Evaluation of Automatic Blood Analyzer as Screening Method in Fetomaternal Hemorrhage.

Screening of fetomaternal hemorrhage (FMH) is essential in management of fetomaternal antigen incompatibilities of blood. The objective in this study was to evaluate the ability of automatic blood analyzer (ABA) to screen FMH, also comparing this method with flow cytometry (FCM). The contents of fetal red blood cells and fetal hemoglobin were evaluated by FCM and ABA, respectively, using both blood samples of male adults laced with umbilical cord blood diluted at 1/10, 1/100, 1/1,000, and 1/10,000, or blood from puerperal women collected within 48 hours following delivery. FCM had better performance (area under curve, AUC = 0.8723) than ABA (AUC = 0.6569) in detecting fetal blood laced with blood from male adults. At a critical level of 0.5%, ABA indicated that 27.5% of puerperal women would have FMH while FCM did not detect FMH. Our results showed that ABA overestimates FMH and disagrees with FCM on indicating puerperal women with FMH. ABA is inadequate for being used to screen for or to measure FMH.

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface.

Hltf is regulated by intron retention, and global Hltf-deletion causes perinatal lethality from hypoglycemia. In heart, full-length Hltf is a transcriptional regulator of Hif-1α that controls transport systems. Thus, we tested the hypothesis that Hltf deletion from placenta caused or exacerbated neonatal hypoglycemia via Hif-1α regulation of nutrient transporters. RNA-seq data analyses identified significant changes in transcript expression and alternative splicing (AS) in E18.5 placentome. iPathwayGuide was used for gene ontology (GO) analysis of biological processes, molecular functions and cellular components. Elim pruning algorithm identified hierarchical relationships. The methylome was interrogated by Methyl-MiniSeq Epiquest analysis. GO analysis identified gene enrichment within biological processes. Protein expression was visualized with immunohistochemistry. Although two Hltf mRNA isoforms are quantifiable in most murine tissues, only the truncated Hltf isoform is expressed in placenta. The responsible intron retention event occurs in the absence of DNA methylation. iPathwayGuide analysis identified 157 target genes of 11,538 total genes with measured expression. These were obtained using a threshold of 0.05 for statistical significance (p-value) and a long fold change of expression with absolute value of at least 0.6. Hltf deletion altered transcription of trophoblast lineage-specific genes, and increased transcription of the Cxcr7 (p = 0.004) gene whose protein product is a co-receptor for human and simian immunodeficiency viruses. Concomitant increased Cxcr7 protein was identified with immunolabeling. Hltf deletion had no effect on transcription or site-specific methylation patterns of Hif-1α, the major glucose transporters, or System A amino acid transporters. There was no measureable evidence of uteroplacental dysfunction or fetal compromise. iPathGuide analysis revealed Hltf suppresses cytolysis (10/21 genes; p-value 1.900e-12; p-value correction: Elim pruning; GO:019835) including the perforin-granzyme pathway in uterine natural killer cells. Our findings 1) prove the truncated Hltf protein isoform is a transcription factor, 2) establish a functional link between AS of Hltf and immunosuppression at the feto-maternal interface, 3) correlate intron retention with the absence of DNA methylation, and 4) underscore the importance of differential splicing analysis to identify Hltf's functional diversity.

Placental Pathologic Features in Fetomaternal Hemorrhage Detected By Flow Cytometry.

