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Introduction: Reducing neonatal deaths in premature infants in low- and middle-income countries is key to reducing global neonatal mortality. International neonatal networks, along with patient registries of premature infants, have contributed to improving the quality of neonatal care; however, the involvement of low-to-middle-income countries was limited. This project aims to form an international collaboration among neonatal networks in Asia (AsianNeo), including low-, middle- and high-income countries (or regions). Specifically, it aims to determine outcomes in sick newborn infants, especially very low birth weight (VLBW) infants or very preterm infants, with a view to improving the quality of care for such infants., Methods and Analysis: Currently, AsianNeo comprises nine neonatal networks from Indonesia, Japan, Malaysia, Philippines, Singapore, South Korea, Sri Lanka, Taiwan and Thailand. AsianNeo will undertake the following four studies: (1) institutional questionnaire surveys investigating neonatal intensive care unit resources and the clinical management of sick newborn infants, with a focus on VLBW infants (nine countries/regions); (2) a retrospective cohort study to describe and compare the outcomes of VLBW infants among Asian countries and regions (four countries/regions); (3) a prospective cohort study to develop the AsianNeo registry of VLBW infants (six countries/regions); and (4) implementation and evaluation of educational and quality improvement projects in AsianNeo countries and regions (nine countries/regions)., Ethics and Dissemination: The study protocol was approved by the Research Ethics Board of the National Center for Child Health and Development, Tokyo, Japan (reference number 2020-244, 2022-156). The study findings will be disseminated through educational programmes, quality improvement activities, conference presentations and medical journal publications., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: The management of patent ductus arteriosus (PDA) is a critical concern in premature infants, and different hospitals may have varying treatment policies, fluid management strategies, and incubator humidity. The Asian Neonatal Network Collaboration (AsianNeo) collected data on prematurity care details from hospitals across Asian countries. The aim of this study was to provide a survey of the current practices in the management of PDA in premature infants in Asian countries., Methods: AsianNeo performed a cross-sectional international questionnaire survey in 2022 to assess the human and physical resources of hospitals and clinical management of very preterm infants. The survey covered various aspects of hospitals resources and clinical management, and data were collected from 337 hospitals across Asia. The data collected were used to compare hospitals resources and clinical management of preterm infants between areas and economic status., Results: The policy of PDA management for preterm infants varied across Asian countries in AsianNeo. Hospitals in Northeast Asia were more likely to perform PDA ligation ( p  < 0.001) than hospitals in Southeast Asia. Hospitals in Northeast Asia had stricter fluid restrictions in the first 24 h after birth for infants born at <29 weeks gestation ( p  < 0.001) and on day 14 after birth for infants born at <29 weeks gestation ( p  < 0.001) compared to hospitals in Southeast Asia. Hospitals in Northeast Asia also had a more humidified environment for infants born between 24 weeks gestation and 25 weeks gestation in the first 72 h after birth ( p  < 0.001). A logistic regression model predicted that hospitals were more likely to perform PDA ligation for PDA when the hospitals had a stricter fluid planning on day 14 after birth [Odds ratio (OR) of 1.70, p  = 0.048], more incubator humidity settings (<80% vs. 80%-89%, OR of 3.35, p  = 0.012 and <80% vs. 90%-100%, OR of 5.31, p  < 0.001)., Conclusions: In advanced economies and Northeast Asia, neonatologists tend to adopt a more conservative approach towards fluid management, maintain higher incubator humidity settings and inclined to perform surgical ligation for PDA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Hsieh, Jeng, Lin, Lin, Rohsiswatmo, Dewi, Chee, Neoh, Velasco, Imperial, Nuntnarumit, Ngerncham, Chang, Kim, Quek, Amin, Kusuda, Miyake and Isayama.)

Advances in perinatal care have led to the increased survival of preterm infants with subsequent neonatal morbidities, such as retinopathy of prematurity (ROP). This study aims to compare the differences of neonatal healthcare systems, resources, and clinical practice concerning ROP in Asia with review of current literature. An on-line survey at the institutional level was sent to the directors of 336 neonatal intensive care units (NICU) in 8 collaborating national neonatal networks through the Asian Neonatal Network Collaboration (AsianNeo). ROP screening was performed in infants born at < 34 weeks in Indonesia and Japan. In South Korea, Malaysia, and Taiwan, most screened for ROP in infants born at < 32 weeks. In all networks, majority of NICUs conducted ROP screening to infants with birth weight < 1500 g. In most NICU's in-hospital ophthalmologists performed indirect ophthalmoscopy and some were supplemented with digital imaging. Both laser photocoagulation and anti-vascular endothelial growth factor injection are performed for treatment and, vitreous surgeries are conducted less frequently in all countries. Despite limited information collected by the survey, this first study to compare ROP practices implemented in eight Asian countries through AsianNeo will enable an understanding of the differences and facilitate quality improvement by sharing better practices., (© 2023. Springer Nature Limited.)

