CLINICAL CHEMISTRY, or metabolite concentrations expected sera ...

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CA 125 Determined by Three. Methods in Samples from. Patients with Human. Anti-Mouse. Antibodies. (HAMA),. Ursula. Turpeinen,'3. Pentti. Lehtovirta,2 and.

1.6-] A).

B)

#{149}

D

I

1.6

S

l.5

i

!

Fig. 1.2

I.’

0.81

0

0.8-

0.71,

.,.,#{149},‘

-25

50

25

COOH-THC

75

100

-100

0

(ltgfL)

100

200

Morphine

-100

300

0

100

for testing

offers a method

serum

samples

at

lower

cutoffs that may be more reasonable for this matrix. The wide variation in signals of negative and positive samples dictates that a laboratory will need to make a pool of negative and cutoff samples from several different individuals to establish separation criteria for determination of presumptive positives when either extracted or unextracted sera are to be tested. These data on fortified sera should now provide approaches for validating the usefulness of KIMS reagents on “real” serum samples. Reagents Roche

and

partial

Diagnostics,

funding

Nutley,

for this

study

were

provided

by

NJ.

References 1. Christophersen purposes cine and

AS,

Morland

in forensic toxicology, related areas. Pharmacol

J.

Drug

analysis

workplace Toxicol

for

control

testing, sports 1994;74:202-10.

medi-

2. Blum

LM, Klinger RA, Rieders F. Direct automated EMITS d.a.u. analysis of N,N-dimethylformamide-modified serum, plasma, and postmortem blood for benzodiazepines, benzoylecgonine, cannabinoids, and opiates. J Anal Toxicol 1989;13:285-8. 3. McCurdy HH, Callahan LS, Williams RD. Studies on the stability and detection of cocaine, benzoylecgonine, and 11-nordelta-9-tetrahydrocannabinol-9-carboxylic acid in whole blood using Abuscreen#{174} radioimmunoassay. J Forensic Sci 1989;34: 858-70. 4.

Huang

say

detection

zolam 5.

W,

in blood.

Armbruster

Enzyme

Moody

DE,

Andrenyak

of nordiazepam, J Anal Toxicol DA,

Schwarzhoff

immunoassay,

DA, triazolam, 1993;17:365-9. RH,

kinetic

6.

Moody

road

DE,

Crouch

DG,

DJ,

Rollins DE. accidents. J Forensic

Smith

RP,

DE.

lorazepam,

Hubster

alpra-

Liserio

MK.

immunoassay,

polarization Clin Chem Cresalia

Drug and alcohol Sci 1991;36:1474-84.

Immunoasand

EC,

microparticle

dioimmunoassay, and fluorescence compared for drugs-of-abuse screening. 46. Wilkins

Rollins

ra-

immunoassay 1993;39:2137CW,

involvement

Francom

correspondence:

fax

[email protected])

972-2-652-0689,

e-mail

P,

in rail-

Enzymatic Activity in Glucose-6-phosphate Dehydrogenase-Normal and -Deficient Neonates Measured with a Commercial Kit, Michael Kaplan,”34 Chava Leiter,2 Cathy Hammerman,”3 and Bernard Rudenskf (Shaare Zedek Med. Ctr., 1 Dept. of Neonatol., and 2 Clin. Hematol. Lab., P.O. Box 3235, Jerusalem 91031, Israel; Med. School of the Hebrew Univ.-Hadassah, Jerusalem, Israel; author for

200

300

(j.tgfL)

Benzoylecgonine

(tIg/L)

or metabolite concentrations expected in serum (positive sera can be reliably distinguished from negatives only at higher concentrations) but may be of use in extreme overdose cases. Extraction of serum is time consuming, but

1. Individual

variation

(#{149}) serum samples and benzoylecgonine.

Sera from 10 individuals were fortified with the three drugs at the concentrations noted and KIMS analysis of extracted and unextracted sera was performed as described in the text. The maximal signals of negative unextracted (. . -) and extracted (- - - -) sera are indicated.

