Abstract The three major causes of anemia in neonates are blood loss, decreased red blood cell production, and increased degradation of erythrocytes. Establishing the cause of anemia in a neonate born prematurely can be challenging. Clinically, fetomaternal hemorrhage (FMH) can be difficult to diagnose—the condition often presents only after the manifestation of severe fetal anemia. FMH can be confirmed by determining the fetal hemoglobin F fraction in the mother, which is traditionally performed using the Kleihauer-Betke test (KBT). Herein, we present a case study of a newborn baby boy of Dutch ethnicity with massive FMH and negative KBT result. The KBT result appeared to be false-negative due to AO antagonism. However, the results of an additional marker alpha-fetoprotein (AFP) test confirmed the diagnosis of massive FMH. Therefore, measuring AFP in maternal blood can be helpful in confirming FMH in unexplained anemia of the neonate. anemia of the neonate, fetal-maternal hemorrhage, hemolytic disease of the neonate, Kleihauer-Betke test, alpha-fetoprotein, AO antagonism Clinical History A 28 year old Dutch woman at 31 weeks gestation was referred to the hospital with general malaise and decreased fetal movements for the previous 3 days. Her obstetric history showed a miscarriage, birth of a healthy child and, during the current pregnancy, a single anti-RhD injection at week 30 because of RhD incompatibility. Because the sinusoidal pattern of the fetus during cardiotocography raised the suspicion of severe fetal anemia, an emergency caesarian section was performed. A 2.075-kg Dutch boy with Apgar scores of 2, 4, and 5 after 1, 5, and 10 minutes, respectively, was delivered. The extremely pale neonate had bradycardia, which subsided after inflation of the lungs. Normal saline and a unit of O RhD-negative packed red blood cells (RBCs) were administered. Laboratory results for the neonate showed a hemoglobin (Hb) concentration of 2.6 g per dL (Table 1), after which 3 more units of O RhD-negative RBCs were administered. After 4 hours, the Hb remained stable, at approximately 14.5 g per dL. Manual differential blood count, blood smear, serum bilirubin, and other laboratory results excluded hemolysis and jaundice as causative factors (Table 1). Table 1. Laboratory Test Results for the Dutch Male Neonate and His 28-Year-Old Dutch Mother Variable Normal Range Results Neonatea Hemoglobin (g/dL) 14–21 2.6 ↓ Hematocrit (%) 45– 65 10 ↓ Reticulocytes (‰) 10– 60 186 ↑ Leukocytes (109/L) 5.0–30.0 16.4 Thrombocytes (109/L) 150–400 165 CRP (mg/L) <5.0 <1.0 LDH (U/L) 300–700 410 Bilirubin (mg/dL) <2.9 <2.9 DAT Negative Negative Motherb Kleihauer-Betke Test Negative Negative ALT (U/L) <45 16 AST (U/L) <40 28 LDH (U/L) 50–250 363 ↑ Haptoglobin (mg/dL) 2.6–17.0 <0.4 ↓ AFP (ng/mL) <200 13,915↑ Variable Normal Range Results Neonatea Hemoglobin (g/dL) 14–21 2.6 ↓ Hematocrit (%) 45– 65 10 ↓ Reticulocytes (‰) 10– 60 186 ↑ Leukocytes (109/L) 5.0–30.0 16.4 Thrombocytes (109/L) 150–400 165 CRP (mg/L) <5.0 <1.0 LDH (U/L) 300–700 410 Bilirubin (mg/dL) <2.9 <2.9 DAT Negative Negative Motherb Kleihauer-Betke Test Negative Negative ALT (U/L) <45 16 AST (U/L) <40 28 LDH (U/L) 50–250 363 ↑ Haptoglobin (mg/dL) 2.6–17.0 <0.4 ↓ AFP (ng/mL) <200 13,915↑ ↓, lower than normal; ↑, higher than normal; CRP, C-reactive protein; LDH, lactate dehydrogenase; DAT, direct antiglobulin test; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AFP, alpha-fetoprotein. aBlood type: A RhD-positive. bBlood type: O RhD-negative. View Large Table 1. Laboratory Test Results for the Dutch Male Neonate and His 28-Year-Old Dutch Mother Variable Normal Range Results Neonatea Hemoglobin (g/dL) 14–21 2.6 ↓ Hematocrit (%) 45– 65 10 ↓ Reticulocytes (‰) 10– 60 186 ↑ Leukocytes (109/L) 5.0–30.0 16.4 Thrombocytes (109/L) 150–400 165 CRP (mg/L) <5.0 <1.0 LDH (U/L) 300–700 410 Bilirubin (mg/dL) <2.9 <2.9 DAT Negative Negative Motherb Kleihauer-Betke Test Negative Negative ALT (U/L) <45 16 AST (U/L) <40 28 LDH (U/L) 50–250 363 ↑ Haptoglobin (mg/dL) 2.6–17.0 <0.4 ↓ AFP (ng/mL) <200 13,915↑ Variable Normal Range Results Neonatea Hemoglobin (g/dL) 14–21 2.6 ↓ Hematocrit (%) 45– 65 10 ↓ Reticulocytes (‰) 10– 60 186 ↑ Leukocytes (109/L) 5.0–30.0 16.4 Thrombocytes (109/L) 150–400 165 CRP (mg/L) <5.0 <1.0 LDH (U/L) 300–700 410 Bilirubin (mg/dL) <2.9 <2.9 DAT Negative Negative Motherb Kleihauer-Betke Test Negative Negative ALT (U/L) <45 16 AST (U/L) <40 28 LDH (U/L) 50–250 363 ↑ Haptoglobin (mg/dL) 2.6–17.0 <0.4 ↓ AFP (ng/mL) <200 13,915↑ ↓, lower than normal; ↑, higher than normal; CRP, C-reactive protein; LDH, lactate dehydrogenase; DAT, direct antiglobulin test; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AFP, alpha-fetoprotein. aBlood type: A RhD-positive. bBlood type: O RhD-negative. View Large During the caesarian section, there were no clinical clues that could explain the fetal blood loss. Pathological evaluation of the placenta and umbilicus showed no abnormalities. A Kleihauer-Betke test (KBT) performed on a blood specimen from the mother shortly after delivery showed no fetal erythrocytes, making fetal-maternal hemorrhage (FMH) unlikely. Ultrasound imaging of the abdomen and brain of the neonate showed no site of possible blood loss. The high reticulocyte count discovered during the severe anemia ruled out decreased erythrocyte production by bone marrow suppression (Table 1). The results of irregular antibody screening (IAT) of the mother showed an anti-RhD, most likely due to prophylactic anti-RhD (1000 IE) administration at 30 weeks gestation. The results of the IAT and direct antiglobulin test (DAT), as well as the eluate of the erythrocytes of the neonate, were negative; the preliminary blood type of the neonate was determined to be A RhD-positive. Because the blood type of the mother was O RhD-negative, we investigated whether AO antagonism could be the cause of the fetal anemia. Immunoglobulin (Ig)G anti-A antibodies in the blood of the mother were present at a high concentration (titer of 1:128). However, the negative DAT result and absence of hemolysis parameters made intravascular hemolysis by AO antagonism a less likely cause of the anemia of the neonate. Discussion in the multidisciplinary neonatology meeting resulted in the following working diagnosis. Three days before presentation, the mother had exercised vigorously at a gym, after which her symptoms had started. The intensity of the exercise possibly provoked the massive FMH. The fetal A erythrocytes that entered the maternal circulation were immediately hemolyzed by the already present maternal anti-A antibodies (IgM and perhaps the newly formed IgG), possibly in combination with the still-present previously administered anti-RhD. Three days later, on the day of presentation, no more fetal erythrocytes were present in the maternal circulation, and the KBT result was negative. To support the working diagnosis, haptoglobin and alpha-fetoprotein (AFP) concentrations were measured in the blood of the mother, which was collected at presentation. Haptoglobin concentration had decreased to unmeasurable levels, indicating intravascular hemolysis (Table 1). AFP concentrations physiologically increase during gestation due to placenta leakage. However, the concentration in blood of the mother was increased to a very high level (Table 1), suggesting a massive FMH (because AFP during gestation must be of fetal origin). In conclusion, the anemia of the neonate was most likely caused by a massive FMH, and the negative KBT result could be explained by vivid intravascular hemolysis of fetal erythrocytes in the maternal circulation by AO antagonism and exogenous administered anti-RhD. The mother was discharged in good condition 5 days after the caesarian