Fetal heart rate evolution patterns in cerebral palsy associated with umbilical cord complications: a nationwide study.

BACKGROUND: The aim of the present study was to clarify fetal heart rate (FHR) evolution patterns in infants with cerebral palsy (CP) according to different types of umbilical cord complications. METHODS: This case-control study included children born: with a birth weight ≥2000 g, at gestational age ≥33 weeks, with disability due to CP, and between 2009 and 2014. Obstetric characteristics and FHR patterns were compared among patients with CP associated with (126 cases) and without (594 controls) umbilical cord complications. RESULTS: There were 32 umbilical cord prolapse cases and 94 cases with coexistent antenatal umbilical cord complications. Compared with the control group, the persistent non-reassuring pattern was more frequent in cases with coexistent antenatal umbilical cord complications (p = 0.012). A reassuring FHR pattern was observed on admission, but resulted in prolonged deceleration, especially during the first stage of labor, and was significantly identified in 69% of cases with umbilical cord prolapse and 35% of cases with antenatal cord complications, compared to 17% of control cases (p < 0.001). CONCLUSION: Hypercoiled cord and abnormal placental umbilical cord insertion, may be associated with CP due to acute hypoxic-ischemic injury as well as sub-acute or chronic adverse events during pregnancy, while umbilical cord prolapse may be characterized by acute hypoxic-ischemic injury during delivery.

A novel assay for serum creatinine using a creatine kinase cycling reaction.

For assaying serum creatinine, the enzymatic method is regarded as accurate. However, a reliable measurement of low levels is problematic. We have developed a new method that utilizes an enzymatic cycling reaction involving creatine kinase (CK) in the presence of excess ATP and IDP and implicated the application to a serum creatinine assay by incorporating with creatininase. Here, we evaluated applying the CK cycling method to a serum creatinine assay. In this study, we focused on assessing whether an accurate measurement could be achieved, especially in the reference interval and the lower concentration range. The effective sensitivity of the assay using 30 U/mL CK was approximately 4-fold greater than that of a commercial reagent. Under these conditions, 0.19 mg/dL of creatinine was accurately detected. The correlation coefficient of the comparison study with an existing commercial reagent was 0.995. Moreover, the effect of the increased signal intensity on accuracy and precision was assured.

Weekend and off-hour effects on the incidence of cerebral palsy: contribution of consolidated perinatal care.

OBJECTIVE: This study estimated the effects of weekend and off-hour childbirth and the size of perinatal medical care center on the incidence of cerebral palsy. METHODS: The cases were all children with severe cerebral palsy born in Japan from 2009 to 2012 whose data were stored at the Japan Obstetric Compensation System for Cerebral Palsy database, a nationally representative database. The inclusion criteria were the following: neonates born between January 2009 and December 2012 who had a birth weight of at least 2000 g and gestational age of at least 33 weeks and who had severe disability resulting from cerebral palsy independent of congenital causes or factors during the neonatal period or thereafter. Study participants were restricted to singletons and controls without report of death, scheduled cesarean section, or ambulance transportation. The controls were newborns, randomly selected by year and type of delivery (normal spontaneous delivery without cesarean section and emergency cesarean section) using a 1:10 case to control ratio sampled from the nationwide Japan Society of Obstetrics and Gynecology database. RESULTS: A total of 90 cerebral palsy cases and 900 controls having normal spontaneous delivery without cesarean section were selected, as were 92 cerebral palsy cases and 920 controls with emergent cesarean section. A significantly higher risk for cerebral palsy was found among cases that underwent emergent cesarean section on weekends (odds ratio [OR] 1.72, 95% confidence interval [CI] 1.06-2.81) and during the night shift (OR 2.29, 95% CI 1.30-4.02). No significant risk was found among normal spontaneous deliveries on weekends (OR 1.63, 95% CI 0.97-2.73) or during the quasi-night shift (OR 1.26, 95% CI 0.70-2.27). Regional perinatal care centers showed significantly higher risk for cerebral palsy in both emergent cesarean section (OR 2.35, 95% CI 1.47-3.77) and normal spontaneous delivery (OR 2.92, 95% CI 1.76-4.84). CONCLUSION: Labor on weekends, during the night shift, and at regional perinatal medical care centers was associated with significantly elevated risk for cerebral palsy in emergency cesarean section.