Background Fetomaternal hemorrhage (FMH) is a poorly understood entity that can have significant clinical effects. Flow cytometry is a reliable and relatively new method for FMH diagnosis. The objective of this study was to correlate placental pathology with FMH detected by flow cytometry. Methods All patients with available placentas and FMH flow cytometric testing performed from 2009 to 2015 were retrospectively reviewed. Cases were defined as ≥0.10% fetal red blood cells (RBCs) in the maternal circulation while controls contained <0.10%. Placental findings associated with FMH were determined. Results In this study, 35 cases and 79 controls were identified. Villous dysmaturity/immaturity was significantly more prevalent among the cases compared to the controls. Placentas with villous edema and nucleated RBCs (nRBCs) in fetal vessels were associated with greater mean volumes of fetal blood in the maternal circulation. Fetal and maternal vascular pathology was more frequent in the controls. When the cases were stratified into mild (<30 mL), moderate (30 mL-100 mL), and severe (>100 mL) FMH, nRBCs, villous dysmaturity/immaturity, and villous edema were all positively correlated with increasing FMH severity. The cases were more likely than the controls to display ≥2 of these 3 features. Fetal nRBCs within fetal vessels were semi-quantified and moderate to severe numbers of nRBCs were associated with higher mean volumes of fetal blood in maternal circulation. Conclusions Villous dysmaturity/immaturity, villous edema, and nRBCs in fetal vessels, findings compatible with fetal anemia, in addition to relatively few chronic placental changes, are the most significant placental findings in FMH detected by flow cytometry.

Placental findings in feto-maternal hemorrhage in livebirth and stillbirth.

Feto-maternal hemorrhage (FMH) is not an uncommon event during pregnancy with important clinical implications for both maternal and fetal outcomes. The diagnosis is often made using Kleihauer-Betke (KB) test. As FMH occurs transplacentally, examination of the placenta may contribute to the diagnosis of FMH. This retrospective case-control study aims to examine the placental features associated FMH in patients with known positive KB test results. When compared with KB negative placentas (n=88), KB positive placentas (n=49) had significantly higher incidence of pallor (6/49 vs 0/88, p=0.0017), IVT (11/49 vs. 5/88, p=0.0032) and nRBCs (12/49 vs. 4/88, p=0.0008). Autopsy cases with fetal or neonatal death due to FMH, (n=13) compared to a cohort of 162 placentas associated with other, non-FMH causes of death also had significantly higher frequency of pallor (5/13 vs 0/162, p<0.0001), IVT (6/13 vs 24/162, p=0.011) and nRBCs (11/13 vs 67/162, p=0.003). Pallor and nRBC were also associated with higher volume of FMH. Placental parenchymal pallor, intervillous thrombi and presence of nRBCs are significantly associated with documented FMH in both normal pregnancies and pregnancies associated with fetal or neonatal death. The presence of these findings, especially in combination, may suggest the need for maternal KB testing to rule out FMH and neonatal monitoring and/or intervention.

[A case of fetal death resulting from a massive fetomaternal hemorrhage]. / LE CAS CLINIQUE DU MOIS. Mort foetale in utero due à une hémorragie foeto-maternelle massive.

We report the case of a late stillbirth which unexpectedly occurred in a patient without any medical history and after a meticulous obstetrical follow up. Stillbirth is unfortunately not unusual and implies a complete etiological work up. In the present observation, the Kleihauer test and anatomoclinical examination concluded that the death was due to an acute cerebral anoxy resulting from a massive fetomaternal hemorrhage (HFM). HFM is rarely considered as the cause of a late stillbirth, but its occurrence is certainly underestimated. Yet, if HFM is identified before fetal death, an .adequate management could considerably improve the fetal prognosis and, sometines, save the child's life.

A system for counting fetal and maternal red blood cells.

The Kleihauer-Betke (KB) test is the standard method for quantitating fetal-maternal hemorrhage in maternal care. In hospitals, the KB test is performed by a certified technologist to count a minimum of 2000 fetal and maternal red blood cells (RBCs) on a blood smear. Manual counting suffers from inherent inconsistency and unreliability. This paper describes a system for automated counting and distinguishing fetal and maternal RBCs on clinical KB slides. A custom-adapted hardware platform is used for KB slide scanning and image capturing. Spatial-color pixel classification with spectral clustering is proposed to separate overlapping cells. Optimal clustering number and total cell number are obtained through maximizing cluster validity index. To accurately identify fetal RBCs from maternal RBCs, multiple features including cell size, roundness, gradient, and saturation difference between cell and whole slide are used in supervised learning to generate feature vectors, to tackle cell color, shape, and contrast variations across clinical KB slides. The results show that the automated system is capable of completing the counting of over 60,000 cells (versus ∼2000 by technologists) within 5 min (versus ∼15 min by technologists). The throughput is improved by approximately 90 times compared to manual reading by technologists. The counting results are highly accurate and correlate strongly with those from benchmarking flow cytometry measurement.