Objectives: To determine the incidence, causative pathogens, morbidities, mortality, and risk factors associated with blood culture-positive early-onset sepsis (EOS, ≤72 hours of age) in symptomatic neonates admitted to the neonatal intensive care units (NICUs) of a middle-income country., Study Design: Retrospective cohort study using data submitted prospectively to the Malaysian National Neonatal Registry (MNNR)., Setting: 44 Malaysian NICUs., Participants: All neonates born in 2015- 2020., Results: EOS was reported in 991 neonates. The annual incidence of EOS increased from 0.46 to 0.49/1000 livebirths over the six years. The most common pathogen was Streptococcus agalactiae or Group B haemolytic streptococcus (GBS) (n=388, 39.2%), followed by Escherichia coli (E. coli) (n=80, 8.1%), Klebsiella spp (n=73, 7.4%), coagulase negative staphylococcus (CONS) (n=73, 7.4%), Pseudomonas spp (n=44, 4.4%) and methicillin-sensitive Staphylococcus aureus (n=34, 3.4%). The incidence of EOS due to GBS increased from 0.17 to 0.22/1000 livebirths. Morbidities and mortality were higher in those with EOS than without EOS. Multiple logistic regression analysis showed that Indian ethnic group, chorioamnionitis, gestation≥37weeks, female, spontaneous vaginal delivery, instrumental delivery, and surfactant therapy were significantly associated with increased risk of EOS due to GBS. Four factors were significantly associated with increased risk of non-GBS EOS (outborns, birthweight lt;1000 g, vaginal delivery, and surfactant therapy). Early continuous positive airway pressure was associated with significantly lower risk of EOS., Conclusion: The incidence of EOS showed an increasing trend in Malaysian NICUs. GBS was the most common causative pathogen. Several modifiable risk factors associated with EOS have been identified.

Objectives: To investigate the types of therapeutic hypothermia (TH) used and risk factors associated with mortality in late-preterm and term neonates (LPTN, gestation of ≥35 weeks) with hypoxic-ischemic encephalopathy (HIE) in a middle-income country., Design: This was an observational retrospective cohort study., Setting: A total of 44 neonatal intensive care units (NICUs) in the Malaysian National Neonatal Registry participated in the study., Patients: All LPTN without major malformations and diagnosed to have HIE were included., Main Outcome Measures: Number of in-hospital mortality, and types of TH used [no TH, TH using commercially available servo-controlled devices (SCDs), passive TH by exposing neonates to NICU's air-conditioned ambient temperature with/without the use of cooled gel packs (P±CGPs)]., Results: Of a total of 2,761 HIE neonates, 66.3% received TH. All NICUs provided TH; 55.4% NICUs had SCDs, which was administered to 43.6% (248/569) of severe, 51.6% (636/1,232) of moderate, and 18.6% (179/960) of mild HIE neonates. P±CGPs was used on 26.9% of severe, 33.4% of moderate, and 21.1% of mild HIE neonates. There were 338 deaths. Multiple logistic regression analysis showed that 5-min Apgar scores <5 (aOR: 1.436; 95% CI: 1.019, 2.023), Cesarean section (aOR: 2.335; 95% CI: 1.700, 3.207), receiving no TH (aOR: 4.749; 95% CI: 3.201, 7.045), TH using P±CGPs (aOR: 1.553; 95% CI: 1.031, 2.338), NICUs admitted <50 HIE cases (aOR: 1.898; 95% CI: 1.225, 2.940), NICUs admitted 50-<100 HIE cases (aOR: 1.552; 95% CI: 1.065, 2.260), moderate HIE (aOR: 2.823; 95% CI: 1.495, 5.333), severe HIE (aOR: 34.925, 95% CI: 18.478, 66.012), Thompson scores of 7-13 (aOR: 1.776; 95% CI: 1.023,3.082), Thompson scores of ≥14 (aOR: 3.641; 95% CI: 2.000, 6.629), pneumothorax (aOR: 3.435; 95% CI: 1.996, 5.914), and foreigners (aOR: 1.646; 95% CI: 1.006, 2.692) were significant risk factors associated with mortality., Conclusion: Both SCD and P±CGP were used for TH. Moderate/severe HIE and receiving passive/no TH were among the risk factors associated with mortality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Boo, Neoh and Chee.)

Introduction: We aimed to determine global professional opinion and practice for the use of therapeutic hypothermia (TH) for treating infants with mild hypoxic-ischaemic encephalopathy (HIE)., Methods: A web-based survey (REDCap) was distributed via emails, social networking sites, and professional groups from October 2020 to February 2021 to neonatal clinicians in 35 countries., Results: A total of 484 responses were obtained from 35 countries and categorized into low/middle-income (43%, LMIC) or high-income (57%, HIC) countries. Of the 484 respondents, 53% would provide TH in mild HIE on case-to-case basis and only 25% would never cool. Clinicians from LMIC were more likely to routinely offer TH in mild HIE (25% v HIC 16%, p < 0.05), have a unit protocol for providing TH (50% v HIC 26%, p < 0.05), use adjunctive tools, e.g., aEEG (49% v HIC 32%, p < 0.001), conduct an MRI post TH (48% v HIC 40%, p < 0.05) and less likely to use neurological examinations as a HIE severity grading tool (80% v HIC 95%, p < 0.001). The majority of respondents (91%) would support a randomized controlled trial that was sufficiently large to examine neurodevelopmental outcomes in mild HIE after TH., Conclusions: This is the first survey of global opinion for TH in mild HIE. The overwhelming majority of professionals would consider "cooling" an infant with mild HIE, but LMIC respondents were more likely to routinely cool infants with mild HIE and use adjunctive tools for diagnosis and follow-up. There is wide practice heterogeneity and a sufficiently large RCT designed to examine neurodevelopmental outcomes, is urgently needed and widely supported., (© 2022 S. Karger AG, Basel.)

