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Articles |
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Osaka City Environment and Public Health Association, Osaka 541-0055, Japan.
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Department of Pediatrics, Osaka City University Medical
School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan.
a Author for correspondence. Fax 81-6-6636-8737; e-mail okano{at}med.osaka-cu.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We modified the macroscopic visual Beutler enzyme spot test by adding extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microplate reader and personal computer. All handling was performed in microplates. The measurement time was 90 min.
Results: Fluorescence intensity (FI) of healthy controls correlated with hematocrit and galactose-1-phosphate uridyltransferase (GALT) activity. Patients with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by FI. FI decreased to 75% of the initial activity after storage at 25 °C for 3 days and to 40% after storage at 37 °C for 7 days. Screening of 46 742 newborns yielded 1 false-positive result (in a heterozygous carrier), 1 patient with glucose-6-phosphate dehydrogenase deficiency, and no apparent false negatives as judged by concurrent measurements of galactose and galactose-1-phosphate.
Conclusions: The quantitative Beutler test can provide precise GALT activity in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, and anemia. This method is clinically useful, simple, automated, and highly reliable for newborn mass screening of galactosemia.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Classic galactosemia causes severe and rapidly progressive symptoms in the neonate, including jaundice, cataracts, hepatomegaly, failure to thrive, and even neonatal death if not treated during the early stages (4). The Beutler enzyme spot test is an effective screening test for galactosemia, offering the following advantages: immediate detection of classic galactosemia, and diagnosis even when the infants is fed a non-milk formula. However, it also has disadvantages, including false-positive results after deterioration of the enzyme activity by exposure to high temperature and humidity, dependence on visual evaluation, and quenching by hemoglobin.
In the present study, we describe a quantitative Beutler test modified by separating the enzyme extraction and reaction, and quantifying and recording the results by a fluorescent microplate reader and personal computer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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methods
A 3.2-mm blood disc from a dried-blood newborn screening
filter paper and 50 µL of galactosemia test reagent were placed in
each well of a microplate with conical bottoms. The plate was allowed
to stand for 1 min, followed by a 5-min centrifugation at
2010g by a plate centrifuge (himac CT5DL; Hitachikoki).
After the plate was sealed and shaken for 1 min, it was incubated for
1 h at 37 °C for the enzyme reaction. The plate was then shaken
for 1 min, and 100 µL of acetone-methanol mixture (1:1, by volume)
was added to each well to precipitate the hemoglobin and protein. The
plate was sealed again and centrifuged for 15 min at 2010g.
In the next step, 35 µL of the supernatant was added to a microstrip
well containing 230 µL of distilled water and shaken continuously for
1 min. Fluorescence intensity (FI) was measured at an emission
wavelength of 360 nm (excitation wavelength, 450 nm) using an
MTP-100F fluorescent microplate reader (Corona Electric), and the data
were automatically digitized into a personal computer for analysis.
samples
Dried blood filter paper specimens collected for routine newborn
mass screening at maternity hospitals in Osaka City were used in these
studies. These filter papers were examined for (a)
distribution of GALT activity as determined by FI in 1494 newborns in
November 1995, (b) pilot mass screening of 15 233 newborns
conducted from May to November 1996, and (c) full-scale mass
screening of 46 742 newborns conducted from August 1997 to March 1999.
The measurement of GALT activity in newborn mass screening in our
institution proceeded (a) from the first routine screening
test, (b) to retesting of specimens with abnormal first test
results, (c) to obtaining and testing of a second dried
blood filter paper specimen from subjects whose initial specimen had
repeatedly abnormal results, and (d) to examination of
subjects with abnormal results in the repeat specimens by a medical
specialist. Resampling of blood and the close examination were
requested with low GALT activity and high galactose and/or Gal-1-P
concentrations; the cutoff values in our institution were 1.17 mmol/L
(7 mg/dL) for Gal-1-P plus galactose and/or 0.17 mmol/L (3 mg/dL) for
galactose. The entire study protocol was approved by the Institutional
Review Board of the Osaka City Environment and Public Health
Association.
statistics
All data are expressed as the mean ± SD. Simple linear
regression analysis was used to examine the relationship between FI and
hematocrit, and correlation coefficients and probabilities were
determined according standard statistical methods.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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effect of reaction time of galt enzyme
In the first series of experiments, we examined the effect of
reaction time (0–180 min) on GALT velocity using dried blood spots
obtained from healthy subjects, carriers, and patients (n = 3 for
each group). The FI in healthy subjects was almost proportional to the
incubation time (0–120 min; Fig. 1
). FI values at 60-min reaction time were markedly different
among patients, carriers, and healthy controls (mean ± SD,
121 ± 6, 352 ± 124, and 818 ± 158, respectively).