Glucose-6-phosphate ciency

(0) and unextracted with COOH-THC, morphine,

in extracted

fortified

is

(G-6-PD)

dehydrogenase

frequently

associated

with

neonatal

defijaundice

kernicterus and death. Infants at Mediterranean variant should be screened as soon as possible after delivery to categorize those likely to develop jaundice. Confirmation of a pathological screening test with a definitive, quantitative analysis is recommended (4). At this Medical Center we have routinely screened high-risk newborns for many years (5, 6), using a commercial qualitative color reduction kit [visual qualitative determination of G-6-PD deficiency in red cells (no. 400); Sigma Diagnostics, St. Louis, MO]. We recently attempted to improve the service offered our G-6-PD-deficient families and investigated the possibility of confirming with quantitative G-6-PD determinations those found to be enzyme deficient by the screening test. While setting up a system utilizing another commercial package [G-6-PD: quantitative, ultraviolet kinetic determination in blood at 340nm (no. 345-UV), Sigma Diagnostics], we found that the values we were reading for normal infants were in excess of the manufacturer-supplied range for adults. Although the package insert implies that values for this test in newborns may be higher than in adults, no actual values are provided. To determine standard values to make this test more meaningful, we studied the range of

(1-3),

which

high

risk

enzyme low-risk

may

for

cause

the

activity in expected population group

G-6-PD-normal and also in

newborns

those

found

to

of a be

G-6-PD deficient by the screening test. The principle of the quantitative test involves the oxidation of glucose 6-phosphate to 6-phosphogluconate, and the concomitant reduction of NADP to NADPH. These reactions occur in the presence of G-6-PD, and the rate of NADPH formation, which is proportional to G-6-PD activity, is measured spectrophotometrically. Formation of additional NADPH by 6-phosphogluconate dehydrogenase from erythrocytes (RBC) is inhibited by the use of maleimide, an inhibitor of this enzyme. One international enzyme unit (1 U) is defined as that amount of G-6-PD activity that will convert 1 Mmol of substrate (glucose 6-phosphate to 6-phosphogluconate) per minute. Term, healthy neonates in the first week postpartum were drawn from the pool of infants in the well-baby nursery. For the study of infants expected to be normal, only babies drawn from the lowest risk group for G-6-PD deficiency in our population, i.e., both of whose parents were of Ashkenazi (Eastern European) Jewish origin (7), were included. Because of the very low incidence of G-6-PD deficiency in this population, babies from this subgroup are not routinely screened. Newborns from our high-risk group (Sephardic Jewish families, who originated in Kurdistan, Iraq, Iran, Syria, Lebanon, and TurCLINICAL

CHEMISTRY,

Vol.

41,

No. 11, 1995

1665

who

key),

protocol the

by

had

been

(5, 6) and who color

routinely were

reduction

screened as per nursery found to be G-6-PD deficient

method,

made

up

the

u/b12

G-6-PD-

deficient group. At the time of routine blood drawing for phenylketonuna testing or bilirubin determination, 250 L of capillary blood was collected from each infant by heel stick into a microtainer containing EDTA anticoagulant. The specimens were stored in the refrigerator and analyzed within 1

week.

Research

The

Ethics

study

was

approved

by

the

800 600

400

institution’s

Committee. the

G-6-PD assay, we determined and the hemoglobin (Hb) concentration by the Coulter method (Coulter T-890; Coulter Electronics, Luton, UK) and performed a reticulocyte count, using freshly prepared reagents. In accordance with the manufacturer’s recommendations, potassium dichromate solution was used instead of water in the reference cell. For quality control, we assayed a G-6-PD-normal control and a deficient control (Sigma Diagnostics, cat. nos. G

