Effects of 14-weeks betaine supplementation on pro-inflammatory cytokines and hematology status in professional youth soccer players during a competition season: a double blind, randomized, placebo-controlled trial.

OBJECTIVE: Systemic elevations in pro-inflammatory cytokines are a marker of non-functional over reaching, and betaine has been shown to reduce the secretion of pro-inflammatory cytokines in vitro. The aim of this study was to investigate the effects of betaine supplementation on tumor necrosis factor alpha (TNF-α), interleukins-1 beta (IL-1ß), - 6 (IL-6) and the complete blood cell (CBC) count in professional youth soccer players during a competitive season. METHODS: Twenty-nine soccer players (age, 15.5 ± 0.3 years) were randomly divided into two groups based on playing position: betaine group (BG, n = 14, 2 g/day) or placebo group (PG, n = 15). During the 14-week period, training load was matched and well-being indicators were monitored daily. The aforementioned cytokines and CBC were assessed at pre- (P1), mid- (P2), and post- (P3) season. RESULTS: Significant (p < 0.05) group x time interactions were found for TNF-α, IL-1ß, and IL-6. These variables were lower in the BG at P2 and P3 compared to P1, while IL-1ß was greater in the PG at P3 compared to P1 (p = 0.033). The CBC count analysis showed there was significant group by time interactions for white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), and mean corpuscular hemoglobin concentration (MCHC). WBC demonstrated increases at P3 compared to P2 in PG (p = 0.034); RBC was less at P3 compared to P1 in BG (p = 0.020); Hb was greater at P2 compared to P1, whilst it was less at P3 compared to P3 for both groups. MCHC was greater at P3 and P2 compared to P1 in BG, whereas MCHC was significantly lower at P3 compared to P2 in the PG (p = 0.003). CONCLUSION: The results confirmed that 14 weeks of betaine supplementation prevented an increase in pro-inflammatory cytokines and WBC counts. It seems that betaine supplementation may be a useful nutritional strategy to regulate the immune response during a fatiguing soccer season.

Single versus Split Dose of Iron Optimizes Hemoglobin Mass Gains at 2106 m Altitude.

PURPOSE: To determine if a single versus a split equivalent daily dose of elemental iron was superior for hemoglobin mass (Hbmass) gains at altitude while minimizing gastrointestinal (GI) discomfort. METHODS: Twenty-four elite runners attended a 3.1 ± 0.3 wk training camp (Flagstaff, AZ; 2106 m). A two-group design, randomized and stratified to baseline Hbmass, sex, and ferritin (>30 µ·L), was implemented daily as: 1) single dose of 1 × 200 mg (PM only, SINGLE) versus 2) split dose of 2 × 100 mg (AM and PM; SPLIT) elemental iron (ferrous fumarate). The Hbmass and venipuncture assessments were completed upon arrival and departure (±2 d) from camp for ferritin, hepcidin, and erythroferrone (ERFE) concentrations. Validated food frequency, GI distress, menstrual blood loss (MBL) and training questionnaires were implemented throughout. Univariate analysis was used to compare Hbmass, with baseline ferritin, dietary iron intake, MBL, and training volume used as covariates. RESULTS: Both conditions increased Hbmass from baseline (P < 0.05), with SINGLE (867.3 ± 47.9 g) significantly higher than SPLIT (828.9 ± 48.9 g) (P = 0.048). The GI scores were worse in SINGLE for weeks 1 and 2 combined (SINGLE, 18.0 ± 6.7 points; SPLIT, 11.3 ± 6.9 points; P = 0.025); however, GI scores improved by week 3, resulting in no between-group differences (P = 0.335). Hepcidin significantly decreased over time (P = 0.043) in SINGLE, with a nonsignificant decrease evident in SPLIT (~22%). ERFE significantly decreased in both groups (~28.5%; P < 0.05). No between-group differences existed for ERFE, hepcidin, food frequency, MBL, or daily training outcomes (P > 0.05). CONCLUSIONS: A single nightly 200-mg dose of elemental iron was superior to a split dose for optimizing Hbmass changes at altitude in runners over an approximately 3-wk training camp.

Time Trial Performance Is Sensitive to Low-Volume Autologous Blood Transfusion.