section. Due to his prematurity and the consequences of his severe anemia, the neonate was hospitalized for 1 month before being discharged. Due to abnormalities in his basal ganglia and hippocampus, the consequences for his further development remain uncertain. Discussion Although the diagnosis of neonatal anemia was confirmed quickly after birth, finding the cause of the anemia was challenging. There were no clinical clues for blood loss, the KBT result was negative, there was absence of hemolysis parameters, and there were no signs of bone marrow suppression. Although the DAT result for the neonate was negative, an eluate of the neonate erythrocytes was made. Due to the relatively weak binding of IgG anti-A or anti-B antibodies to the erythrocyte, ABO antagonism may result in a negative DAT result.1 If IgG anti-A or anti-B antibodies are present in the blood of the mother, further investigation should be performed in the eluate of the erythrocytes from the neonate. The elution and increased concentration in the eluate of antibodies attached to the erythrocytes increase the sensitivity of the assay.2 In some cases of ABO antagonism, the IgG anti-A or anti-B antibodies could be detected in the eluate from the infant despite a negative DAT result.3 However, in our case, the eluate of the neonate also tested negative for IgG anti-A antibodies. These results, together with the absent hemolysis parameters, made hemolytic disease of the newborn (HDN) caused by AO incompatibility very unlikely. Also, AO antagonism is not known to cause severe HDN because the ABO blood group is only weakly developed in infants.2 Because the KBT result was negative and there was AO incompatibility, FMH was initially ruled out as the working diagnosis. In our laboratory, the KBT result is always double checked, and positive and negative controls are included. However, visual discrimination of fetal hemoglobin (HbF) cells can be complicated by the presence of adult HbF cells.4 Adults normally have approximately 0.5% HbF cells that contain 20% to 25% HbF. However, the HbF-cell percentage can increase physiologically during pregnancy. In 25% of pregnancies, the number of HbF cells increases after 8 weeks of gestation, with maximum concentrations of 7% between 18 and 22 weeks.5 In these cases, the KBT can produce false-positive results or can result in overestimation of FMH. Flow cytometry is another quantitative test for measuring HbF cells in the maternal circulation and has been shown to have greater precision and accuracy than the KBT.5 However, flow cytometry is not without its limitations and has a tendency to underestimate the possibility of massive FMH. Underestimation of FMH is also reported using the KBT in the case of ABO antagonism.6 However, false-negative results are not previously described in the literature, to our knowledge. These results make it highly likely that the negative KBT result described in this case was correct and cannot be attributed to the test characteristics. Fetal erythrocytes entering the maternal circulation often are hemolyzed through IgM anti-A or anti-B antibodies so rapidly that no RhD immunization occurs in the mother.7 Therefore, it is possible that small volume FMH can result in a false-negative KBT result, especially when the KBT is performed a number of days after the FMH occurred, as described in our case individuals. AFP has been previously shown to be a good alternative method for detecting FMH.8 AFP is mainly produced by the fetal liver and is excreted by the fetal kidneys in the amnionic fluid. In the fetus, AFP reaches maximum levels around the fourteenth week of gestation, with concentrations as high as 3 × 106 ng per mL, after which it decreases to 50 ng per mL at birth.9 During pregnancy, the maternal AFP concentrations are approximately 35 ng per mL and 200 ng per mL in the first and late third