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precision and reproducibility
The relationship between FI and the hematocrit of dried blood
spots was examined. Blood samples with hematocrit values of 20%, 30%,
and 55% were prepared by diluting red blood cells (RBCs) with
the plasma, and then spotting the diluted RBCs on filter papers
(n = 12). There was a significant relationship between hematocrit
and FI values (r2 = 0.98;
P = 0.0001; Fig. 2
). These results demonstrated that FI reflected hematocrit
values as well as the amount of GALT enzyme, and that the FI of dried
blood spots from subjects with anemia was markedly lower than for the
controls.
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To examine reproducibility, dried blood spots on filter paper were prepared from the blood of healthy controls adjusted to a hematocrit of 55% (n = 24). The mean FI was 766 ± 68, and the CV was 8.9%. Dried blood spots for use as blanks were prepared by adding 0.3 U of alkaline phosphatase (to decompose the Gal-1-P and phosphorylated substrates) to 1 mL of blood from healthy controls adjusted to a hematocrit of 55% (n = 19). The mean FI was 112 ± 5.8, and the CV was 5.2%. Thus, the procedure showed satisfactory reproducibility in samples of healthy controls and in blank samples in which GALT activity was considered to be 0.
effects of different temperatures and storage duration
The effects of storage temperature and duration on FI values for
dried blood spots from seven healthy control subjects are shown in Fig. 3
. The dried blood spots stored at 4 °C were relatively
stable, i.e., GALT activity remained steady. FI values were >80% of
the initial intensity during the first week of storage and were 66% ±
3% of the initial reading after storage for 30 days. Storage at 25 or
37 °C was associated with a clear deterioration of FI values
relative to the initial values. For example, FI values decreased to
75% and 40% of the initial intensity when the samples were stored at
25 °C for 3 days and 37 °C for 7 days, respectively, a period and
temperature representative of that between blood sampling and testing
in the summer.
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distribution of fi in dried blood spots from newborn mass screening
We examined the distribution of FI in dried blood spots of 1494
newborns collected by routine newborn mass screening. There were no new
cases of GALT deficiency in the screened population. The distribution
of FI was not significantly different from a gaussian
distribution, as determined by the Kolmogorov–Smirnov fit test
(P = 0.659; Fig. 4
). FI values were 139–1128 (mean ± SD, 541 ± 132;
median, 538). The FI at the third percentile was 296. The cutoff value
in the pilot mass screening was set at 300.
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newborn mass screening
Table 1
shows the results of newborn mass screening by the visual
Beutler enzyme spot test and the quantitative Beutler test, and
compares the proportions of samples designated for retesting,
resampling, and close examinations. From 1986 to 1989, the
method used by our institutions for the Beutler enzyme spot test
entailed only one general visual determination after a 90-min
incubation; from 1990 to 1995, this was changed to visual
determinations at 40 and 80 min of incubation.
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In the pilot mass screening, which involved a quantitative Beutler spot
test of 15 233 newborns, with the cutoff value of the first screening
test and retest set at 300, retest was performed in 396 newborns
(2.6%), yielding FI values of 107–325 on retesting. Forty-nine
subjects (0.32%) were requested to undergo resampling of dried blood
spots because of FI values 
250 and Gal-1-P and galactose of 
1.17
mmol/L (7 mg/dL). Among these subjects, seven were requested for close
examination. Six were found to be heterozygous carriers of GALT
deficiency (G/N), and one was a compound heterozygote (G/D), based on
the results of GALT activity using the consumption test (5).
Table 1
shows the results of the full-scale mass screening of 46 742
newborns. The cutoff FI value of the first test and retest was
lowered to 250. Resampling was requested when the FI value was 250
and Gal-1-P and galactose were 1.17 mmol/L (7 mg/dL), whereas close
examination was requested when the FI value was 200 and Gal-1-P and
galactose were1.17 mmol/L (7 mg/dL). The full-scale mass screening by
quantitative Beutler test based on the cutoff points described above
did not substantially reduce the proportion of retesting and
resampling of subjects. However, the proportion requiring close
examination was reduced to only one subject, who was found to be a
heterozygous carrier of GALT deficiency (G/N).