PURPOSE: This study tested the hypothesis that autologous blood transfusion (ABT) of ~50% of the red blood cells (RBC) from a standard 450-mL phlebotomy would increase mean power in a cycling time trial. In addition, the study investigated whether further ABT of RBC obtained from another 450-mL phlebotomy would increase repeated cycling sprint ability. METHODS: In a randomized, double-blind, placebo-controlled crossover design (3-month wash-out), nine highly trained male subjects donated two 450-mL blood bags each (BT trial) or were sham phlebotomized (PLA trial). Four weeks later, a 650-kcal time trial (n = 7) was performed 3 d before and 2 h after receiving either ~50% (135 mL) of the RBC or a sham transfusion. On the following day, transfusion of RBC (235 mL) from the second donation or sham transfusion was completed. A 4 × 30-s all-out cycling sprint interspersed by 4 min of recovery was performed 6 d before and 3 d after the second ABT (n = 9). RESULTS: The mean power was increased in time trials from before to after transfusion (P < 0.05) in BT (213 ± 35 vs 223 ± 38 W; mean ± SD) but not in PLA (223 ± 42 vs 224 ± 46 W). In contrast, the mean power output across the four 30-s sprint bouts remained similar in BT (639 ± 35 vs 644 ± 26 W) and PLA (638 ± 43 vs 639 ± 25 W). CONCLUSIONS: ABT of only ~135 mL of RBC is sufficient to increase mean power in a 650-kcal cycling time trial by ~5% in highly trained men. In contrast, a combined high-volume transfusion of ~135 and ~235 mL of RBC does not alter 4 × 30-s all-out cycling performance interspersed with 4 min of recovery.

[Iron, a "miralcle cure" for chronic cardiac insufficiency?] / Eisen, ein Wundermittel bei Herzinsuffizienz?

Anemia and chronic heart failure are frequent comorbidities in geriatric patients. In approximately one third of older adults the cause of the anemia is an iron, vitamin B12 and/or folate deficiency and in another third a chronic inflammatory process is present. In the case of iron deficiency a differentiation must be made between the absolute and functional forms. Although in functional iron deficiency ferritin, as a parameter of iron metabolism, is within the normal range or can even be higher, an iron-deficient erythropoiesis is present. In cardiac insufficiency a chronic inflammatory process is assumed. According to the recent guidelines of the Deutsche Gesellschaft für Kardiologie (DGK, German Cardiac Society) and European Society of Cardiology (ESC) a routine contol of the iron status should be performed and, if necessary, initiation of adequate supplementation is recommended.

[Delayed umbilical cord clamping in preterm infants born before 37 weeks of gestation: A prospective observational study]. / Clampage tardif du cordon ombilical chez les enfants prématurés nés avant 37 semaines d'aménorrhée : étude observationnelle prospective.

INTRODUCTION AND OBJECTIVES: Many international studies have demonstrated that delayed umbilical cord clamping reduces neonatal morbidity. However, in France, delayed umbilical cord clamping is still not performed in many neonatal units. The aims of this study were to evaluate the feasibility of developing a protocol of delayed umbilical cord clamping in the maternity ward of the Toulouse university hospital and to evaluate the impact of this new protocol on neonatal mortality. METHODS: We conducted a prospective observational study including 123 preterm infants born before 37 weeks of gestation between June 2012 and June 2013 and hospitalized at birth. Delayed cord clamping was performed for at least 30s after birth; otherwise, it was evaluated as early cord clamping. We excluded twin-to-twin transfusion syndrome, congenital abnormalities, alloimmunization, and perinatal asphyxia. We analyzed the reasons why delayed umbilical cord clamping was not performed and then neonatal morbidity in our population. RESULTS: Delayed umbilical cord clamping was performed on 79 infants and 44 infants had early umbilical cord clamping. The two groups had similar baseline characteristics. Preterm infants in the delayed cord-clamping group had a higher level of hemoglobin during the first 24h of life (17.9g/dL versus 16.6g/dL, P=0.005), fewer of them required transfusion (14% versus 35%, P=0.03), and fewer presented late-onset sepsis (8% versus 26%, P=0.02) or bronchopulmonary dysplasia (9% versus 26%, P=0.03). There was no statistically significant increase of hyperbilirubinemia requiring phototherapy. DISCUSSION AND CONCLUSION: Implanting a new protocol of delayed umbilical cord clamping in our maternity ward proved to be possible without difficulty. The advantages of delayed umbilical cord clamping were observed in this prospective study. Today, delayed cord clamping has become a common practice in our maternity unit.

