Kleihauer-Betke Stain Research Articles

Effect of Hydroxyurea on Patient with High Fetal Hemoglobin Expression

High Fetal Hemoglobin (Hb F) expression in Sickle Cell Disease (SS), when first described, was defined as an "entirely benign disorder", due to its less severe manifestations; protective levels were described as 10 pg or more (AJCD, 133:1248, 1979). Continued high expression of Hb F as hereditary persistence of fetal hemoglobin (HPFH) occurs in 0.1% of African-American individuals. Other individuals may not have compound heterozygosity for Hemoglobin S and gene deletion HPFH but still have haplotypic differences resulting in higher HbF levels. We performed an IRB-approved chart review looking at SS patients with High HbF, followed at Children's Hospital from 2005-2020. We identified 13 children, 4-20 years, having mean HbF levels of 22.14 +/- 4.9%. Two children were considered inevaluable for analysis due to one being on voxelotor and the other being on chronic transfusion at the time of analysis. Of the 11 children remaining for analysis, 8 were girls and 3 boys. Complications requiring hospitalization included: acute chest syndrome, VOE, stroke including occult, osteomyelitis, aplastic crisis. Surgeries included osteotomy, cholecystectomy, and tonsillectomy/adenoidectomy. Eight children were placed on hydroxyurea (HU). After HU, total Hb and HbF did not significantly change (Hb 7.08+/-1.27 vs 8.97+/-1.55;HbF 22.4 +/-4.9 pre to 18.9+/-6.6, respectively). Admission or hospitalization frequency (admits or hospital stays/years studied) were evaluated, as were ED visits/years studied for SS-related problems. These data were: 1.36+/-0.51 pre vs. 0.4+/-0.46 post (for admits); 1.52+/-0.32 pre vs 0.33+/-0.21 post (for ED visits) p<0.05 for both). Three individuals were not on HU with an annual admission frequency of .75-1.2 and 1-3.2 for ED visits. The limitations of this study are: small n, lack of serologic proof of HU compliance, difficulties with data retrieval. Although the sample size is small, however, there does appear to be a salutary effect of HU on hospitalizations or vasoocclusive events after placement of these children on HU. Current recommendations to begin HU in children with SS at 9 months of age appear to have validation even in children with high HbF. We used the Kleihauer-Betke stain for HPFH diagnosis in most instances since gene analysis was not available to us due to insurance restraints; we acknowledge the need for genetic analysis to determine whether these individuals are "high F" or HPFH patients. Moderation of clinical course is dependent not only on HbF levels (which did not show significant increment after HU), but also on decreased WBC and platelet count with associated decreased inflammation and hypercoagulability.

Enumeration of Fetal Red Blood Cells, Hemoglobin-Specific RBC Cells, and F Reticulocytes in Human Blood.

Recent advances in analytical cytometry have improved diagnostic tools for the study of erythropoiesis in anemic patients and resolution of differential diagnosis in diseases of the erythron. This article presents three applications of red blood cell (RBC) analysis-quantitation of fetal red cells, F-cell enumeration, and F-reticulocyte analysis-which improve diagnostic precision, sensitivity, and specificity, and provide better laboratory indicators of therapeutic efficacy in a variety of hematologic and obstetric disorders. Such advances also include the measurement and quantitation of RBC hemoglobins and their relative ribonucleic acid levels. These advances not only promise to improve diagnostic accuracy and laboratory precision over techniques such as the traditional manual reticulocyte counting method and the Kleihauer-Betke stain method for evaluating fetomaternal hemorrhage (FMH), but also serve as tools for newer assays of anemia diagnosis and improved clinical outcomes. In addition to the primary methods, supporting techniques for preparing spiked controls, automating data analysis, setting up a fetal hemoglobin acquisition protocol, and assaying reticulocytes using thiazole orange are also presented. © 2019 by John Wiley & Sons, Inc.

Clinically Silent Massive Fetomaternal Hemorrhage: Important Lessons from an Illustrative Case

Massive fetomaternal hemorrhage (FMH) >150 mL is rare and may occur in the absence of high-risk obstetrical events. The significance of FMH in Rh D-negative women is alloimmunization with an increased risk of hemolytic disease of the newborn in subsequent Rh D-positive pregnancies and adverse outcomes for the fetus/neonate. The Kleihauer-Betke (KB) acid elution test is used to quantify fetal erythrocytes in the circulation of Rh D-negative women postpartum and to calculate the dose of Rh immune globulin (RhIG) needed for prophylaxis against alloimmunization. In this case, the KB stain unexpectedly revealed 4.5% fetal cells, a finding consistent with a massive FMH of 225 mL, in the absence of a predisposing cause and clinical signs in the infant. This case underscores the importance of FMH quantification in all Rh D-negative women with Rh D-positive fetuses, uncomplicated pregnancies, and healthy newborns. We discuss factors that can affect KB test performance and caveats in interpretation.