Complete hydatidiform mole and live fetus in a singleton pregnancy with confined placental mosaicism and fetomaternal hemorrhage: a case report.

BACKGROUND: Coexistence of complete mole and a live fetus is uncommon (1:22,000-100,000), more so with euploidy. CASE: We present a case of a molar pregnancy with a euploid fetus who had close fetal evaluation for second trimester bleeding. The patient presented at 29 weeks' pregnancy with decreased fetal movements, a result of fetomaternal hemorrhage. She underwent cesarean section and delivered a live infant. By close follow-up and a multidisciplinary approach, the appropriate diagnosis and a favorable outcome were achieved. Both mother and the child at 5 years of age are doing well. CONCLUSION: Detailed anatomic and molecular studies demonstrated a complete mole resulting from confined placental mosaicism, with molar tissue showing a single paternal allele at 8/8 informative loci, all shared with the fetus, thus this coexistent molar pregnancy was not that of a separate conceptus.

Quantitation of fetomaternal haemorrhage and F cells in unusual maternal blood samples by flow cytometry using anti-D and anti-HbF.

BACKGROUND: Fetomaternal haemorrhage (FMH) assessment by the Kleihauer-Betke test (KBT) is rapid but semi-quantitative and liable to false positive results. OBJECTIVES: To compare FMH estimated by KBT with flow cytometry (FC) quantitation for 37 patients with massive FMH, obstetric risk factors or technical problems. METHODS: Maternal blood was sent for analysis by FC after KBT. A variety of reagents including anti-haemoglobin F (HbF), anti-D and combined anti-HbF/anti-carbonic anhydrase (CA) were used. RESULTS: Eight cases of massive FMH (>100 mL fetal cells) causing fetal death or severe neonatal anaemia in late gestation were confirmed by FC. Anti-HbF FC identified maternal F cells and fetal cells. In some cases these red cell populations merged but they could be differentiated by anti-CA, labelling F cells only. Using KBT, false positive FMH results were obtained for 12 patients, who had strongly stained cells that were then shown by FC to be maternal F cells. All these patients had increased F cells (>5% of total red cells) whereas only 16% of patients with FMH and 22% of donors had elevated F cells. In contrast, anti-D FC was simple and rapid, quantitating D-positive FMH in all 15 D-negative patients except one with massive FMH of weak D fetal cells. Leucocytes in four samples bound anti-D, variably, giving erroneously high FMH, but they could be eliminated from FC analysis using combined anti-D/anti-CD45. CONCLUSION: FMH quantitation using anti-D by FC is suitable for the majority of maternal samples and could enable accurate targeted dosing of anti-D prophylaxis.

Massive idiopathic feto-maternal transfusion associated with dilatation of umbilical vein: case report and review of literature.

Feto-maternal transfusion (FMT) or haemorrhage occurs when there is an entry of fetal blood into the maternal circulation in pregnancy or during delivery. It has been stated that very small amount of fetal red cells are normally detectable in maternal circulation in all pregnancies. However, massive FMT is rare and even rarer is the resultant severe anaemia which may cause severe fetal morbidity or early neonatal death in apparently uneventful normal pregnancy. Massive FMT is regarded as a pathological condition with a variety of clinical presentations essentially secondary to the fetal anaemia. We present a case of FMT associated with umbilical vein dilation and speculate whether this finding is of prognostic value.

Fetomaternal hemorrhage in normal vaginal delivery and in delivery by cesarean section.