Altitude Training in Elite Swimmers for Sea Level Performance (Altitude Project).

INTRODUCTION: This controlled, nonrandomized, parallel-groups trial investigated the effects on performance, V˙O2 and hemoglobin mass (tHbmass) of four preparatory in-season training interventions: living and training at moderate altitude for 3 and 4 wk (Hi-Hi3, Hi-Hi), living high and training high and low (Hi-HiLo, 4 wk), and living and training at sea level (SL) (Lo-Lo, 4 wk). METHODS: From 61 elite swimmers, 54 met all inclusion criteria and completed time trials over 50- and 400-m crawl (TT50, TT400), and 100 (sprinters) or 200 m (nonsprinters) at best stroke (TT100/TT200). Maximal oxygen uptake (V˙O2max) and HR were measured with an incremental 4 × 200 m test. Training load was estimated using cumulative training impulse method and session RPE. Initial measures (PRE) were repeated immediately (POST) and once weekly on return to SL (PostW1 to PostW4). tHbmass was measured in duplicate at PRE and once weekly during the camp with CO rebreathing. Effects were analyzed using mixed linear modeling. RESULTS: TT100 or TT200 was worse or unchanged immediately at POST, but improved by approximately 3.5% regardless of living or training at SL or altitude after at least 1 wk of SL recovery. Hi-HiLo achieved greater improvement 2 (5.3%) and 4 wk (6.3%) after the camp. Hi-HiLo also improved more in TT400 and TT50 2 (4.2% and 5.2%, respectively) and 4 wk (4.7% and 5.5%) from return. This performance improvement was not linked linearly to changes in V˙O2max or tHbmass. CONCLUSIONS: A well-implemented 3- or 4-wk training camp may impair performance immediately but clearly improves performance even in elite swimmers after a period of SL recovery. Hi-HiLo for 4 wk improves performance in swimming above and beyond altitude and SL controls through complex mechanisms involving altitude living and SL training effects.

The course of anaemia in children and adolescents with Crohn's disease included in a prospective registry.

AIM: The aim of this study is to determine the prevalence and evolution of anaemia in prospectively followed children and adolescents diagnosed with Crohn's disease (CD). METHODS: The BELCRO registry (inclusion May 2008-April 2010), describing current clinical treatment practice of children diagnosed with CD, provided data on age, height, body mass index (BMI), paediatric Crohn's disease activity index (PCDAI), therapy and haemoglobin (Hb) at diagnosis 12 and 24 months follow-up. Anaemia was defined as Hb < -2 sd, while severe anaemia was defined as Hb < -4 sd. Patients were classified as child ≤13 and adolescent >13 years of age. RESULT: Ninety-six were included, 13 dropped out due to insufficient Hb data (37 females/46 males; median age 13.3 years, range 2.2-17.8 years). At diagnosis, the median Hb sd was -2.66 (-8.4; 1.07) and was correlated with the PCDAI (p = 0.013). At diagnosis, 51/83 (61%) were anaemic and all had active disease. Hb z-score significantly improved (p < 0.0001) but 26/68 (38%) remained anaemic at 12 months and 29/76 (38%) at 24 months of follow-up. The correlation to the PCDAI disappeared. At 24 months, children were more likely to be anaemic. There was no difference in iron dose nor duration of iron supplements between children and adolescents. Iron treatment was more readily given to patients presenting with anaemia. Hb did not differ between patients with (n = 28) or without iron supplements. Half of the patients with persisting anaemia were given iron supplements, of which, only three were given intravenously. CONCLUSION: Anaemia remains an important extra-intestinal manifestation of CD in children. Physicians, lacking optimal treatment strategies, undertreat their patients.

Monitoring recovery from iron deficiency using total hemoglobin mass.