Comparision in the yield of fetal nucleated red blood cell between the first-and second-trimester using double density gradient centrifugation

Objective: The aim of our study was to make a practical comparative evaluation of the first and second trimesters in order to determine the period during which a higher yield of fetal nucleated red blood cells (FNRBCs) can be obtained. Methods: NRBCs were isolated from maternal blood during the first and second trimesters of pregnancy using double Percoll gradients with different osmolarities. Magnetic activated cell sorting was performed with Kleihauer-Betke stain. We isolated fetal NRBCs from 10 mL of samples of maternal blood and determined fetal sex and fetal aneuploidy by fluorescence in situ hybridization (FISH). Results: The average number of NRBCs was 9.85 in samples obtained during the first trimester and 14.88 in samples obtained during the second trimester (P=0.07). The average number of NRBCs with Y chromosome signals was 5.73 in the first trimester and 8.22 in second trimester (P=0.56). However, the percentage of NRBCs with Y chromosome signals in the first trimester (70.6%) was significantly higher than in the second trimester (59.8%) (P=0.049). We diagnosed the blood samples from 7 pregnant women having fetal aneuploidy using this method and the number of NRBCs was 18.4. Conclusion: The method using Percoll osmolarity and a double density gradient system may be a very useful method for separation of NRBCs in the first trimester of pregnancy and also in the second trimester.

Massive Fetomaternal Hemorrhage and Severe Anemia in the Newborn – Two Case Reports

Fetomaternal hemorrhage (FMH) is defined as the entrance of fetal erythrocytes into the maternal circulation. In late gestation, it occurs in 40 – 50 % of all pregnancies. In 98 % of the cases the amount of bleeding is less than 0.1 mL. Massive FMH is defined as a loss of more than 150 mL or approximately 50 % of the fetal blood volume. It occurs in one out of 1000 deliveries (Akanli LF et al. Am J Perinatol 1997; 14: 271 – 273). The most common symptoms are anemia, decreased or absent fetal body movement, and unexpected stillbirth (Giacoia GP. Obstet Gynecol Surv 1997; 52: 372 – 380). Specific symptoms do not exist. In most cases, the cause for massive FMH remains unknown. The extent of FMH can be measured with the Kleihauer-Betke stain.

A high yield of fetal nucleated red blood cells isolated using optimal osmolality and a double‐density gradient system

To increase the yield of fetal nucleated red blood cells (NRBCs) from maternal blood using a discontinuous Percoll gradient and to determine the effects of osmolality on NRBC yield. Fetal NRBCs were isolated from combined umbilical cord blood and adult female blood, or from maternal blood using single or double Percoll gradients with different osmolalities. Magnetic activated cell sorting was used to enrich isolated NRBCs, and morphological differentiation was performed with Kleihauer-Betke stain. We also isolated fetal NRBCs from 25 10 mL samples of maternal blood and determined fetal sex by fluorescence in situ hybridization (FISH), using X-Y probes. For single-density Percoll columns, the greatest number of NRBCs was isolated using 280 mOsm/kg H(2)O with 1.077 g/mL Percoll and 520 mOsm/kg H(2)O with 1.119 g/mL Percoll. Significantly more fetal NRBCs were isolated with double Percoll density gradients than with double-Histopaque gradients (p = 0.043). FISH analysis on NRBC in 25 cases correctly identified 15 male and 9 female euploid fetuses and one Trisomy 21 fetus. The NRBC enrichment method we present requires less maternal blood and yields more NRBCs compared to previous methods.

Placental sonolucency and pregnancy outcome in women with elevated second trimester serum alpha-fetoprotein levels.

Women with unexplained elevation of serum alpha-fetoprotein (AFP) are at increased risk for adverse pregnancy outcomes, including small for gestational age neonate, preterm labor, abruptio placentae, preeclampsia, intrauterine fetal death, and congenital malformations. This study investigated the association between placental sonolucency, elevation of maternal serum AFP, and pregnancy outcomes. Singleton pregnancies (n = 168) with second trimester serum AFP level >/= 2.0 weight-adjusted multiples of the median (MoM) were recruited as the study group. Women with second trimester serum AFP level between 0.4 and 2.0 weight-adjusted MoM (n = 150) served as controls. A maternal Kleihauer-Betke stain was obtained for all participants. All participants were prospectively evaluated and the pregnancy complications were assessed by chart analysis after delivery. Compared with control subjects, women with placental sonolucent areas were not at increased risk for pregnancy complications, while women without sonolucent areas had higher risk of pregnancy complications. Singleton pregnancies with elevated serum AFP level had increased incidence of feto-maternal hemorrhage when placental sonolucency was observed. Our data suggest that feto-maternal hemorrhage may be the major factor contributing to elevated maternal serum AFP levels in pregnancies carrying placental sonolucencies. Screening for pregnancies with both elevated serum AFP and placental sonolucencies would help to identify the low-risk cases and facilitate cost-effective obstetric management.

