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Periphyton standing crop is often measured as chlorophyllabut there is increasing interest in using visual assessments of periphyton cover. Visual methods are rapid and require no sample analysis, but can be regarded as subjective with high inter-operator variability. We investigated variability in periphyton standing crop across operators, rivers and time, as determined visually and from chlorophyllameasurements. We found that visual assessments (comprising percentage cover estimates of up to eight periphyton categories) distinguished sites and occasions as effectively as chlorophylla. Furthermore, an estimate of chlorophyllacould be derived from the visual assessments. Because our surveys were conducted in only three rivers, general applicability of the derivation of estimated chlorophyllafrom the visual assessments warrants further investigation. The current recommendation of 20 views was sufficient for realistic visual assessments of average cover. Overall, our results indicated that inter-operator variability in visual assessments need not be a major concern, given adequate training. [ABSTRACT FROM PUBLISHER]

Monitoring for adaptive management (AM) involves collection of data with the aim of reducing uncertainty about links between human pressures (e.g. water abstraction from rivers), consequent stressors (e.g. low river flows) and environmental state (e.g. biodiversity). 'Surveillance monitoring' involves documenting trends in state, without the aim of understanding relationships between state, stressors, and pressures. Critics have highlighted that surveillance monitoring dominates monitoring investments but is not supporting AM. Decision-makers continue to be disappointed by monitoring data that are unsuitable for AM, yet designers of monitoring programs tend to make decisions that reinforce rather than reimagine the status quo. We argue that a structured, collaborative approach to objective-setting is required to break the status quo. We collaborated with regional management authorities to develop monitoring objectives and implementation strategies to support AM of New Zealand's rivers. Our collaborative approach discouraged 'failure fearing' and encouraged reimagining 'what could be' as opposed to 'what is.' Seventeen monitoring objectives were identified based on the AM requirements of national policy and regional authorities. Several objectives-particularly those arising from national policy-stretch the limits of what environmental science can currently provide. There were also strong trade-offs among objectives. We offer practical implementation strategies for overcoming the technical challenges of, and reducing trade-offs among, monitoring objectives. These strategies point to a monitoring program that contrasts strongly with one aimed at surveillance. Monitoring for AM is more complex than monitoring for surveillance, so strong leadership is required for successful implementation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Habitat modification and introduced mammalian predators are linked to global species extinctions and declines, but their relative influences can be uncertain, often making conservation management difficult. Using landscape-scale models, we quantified the relative impacts of habitat modification and mammalian predation on the range contraction of a threatened New Zealand riverine duck. We combined 38 years of whio (Hymenolaimus malacorhynchos) observations with national-scale environmental data to predict relative likelihood of occurrence (RLO) under two scenarios using bootstrapped boosted regression trees (BRT). Our models used training data from contemporary environments to predict the potential contemporary whio distribution across New Zealand riverscapes in the absence of introduced mammalian predators. Then, using estimates of environments prior to human arrival, we used the same models to hindcast potential pre-human whio distribution prior to widespread land clearance. Comparing RLO differences between potential pre-human, potential contemporary and observed contemporary distributions allowed us to assess the relative impacts of the two main drivers of decline; habitat modification and mammalian predation. Whio have undergone widespread catastrophic declines most likely linked to mammalian predation, with smaller declines due to habitat modification (range contractions of 95% and 37%, respectively). We also identified areas of potential contemporary habitat outside their current range that would be suitable for whio conservation if mammalian predator control could be implemented. Our approach presents a practical technique for estimating the relative importance of global change drivers in species declines and extinctions, as well as providing valuable information to improve conservation planning., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Whitehead et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A method for objectively estimating reference states for suspended fine sediment (turbidity) is presented. To be fit for water policy development and implementation the method had to satisfy four requirements: (1) the method must not be dependent on data from minimally-disturbed reference sites; (2) the method must facilitate characterization of reference states throughout heterogeneous river networks, given patchy data; (3) the classification of reference states must be relevant and legitimate to end-users; (4) the method should provide several classifications of reference states at different spatial resolutions allowing selection of the resolution yielding the most parsimonious classification of reference states throughout the network. Implementing the method involves two stages: (1) Development of a river classification based on sediment supply and retention regimes (defining 'turbidity classes') at multiple spatial resolutions. (2) At each resolution, for each turbidity class, estimation of a reference state based on relationships between turbidity and anthropogenic stressors, then objective selection of the resolution yielding the most parsimonious classification of reference states throughout the network. Implementing the method requires a river network GIS and turbidity data within classes, preferably from monitoring sites spanning the domains of the anthropogenic stressor variables used for model-based estimation of reference states.•A method is presented for estimating reference states for suspended fine sediment (turbidity) throughout spatially heterogeneous river networks.•Development of the method was guided by the requirements of policy analysts during reform of water policy in New Zealand.•The method presented was used to develop fine sediment regulatory thresholds of national water policy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