Objectives: To determine a 10-year trend of survival, morbidities and care practices, and predictors of in-hospital mortality in very preterm neonates (VPTN, gestation 22 to <32 weeks) in the Malaysian National Neonatal Registry., Design: Retrospective cohort study., Setting: 43 Malaysian neonatal intensive care units., Patients: 29 010 VPTN (without major malformations) admitted between 1 January 2009 and 31 December 2018., Main Outcome Measures: Care practices, survival, admission hypothermia (AH, <36.5°C), late-onset sepsis (LOS), pneumothorax, necrotising enterocolitis grade 2 or 3 (NEC), severe intraventricular haemorrhage (sIVH, grade 3 or 4) and bronchopulmonary dysplasia (BPD)., Results: During this 10-year period, there was increased use of antenatal steroid (ANS), lower segment caesarean section (LSCS) and early continuous positive airway pressure (eCPAP); but decreased use of surfactant therapy. Survival had increased from 72% to -83.9%. The following morbidities had decreased: LOS (from 27.9% to 7.1%), pneumothorax (from 6.0% to 2.7%), NEC (from 8.1% to 4.7%) and sIVH (from 12.2% to 7.5%). However, moderately severe AH (32.0°C-35.9°C) and BPD had increased. Multiple logistic regression analyses showed that lower birth weight, no ANS, no LSCS, admission to neonatal intensive care unit with <100 VPTN admissions/year, no surfactant therapy, no eCPAP, moderate and severe AH, LOS, pneumothorax, NEC and sIVH were significant predictors of mortality., Conclusion: Survival and major morbidities had improved modestly. Failure to use ANS, LSCS, eCPAP and surfactant therapy, and failure to prevent AH and LOS increased risk of mortality., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Early nasal continuous positive airway pressure (EnCPAP) therapy after birth for very low birth weight (VLBW; <1,500 g) neonates has been reported to be beneficial in developed countries. Its benefits in developing countries, such as Malaysia, are unknown., Objectives: This study aimed to determine EnCPAP rates in 36 neonatal intensive care units of the Malaysian National Neonatal Registry (MNNR) in 2013, to compare the outcomes of VLBW neonates with and without EnCPAP, and to determine whether the availability of CPAP facilities and unit policies played a significant role in EnCPAP rates., Methods: First, a retrospective cohort study was conducted of VLBW neonates born in the hospitals participating in the study without major congenital abnormalities in the MNNR. This was followed by a questionnaire survey of these hospitals focussed on CPAP facilities and unit policies., Results: Of the 2,823 neonates, 963 (34.1%) received EnCPAP. Amongst EnCPAP neonates significantly fewer deaths were recorded (10.9 vs. 21.7%; p < 0.001), less bronchopulmonary dysplasia was observed (BPD; 8.0 vs. 11.7%; p = 0.002) and fewer mechanical ventilation days were necessary (p < 0.001) than in non-EnCPAP neonates. Logistic regression analysis showed that EnCPAP was significantly associated with a lower mortality (adjusted OR 0.623; 95% CI 0.472, 0.824; p = 0.001) and BPD among survivors (adjusted OR 0.585; 95% CI 0.427, 0.802; p = 0.001). The median EnCPAP rate of the 36 hospitals was 28.4% (IQR 14.3-38.7). Hospitals with CPAP facilities in the delivery suites (p = 0.001) and during transport (p = 0.001) and a policy for EnCPAP (p = 0.036) had significantly higher EnCPAP rates., Conclusion: EnCPAP reduced mortality and BPD in Malaysian VLBW neonates. Resource-strapped developing countries should prioritise the use of this low-cost therapy., (© 2016 S. Karger AG, Basel.)

Parotid abscess is uncommon in neonates. It is frequently related to prematurity, prolonged gavage feeding and dehydration. We report a case of a late preterm infant who developed the classical manifestation of unilateral acute Staphylococcus aureus suppurative parotitis progressing to formation of abscess which responded to surgical drainage and antibiotic therapy.

