AMERICAN JOURNAL OF PERINATOLOGY/VOLUME 9, NUMBER 4

July 1992

SERUM ERYTHROPOIETIN QUANTITATION IN PREGNANCY USING AN ENZYME-LINKED IMMUNOASSAY Timothy W. Harstad, M.D., Ruble A. Mason, M.T., and Susan M. Cox, M.D.

ABSTRACT

As normal pregnancy advances, there is a disproportionate increase in plasma volume compared with the expansion of erythrocyte volume. This causes a decrease in hematocrit that reaches a nadir near 28 to 30 weeks. Mechanisms that stimulate erythropoiesis during pregnancy are unclear, but because erythropoietin (EPO) is the major hormonal regulator of human erythropoiesis, it seems reasonable that it would increase during pregnancy. EPO concentrations during normal pregnancy have been quantitated by immunoreactive assay1 and mouse bioassay,2 but, to our knowledge, this is the first report in which the enzyme immunoassay was used. We therefore sought to determine serum EPO levels during normal pregnancy and in third trimester pregnancies complicated by anemia using this sensitive and specific enzyme-linked immunosorbent assay. MATERIALS AND METHODS We studied cross-sectionally 86 nonanemic healthy women with a mean age of 23.8 ± 5.4 (SEM) years and mean gravidity of 3.3 ± 5.6. Eight women with uncomplicated pregnancies were studied during the first, 32 during the second, and 46 during the third trimesters. Serum EPO was also determined in nine anemic women whose hematocrit was less than 30 vol% in the third trimester (mean hematocrit, 27.4 ± 2.6 vol%). After informed consent was obtained, venous blood was collected into two Vacutainer tubes and immediately refrigerated. The hematocrit and reticulocyte count were determined shortly after venipuncture. Serum was extracted within 2 hours

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During normal pregnancy there is a decrease in the hematocrit due to a disproportionate increase in the blood volume compared with the red cell mass. Using a new enzyme-linked immunoassay (Amgen Diagnostics), serum erythropoietin was quantified in normal nonanemic pregnancies throughout gestation and in third trimester anemic patients. We found that the mean hematocrit in normal pregnancy reached a nadir late in the second trimester and the serum erythropoietin plateaued at a 50% increase. Those pregnancies complicated by anemia defined by a hematocrit less than 30 vol% demonstrated a statistically significant increase in serum erythropoietin above those not anemic.

from the nonanticoagulated tube and frozen at — 20°C until analysis. A wash solution of buffered 0.2% Triton X-100 and a substrate solution of 0.4 gm/liter tetramethylbenzadine and 0.02% buffered hydrogen peroxide were prepared. Serum specimen of 200 (JLI, EPO standards, and serum controls each were placed into pretreated microliter wells. The wells were sealed and incubated at 18° to 25°C for 3 hours, at which time the contents of the wells were discarded. Two hundred microliters of anti-EPO polyclonal (rabbit) antibody: horseradish peroxidase conjugate was added to each well, incubated for 2 hours at 37°C, and then removed. The wells were washed four times with the wash solution. The substrate solution, 200 |JL1, was added to the wells and incubated for 30 minutes, at which time the reaction was stopped by the addition of 100 |xl of 0.5 N sulfuric acid. The spectrophotometric absorbance was measured in each well at 450 nm as the primary wavelength and at 630 nm as the reference wavelength. All samples and standards were run in duplicate and the EPO concentration was determined from the standard curve generated. Known serum controls were determined concurrently.

RESULTS

As shown in Figure 1, the mean hematocrit reached a nadir near the end of the second trimester, and the mean serum EPO concentrations increased by about 50% and plateaued with a slight decrease near term in nonanemic pregnancies. The reticulocyte count ranged from a mean

Department of Obstetrics & Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas Reprint requests: Dr. Cox, Department of Obstetrics & Gynecology, University of Kentucky Medical Center, 800 Rose Street—MN 318, Lexington, KY 40536 Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.

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AMERICAN JOURNAL OF PERINATOLOGY/VOLUME 9, NUMBER 4 July 1992 20

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Figure 1. Relationship of mean hematocrit, mean serum erythropoietin, and mean reticulocyte count throughout pregnancy in the 86 nonanemic women.

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28

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Table 1. Mean Hematocrit, Reticulocyte Counts, and Serum Erythropoietin Values for Specific Gestational Ages Gestational Age (weeks)

Hematocrit Mean (SE)

9-11 12-15 16-19 20-23 24-27 28-31 32-35 36-41

37.5 36.8 35.8 35.2 32.5 33.6 33.8 37.3

Reticulocyte Count Mean (SE)

Serum Erythropoietin Mean (SE)

1.3(0.5) 0.9 (0.2) 2.0 (0.2) 2.1 (0.54) 2.0 (0.16) 1.3 (0.25) 1.8 (0.23) 1.5 (0.2)

8.1 (0.9) 11.8 (1.13) 11.0(1.3) 9.4 (0.87) 13.4 (1.63) 17.1 (3.85) 15.3 (2.31) 14.5 (1.68)

(0.85) (1.1) (0.95) (0.54) (0.66) (1.45) (1.17) (0.56)

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Serum erythropoietin quantitation in pregnancy using an enzyme-linked immunoassay.

During normal pregnancy there is a decrease in the hematocrit due to a disproportionate increase in the blood volume compared with the red cell mass. ...
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