Pyruvate kinase from Geobacillus stearothermophilus displays an unusual preference for Mn2+ in a cycling reaction.

Previously, we developed a kinase cycling method using creatine kinase and pyruvate kinase (RMPK) both from rabbit muscle in the presence of an excess amount of ATP and IDP for the quantitative determination of substrate. To our surprise, the RMPK cycling reaction was 10-fold more efficient using Mn2+ rather than Mg2+. Here, we investigated PK from Geobacillus stearothermophilus (GSPK) as an alternative source of enzyme. Spectrophotometric real-time detection was accomplished by coupling the reaction to ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase (G6PD). The rate of increase in absorbance of NADH at 340 nm was monitored. GSPK displayed an even greater preference than RMPK for Mn2+ over Mg2+ in the cycling reaction with ATP and GDP or ATP and IDP. The much lower Km values for the substrate in the presence of Mn2+ rather than Mg2+ are consistent with the results of the cycling reaction observed in this study.

Japan Obstetric Compensation System for Cerebral Palsy: Strategic system of data aggregation, investigation, amelioration and no-fault compensation.

The Japan Obstetric Compensation System for Cerebral Palsy (JOCS-CP), which investigates, develops preventive mechanisms and awards monetary compensation, to cases of cerebral palsy was urgently introduced in 2009 in response to growing concern about Japan's deteriorating perinatal care and low birthrate. Under the political leadership, the Japan Council for Quality Health Care launched the JOCS-CP with support of various stakeholders. The JOCS-CP features of no-fault-based compensation which was discussed decades ago in the Japan Medical Association aiming at financial aid to patient and family and early settlement of dispute. As of 2017, 2233 petitions had been approved by the Review Committee for compensation. All the approved cases were consecutively put on analysis in the Investigation Committee which has compiled more than 1000 Investigative Reports. The reports were delivered not only to the childbirth facility but to the guardians/families. Survey revealed that most of childbirth facility and the guardians/families responded in favor of the reports. With regard to amelioration of profound cerebral palsy, the Prevention Report has been published on annual basis through analysis of all the Investigative Reports. The Prevention Reports and other educational materials were produced and distributed not only among medical professionals but among pregnant women. It is notable that the number of lawsuit filing related to obstetrics demonstrated rapid decrease compared to that of other medical specialties. The JOCS-CP could be described as a social experiment. It was overhauled in 2015 but deserves further discussion on reform for evolving into better system.

Relevant obstetric factors associated with fetal heart rate monitoring for cerebral palsy in pregnant women with hypertensive disorder of pregnancy.

AIM: The study identifies the relevant obstetric factors associated with fetal heart rate (FHR) monitoring for cerebral palsy (CP) in pregnant women with hypertensive disorders of pregnancy (HDP). METHODS: The subjects were neonates with CP (birth weight ≥ 2000 g, gestational age ≥ 33 weeks) who were approved for compensation for CP by the Operating Organization of the Japan Obstetric Compensation System between 2009 and 2012. After selection of women with antepartum HDP, obstetric characteristics associated with FHR monitoring were analyzed. RESULTS: The subjects included 33 neonates with CP whose mothers suffered from HDP during pregnancy and 450 neonates whose mothers did not develop HDP. The rates of placental abruption (48.5% vs. 20%; P < 0.001) and light-for-gestational age (12.1% vs. 2.2%; P = 0.011) were significantly higher in women with HDP than in those without HDP. Regarding FHR pattern analysis, fetal bradycardia was observed on admission to hospital in 94% of women with placental abruption. In women without placental abruption, FHR was likely to indicate a favorable pattern on admission, but became worse with the progression of labor. CONCLUSION: This is first study to clinically demonstrate FHR patterns in CP cases in association with HDP. Although antepartum CP is undetectable, pregnant women with HDP should be placed under strict observation and management to minimize fetal hypoxic conditions during labor.