The level of minimal residual disease (MRD) early in treatment of acute lymphoblastic leukemia (ALL) strongly predicts the risk of marrow relapse. As a variety of methods of varying complexity have been separately used for detecting and quantifying MRD, we compared the prognostic utility of three methods measurement of blast percentage on day 14 of treatment, detection of monoclonality on day 14 or day 35, and measurement of MRD by PCR-based limiting dilution analysis on day 14 or day 35. The study group comprised 38 children aged 1-15 with Philadelphia-negative B-lineage ALL who were uniformly treated and followed until relapse or for a minimum of 5 years. We also studied some of the technical factors which influence the ability to detect MRD. Measurement of blast percentage on day 14 by an expert morphologist, detection of monoclonality on day 35, and PCR-based measurement of MRD levels on days 14 and 35 all showed significant ability to divide patients into prognostic groups. Measurement of blast percentage on day 14 by routine morphology or detection of monoclonality on day 14 were not useful. The quality of DNA samples varied greatly, as determined by amplifiability in the PCR. However, virtually all amplifiable leukemic targets in a sample were detectable which suggests that the level of detection achieved by limiting dilution analysis is essentially determined by the amount of DNA which it is practicable to study. We conclude that quantification of MRD at the end of induction provides the full range of prognostic information for marrow relapse but is complex; detection of monoclonality on day 35 is simple and has good positive predictive value; and quantification of MRD on day 14 merits further study. PCR-based methods for measurement of MRD levels should incorporate a correction for variation in DNA amplifiability.

Many patients with acute lymphoblastic leukemia (ALL) are not cured by current therapy because of the development of drug resistance. It is not clear when resistance develops during the growth of the leukemic clone and whether resistant cells are already present at diagnosis or develop later during treatment. Twenty-two uniformly treated children with ALL were studied throughout induction treatment. The size of the leukemic clone in blood and marrow was estimated by limiting dilution PCR analysis, using the rearranged immunoglobulin heavy chain gene as a molecular marker. The decline in the number of leukemic cells was biphasic in virtually all patients. For both marrow and blood, the logarithmic mean of the number of leukemic cells fell by approximately four orders of magnitude during the first 2 weeks, one order of magnitude during the third week, and not at all during the last two weeks of induction treatment. For marrow, the median of the fraction of leukemic cells in each patient that survived per week of treatment was 0.008 for the first 2 weeks, 0.12 for the third week, and 1.4 for the last 2 weeks; for blood, the corresponding figures were 0.003, 0.14, and 0.69, respectively. In individual patients, the results for marrow and blood showed good correlation. The biphasic decline of leukemic cell number suggests that most leukemic cells were sensitive to treatment and were rapidly killed, leaving behind a minor but substantial population of drug-resistant cells. The most likely explanation for this phenomenon is that these resistant cells were already present at diagnosis, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemic clone.

A rapid method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) was used to identify the haemochromatosis genotype in 112 individuals and the factor V genotype in 134 individuals. The results were compared with conventional methods based on restriction enzyme digestion of PCR products. The two methods agreed in 244 of the 246 individuals; for the other two individuals, sequencing showed that they had been incorrectly genotyped by the standard method but correctly genotyped by FRET. The simplicity, speed, and accuracy of real time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping the haemochromatosis and factor V genes.

The level of minimal residual disease (MRD) in marrow early in treatment strongly predicts outcome in childhood acute lymphoblastic leukaemia (ALL). Using PCR we studied 30 pairs of aspirates and trephines taken during induction treatment. Consensus PCR primers showed a monoclonal gene rearrangement in eight pairs, polyclonal rearrangement in 18 pairs and a monoclonal rearrangement only in the trephine in four pairs. MRD was quantified by leukaemia-specific primers in 22 pairs. There was a linear relationship between the logarithms of MRD levels of aspirate and trephine, with a residual variance which increased as the level of MRD fell. The mean level of MRD in the trephines was 4.1-fold greater than that in the aspirates, probably due to greater dilution of the aspirates with peripheral blood. The high variance at low levels of MRD could not be explained by measurement variation, which had an MRD-independent value of 0.42 log10 units, and was attributed to sampling variation due to patchiness of disease at low MRD levels. The magnitude of the variation was such that predictions of outcome could well be confounded for many patients. We suggest that MRD sampling variability could be minimized either by taking multiple marrow samples or by measuring MRD in peripheral blood.

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 x 10(-2) to < 9.4 x 10(-7) leukaemic cells/total cells, corresponding to 1.3 x 10(7) to < 2 x 10(3) leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10(-4), but in only two of 13 pairs in which the level in marrow was < 10(-5). Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.

The Philadelphia translocation is associated with a poor prognosis in adults and children with acute lymphoblastic leukemia, even though the majority of patients achieve remission. To test the hypothesis that the translocation leads to drug resistance in vivo, we studied 61 children and 20 adults with acute lymphoblastic leukemia and used the level of minimal residual disease at the end of induction as the measure of drug resistance in vivo. In children the presence of the translocation was associated with a significant increase in residual disease, indicating higher drug resistance in vivo; five of seven Philadelphia-positive children but only five of 54 Philadelphia-negative children had a minimal residual disease level >10(-3), a level which is associated with a high risk of relapse in childhood acute lymphoblastic leukemia of standard risk. By contrast, in adults, residual disease and hence drug resistance was already higher than in children, and the presence of the Philadelphia translocation in seven patients had no obvious additional effect. We conclude that the Philadelphia chromosome may increase resistance to drugs in vivo in children, but not detectably in adults.