Postpartum chills phenomenon: is it a feto-maternal transfusion reaction?

To examine the theory that the postpartum shivering phenomenon is related to feto-maternal bleed during the third stage of labor. One hundred laboring low-risk women who had a normal vaginal delivery were observed for the presence of postpartum chills. The duration of the first and second stages of labor changes in body temperature, maternal and fetal blood types and the use of epidural anesthesia were recorded. Following the delivery maternal blood was examined for the presence of fetal red blood cells using the Kleihauer-Betke stain. Complete data was available in 97 patients. Post-partum chills occurred in 31 of them (32%). Women with and without chills were similar in their maternal and gestational age, the use of epidural anesthesia, and length of second stage of labor. Women with chills delivered smaller babies but the difference did not reach significance. Maternal-fetal blood group incompatibility was significantly more common among shivering than non-shivering women (48% vs. 20% respectively, p=0.006). Kleihauer-Betke test was positive in 11 women. The only two women in this group who experienced chills had maternal-fetal blood group incompatibility. Post-partum chills are a common phenomenon. It may be the clinical presentation of feto-maternal transfusion reaction. The small number of positive Kleihauer-Betke tests may reflect its low sensitivity in the detection of small feto-maternal bleeds.

Postpartum chills phenomenon: Is it a feto-maternal transfusion reaction?

Objective. To examine the theory that the postpartum shivering phenomenon is related to feto-maternal bleed during the third stage of labor. Methods. One hundred laboring low-risk women who had a normal vaginal delivery were observed for the presence of postpartum chills. The duration of the first and second stages of labor changes in body temperature, maternal and fetal blood types and the use of epidural anesthesia were recorded. Following the delivery maternal blood was examined for the presence of fetal red blood cells using the Kleihauer-Betke stain. Results. Complete data was available in 97 patients. Post-partum chills occurred in 31 of them (32%). Women with and without chills were similar in their maternal and gestational age, the use of epidural anesthesia, and length of second stage of labor. Women with chills delivered smaller babies but the difference did not reach significance. Maternal-fetal blood group incompatibility was significantly more common among shivering than non-shivering women (48% vs. 20% respectively, p=0.006). Kleihauer-Betke test was positive in 11 women. The only two women in this group who experienced chills had maternal-fetal blood group incompatibility. Conclusion. Post-partum chills are a common phenomenon. It may be the clinical presentation of feto-maternal transfusion reaction. The small number of positive Kleihauer-Betke tests may reflect its low sensitivity in the detection of small feto-maternal bleeds.

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture.

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1 x 10(6)-1.0 x 10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4 x 10(4)-9.8 x 10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.

Is adverse pregnancy outcome predictable after blunt abdominal trauma?

Objective: The aim of the study was to evaluate the following: (1) pregnancy outcome after blunt abdominal trauma and (2) factors that may predict preterm birth and adverse peripartum outcomes. Study Design: All women who had noncatastrophic abdominal trauma and came to the labor and delivery suite July 1994-August 1997 were prospectively evaluated and admitted for continuous uterine and fetal monitoring. A complete blood cell count, coagulation profile, and Kleihauer-Betke stain were performed. Ultrasonographic examination was performed to rule out hematoma. Tocolytic agents were administrated in cases with persistent contractions. Pregnancy outcomes and risk factors were compared between those with preterm birth before 37 weeks’ gestation and those who were delivered after 37 weeks’ gestation. Results: Delivery information was available for 85 women with blunt abdominal trauma from motor vehicle accident (28), falls (27), and direct assault (30, which included 17 cases of domestic abuse). Four women, 3 of whom were exposed to domestic abuse, were hospitalized twice. Thirteen patients had preterm birth and 72 patients were delivered at term. In all cases the results of Kleihauer-Betke stains, maternal vital signs, blood cell count, coagulation profile, and placental ultrasonographic examinations were normal. The differences between the 2 groups with respect to gestational age at the time of trauma, length of hospital stay, subjective reports of abdominal pain, objective findings of abdominal tenderness, patterns of uterine contractions, interval between trauma and delivery, and Apgar scores were not statistically significant. However, the preterm birth group received magnesium sulfate tocolysis more frequently (31% vs 7%) and had a significantly greater rate of peripartum complications, such as rupture of membranes and abruptio placentae, than the group of patients who delivered at term (46.2% vs 12.5%, P < .05). Women with domestic abuse had increased uterine contractions at the time of abdominal trauma (52.9% vs 19.1%, P = .01) but did not require increased use of tocolysis. Women with domestic abuse had more peripartum complications (41.8% vs 11.8%, P < .01). Conclusions: Women with noncatastrophic blunt abdominal trauma in pregnancy tend to have favorable neonatal outcomes. Findings or reports of abdominal tenderness and uterine contractions are not predictive of preterm birth. Preterm birth was associated with increased peripartum complications. However, domestic abuse was associated with repeated trauma in the index pregnancy and increased peripartum complications. (Am J Obstet Gynecol 1998;179:1140-4.)

