
(5) 1.55 Included, unless C is present and [(5)] <[C]/4, in which case (5) is deleted
from the pattern
(6) 1.85 Always included