Occasional cases of transient marrow hypoplasia in childhood evolve into acute leukaemia. We studied two children who presented with marrow hypoplasia following infection and who developed acute lymphoblastic leukaemia 2-3 months later. A simple polymerase-chain-reaction (PCR) test for monoclonality showed that immunoglobulin heavy-chain gene rearrangements of the same size were present at the times of both hypoplasia and leukaemia, and DNA sequencing confirmed identity of these rearrangements. PCR-based quantification, using patient-specific primers, showed in both patients that the leukaemic clone made up 20-25% of the marrow cells during hypoplasia. In contrast, four patients with typical aplastic anaemia showed only polyclonal B-cell populations in the marrow. We conclude that the leukaemic clone was already present at the time of hypoplasia in the two index patients and that in future a simple PCR test for monoclonality could be used to screen patients with marrow aplasia or hypoplasia for the presence of a monoclonal B-cell population. Patients with monoclonal populations could then be monitored carefully for subsequent development of leukaemia.

Sensitive quantification of minimal residual disease (MRD) using the polymerase chain reaction (PCR) is strongly predictive of outcome in childhood acute lymphoblastic leukemia (ALL), with MRD levels at the end of induction therapy of >10(-3) predicting a poor outcome. Methods for sensitive quantification are, however, complicated and time-consuming. Detection by PCR of monoclonal immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements is simple and can be used in routine laboratories but is non-quantitative and of lower but uncertain sensitivity. The aim of this study was to determine the value of detection of monoclonality in identification of different levels of MRD. We looked for monoclonality in 64 bone marrow aspirates which had been obtained from 31 patients with B lineage ALL at various times during induction therapy and for which levels of MRD had been determined by limiting dilution analysis using patient-specific PCR primers. Detection of monoclonality identified levels of MRD of > or =10(-3) during induction with a sensitivity of 78% and a specificity of 93%. The positive and negative predictive values were 0.86 and 0.88, respectively. The sensitivity of detection of a monoclonal IgH rearrangement was greater than that for the TCRgamma locus during induction as an IgH rearrangement was detected more often than a TCRgamma rearrangement in patients who had both IgH and TCRgamma rearrangement at diagnosis. Detection of monoclonality is therefore a simple and quick test applicable to the majority of patients with ALL and it may be useful in identifying high-risk patients at the end of induction and in identifying relapsing patients later during therapy.

Surface molecules present in low copy numbers can be detected with high-sensitivity fluorescence flow cytometry. Many cells previously thought not to express certain molecules on their surface can now be shown to have these molecules in very low copy numbers by high-sensitivity fluorescent cytometric methods. Detection of molecules by immunoperoxidase staining methods has not previously been compared with high-sensitivity flow cytometry techniques. Computerized video image analysis (VIA) is a method that allows measurement of area and density of the immunostain chromogen reaction product in a standardized fashion analogous to flow cytometry. In this study, we compared immunoperoxidase reaction products measured by VIA methods with high-sensitivity flow cytometric measurements for cells with 10,000 down to 50 antibody molecules bound to their surfaces. Detection of 100-200 surface molecules was possible with heavy metal-enhanced immunoperoxidase methods, whereas standard immunoperoxidase methods were not as sensitive. The sensitivity of the nickel-enhanced immunoperoxidase staining method was confirmed for detection of an epitope (Tac-IL2 receptor alpha-chain) present in low numbers on the surface of peripheral blood lymphocytes.

Methods to detect and quantify minimal residual disease (MRD) after chemotherapy for acute lymphoblastic leukaemia (ALL) could improve treatment by identifying patients who need more or less intensive therapy. We have used a clone-specific polymerase chain reaction to detect rearranged immunoglobulin heavy-chain gene from the leukaemic clone, and quantified the clone by limiting dilution analysis. MRD was successfully quantified, by extracting DNA from marrow slides, from 88 of 181 children with ALL, who had total leucocyte counts below 100 x 10(9)/L at presentation and were enrolled in two clinical trials, in 1980-84 and 1985-89. Leukaemia was detected in the first remission marrow of 38 patients, in amounts between 6.7 x 10(-2) and 9.9 x 10(-7) cells; 26 of these patients relapsed. Of 50 patients with no MRD detected, despite study of 522-496,000 genomes, only 6 relapsed. The association between MRD detection and outcome was significant for patients in each trial. In the first trial, patients relapsed at all levels of detected MRD, whereas in the later trial, in which treatment was more intensive and results were better, the extent of MRD was closely related to the probability of relapse (5 of 5 patients with > 10(-3) MRD, 4 of 10 with 10(-3) to 2 x 10(-5), 0 of 3 with levels below 2 x 10(-5), and 2 of 26 with no MRD detected). Early quantification of leukaemic cells after chemotherapy may be a successful strategy for predicting outcome and hence individualizing treatment in childhood ALL, because the results indicate both in-vivo drug sensitivity of the leukaemia and the number of leukaemic cells that remain to be killed by post-induction therapy.