INTRODUCTION: Using hemoglobin concentration ([Hb]) to diagnose borderline iron deficiency and monitor the progress of its treatment is difficult because of the confounding effects of plasma volume. Because hemoglobin mass (Hbmass) is not affected by plasma volume, it may be a more sensitive parameter. The aim of this study was to monitor Hbmass, iron storage, and maximal oxygen consumption (V˙O2max) during and after oral iron therapy in subjects with severe and moderate iron deficiency. METHODS: Three groups of female recreational athletes were monitored for at least 22 wk, as follows: 1) severe iron deficiency group (SID) (n = 8; ferritin, ≤12 ng·mL), 2) moderate iron deficiency group (MID) (n = 14; ferritin, ≤25 ng·mL), and 3) control group (n = 8; ferritin, >25 ng·mL). Hbmass and iron status were determined before, during, and up to 12 wk after at least 10 wk of oral iron supplementation. In total, five V˙O2max tests were performed before, during, and after the supplementation period. RESULTS: Hbmass increased markedly in the SID group (15.6% ± 11.0%, P < 0.001) and slightly in the MID group (2.2% ± 3.7%, P < 0.05) by the end of the supplementation period and remained at this level for the following 12 wk. [Hb] and Hbmass were similarly affected, but Hbmass was more closely related to mean corpuscular volume and mean corpuscular hemoglobin than [Hb]. The SID group incorporated 534 ± 127 mg of iron into ferritin and hemoglobin, whereas the MID group incorporated 282 ± 68 mg of iron. V˙O2max increased only in the SID group by 0.20 ± 0.18 L·min (P < 0.05) and was closely related to Hbmass (P < 0.01). CONCLUSIONS: Hbmass is a sensitive tool for monitoring recovery from iron deficiency anemia and assessing the effectiveness of iron supplementation in individuals with severe or moderate iron deficiency.

[Development and implementation of an integrated care pathway at the POLIKUM health centers using the example of anemia]. / Entwicklung und Umsetzung eines integrierten Behandlungspfads in den POLIKUM-Gesundheitszentren am Beispiel der Anämie.

BACKGROUND: Integrated treatment pathways are an appropriate means for increasing the quality of treatment and outcome via process optimization. Taking the POLIKUM Health Centers as an example, we intend to demonstrate how the implementation can be effected for the indication of anemia. METHOD: The development and implementation were executed by an interdisciplinary workgroup in several workshops. In addition, the diagnoses and hemoglobin values of all patients with requests for hemograms were obtained and analyzed at two locations. RESULTS: Developing the pathway required significantly greater efforts than initially planned. The biggest challenge was to adequately map the complexity of the different forms of anemia and, concomitantly, to design a pathway that can actually be realized in everyday life. Moreover, evaluation of the patient data demonstrated that there are a large number of cases where existing anemias are not reflected in the respective diagnoses. CONCLUSION: While the ultimate effects of the new pathway cannot yet be assessed conclusively, it was possible to obtain valuable findings for practical use even at this point. Despite the limitations of the sample, the surprisingly high number of undetected anemias should give physicians cause for taking diagnostic measures even in patients with mild anemia.

[Iron substitution in outpatients in Switzerland: Increase of costs associated with intravenous administration]. / Ambulante Eisensubstitution in der Schweiz - Kostensteigerung infolge venöser Applikation.

Iron anaemia and iron-deficient erythropoiesis are treated with oral iron supplements. For chronic haemodialysis or in the case of therapy failure or intolerance to oral iron therapy, intravenous supplements are administered. The costs of iron supplements borne by statutory health care insurance had strongly increased during the observation period from 2006 to 2010. Based on the invoice data of a large health insurance company with a market share of around 18 %, prescription data of iron preparations and laboratory tests were analysed and extrapolated to the Swiss population. During the 5-year observation period, costs of intravenous iron substitution increased by 16.5 m EUR (340.3 %) and the number of individuals treated by 243.5 %. A sharp rise was observed in women of menstruating age, which was mainly due to prescriptions issued by primary care physicians. More than 8 % of intravenous iron substitutions were administered without prior laboratory analysis,and must therefore be regarded as off-label use. A cost-benefit analysis is needed to demonstrate the additional value of intravenous over oral iron supplementation, and intravenous iron supplementation should be administered only to patients with proven iron deficiency.

Randomized trial of iron supplementation versus routine iron intake in VLBW infants.