[A Case Involving the Re-Application of TAE for the Rupture of Recurrent Dissemination Lesion after Initial TAE and Hepatectomy Was Performed for A Previous Rupture of HCC].

The patient was a 65-year-old man who had been previously diagnosed with chronic hepatitis B, but the patient had discontinued treatment while in his thirties. The patient was admitted to the emergency department after losing consciousness due to abdominal pain. Emergency contrast CT was performed in the shock state, and the diagnosis was hemorrhagic shock due to rupture of hepatocellular carcinoma(HCC). Emergency TAE was performed, and hemostasis was successful due to left hepatic arterial embolism. The tumor was confined to the liver lateral area and it was judged to be resectable curatively, upon state restoration. Ten days after TAE, lateral segmentectomy of the liver was performed. Pathological findings indicated moderately differentiated HCC, mostly necrotic and partially viable. He was discharged on 11POD. On 69POD, the patient reexperienced sudden abdominal pain after lunch. The abdominal pain continued while emergency contrast CT was performed at the time of visit in the shock state. Recurrence of multiple dissemination via high-absorption ascites was found around the largest tumor nest with lower left diaphragm diameter of 15cm, and it was judged that the HCC disseminated recurrence had ruptured. Emergency TAE was performed again, and hemostasis was successful by embolization of the left gastric artery and lower left diaphragm artery. Subsequently, tumor growth slowed after initiating oral administration of sorafenib, and the patient is alive 8 months after re-TAE.

Enzymatic cycling method using creatine kinase to measure creatine by real-time detection.

We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP-GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay.

[An Examination of Colon Cancer Treatment Policies for the Elderly].

Currently, the rate of aging is increasing rapidly and the number of elderly patients in hospitals is rising in Japan. Under such circumstances, we established an emergency care unit for the elderly at our hospital in July 2014 to provide acute healthcare of the community. Our unit has been active in providing treatment for acute diseases in elderly individuals in the community. We are also pursuing continuous medical care for elderly individuals in collaboration with the community comprehensive care unit at our hospital and other facilities in the community, such as care facilities for the elderly and specialist elderly nursing homes. From January to December 2015, we examined treatment policies for colon cancer at our hospital. The presence or absence of dementia and patient's age are factors considered before treatment at our hospital; however, in many cases colon cancer was treated using surgery if the patient's generalcondition permitted it. In colon cancer patients, this takes into consideration the decreased quality of life resulting from losing the ability to eat due to bleeding and intestinal obstruction. We hereby report specific cases of colon cancer treatment policies for elderly patients at our hospital, together with a discussion of the literature.

[Advanced Digestive Cancer in Super Elderly Patients More Than 85 Years Old - Two Cases That Required Major Invasive Surgery].

Case 1 involved an 87-year-old woman. She had increased serum amylase levels, and intraductal papillary mucinous adenocarcinoma( IPMC)in the accessory pancreatic duct was diagnosed following various medical examinations. Radical surgery is possible for such a lesion when there are no issues detected following evaluation. The patient and her family decided that she should undergo surgery; therefore, pancreatoduodenectomy was performed for Stage III IPMC. Various morbidities required medical treatment, but she was finally discharged from hospital 89 days after surgery. She has had no recurrence 2 years and 11 months after surgery. Case 2 involved a 90-year-old man. He had liver dysfunction, and progressive hepatocellular carcinoma( HCC)was diagnosed based on computed tomography(CT)findings. The tumor size reduced to around 10 cm with enforced 2 degrees TACE. The patient and his family chose for him to undergo radical surgery; therefore, right trisegmentectomy of the liver was performed for Stage III HCC. He was discharged from the hospital 22 days after surgery, and has had no recurrence for the past 1.6 years. More careful examination and a general assessment are necessary to make decisions on surgical indications in super elderly patients.