Techniques based on the polymerase chain reaction (PCR) to detect rearrangement of the immunoglobulin or T-cell receptor genes can detect residual disease in leukemia and hence have the potential to improve prognosis and treatment. Such techniques may involve either detection of monoclonality, which is simple and quick but has limited sensitivity, or specific detection of the leukaemic clone, which is complex and time-consuming but has high sensitivity. The PCR was used to detect monoclonal rearrangements of the immunoglobulin heavy chain and/or T-cell receptor gamma chain genes in archival marrow specimens from 185 children with acute lymphoblastic leukemia who achieved remission during two consecutive Australasian trials of treatment. A monoclonal rearrangement was detected at diagnosis in 152 (84%) patients and in these patients detection of the same rearrangement in the remission marrow at the end of induction therapy was highly significantly correlated with outcome. There were nine patients in whom polymerase chain reaction showed only the monoclonal rearrangement and eight (89%) relapsed; there were 26 patients in whom PCR showed the leukemic monoclonal rearrangement as well as polyclonal rearrangements from normal lymphocytes and 12 (46%) relapsed; and there were 117 patients in whom only polyclonal rearrangements could be detected and only 29 (25%) relapsed. In patients who relapsed, remissions were shorter in those patients in whom the leukemic rearrangements had been detected in the remission marrow. Treatment in the later trial was more intensive than in the earlier trial, the results were better and the PCR detected the leukemic rearrangement in the remission marrow in significantly fewer patients. We conclude that detection by PCR of the monoclonal gene rearrangement of the leukemic clone in remission marrow indicates that numerous leukemic cells have survived induction therapy and is a good predictor of relapse. However, due to limited sensitivity of the test, failure to detect the leukemic clone by PCR is not a sufficiently good predictor of ultimate cure.

We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.

We report a simple and robust method for sensitive quantitation of leukaemic cells in acute lymphocytic leukaemia. Chain determining region 3 (CDR3) of the immunoglobulin heavy chain gene is a precise genetic marker for a patient's leukaemic clone. Quantitation of the leukaemic lymphocytes was achieved by use of the polymerase chain reaction to detect CDR3 at limiting dilution of DNA samples. Five patients were studied and high levels (1 in 1 to 1 in 10) of leukaemic cells were detected at diagnosis or relapse. Leukaemic cells were detected in remission marrows from three patients, at levels of 1 in 1000 to 1 in 100,000. All five patients showed a 1000 to 100,000-fold reduction in the levels of leukaemic cells after induction therapy. This technique should prove useful for monitoring therapy and may help predict outcome.

Receptors for lymphokines and growth factors are present on cell surfaces often at concentrations of 100-500 copies per cell. Although conventional immunofluorescence cannot detect such low levels, cell membrane antigens present at these concentrations can be detected using an optimally set up flow cytometer together with a three-layer immunofluorescence technique, consisting of monoclonal antibody reacted with selected batches of biotinylated horse anti-mouse immunoglobulin and phycoerythrin-streptavidin. In this study we purified and radiolabelled a number of monoclonal antibodies, determined the specific radioactivity by self-displacement analysis, and used the radiolabelled antibody in experiments where the number of molecules of antibody bound per cell and the fluorescence intensity were measured on the same sample. This permitted us to determine the sensitivity of the fluorescence procedure in molecules per cell, using several different antibody/target cell combinations. The method was consistently capable of detecting fewer than 100 molecules of antibody bound per cell.

Circulating low molecular weight (LMW) IgM was demonstrated in five of 38 patients with B cell lymphoproliferative disorders. These five patients all had malignant disease and could be subdivided into two groups. In the first group were three patients, each with an associated serum IgM paraprotein; two had Waldenström's macroglobulinemia, and one lymphocytic lymphoma. The two patients of the second group did not have IgM paraproteins; one had lymphocytic lymphoma and one chronic lymphocytic leukemia. Both these patients also had acquired C1 esterase inhibitor deficiency, a previously recognised association with circulating LMW IgM. None of the 16 patients with benign IgM macroglobulinemia had circulating LMW IgM. In those positive sera with LMW IgM this moiety contributed between 10.5% and 37.5% of the total IgM. There was no apparent association between LMW IgM and total IgM levels, kappa/lambda typing or the presence of Bence Jones proteinuria, but rheumatoid factor, immune complexes and cryoglobulins occurred in many of the sera which contained LMW IgM. Pokeweed mitogen stimulated peripheral blood mononuclear cells from two patients with circulating LMW IgM secreted considerable quantities of this moiety in vitro but this did not occur in two patients with benign IgM macroglobulinemia. We conclude that LMW IgM is found in the malignant but not the benign forms of B cell lymphoproliferative disorders and is frequently associated with other serological abnormalities. The basic abnormality causing defective IgM polymerisation in these disorders is obscure.

Radiologic contrast materials activate complement by both the classical and alternative pathways. This activation is time, dose, and temperature dependent and is able to proceed with equal facility in either the presence or absence of Ca++ or Mg++ chelating reagents (EGTA, EDTA). All the components examined (C1, C4, C2, Factor B, C3, and C5) were consumed during complement activation. Immune complexes are produced during interaction of serum with contrast materials. The activation of complement by contrast materials appears to be principally initiated by the activation of plasminogen to plasmin. Inhibition of plasminogen activators by epsilon-aminocaproic acid affects complement activation markedly.