200

Prior to performing both the RBC count

and G 6888, respectively) prior to running each batch obtained results within the given range. NADPH activity was determined by comparing readings of the reagents incubated at 30 #{176}C in a narrow-width spectrophotometer (Ocean Scientific Co., Garden Grove, CA) 5 mm after obtaining baseline readings. G-6-PD activity was calculated in relation to both RBC count and Hb 5888 and

value. The test was validated by testing healthy volunteer Ashkenazi Jewish adults, five men and five women. Values for the men (mean ± SD) were 237.8 ± 76.2 U/b’2 RBC or 8.18 ± 2.60 U/g Hb; for the women they were 241.1 ± 38.1 U/b’2 RBC or 8.08 ± 1.4 U/g Hb. All these results fell within the expected range for men and women combined, as supplied by the manufacturer (146-376 U/b’2 RBC or 4.6-13.5 U/g Hb). The expected normal group comprised 100 (50 male, 50 female) neonates. One infant of each sex was found to be G-6-PD deficient by the quantitative testing and was therefore excluded from the analysis; the families of these two infants were informed of their condition. Enzyme activity in the remaining males (mean ± SD) was 504.8 ± 101.5 U/b’2 RBC (range 361-905) or 14.35 ± 2.52 U/g Hb (10.0-2 1.2). The mean reticulocyte count in the males was 4.3% ± 1.3%. In the remaining females, enzyme activities were 503.8 ± 109.4 U/10’2 RBC (250-934) or 14.25 ± 3.0 U/g Hb (9.3-26.2). The mean reticulocyte count in the females was also 4.3% ± 1.3%. The G-6-PD-deficient group studied comprised 29 males and 10 females. The mean enzyme activity value for the pooled sample was 26.5 ± 33.6 U/b12 RBC (range 0-111) or 0.82 ± 0.98 U/g Hb (2.9-7.1); the mean reticulocyte count was 4.8% ± 1.1%. Enzyme activity for the males was 24.5 ± 33.7 U/10 RBC (0-110) or 0.77 ± 0.99 U/g Hb (0-3.2). Values for the females were 32.1 ± 34.8 U/10’2 RBC (0-115) or 1.0 ± 0.99 U/g Hb (0-33). Comparison of corresponding values of enzyme activity as U/10’2 RBC vs U/g Hb showed excellent correlation (r2 = 0.98), shown graphically in Fig. 1. Because the results were similar for males and females, the data were pooled. Note that there was no overlap between assay results for the deficient and the normal groups. By design, we studied only Ashkenazi Jewish infants for our “normal” determinations, this being the population group with the lowest frequency of G-6-PD deficiency available to us. High-risk population groups were avoided 1666

CLINICAL

CHEMISTRY,

Vol.

41,

No.

11,

1995

RBC

1 000

00

5

10

15

UIg

20

30

25

Hb

Fig. 1. Correlation between enzyme activity values expressed as U/lU12 RBC vs U/g Hb in G-6-PD-normal (n = 98) and -defi#{225}ient (n = 39) neonates (males and females combined).

because of the large number that would have been found. erozygotes with intermediate

of G-6-PD-deficient infants Furthermore, female hetvalues would have made for normal values impossible.

determination of standards No chemical test, screening or quantitative, can separate female heterozygotes from normal homozygotes; only DNA analysis can do this (1). Our results clearly show that normal values for the newborns quently in excess of the manufacturer’s adults, or of those found by Ciulla

studied

were

fre-

range for normal et al. (8) (145-395 RBC). On the other hand, the range of G-6-PD in the enzyme-deficient infants we studied was below the normal range even for adults, with no

U/b’2 activity clearly

overlap. Sheba et al. (7) documented the incidence of the condition as 0.4% in males of the Ashkenazi Jewish population group. The finding of one G-6-PD-deficient infant of each sex among the two groups of Ashkenazi Jewish infants tested results in an observed incidence of 1 in 50. How-