Water abstraction from rivers and aquifers has considerable potential to alter flow regimes, thereby influencing the physical, chemical, and ecological well-being of freshwater ecosystems. The economic and social benefits of abstraction need to be balanced against its potentially deleterious consequences for hydrologically-driven ecological functions, ecosystem services, cultural values, and recreation. In New Zealand, recent legislation states that limits for the use of water resources should be set for all waterbodies to manage the potential cumulative impacts of abstraction and reduce allocation of the hydrological resource in over-allocated catchments. These limits must comprise at least a predefined minimum flow (the flow at which all abstraction must cease) and a total allocation (the maximum rate of abstraction summed across upstream abstractions). Over-allocation occurs when the sum of all upstream abstractions exceeds the total allocation. A national database describing consents to abstract water was collated. A replicable, transferable, and objective method was applied to calculate total allocation at the national, catchment, and reach scales across the entire country. Total allocation for each catchment was expressed by mapping Weighted Allocation Impact; an index that integrates magnitude and spread of water resource allocation across entire catchments. Results show that existing consents have caused over-allocation in several catchments, prompting questions about how to reduce abstraction in these locations.

Despite many studies highlighting the widespread occurrence and effects of resource movement between ecosystems, comparatively little is known about how anthropogenic alterations to ecosystems affect the strength, direction and importance of such fluxes. Hydrological regime and riparian land use cause well-documented changes in riverine larval invertebrate communities. Using a dataset from 66 sites collected over 20 years, we showed that such effects led to spatial and temporal differences in the density and type of larvae with winged adults within a river reach, altering the size and composition of the source pool from which adult aquatic insects can emerge. Mean annual larval densities varied 33-fold and the temporal range varied more than 20-fold between sites, associated with the hydrological regime and land cover and antecedent high and low flows, respectively. Densities of larvae with winged adults were greater in sites that had more algal coverage, agricultural land use, seasonally predictable flow regimes and faster water velocities. More interestingly, by influencing larval communities, riparian land use and the magnitude and frequency of high and low flows affected the size structure, dispersal ability and longevity of adults available to emerge from river reaches, potentially influencing the spatial extent and type of terrestrial consumers supported by aquatic prey. This suggests that anthropogenic alterations to land use or river flows will have both spatial and temporal effects on the flux and potential availability of adult aquatic insects to terrestrial consumers in many rivers.

The timing of Anguilla spp. glass eel recruitment into the Waikato River, North Island, New Zealand, was studied over a 2 year period (2004-2005). While glass eels of both the shortfin eel Anguilla australis and the endemic longfin eel Anguilla dieffenbachii were caught, the former comprised >97% of the species composition. There was a positive correlation of glass eel migrations with spring tides, with peak migration periods typically occurring within a few hours of the peak of high tide, and between 2 and 4 days after the day of spring tide. Both water temperature and discharge had significant inverse relationships with glass eel catches, with temperature explaining >30% of the variance in catch periodicity. Comparison of catch data 30 years apart showed that main migration periods appear to occur several weeks earlier today than previously. Reduced catch per unit effort and duration of runs from recent years' sampling (compared with the 1970s) indicate that a reduction in recruitment may also have occurred during this period, something recorded in other temperate species of Anguilla.

A bioenergetic model of marine phase, wild Atlantic salmon was constructed to investigate the potential effects on post-smolt growth of predicted changes in oceanic conditions. Short-term estimates of growth in weight were similar to measurements in captivity and simulated growth varied with water temperature and swimming speed as expected. Longer-term estimates of growth in length were less than that achieved by wild salmon, particularly with constant swimming assumed. The model was sensitive to parameters relating to maximum daily food consumption, respiration and the relationships between body energy content, length and weight. Some of the sensitive parameters were based on substantive information on Atlantic salmon and their realistic ranges are likely to be much narrower than those tested. However, other parameter values were based on scant data, farmed Atlantic salmon or other salmonid species, and are therefore less certain and indicate where future empirical research should be focussed.