One boy was identified to have G6PD deficiency. The boy had a FI value of 141, which was in the same range as those for complete GALT-deficient patients, and his Gal-1-P concentration was 0.08 mmol/L (1.5 mg/dL). Thus, the Beutler test identified one other RBC enzyme deficiency. G6PD activity in this boy was reduced to 0 unit/g hemoglobin (reference range, 6.33–7.91 units/g hemoglobin). The child did not have a hemolytic diathesis under routine conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Shih et al. (13) identified an increased frequency of false
positives in the Beutler spot test during the summer season. The
deterioration of the GALT enzyme in dried blood spots was attributed to
high humidity and high temperature, and was recognized as a weak point
of Beutler enzyme spot tests (8)(11). In our
experiments, FI values decreased markedly when samples were stored at
25 or 37 °C for 3–7 days, respectively (Fig. 3
). Furthermore, these
conditions are fully conceivable in Osaka, Japan, where the mean
temperatures in July and August are 27.6 and 29.6 °C, respectively,
and the mean humidity is 71% in July and 66% in August. In fact, the
rate of resampling of dried blood spots was the highest between May and
August (data not shown). Thus, it is important to take into
consideration the number of days from blood sampling to testing when
interpreting the results of samples showing low FI values. In contrast,
the quantitative Beutler test described in our study allows prediction
of the influence of high temperature and humidity because GALT activity
is reported in a numerical value.
When carrying out newborn mass screening using the quantitative Beutler
test, the false-negative results are a matter of highest importance.
Therefore, the cutoff value for retesting in the pilot mass screening
was set to 300, based on the third percentile for FI (Fig. 4
), although
the FI values in patients with GALT deficiency were much lower,
112 ± 4.7. In the pilot mass screening, seven subjects were
requested to have close examination by medical specialists, which
identified six heterozygous carriers and one compound heterozygous
carrier of G/D. However, in the pilot study, the percentages of
individuals requiring retesting, resampling, or close examination were
higher than those reported in previous studies using the Beutler enzyme
spot test because of the high cutoff value for FI (Table 1
). Therefore,
in the full-scale newborn mass screening, the cutoff value for
retesting and resampling of dried blood spots was set at 250 and that
for close examination at 200. With these cutoff values, we could not
substantially reduce the proportions that required retesting and
resampling. However, close examination was required for only one
heterozygous carrier. We simulated the retesting and resampling rates
for a cutoff of 200 in a screening of 46 742 subjects. In this
simulation, the proportions of retesting and resampling were reduced
from 765 (1.6%) to 168 (0.39%) and from 235 (0.5%) to 79 (0.17%),
respectively, which are similar to the results obtained with the visual
Beutler enzyme spot test. We recently have set the cutoff values to 200
in newborn mass screening.
The Beutler enzyme spot test utilizes the phosphoglucomutase, G6PD, and 6-phosphogluconate dehydrogenase present naturally in RBCs as the enzyme reactions subsequent to the GALT enzyme. GALT activity in the Beutler enzyme spot test is determined by the fluorescence of NADPH converted from NADP+ in the G6PD reaction. On the basis of this principle, it is suspected that G6PD deficiency appears as a positive result. In fact, the quantitative Beutler test could detect a patient with G6PD deficiency showing almost null G6PD activity with the same FI values as those given by patients with GALT deficiency. On the other hand, Mediterranean G6PD patients with an activity 2–7% of the activity of healthy subjects and G6PD A patients with an activity 8–20% of that of healthy subjects showed almost normal values in a mass screening test for GALT activity, as reported by Frazier and Summer (8). These results are attributable to the fact that the enzyme reaction rates of phosphoglucomutase, G6PD, and 6-phosphogluconate dehydrogenase are 7- to 20-fold higher than GALT. The above findings suggest that, although our quantitative Beutler test may not detect partial G6PD deficiency, this test can detect complete deficiency of G6PD with a normal Gal-1-P concentration and low FI value similar to those of patients with GALT deficiency.
These results demonstrate that because it uses quantitative analysis, the quantitative Beutler test can take into account the influence of high temperature and humidity, the interval between sampling to test, and anemia in the dried blood spot. Thus, the quantitative Beutler test is a clinically useful, simple, automated, and highly reliable test for mass screening of newborns for galactosemia.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), at 25 °C
and 43% humidity (
), and at 37 °C and 55% humidity (
).