ever, we believe this apparently high incidence to be artifactual, the number of infants studied being too small to reach any conclusions regarding population statistics. Ours was not intended to be a genetic study, and the small number of subjects involved precludes any comparison with the large population group studied by Sheba et al. (7). The known increased G-6-PD activity in newborn blood (9) may be due in part to the large number of immature RBCs and reticulocytes found in newborns, which contain higher amounts of G-6-PD activity than do mature cells (10). Furthermore, the mean cell age of cord RBCs is younger than that of normal adult blood, a factor that results in higher activities of many enzymes in neonates. However, the number of circulating reticulocytes cannot explain the increased enzyme activity in newborns entirely. Konrad et al. (10) compared cord blood samples, for which the reticulocyte count ranged from 2.3% to 7.0%, with nonneonatal subjects undergoing hemolysis, whose reticulocyte count ranged from 3.0% to 8.9%. Despite the similarity in reticulocyte counts in both groups, RBC G-6-PD assay results were higher in the cord blood samples

than

another,

for

the No

in

the

as yet higher agreement

nonneonatal

control

undetermined, neonatal exists

factor enzyme

group.

Apparently,

part

is responsible

in

the unit

for G-6-PD

values.

as to whether

activity should be expressed per 1012 RBC or per gram of Hb (11). The excellent correlation we found between the two implies that, at least in the patient group we studied, either method can be utilized with equal accuracy. We thank samples,

Chana Arnsalem

and

Ilan

Rozenberg

for assistance for

data

in obtaining

the blood

computerization.

high values It should

References

Beutler 2. Valaes 1.

because the same monoclonal antibody (MAb) is used both on the solid phase and as a tracer and because the idiotype of OC125 appears to be highly immunogenic. In the first-generation Byk-IRMA (IRMA-mat#{174}; Byk-Sangtec Diagnostica, Dietzenbach, Germany), anti-idiotypic antibodies, which bind to an idiotype on OC125, can cross-link solid-phase and detector antibody, resulting in falsely

E. G6PD deficiency [Review]. Blood b994;84:3613-36. T. Severe neonatal jaundice associated with glucose-6dehydrogenase deficiency: pathogenesis and global

anti-idiotypic

for CA b25 (1-3). be possible to reduce antibodies

by

the

developing

interference assays

based

by on

ardic-Jewish neonates: incidence, severity and the effect of phototherapy. Pediatrics 1992;90:401-5. 6. Kaplan M, Hammerman C, Rudensky B, Kvit R, Abramov A. Neonatal screening for glucose-6-phosphate dehydrogenase deficiency: sex distribution. Arch Dis Child 1994;71:F59-60. 7. Sheba C, Szeinberg A, Ramot B, Adam A, Ashkenazi I. Epidemiologic surveys of deleterious genes in different population groups in Israel. Am J Public Health 1962;52:1101-5. 8. Ciulla AP, Kaster JM, Tetlow AL. Determination of glucose-

antibodies other than that used for injection. The recently introduced Truquant#{174} OV2TM (Biomira, Edmonton, Canada) is a two-step heterologous double-determinant solidphase assay that utilizes the B27.1 mouse MAb as a capture antibody to bind molecules containing 0C125reactive determinants (6). Quantification is obtained by using B43. 13 as a detector antibody, and assay calibration is based on a reference preparation maintained by Biomira. The Centocor CA b25 II assay (Centocor, Malvern, PA) is a one-step assay using the Mu antibody as a capture antibody and the OC 125 antibody as a detector antibody (7). Assay calibration is based on a Centocormaintained reference preparation. In our two-step modification of the assay, the sample is first incubated overnight with the capture antibody and, after washing, incubated for 2 h with the second antibody. To reduce the HAMA interference in the modified Centocor CA 125 II assay, we preincubated serum samples for 2 h at room

6-phosphate methods.

temperature Glostrup,

phosphate epidemiology

[Review].

Acta

Paediatr

1994;394(Suppl):58-76.

3. Slusher TM, Vreman HJ, McLaren DW, AK, Stevenson DK. Glucose-6-phosphate ciency and carboxyhemoglobin concentrations bilirubin-related morbidity and death in Pediatr 1995;126:102-8.

Brown

Lewison U, dehydrogenase associated

Nigerian

defiwith

infants.