OBJECTIVE: To determine if iron supplementation of 2 mg/kg per day, in addition to routine iron-fortified formula or mother's milk, increased the hematocrit (Hct) at 36 weeks' postmenstrual age (PMA). METHODS: : Infants with a birth weight <1500 g who reached 120 mL/kg per day of feedings before 32 weeks' PMA were randomly assigned to iron (multivitamin with iron) or control (multivitamin) from enrollment until 36 weeks' PMA (or discharge, if sooner). Investigators and caregivers were masked. Transfusion guidelines were used. The primary outcome was Hct at 36 weeks' PMA. A nonparametric rank sum analysis was performed so that infants who died before 36 weeks and infants who were transfused could be included in an intention-to-treat analysis. Infants were ranked by death (lowest rank) then by number of transfusions (next lowest ranks). For infants who survived and were not transfused, the 36-week PMA Hct was used for the rank. RESULTS: One hundred fifty infants were enrolled (76 iron, 74 controls). There were 2 deaths (1 in each group). One hundred other infants (47 iron, 53 controls) received transfusion(s). There was no significant difference in the primary outcome ranking (P = .59), in the number of transfusions per subject (P = .64), or in 36-week Hct (iron mean ± SD, 29.2% ± 4.0%; control, 28.3% ± 4.5%; mean difference and 95% confidence interval 0.9 [-0.5 to 2.3]; P = .21) or reticulocyte count among survivors. No short-term adverse effects of iron supplementation were observed. CONCLUSIONS: Among infants <1500 g birth weight, iron supplementation, in addition to routine iron intake, did not significantly increase the 36-week Hct or the decrease number of transfusions.

A field study on feed supplementation, body weight and selected blood parameters in local pigs in Laos.

The aim of the present study was to investigate feed allowances, body weight (BW), haematocrit, haemoglobin, plasma ionised calcium (iCa), sodium, potassium, pH and glucose concentration and faecal K/Na ratio in local growing pigs, sows and piglets kept by small-holder farmers in Laos. Starting hypotheses were that (1) local pigs are under fed, (2) BW is higher in pigs receiving supplementary feed and (3) the blood profile of pigs subjected to very poor nutrition is outside the normal range. On 54 pig-keeping smallholdings in Borikhamxay province, Lao PDR, daily feed allowances were weighed and BW recorded for 27 lactating sows, 54 piglets and 27 growing pigs. Blood samples were collected from the vena jugularis in all pigs. Feed supplementation did not affect BW, but plasma iCa concentration was outside the normal range for all pigs. There was a tendency for lower faecal K/Na ratio in Na-supplemented sows. The results confirm that local pigs in small-scale production systems in Laos suffer from poor nutrition. The most important challenge for farmers appears to be provision of more feed, particularly feed with a high Ca content.

Timing of the introduction of complementary foods in infancy: a randomized controlled trial.

OBJECTIVE: To increase knowledge on iron status and growth during the first 6 months of life. We hypothesized that iron status would be better in infants who received complementary foods in addition to breast milk compared with those exclusively breastfed. METHODS: One hundred nineteen healthy term (≥37 weeks) singleton infants were randomly assigned to receive either complementary foods in addition to breast milk from age 4 months (CF) or to exclusive breastfeeding for 6 months (EBF). Dietary data were collected by 3-day weighed food records, and data on iron status and growth were also collected. RESULTS: One hundred infants (84%) completed the trial. Infants in the CF group had higher mean serum ferritin levels at 6 months (P = .02), which remained significant when adjusted for baseline characteristics. No difference was seen between groups in iron deficiency anemia, iron deficiency, or iron depletion. The average daily energy intake from complementary foods of 5-month-olds in the CF group was 36.8 kJ per kg body weight. Infants in both groups grew at the same rate between 4 and 6 months of age. CONCLUSIONS: In a high-income country, adding a small amount of complementary food in addition to breast milk to infants' diets from 4 months of age does not affect growth rate between 4 and 6 months, but has a small and positive effect on iron status at 6 months. The biological importance of this finding remains to be determined.

BCQ-: A Body Constitution Questionnaire to assess Yin-Xu. Part II: evaluation of reliability and validity.