Background: Immune-mediated hemolysis is a well-recognized complication of transplantation, but few reports have drawn together the different mechanisms that could be involved., Study Design and Methods: The clinical and laboratory records of three patients are used to illustrate different types and complexities of posttransplant immune-mediated RBC destruction., Results: Patient 1 received bone marrow from an HLA-matched, unrelated donor. At 7 months after transplant, his Hb level fell to 50 g per L. The serum contained warm autoantibodies, and the DAT was strongly positive for IgG, IgM, and C3d; an eluate yielded IgG and IgM autoantibodies. Autoimmune hemolytic anemia was diagnosed. Patient 2, blood group A, experienced severe hemolysis 14 days after receiving a lung from a group O donor. The DAT was positive for IgG. Serum and RBC eluate contained anti-A produced by immunocompetent B cells in the transplanted lung-this was the passenger lymphocyte syndrome. Patient 3 experienced posttransplant hemolysis caused by two different immune mechanisms. Originally group A, D- with anti-C, -D, -E, she received a peripheral blood progenitor cell (PBPC) transplant from her HLA-identical group A, D+ son. Six months later, chimerism was evident; the remaining recipient marrow was still producing antibodies that destroyed D+ RBCs made by the transplant. Later, autoimmune hemolytic anemia also developed; the DAT became positive for IgG, and warm autoantibodies were eluted from D- RBCs., Conclusion: An understanding of the causes and circumstances under which posttransplant immune hemolysis arises is required for proper management. As more patients become long-term survivors of unrelated bone marrow and/or PBPC transplants, chimerism and complex serologic problems will become more common.

Aim: To examine the role of IgG2 red cell autoantibodies in autoimmune haemolysis., Methods: Study of immunohaematology case records., Results: Six patients had only IgG2 autoantibodies detected by direct antiglobulin testing and in red cell eluates; two individuals, whose red cells were also coated with complement, suffered from autoimmune haemolytic anaemia., Conclusions: IgG2 antibodies are found alone in <1% of patients with warm autoantibodies and even more rarely cause red cell destruction. Several factors are important for inducing haemolysis. They include allele differences in the FCRIIA genes encoding for the FcyRII receptors--an allele with high affinity for IgG2 is needed for haemolysis. Topography of red cell antigens may also be significant; IgG2 is a relatively inflexible molecule and access of effector cell Fc receptors to the recognition sites on the IgG2 might be impossible unless the antigens are on, or proud to, the red cell surface. On rare occasions, IgG2 activates complement (as in our patients with active haemolysis); the synergistic effect between red cell bound immunoglobulins and C3 in causing haemolysis is well recognised.

Two hundred and twenty-one patients with cold haemagglutinins of thermal amplitude > or = 30 degrees C (considered to be a reasonable indicator of clinical significance) were classified by in vitro haemolysin activity into three groups. Group 1 contained 116 individuals in whom haemolysins were never detected; the 74 patients in Group 2 had monophasic haemolysins alone; whereas both monophasic and biphasic haemolysins were detected in the 31 Group 3 patients. There was a significantly higher proportion of patients in Groups 2 and 3 with haptoglobin levels < 0.1 g/l compared with Groups 1 and 2, respectively (P < 0.005 and P < 0.001). Direct antiglobulin test results showed that the autoimmune response became more complex and IgM predominant through Groups 1-3, resulting in an increasing ability to activate complement which was reflected in increasing haemolysin activity and number of patients with active haemolysis. The 31 patients in Group 3 were mostly elderly (median age 71 years at presentation) and the majority had chronic cold haemagglutinin disease (CHAD), several in association with lymphoid neoplasms or carcinomas; only four had acute CHAD. The natural history of idiopathic chronic CHAD was of mild, well compensated haemolysis, punctuated by severe acute episodes necessitating intensive therapy. The condition often remained active for long periods and did not appear to affect natural lifespan. In some cases, no treatment (or just warmth) was needed; in others continuous or intermittent prednisolone and/or chlorambucil were effective; yet others required a greater variety and more intense therapy, or treatment of associated conditions. Blood transfusion support was frequently required when haemolysis was severe.

Hairy cell leukaemia (HCL) is a rare lymphoproliferative disorder associated with pancytopenia, splenomegaly and the presence of typical hairy B lymphocytes in the bone marrow and/or peripheral blood. The most significant complication relates to opportunistic infections that arise as a consequence of neutropenia and monocytopenia. HCL is occasionally associated with systemic autoimmune disorders including polyarteritis nodosa and rheumatoid disease. Secondary autoimmune haemolytic anaemia (AIHA) appears to be rare. We report on two cases of HCL complicated by fatal cold anti-i AIHA. Fulminant haemolysis causing death is rare in cold AIHA and only a few individual cases have been reported, none having anti-i specificity.