J

Luzzatto L. G6PD deficiency and hemolytic anemia. In: Nathan DC, Oski FA, eds. Hematology of infancy and childhood. Philadelphia: WB Saunders, 1993:674-95. 5. Kaplan M, Abramov A. Neonatal hyperbilirubinemia associ4.

ated with glucose-6-phosphate

Lab

dehydrogenase

dehydrogenase deficiency: Med b983;14:299-302.

deficiency

comparison

in Seph-

of

three

9. Beutler E, Blume KG, Kaplan JC, Lohr GW, Ramot B, Valentine WN. International Committee for Standardization in Haematology: recommended methods for red-cell enzyme analysis. Br J Haematol 1977;35:331-40. 10. Konrad PN, Valentine WN, Paglia DE. Enzymatic activities and glutathione content of erythrocytes in the newborn: comparison with red cells of older normal subjects and those with comparable reticulocytosis. Acta Haematol 1972;48:193-201. 11. Kachmar JF, Moss DW. Enzymes. In: Tietz N, ed. Fundamentals 666-72.

of clinical

chemistry.

CA 125 Determined Patients with Human Ursula Turpeinen,’3 Stenman1 (Depts. of Gynecol. at Helsinki Haartmaninkatu 2, for correspondence:

Philadelphia:

WB

Saunders,

1976:

by Three Methods in Samples from Anti-Mouse Antibodies (HAMA), Pentti Lehtovirta,2 and Ulf-Hdkan 1 Clin. Chem. and 2 Obstet. and Univ. Central Hosp., 00290 Helsinki, Finland; author fax

+358-0-471-4804)

Infusion of OCb25 fragments in connection with radioimaging of ovarian tumors often causes formation of anti-mouse IgG antibodies (HAMA), which can lead to falsely high results in OC125-based homologous immunoassays for CA 125 (1-5). The interference can be reduced by addition of mouse IgG. However, the antibodies are often anti-idiotypic, i.e., directed against idiotypes within the hypervariable region of OC125, such that interference by these is not blocked by mouse IgG (1, 2). We recently described a chromatographic method for separating immunoglobulins from CA 125 before assay (2). However, the method is time consuming and dilutes the samples, which reduces the accuracy of the assay. The CA b25 assay is especially prone to HAMA effects

with Denmark)

20 iL of mouse serum (Dakopatts, per 100 L of serum. second-generation CA 125 assays have

Because the replaced the original CA 125 tests, it is important to assess the effect of HAMA on these new assays. To determine the real CA 125 concentration in discrepant samples, we used cationexchange chromatography to remove interfering antibodies (2). HAMA was determined by an immunofluorometric method (2) and by the Truquant#{174} HAMA-RIA (Biomira). Immunoscintigraphy was performed with a ‘3’I-labeled F(ab’)2-fragment of the OC125 antibody Imacis-2 (ORIS, Gif-sur-Yvette, France) (8). Figure bA shows a comparison between the Truquant 0V2 (y) and Byk-IRMA (x) CA 125 methods for 35 control samples and 29 samples drawn 0.5-2 years after immunoscintigraphy.

For

the

control

samples,

the

methods

is good:

CLINICAL

CHEMISTRY,

r

=

1.0, y

the

Vol.

41,

=

0.95

No.

11,

correlation

x + 1.52. However, in 72% of the samples obtained after immunoscintigraphy, results were clearly higher with the BykIRMA than with the Truquant 0V2. This suggests that bridging of the two MAbs by HAMA was responsible for the increase in CA 125 concentrations in the Byk-IRMA. To determine the true CA 125 concentration of the discrepant samples, we removed HAMA by cation-exchange chromatography (2). Fig. lB shows the effect of HAMA removal on the apparent CA 125 concentrations measured by the Byk-IRMA and by the Centocor one-step assay (Fig. 1C) in samples from five patients obtained after immunoscintigraphy. After HAMA removal, the apparent CA 125 concentrations were markedly reduced in practically all samples. When the Centocor assay was performed in two steps and mouse serum was added to the samples before assay, the interference of llAMA seen in the one-step assay was strongly reduced and apparently completely eliminated. The concentrations obtained with the two-step modification were in the most cases similar to those obtained by Byk-IRMA after HAMA removal. Howbetween

1995

1667

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