Massive fetomaternal hemorrhage.

This report describes a full-term newborn with massive fetomaternal hemorrhage. Fetal movements were decreased 48 hr prior to delivery. On the day of delivery, they were absent. The nonstress test was abnormal with low biophysical profile and decreased beat-to-beat variability. The infant presented with extreme pallor, hypotonia, hepatosplenomegaly, and ascites. The initial hemoglobin was 2.2 g/dL, the Kleihauer-Betke stain was 27.6% (highest level ever reported). Right temporal and cerebellar hemorrhages were present. Sequelae include severe developmental delay and asymmetric double hemiplegia.

Twin-to-twin transfusion syndrome: A subset of the twin oligohydramnios-polyhydramnios sequence

Our purpose was to assess the adequacy of currently accepted criteria for the diagnosis of twin-to-twin transfusion syndrome. Between April 1991 and February 1992 nine consecutive women with twin gestations and ultrasonographic findings of marked growth discordance, coexistent oligohydramnios-polyhydramnios, monochorionic placenta, and same sex of the fetus in the second trimester were evaluated. Mean gestational age at diagnosis was 19 weeks 5 days +/- 5 weeks, and mean growth discordance was 34% +/- 9%. Seven women underwent attempted sequential amniocentesis and cordocentesis at 23 weeks 6 days +/- 2 weeks 4 days to obtain amniotic fluid and cord blood from each fetus. Type O negative, leucocyte-poor, washed adult red blood cells were transfused into the small, oligohydramnic twin during cordocentesis; immediately afterward blood from the polyhydramnic twin was tested with the Kleihauer-Betke stain. The combined procedure was successful in six (86%) of the cases attempted. True twin-to-twin transfusion was confirmed in only four (44%) of those initially identified by ultrasonographic criteria. Hemoglobin difference > 5 g/dl was present in only one of the four cases, and in this case the recipient was anemic. Currently accepted criteria are insufficient for the diagnosis of true twin-to-twin transfusion. Ultrasonographic findings of marked growth discordance, coexistent oligohydramnios-polyhydramnios, monochorionic placenta, and same sex identify the twin oligohydramnios-polyhydramnios sequence.

Fetal and neonatal hematologic parameters in red cell alloimmunization: predicting the need for late neonatal transfusions.

The purpose of this study was to determine whether the need for late neonatal transfusion can be predicted in fetuses who received intrauterine transfusions. The records of 36 patients who underwent intrauterine transfusion were reviewed. Newborns who required late transfusions had a lower reticulocyte count at their last intrauterine transfusion, a longer duration between their lowest fetal reticulocyte count and delivery, and a higher umbilical cord blood hemoglobin and percent adult red cells on Kleihauer-Betke stain at delivery. The need for late transfusions appears to be related to the extent and duration of fetal bone marrow suppression.

492 The Use of the Kleihauer-Betke Stain in Pregnant Patients with Abdominal Trauma

492 The Use of the Kleihauer-Betke Stain in Pregnant Patients with Abdominal Trauma

Ongoing fetomaternal hemorrhage treated by serial fetal intravascular transfusions

Nonimmune hydrops secondary to atraumatic, massive fetomaternal hemorrhage was diagnosed at 33.5 weeks' gestation by a maternal Kleihauer-Betke stain, confirmed by funipuncture. The initial fetal hematocrit was 6.5%. Kleihauer-Betke testing revealed a fetomaternal hemorrhage of approximately 230 mL. Fetal karyotype, total immunoglobulin M, and liver function tests were normal. Maternal parvovirus serology was negative. Treatment with two fetal intravascular transfusions provided only transient improvement because the fetomaternal hemorrhage proved to be unrelenting.

ONGOING FETOMATERNAL HEMORRHAGE TREATED BY SERIAL FETAL INTRAVASCULAR TRANSFUSIONS

Nonimmune hydrops secondary to atraumatic, massive fetomaternal hemorrhage was diagnosed at 33.5 weeks' gestation by a maternal Kleihauer-Betke stain, confirmed by funipuncture. The initial fetal hematocrit was 6.5%. Kleihauer-Betke testing revealed a fetomaternal hemorrhage of approximately 230 mL. Fetal karyotype, total immunoglobulin M, and liver function tests were normal. Maternal parvovirus serology was negative. Treatment with two fetal intravascular transfusions provided only transient improvement because the fetomaternal hemorrhage proved to be unrelenting.