Paroxysmal cold haemoglobinuria is an autoimmune haemolytic anaemia characterized by a biphasic polyclonal IgG autoantibody, the Donath-Landsteiner (D-L) antibody. Although classically described in association with chronic syphilis, it is most commonly seen after acute viral infections in children. We describe a case of high-grade B-cell non-Hodgkin's lymphoma which presented with paroxysmal cold haemoglobinuria. The lymphoma responded promptly to combination chemotherapy whilst, at the same time, the haemolytic process rapidly resolved. Subsequent investigations showed that the D-L antibody originated from the lymphoma cells.

52 patients (30 male, 22 female) with paroxysmal cold haemoglobinuria (PCH) were identified by critically examining the records of all cases with Donath-Landsteiner antibodies seen over a 37 year period. Although ages ranged from 1-82 years, PCH was much commoner in young children; the median age at presentation was 5 and the peak incidence, 0.4 per year per 100,000 of the population at risk, was in the 4 years and under group. 44 patients had acute transient PCH, 3 chronic non-syphilitic PCH and 1 chronic syphilitic PCH; 4, in whom the positive Donath-Landsteiner tests were incidental findings, could not be classified. Acute PCH typically presented in young children as sudden onset of malaise, haemoglobinuria and pallor, often associated with mild jaundice - all 30 patients who were 13 or younger had this type. There was usually a history of a recent viral type infection, most commonly of the upper respiratory tract. The occurrence of acute PCH had no obvious relation to exposure to cold. Dramatic and rapid falls in haemoglobin level were common, often accompanied initially by relative or absolute reticulocytopenia. The illness was severe, but the prognosis was generally good and the majority of patients had completely recovered within one month, some requiring no treatment. In approximately 68% of patients, blood transfusion was needed; the P blood group was not taken into account, but the patients were kept warm throughout. Steroids (usually prednisolone) were given in many cases; but since there was no evidence to support their benefit, it was recommended that they were stopped as soon as the diagnosis was confirmed. Chronic non-syphilitic PCH was much rarer; the patients had a characteristic presentation of severe systemic symptoms (paroxysms) and haemoglobinuria brought on by exposure to cold. The clinical manifestations varied in intensity between individuals; at the extreme, severe debility was experienced over many years. Warmth and avoidance of cold were an effective treatment, though in a severely afflicted patient, an attack could be precipitated by relatively little exposure to cold. With chronic syphilitic PCH there was the added need to treat the specific infection. The direct antiglobulin test was almost always positive (50 out of 51 cases tested), with C3d coating the red cells. The Donath-Landsteiner antibodies were of IgG class, but this was rarely demonstrated unless direct antiglobulin tests were carried out at 4 degrees C. The antibodies showed the classical anti-P specificity in 27 of the 30 patients tested; other specificities were unusual. Although acting much better as haemolysins, Donath-Landsteiner antibodies could also cause weak agglutination at room temperature. This was paralleled in vivo by predominantly intravascular haemolysis with an extravascular component. Diagnosis was usually easy when PCH was suspected, though in some patients Donath-Landsteiner tests did not become positive until more sensitive techniques involving papainised red cells or two-stage procedures were employed. Of particular interest was the association in some cases with lymphoproliferative disorders, collagen disease, myelodysplastic syndrome, delayed haemolytic transfusion reaction and other types of autoimmune haemolytic anaemia. In one patient, an aetiological relationship was confirmed by a saline extract of lymphoma tissue behaving as a strong Donath-Landsteiner antibody with the same anti-P specificity as the serum. 4 patients had other types of autoimmune haemolysis concomitant with, but distinct from, the PCH; in 3 cases this was cold haemagglutinin disease and in one it was warm type autoimmune haemolytic anaemia.