BACKGROUND: The objective was to determine the incidence and volume of fetomaternal hemorrhage (FMH) in normal vaginal delivery and in delivery by cesarean section. Determination of these variables would enable optimalization of guidelines for D alloimmunization prophylaxis. STUDY DESIGN AND METHODS: In a prospective cohort study, a total of 3457 examinations were performed, 2413 after normal vaginal delivery and 1044 after cesarean delivery. FMH was assessed by flow cytometry. (FMH is fetal red blood cell [RBC] volume; fetal blood volume is double [expected fetal hematocrit is 50%].) RESULTS: The fetal RBC volume diagnosed in maternal circulation after delivery ranged from insignificant FMH of not more than 0.1 mL to excessive FMH of 65.9 mL (median, 0.7; mean, 0.78; SD, 1.48). FMH of more than 2.5 mL (immunoglobulin [Ig] G anti-D insufficient dose 50 µg) was observed in 1.4% (49/3457) and excessive volumes of FMH of more than 5 mL (insufficient dose, 100 µg) in 0.29% (10/3457). Delivery by cesarean section presented a higher risk of incidence of FMH of more than 2.5 mL (odds ratio, 2.2; p = 0.004) when compared with normal vaginal delivery. It did not, however, present a significant risk factor for the incidence of excessive volumes of FMH of more than 5 mL. CONCLUSION: During normal vaginal delivery as well as during delivery by cesarean section, FMH of less than 5 mL occurs in the great majority of cases, and thus for the prevention of D alloimmunization, an IgG anti-D dose of 100 µg should be sufficient. Contrarily, only rarely does greater FMH occur and delivery by cesarean section does not present a risk factor.

Scleroderma.

The prototypic autoimmune diseases involving skin (lupus, dermatomyositis) typically result in epithelial injury and autoantibodies to characteristic cellular antigens. Disease-specific autoantibodies are also found in scleroderma, but scleroderma is different from other cutaneous autoimmune diseases because epithelial injury does not occur. Multiple factors and combinations of factors (immune system, vascular and extracellular matrix abnormalities) are the most likely triggers in an individual with a genetic predisposition to scleroderma. These lead to increased synthesis of normal collagen in skin, lungs and gut in the systemic form of scleroderma, systemic sclerosis. The hypotheses for the pathophysiology of scleroderma are diverse and include abnormal immunologic processes such as cytokine and chemokine dysregulation, abnormal T cell signaling, B cell dysfunction, injury due to autoantibodies to endothelial cells, persistent wound healing condition due to dysregulation of matrix homeostasis, abnormalities in the fibrinolytic system, polymorphisms in critical molecules of the immune system and matrix homeostasis, and microchimerism due to fetal/maternal placental exchange of HLAcompatible cells. Systemic sclerosis/scleroderma is chronic and progressive. To date, no definitive therapy is effective for any of the scleroderma variants, although agents for the vascular dysfunction have some utility. Hematopoietic bone marrow or stem cell transplantation before significant tissue fibrosis has occurred may be the most effective treatment.

Evaluation of F cells in sickle cell disorders by flow cytometry -- comparison with the Kleihauer-Betke's slide method.

Adult F cell numbers are raised in inherited haemoglobin disorders, such as beta-thalassaemia and sickle cell anaemia, hereditary persistence of foetal haemoglobin, and some acquired conditions, such as juvenile myelomonocytic leukaemia, during acute erythropoietic stress and pregnancy. True foetal erythrocytes containing foetal amounts of HbF can also occur in the adult circulation during the leakage of HbF-containing cells from the foetus to the maternal circulation. In normal adults, HbF is restricted to a small proportion (3-7%) of red blood cells (RBC), termed 'F cells'. Techniques estimating the amount of HbF use lysates prepared from RBC, whereas those that estimate the adult F cell count use intact RBC. An accurate assessment of adult F cells in sickle cell disorders is important because increased adult F cells are associated with decreased morbidity in these disorders. In the present study, HbF levels were measured and adult F cell numbers were estimated in 100 blood samples (25 normal individuals, 25 sickle heterozygotes, 25 sickle homozygotes and 25 sickle beta-thalassaemia cases), using high pressure liquid chromatography for HbF levels, and flow cytometry and the Kleihauer-Betke (KB) acid elution microscope slide method for cell counts. Flow cytometry gave a more accurate assessment of adult F cells, eliminating any manual error, as compared to KB, which was less sensitive and precise as it is based on subjective visual interpretation.