A simple method is described for the preparation of highly purified IgA and IgM from small volumes of human serum. Enriched IgM and IgA fractions were prepared by precipitation with 7% (w/v) and 14% (w/v) polyethylene glycol respectively. This was followed by affinity chromatography and gel filtration. The final recovery of both IgA and IgM was approximately 30%. The purified preparations obtained were characterized by immunoelectrophoresis, double immunodiffusion, radial immunodiffusion, radioimmunoassay and gel filtration.

In this article we discuss our choices of monoclonal antibody separation methods for the applications which face us most frequently. We explain the rationale behind these choices, to help other users make their own choices. The review is intended to be brief and selective; references to detailed reviews are provided.

Sera from patients with rheumatoid arthritis inhibited the migration of human neutrophils in 63% (twenty-two out of thirty-five) of the cases tested. The inhibition was not due to a toxic effect of the serum as it was reversed by a chemotactic stimulus. There was a strong correlation between the degree of inhibition of neutrophil migration and the amount of immune complexes present in the sera as determined by the C1q binding activity. It is suggested that the inhibition of neutrophil migration is due to the presence of circulating immune complexes, and that the capacity of immune complexes to inhibit neutrophil migration in vitro may also contribute to the accumulation of neutrophils at sites of immune complex formation in vivo.

Human tonsil B cells include a subpopulation (30 per cent) of cells which lack LFA-1 antigen. Activation of tonsil B cells by culture with anti-IgM and interleukin-4 led to an increase in staining intensities and in the proportion of cells staining, until by 48 h the majority of B cells were positive. Culture of activated cells with low-molecular weight B cell growth factor, which induces a proportion of cells to proliferate, led to a minor further increase in expression of the LFA-1 antigen. Inclusion of a monoclonal antibody against the LFA-1 beta chain in culture did not affect either proliferation or immunoglobulin secretion. The expression of LFA-1 by B cells thus changes as B cells are activated, perhaps reflecting the changing requirements of B cells for interaction with other cells and tissue components. On the other hand, our results did not provide any support for the idea that the LFA-1 antigen is directly involved in the interaction of B cells with lymphokines which control proliferation and differentiation.

FMC7, a monoclonal antibody used extensively to characterize B cell leukaemias of differentiated phenotype (prolymphocytic, hairy cell, and similar leukaemias) was compared directly with antibodies of the CD22 cluster, which also react with B cells at a late stage in differentiation. Detailed comparison shows that the reaction spectrum, though similar, is not identical. Differences were particularly prominent in chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL). Binding studies show that the antibodies react with different antigenic determinants, and immunochemical studies show that they react with different molecules. The FMC7 antigen, not previously characterized, was shown to be a protein of apparent molecular weight 105,000, by immunoblotting after electrophoresis of membrane extracts.

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoites were fractionated into a water-soluble and a deoxycholate-soluble fraction. Polyclonal immune mouse serum was prepared by challenging chronically-infected mice with viable RH strain tachyzoites. The parasite fractions were labelled with 125I, and the radio-labelled antigens were precipitated by the immune mouse serum or a monoclonal anti-Toxoplasma antibody (FMC 20), that reacts only in the indirect hemagglutination antibody test. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates showed that the water-soluble fraction contained 10 antigenic polypeptides, and the deoxycholate-soluble fraction contained seven antigenic peptides. The FMC 20 reacted against a 98,000-dalton antigen that was present in the water-soluble fraction only.

Groups of mice were given an intraperitoneal injection of one of six monoclonal antibodies to Toxoplasma gondii, a mixture of equal amounts of five monoclonal antibodies to T. gondii, or the murine myeloma protein MOPC 21, and challenged with either a highly virulent or moderately virulent parasite strain. Two monoclonal antibodies (FMC 19 and FMC 22) conferred total protection against the moderately virulent challenge, with all mice surviving, whereas 90% of control mice died. FMC 19 and FMC 22 also conferred significant protection against the highly virulent challenge as indicated by a prolonged mean time to death (MTD) of immunized compared with control groups of mice. One monoclonal antibody (FMC 23) and the mixture of five antibodies gave significant protection against the moderately virulent challenge only. Passive immunization with dilutions of FMC 22 antibody indicated that the lowest serum titer needed to confer significant protection to mice against a moderately virulent Toxoplasma challenge was 1/640. Mice challenged with highly virulent tachyzoites that had been preincubated with FMC 22 had a significantly longer MTD than mice challenged with highly virulent tachyzoites that had been preincubated with MOPC 21 or phosphate buffered saline, pH 7.2 (PBS). Immunoprecipitation and autoradiography of radiolabeled tachyzoites confirmed that FMC 19 was directed against a 35,000 molecular weight (mol. wt.) antigen and FMC 22 was directed against a 14,000 mol. wt. fraction. The potential for use of single antigens as protective immunogens in preventing toxoplasmosis is raised.