BACKGROUND: Based on the Traditional Chinese Medicine (TCM) theory and guided by the principles of modern medicine (MM), we aimed to develop an effective and reliable diagnostic tool using self-reported data in order to assess the Yin-Xu body constitution. In this study, we further evaluated a provisional 22-item version of the Yin-Xu Body Constitution Questionnaire (BCQ-) previously derived from the Delphi process. METHODS: A total of 1,272 eligible participants aged between 20 and 60 years was recruited. The participants had received health examination in teaching hospitals and had not shown any disease onset in the prior month. The factor structure of the BCQ- was explored, and the reliability and validity of each derived factor (scale) were evaluated. RESULTS: Exploratory factor analysis revealed 5 factors corresponding to the manifestations of the diminishing Yin-Xu level in 5 different body areas: head, 4 limbs, gastrointestinal tract, body surface, and abdominal cavity. 3 items with low factor loading (<0.4) were dropped, resulting in a 19-item BCQ-. Cronbach's α ranged from 0.57 to 0.85, and the intra-class correlation coefficients were greater than 0.7 for most items. The diagnoses of Yin-Xu by both the BCQ- and experienced TCM doctors were consistent. The concurrent validity showed that higher Yin-Xu scores were associated with lower hemoglobin. CONCLUSIONS: The final BCQ- measures the Yin-Xu constitution of 5 different body areas. The questionnaire is a reliable, valid, and potentially useful instrument for evaluating the Yin-Xu constitution in clinical research and practice.

Diospyros lotus L. fruit extract protects G6PD-deficient erythrocytes from hemolytic injury in vitro and in vivo: prevention of favism disorder.

OBJECTIVE: The aim of this study was to evaluate the protective effect of Diospyros lotus L. fruit extract against the hemolytic damage induced by Vicia faba beans extract in both G6PD enzyme-deficient human and rat erythrocyte in vitro and in vivo. MATERIALS AND METHODS: In the former model, venous blood samples were obtained from five subjects with known G6PD deficiency and erythrocyte hemolysis induced by Vicia faba L. bean extract was asessed spectrophotometrically in the presence and absence of Diospyros lotus L. fruits extract. In the in vivo model, G6PD-deficient rats (induced by intraperitoneal injection of dehydroepiandrosterone for 35 days) pre-treated with different doses of Diospyros lotus L. (500, 750, 1000, and 1500 mg/kg, p.o for 7 days) were challenged with Vicia faba beans extract and the protective effect of the fruit extract against hemolysis was evaluated as above. RESULTS AND CONCLUSIONS: The results have shown that Diospyros lotus L. fruits extract has antioxidant activity that may protect against hemolytic damage induced by Vicia faba bean extract in both G6PD-deficient human and rat erythrocytes. The study gives a scientific basis for the efficacy of the fruit extract as used in Iran. The fact that this was shown in human erythrocytes in vitro is significant and provides a rationale for further testing in vivo in G6PD-deficient human populations.

Intravenous immunoglobulin in neonates with rhesus hemolytic disease: a randomized controlled trial.

BACKGROUND: Despite limited data, international guidelines recommend the use of intravenous immunoglobulin (IVIg) in neonates with rhesus hemolytic disease. OBJECTIVE: We tested whether prophylactic use of IVIg reduces the need for exchange transfusions in neonates with rhesus hemolytic disease. DESIGN AND SETTING: We performed a randomized, double-blind, placebo-controlled trial in neonates with rhesus hemolytic disease. After stratification for treatment with intrauterine transfusion, neonates were randomly assigned for IVIg (0.75 g/kg) or placebo (5% glucose). The primary outcome was the rate of exchange transfusions. Secondary outcomes were duration of phototherapy, maximum bilirubin levels, and the need of top-up red-cell transfusions. RESULTS: Eighty infants were included in the study, 53 of whom (66%) were treated with intrauterine transfusion(s). There was no difference in the rate of exchange transfusions between the IVIg and placebo groups (7 of 41 [17%] vs 6 of 39 [15%]; P = .99) and in the number of exchange transfusions per patient (median [range]: 0 [0-2] vs 0 [0-2]; P = .90) or in duration of phototherapy (4.7 [1.8] vs 5.1 [2.1] days; P = .34), maximum bilirubin levels (14.8 [4.7] vs 14.1 [4.9] mg/dL; P = .52), and proportion of neonates who required top-up red-cell transfusions (34 of 41 [83%] vs 34 of 39 [87%]; P = .76). CONCLUSIONS: Prophylactic IVIg does not reduce the need for exchange transfusion or the rates of other adverse neonatal outcomes. Our findings do not support the use of IVIg in neonates with rhesus hemolytic disease.