trimester, respectively.9 Because of the large differences between AFP concentrations in the mother and fetus, AFP can be useful as a marker of FMH. Assay of AFP, in maternal blood, has been shown to be at least as sensitive as the KBT for detecting FMH.8,9 In our case, the FMH induced a significantly increased AFP level but showed a negative KBT result in the mother. We speculate that due to extreme hemolysis, the fetal erythrocytes were not detectable at the time of presentation. However, hemolysis parameters such as lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were not significantly increased in the blood of the mother. The biological half-life of LDH and ASAT is 10 hours and 17 hours, respectively, whereas the half-life of AFP is 5 days.10 Because 3 days had passed between the emergence of symptoms and laboratory testing, the different half-lives could explain why AST and LDH levels were low in the mother and the AFP level was still increased. Our case illustrates that with a strong clinical suspicion of FMH, a negative KBT result does not always rule out FMH. At least in the case of additional ABO antagonism, maternal AFP obtained in the first few days after giving birth can be helpful in confirming the FMH diagnosis, especially when the time between emergence of symptoms and obtaining maternal specimens is delayed. Personal and Professional Conflicts of Interest None declared. Abbreviations RBCs red blood cells Hb hemoglobin KBT Kleihauer-Betke test FMH fetal-maternal hemorrhage IAT irregular antibody screening DAT direct antiglobulin test Ig immunoglobulin AFP alpha-fetoprotein HDN hemolytic disease of the newborn HbF fetal hemoglobin LDH lactate dehydrogenase AST aspartate aminotransferase ↓ lower than normal ↑ higher than normal CRP C-reactive protein References 1. van Rossum HH , de Kraa N , Thomas M , Holleboom CAG , Castel A , van Rossum AP . Comparison of the direct antiglobulin test and the eluate technique for diagnosing haemolytic disease of the newborn . Pract Lab Med . 2015 ; 3 : 17 – 22 . 2. Romano EL , Hughes-Jones NC , Mollison PL . Direct antiglobulin reaction in ABO-haemolytic disease of the newborn . Br Med J . 1973 ; 1 ( 5852 ): 524 – 526 . 3. Voak D , Williams MA . An explanation of the failure of the direct antiglobulin test to detect erythrocyte sensitization in ABO haemolytic disease of the newborn and observations on pinocytosis of IgG anti-A antibodies by infant (cord) red cells . Br J Haematol . 1971 ; 20 ( 1 ): 9 – 23 . 4. Bayliss KM , Kueck BD , Johnson ST , et al. Detecting fetomaternal hemorrhage: a comparison of five methods . Transfusion . 1991 ; 31 ( 4 ): 303 – 307 . 5. Kim YA , Makar RS . Detection of fetomaternal hemorrhage . Am J Hematol . 2012 ; 87 ( 4 ): 417 – 423 . 6. Cohen F , Zuelzer WW . Mechanisms of isoimmunization. II. Transplacental passage and postnatal survival of fetal erythrocytes in heterospecific pregnancies . Blood . 1967 ; 30 ( 6 ): 796 – 804 . 7. Cohen F , Zuelzer WW , Gustafson DC , Evans MM . Mechanisms of isoimmunization. I. the transplacental passage of fetal erythrocytes in homospecific pregnancies . Blood . 1964 ; 23 : 621 – 646 . 8. Banerjee K , Kriplani A , Kumar V , Rawat KS , Kabra M . Detecting fetomaternal hemorrhage after first-trimester abortion with the Kleihauer-Betke test and rise in maternal serum alpha-fetoprotein . J Reprod Med . 2004 ; 49 ( 3 ): 205 – 209 . 9. Klein H , Anstee D. Blood Transfusion in Clinical Medicine . 11th ed . Malden, MA : Blackwell Publishing; 2005 . 10. Burtis CA , Ashwood ER , Bruns DE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics . 5th ed . St. Louis, MO : Elsevier ; 2012 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: email@example.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Laboratory Medicine – Oxford University Press
Published: Jun 2, 2018
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