We describe our 10 years experience in assaying over 15,000 clinical specimens for immune complexes (IC) using the C1q binding assay. Normal ranges were initially established using a large panel of blood donor sera and precision of the assay was optimized by inclusion of heat aggregated IgG (HAGG) as standards. Nevertheless some variability was observed due to variation in C1q binding from batch to batch and with aging of this reagent. In an empirically selected 2 year period involving over 3,000 clinical specimens, 25% had elevated concentrations of IC. Of these the majority were from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), other connective tissue disorders, infective endocarditis (IE), diffuse interstitial lung disease (DILD) and vasculitis (VASC). In RA, IE and VASC, significant correlations were observed between concentrations of IC and rheumatoid factor (RF) and the addition of a purified monoclonal RF to normal serum caused increased C1q binding. Longitudinal studies in RA and IE demonstrated a striking decline in IC in response to effective treatment. We conclude that the measurement of IC provides little additional useful diagnostic information in those diseases associated with high levels of RF but appears more useful in disorders such as SLE, IE and DILD in which RF is absent or present in low concentration. Sequential monitoring of IC in RA and IE reflects response to treatment.

The C1q binding assay and the nephelometric monoclonal rheumatoid factor assay were able to discriminate 79% and 57% respectively of rheumatoid arthritis (RA) patients from healthy blood donors. In addition these assays could distinguish those patients with active arthritis from those with inactive disease, and the C1q binding assay correlated significantly with other laboratory indices of the rheumatoid process, including the erythrocyte sedimentation rate, low molecular weight or 7S IgM, and the rheumatoid factor titre. High levels of C1q binding were also seen in rheumatoid vasculitis. Both assays gave higher mean values in synovial fluid compared with the corresponding serum, but it appeared from ultracentrifugal analysis and from a lack of a consistent correlation between these assays that each assay was measuring different forms of immunecomplex-like material which may be involved in the immunopathogenesis of this disease. The C1q binding assay is of some value in the laboratory assessment of rheumatoid arthritis and appears to offer greater advantages than the monoclonal rheumatoid factor assay, although the usefulness of this latter assay may be very dependent on the monoclonal rheumatoid factor used.

The unstimulated random migration and the serum-induced chemokinesis of neutrophils obtained from the peripheral blood of patients with rheumatoid arthritis (n = 19) was not different from those of controls (n = 20). However, neutrophils obtained from the joint fluid of rheumatoid patients (n = 10) demonstrated a reduced serum-induced chemokinesis which was correlated with the amount of immune complexes present in the synovial fluid. The chemotactic response of peripheral blood neutrophils from subjects with rheumatoid arthritis taking aspirin (n =11) was increased while that of those rheumatoid subjects not taking aspirin (n = 8) was the same as controls. It is concluded that although there is no impairment of the in vitro migratory capacities of peripheral blood neutrophils obtained from patients with rheumatoid arthritis, neutrophils obtained from synovial fluids exhibit a marked defect in chemokinesis which may be related to the ingestion of immune complexes within the joint space.

Laser nephelometric estimation of IgM in the eluate fractions following Sepharose 6B chromatography has enabled the calculation of the proportion of low molecular weight IgM (7S IgM) in normal and pathological sera. This figure has then been used to determine the absolute amount of 7S IgM. Twenty-seven of 36 (75%) patients with rheumatoid arthritis had 7S IgM with a mean value of 17 mg/100 ml (170 mg/l) (range 2.5-59 mg/100 ml). No sera from 10 healthy controls were found to contain 7S IgM. Patients with active rheumatoid arthritis had significantly more 7S IgM than those with inactive disease, but there was no significant difference between those patients with and without rheumatoid vasculitis. Significant correlations occurred between 7S IgM and the absolute IgM level (P < 0.01), the Rose-Waaler titre (P < 0.01), and the erythrocyte sedimentation rate (P = 0.01). However, there was no significant correlation with the age of the patient, the duration of the disease, or the level of circulating immune complexes as measured by the Clq binding assay. It is concluded that 7S IgM commonly occurs in rheumatoid arthritis, and it is postulated that a common immunological stimulus is responsible for the production of 7S IgM and rheumatoid factors, serological abnormalities that characterise this disease.

A method is described for the purification of monoclonal antibody from mouse ascitic fluid. The fluid is clarified and the lipid removed using silicon dioxide powder, before the immunoglobulin is precipitated using polyethylene glycol. The method provides IgM antibody in high yield and good purity. In the case of IgG antibodies the purity is 30-40% after PEG precipitation and the yield is high. The enriched IgG is adequate for many purposes and is suitable for further purification on an ion exchange column.

Suppression of growth of a number of different murine tumours, injected subcutaneously or into the foot pad, was induced by prior intraperitoneal or intravenous immunisation with Salmonella enteritidis 11RX. The effect of immunisation on tumour growth was relatively minor, but could be enhanced by addition of 11RX antigen preparations to the suspensions of tumour cells used for challenge. The significance of these findings is discussed.

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with [125I]-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.