[A Case That in Most Part of the Metastatic Liver Tumor Fell into Spontaneous Necrosis after Surgery of Rectal Cancer].

We present a case of a 74-year-old woman treated with maintenance dialysis for chronic renal failure. She had undergone intravascular treatment of lower limb arteriosclerosis obliterans 3 times. Low anterior resection was performed for rectal cancer StageⅡ, but adjuvant chemotherapy was not administered after the operation. S5/S6 partial hepatectomy was performed after confirming a liver metastatic recurrence 7 months after the first operation. Tumor pathology examination of the resected specimen revealed necrosis in most parts of the specimen. Residual primary lesion similar to adenocarcinoma tissue was found only in a small part of the specimen. Artery wall embolism and recanalization image with cholesterol crystals indicated a Grison's lesion, which was considered to have likely developed into tumor necrosis from the embolization. Spontaneous regression of a malignant tumor is a rare condition found 1 case in 6-10 ten thousand cases. In hepatocellular carcinoma, secondary tumor infarction from tumor invasion and tumor thrombus may occur. The present case was a metastatic liver cancer, in which the cholesterol crystal embolism possibly reached the tumor necrosis. The suggested cause of the cholesterol crystal embolism in this case was intravascular treatment related.

[Relapse-Free Survival Following Multidisciplinary Therapy for Hepatocellular Carcinoma with Multiple Lung Metastases--A Case Report].

Chemotherapy is not effective for metastatic hepatocellular carcinoma(HCC); however, prolonged survival can be expected for patients with multiple metastases who are treated with surgical resection in addition to chemotherapy. We present a case of a 36-year-old woman with hepatitis B who developed HCC with multiple intrahepatic and lung metastases after undergoing resection of HCC in 2010 in Canada. The patient returned to Japan for additional treatment. She was treated with TACE therapy and systemic chemotherapy, but her lung metastases did not improve. The patient's PIVKA-Ⅱ levels remained moderately elevated after initiation of chemotherapy. Therefore, we performed surgical resection of the lung metastases in March 2014. Five months later, the patient received additional TACE therapy when an isolated metastasis was found in the residual liver. Since then, no recurrence of HCC has been found, and the patient's PIVKA-Ⅱ levels have remained in the normal range. This case demonstrates that surgical resection for multiple lung metastases is possible in select patients.

An ultrasensitive enzymatic method for measuring mevalonic acid in serum.

We have developed a simple, precise, and ultrasensitive enzymatic method for measuring serum mevalonic acid (MVA) concentration, which is thought to be a good indicator of the in vivo cholesterol biosynthesis rate. This assay is based on an enzyme cycling reaction and makes use of HMG-CoA reductase (HMGR), thio-NAD, NADH, and CoA. MVA participates in the HMGR cycling reaction, and its level is measured based on the production of thio-NADH, which is determined from the change in absorbance at 405 nm. To achieve high specificity, we used mevalonate kinase (MVK) in addition to HMGR. Only substrates able to participate in both the HMGR cycling reaction and the MVK reaction are measured as MVA. The detection limit for MVA is 0.4 ng/ml (2.7 nmol/l), and the calibration curve for MVA is linear up to 44 ng/ml (300 nmol/l). Regression analysis with 40 serum samples showed the accuracy of quantifying MVA with this enzymatic assay to be comparable to that using LC-MS/MS (correlation: y = 0.83x + 0.24; r = 0.97). This procedure is simple, precise, and robust. It is also rapid and has a high throughput, making it potentially useful for clinical applications.