The complement system in the pathophysiology of pregnancy.

A fully active complement system deriving from the maternal circulation as well as from local production by various cell source is present in the placenta. The role of this system at the placental level, as in any other tissue in the body, is to protect both the fetus and the mother against infectious and other toxic agents. As fetal tissues are semi-allogeneic and alloantibodies commonly develop in the mother, the placenta is potentially subject to complement-mediated immune attack at the feto-maternal interface with the potential risk of fetal loss. Uncontrolled complement activation is prevented in successful pregnancy by the three regulatory proteins DAF, MCP and CD59 positioned on the surface of trophoblasts. The critical role played by these complement regulators is supported by the embryonic lethality observed in mice deficient in the complement regulator Crry. Excessive complement activation in the placenta places the fetus at risk for growth restriction or death. The role played by the complement system in the fetal damage induced by anti-phospholipid antibodies in a mouse model will be examined.

Autopsy findings in a series of five cases of fetomaternal haemorrhages.

AIMS: Fetal blood cells enter the maternal circulation in up to 95% of pregnancies, but usually in minute volumes. Haemodynamically significant fetomaternal haemorrhage (FMH) is a much rarer event reported in approximately 1 in 2800 pregnancies. Most of the literature on this phenomenon emphasises the clinical aspects, and there is no comprehensive description of the autopsy findings. We present a series of five fatal FMH. The aim of this series is to highlight some of the autopsy findings that may prompt consideration of a diagnosis of FMH and lead to appropriate confirmatory testing and counselling of the affected couple. METHODS: The five cases were referred to the Children's Hospital at Westmead for full autopsy. A Kleihauer-Betke test was performed on the mother's blood within one week of delivery in each case. RESULTS: The infants ranged in age from 27 to 40 weeks gestation (mean 36.6 weeks) with a mean birth weight of 2793 g. The estimated volumes of fetal blood lost ranged from 443 to 104 mL (mean loss 243 mL). The estimated percentage of fetal blood volume loss was an average of 107% (i.e., greater than the entire blood volume of the fetus). No other causes of hydrops were identified. Pallor was often noted, and in most cases the autopsies were markedly bloodless with large vessels collapsed. Where the brain:liver ratio could be applied, two fetuses showed a mild increase in ratio, while one infant showed moderate growth restriction with a ratio of 6.2:1 (normal ratio 2.8:1 on non-macerated fetuses over 28 weeks gestation). Placental abnormalities included thrombosis of the umbilical vein and intervillous 'haematomas' in two cases. The most striking microscopic feature was the presence of intravascular nucleated RBC within virtually all organs. Placental intervillous (i.e., within the maternal vascular compartment) nucleated red blood cells were also seen in all cases. CONCLUSIONS: The autopsy findings of FMH can be subtle and easily overlooked unless a high index of suspicion is maintained. The most reliable autopsy features are pallor, subcutaneous oedema or serous effusions, and intravascular nucleated red blood cells (RBC) in organs or more specifically in the placental intervillous space. In all cases of unexplained fetal death a Kleihauer-Betke test should be performed.

[Massive fetomaternal transfusion after induction of labor by oxytocin]. / Transfusion foetomaternelle massive au décours de l'induction du travail par ocytocique.

UNLABELLED: Severe fetomaternal transfusions are seldom, but may lead to neonatal morbidity or death. CASE REPORT: We report a case of lethal fetomaternal transfusion in which a failure to induce labour, at term, can be suspected. COMMENTS: We emphasize the need of a close monitoring of the fetal well-being in cases of failed induced labour at term.