Hypoxanthine-guanine phosphoribosyltransferase is activated via positive cooperativity between guanine and IMP.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway. Here, the reverse reaction of HGPRT from the thermophilic bacterium Hungateiclostridium thermocellum was studied in the presence of IMP and pyrophosphate. As for the human enzyme, the bacterial HGPRT was activated by guanine. Furthermore, guanine was found to operate as both an activator and an inhibitor. Intriguingly, within the concentration range of guanine where it functions as the activator, the Km value for IMP was not influenced by guanine. Consequently, guanine was found to noncompetitively activate the reverse reaction toward IMP. Here, we propose a reaction scheme that explains the activation mechanism in which the enzyme forms a chimeric oligomer bound to both IMP and guanine.

Recovery of high-quality phosphate from steelmaking slag by a hydrometallurgical process.

Phosphorus recovery from wastes has become a worldwide concern. The P-bearing steelmaking slag generated from steel plant is considered a potential phosphorus source. In this study, a novel process of selective leaching-precipitation-alkaline wash was proposed to recover high-quality phosphate from steelmaking slag. During leaching, most of the P was dissolved from slag and Fe was almost insoluble. Increasing temperature and solid-liquid ratio significantly suppressed the dissolution of Si due to the formation of silica sols. An excellent selective leaching of P was achieved at pH 3 and 333 K. The dissolution ratio of P reached 83.5% while only 22.6% of Si was dissolved. The residue containing 49.5% Fe2O3 and 0.9% P2O5 can be reused as a steelmaking feedstock, achieving the recycling in plant. In the dilute leachate, the precipitation of Si and Ca was significantly suppressed as the pH increased and a precipitate with higher P2O5 content and lower SiO2 content was extracted. A large amount of SiO2 was removed from the precipitate by alkaline wash. A precipitate containing 30.1% P2O5 and 45.5% CaO was recovered in this process. This study provided a cost-effective approach to recovering high-quality phosphate.

Evaluation of ERCC1 expression for cisplatin sensitivity in human hepatocellular carcinoma.

BACKGROUND: Excision repair cross-complementation group 1 (ERCC1) is one of the key enzymes in DNA repair. This study was designed to investigate the correlation between ERCC1 expression and chemosensitivity to cisplatin (CDDP) in patients with hepatocellular carcinoma (HCC). METHODS: Eighty-seven HCC samples were analyzed by immunohistochemistry for ERCC1 and chemosensitivity was assessed by the succinate dehydrogenase inhibition (SDI) test for four anti-cancer agents, including CDDP. The ERCC1 expression was examined in HCC cell lines. ERCC1 siRNA was transfected to PLC/RPF/5 to investigate the correlation of ERCC1 expression and CDDP sensitivity. RESULTS: ERCC1 expression was observed in 33% of nuclei in immunohistochemical examination. Patients were divided into two groups as follows: ERCC1 high expression group (n = 43): more than 33% of the nuclei were stained; ERCC1 low expression group (n = 44): 33% or fewer of the nuclei were stained. Tumor size of low expression group was larger than that in the high expression group (p = 0.02). The succinic dehydrogenase (SD) activity only for CDDP was significantly higher in the high expression group than that in the low expression group (p = 0.02). An increased expression of ERCC1 was shown by immunohistochemical and Western blot analyses in PLC/RPF/5. ERCC1 expression was inhibited by ERCC1 siRNA transfection and the LC50 value (nM) of CDDP was reduced from 25.7 to 12.5 (p = 0.01). CONCLUSIONS: Increased ERCC1 expression is associated with CDDP resistance in HCC specimens and cell lines. Therefore, immunohistochemical analysis for resected HCC tissues may be a useful predictor for the effectiveness of adjuvant chemotherapy, using CDDP.

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method.

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 µM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.

1,2-Dioleoylglycerol method for pancreatic lipase catalytic activity in serum.

BACKGROUND: There is a need for a pancreatic lipase (LIP) reference assay to provide an accurate base to which routine methods can be traceable. METHODS: This study developed a novel LIP assay method in which 1,2-dioleoylglycerol (DODG) is the substrate and LIP activity is measured in a coupled enzymatic reaction from the increase in absorbance at 340 nm with production of NADPH. RESULTS: With this method, LIP activity was linear up to 440 U/L (8-times expected upper limit of physiological concentration). When assayed manually, the between-laboratory variation for six samples surveyed at five laboratories was 3.80-26.4% (CV) for samples containing about 20-290 U/L LIP activity; when assayed using an automated analyzer, the range was 1.86-4.86% (four laboratories). Interference by >5 mmol/L glycerol and low specificity with post-heparin samples were noted, but in practice these are avoidable. Precision analyzed by automated assay of 49 samples twice in random order produced a covariance of 2.27 U/L, which is comparable to routine methods, and good correlations were obtained with five routine methods. CONCLUSIONS: Although further studies are required, the DODG method may be likely applicable as one candidate reference method.

Obstetric factors associated with uterine rupture in mothers who deliver infants with cerebral palsy.

Objective: The aim of the present study was to clarify the obstetric factors associated with uterine rupture in mothers who deliver infants with cerebral palsy (CP) in Japan.Methods: This retrospective case-cohort study reviewed the obstetric characteristics and clinical courses of mothers who experienced uterine rupture and compared those who delivered an infant with CP (cases) with those who delivered an infant without CP (cohort). Data were obtained from the Japan Obstetric Compensation System for CP database (27 cases) and the perinatal database of the Japan Society of Obstetrics and Gynecology (312 cohorts). The subjects included live singleton infants delivered between 2009 and 2014 with a birth weight ≥2000 g and gestation ≥33 weeks.Results: Augmentation was performed 33% in cases and 8% in cohorts (p < .001). The amount of bleeding during surgery was 1819 g in cases and 1096 g in cohorts (p < .001). Length of gestational weeks and neonatal birth weight were significantly higher and Apgar scores and umbilical arterial pH were lower in cases compared to cohorts (p < .001). In cases with CP, 11 cases of uterine rupture involved scarred uteruses. Seven were trial of labor after a previous cesarean. On one hand, 16 cases occurred in unscarred uteruses. Five of the uterine fundal pressure maneuvers and four of tachysystole due to excessive augmentation were reported in association with uterine rupture.Conclusion: Two-third of the relevant obstetric factors for CP associated with uterine rupture were iatrogenic. At least, to reduce CP resulting from delivery-related uterine rupture, reckless delivery management should be avoided.

A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin.

BACKGROUND: In vitro diagnostic bilirubin reagents based on oxidation with bilirubin oxidase or vanadic acid for total and direct-reacting bilirubin are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin and delta bilirubin has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four in vitro diagnostic bilirubin reagents. METHODS: Porcine unconjugated bilirubin solution, chemically synthesized ditaurobilirubin solution, and chemically synthesized delta bilirubin solution were used as surrogates of naturally occurring unconjugated bilirubin, conjugated bilirubin, and delta bilirubin, respectively. The total bilirubin and direct-reacting bilirubin concentrations were measured by three bilirubin oxidase methods and one vanadic acid method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure. RESULTS: The unconjugated bilirubin and delta bilirubin concentrations were similar when any of the four in vitro diagnostic bilirubin reagents were used during total bilirubin measurement. This was consistent with reference measurement procedure and exhibited a converged inter-method variation. Compared with reference measurement procedure, significantly low ditaurobilirubin concentrations were observed by the in vitro diagnostic bilirubin reagents despite the converged inter-method variation. In delta bilirubin measurement, some reagents reacted doubtfully with unconjugated bilirubin, while showed lower ditaurobilirubin concentrations than its corresponding total bilirubin concentration. Reactivity with delta bilirubin was different for each method including reference measurement procedure. Some reagents were developed to react less with delta bilirubin and others to strongly react with delta bilirubin. CONCLUSIONS: We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with reference measurement procedure. The delta bilirubin data results vary depending on the reagents used.