Four patients with paroxysmal cold hemoglobinuria (PCH) illustrate some of the difficulties in making the diagnosis. A 46-year-old male presented with anemia, a weakly positive direct antiglobulin test (DAT) with anti-IgG, a haptoglobin < 0.1 g/L, and a cold autoagglutinin showing anti-P specificity. A 9-year-old female had a 4-day coryzal illness, a 20 g/L fall in hemoglobin over 24 hours, and a haptoglobin < 0.1 g/L; the DAT was positive with anti-C3d. A 3-year-old female was referred following a rapid drop in hemoglobin of 30 g/L; the DAT was positive with anti-C3d. A 17-month-old female, unwell for 2 weeks, had a hemoglobin of 41 g/L; the DAT was strongly positive with anti-C3d and weakly positive with anti-IgG and -C3c. In all patients, PCH was confirmed by positive indirect Donath- Landsteiner tests, and the autoantibodies demonstrated P specificity. In two patients, the test was strongly positive; in the third patient, it was only positive using papainized red cells; and in the fourth patient, a two-stage papainized procedure was needed before a positive result was obtained. PCH must always be considered in a child with a rapid drop in hemoglobin, even if initial tests are negative.

Warm IgM autoantibodies occur in association with IgG-class and/or IgA-class immunoglobulins in approximately 30 percent of patients with warm-type autoimmune hemolysis. They may be classified as agglutinins or hemolysins, which may be incomplete or complete, depending on in vitro serology; they almost always bind complement. Autoimmune hemolytic anemia solely due to warm IgM autoantibodies is exceedingly rare. We report two cases of the incomplete agglutinin type. The autoantibodies were confirmed as IgM by their ability to rebind to normal red blood cells (RBCs) after elution; the absence of small increases in RBC-bound IgG and IgA was shown by a sensitive enzyme-linked antiglobulin test. Patient 1 was a 64-year-old female with non-Hodgkin's lymphoma, with a hemoglobin of 50 g/L and haptoglobin of < 0.1 g/L. Direct antiglobulin tests were positive for IgM, C3d, and C3c; only IgM was present in an eluate. The serum contained a weak autoantibody at 37 degrees C and tests for hemolysins were negative. The patient suffered chronic hemolysis and required intensive treatment, including splenectomy. Patient 2 was a 65-year-old female; the hemoglobin was 78 g/L and the haptoglobin was < 0.1 g/L. Direct antiglobulin tests were positive for IgM and C3d; an eluate contained only IgM. No free autoantibody was present in the serum and tests for hemolysins were negative. Two serious infections occurred and the hemolysis remained chronic, requiring continuous treatment during the 4 months she was followed.

A 37-year-old male presented with severe anemia, mild jaundice, and hemoglobinuria during his second course of diclofenac for gout. The peripheral blood showed microspherocytes and nucleated red blood cells (RBCs). The reticulocyte count was 21 percent and haptoglobin was < 0.1 g/L. A presumptive diagnosis of diclofenac-induced immune hemolysis was made and blood, urine, and drug samples were referred for investigation. Direct antiglobulin testing showed the RBCs to be coated with IgG1, IgG4, and C3d, but an eluate only yielded weakly reacting IgG antibodies. In tests for drug-dependent antibodies, group O, R1R2 red cells were incubated with the patient's serum that had been mixed with either urine (which contained diclofenac metabolites) or diclofenac solution and then tested by an antiglobulin method. Strongly positive reactions with anti-IgG occurred in the tests using urine but only weak reactions in those tests employing diclofenac solution. All controls gave negative results. These findings support the role of diclofenac in causing hemolysis and the importance of employing urine as a source of drug metabolites. The findings also showed that an immune complex mechanism predominated and that the eluted IgG (detectable independently of the presence of the drug or its metabolites) confirmed a minor autoimmune component. Diclofenac was stopped and treatment with prednisolone and folic acid instituted; this resulted in complete recovery.

Complement has a complex role in immune mediated red blood cell (RBC) destruction and usually induces extravascular hemolysis of C3b-coated RBCs by erythrophagocytosis and by acting synergistically with cell-bound immunoglobulins. A sensitive two-stage enzyme-linked direct antiglobulin test (ELDAT) was developed and used to measure RBC-bound C3b and C3d in 120 healthy adult individuals and in 60 patients suffering from a variety of conditions, including warm- and cold-type autoimmune hemolytic anemia, neoplasia, and collagen diseases. The results were compared with those of standard agglutination tests employing polyclonal and monoclonal antiglobulin reagents. Small amounts of C3b and C3d were detected on RBCs of the healthy individuals only by the ELDAT and probably reflected the continuing low-grade activation of complement necessary for the maintenance of homeostasis of a variety of physiological systems. The quantity did not vary with age or gender. In the patients, increased amounts of RBC-bound C3b and C3d were relatively common and probably resulted from autoantibody activity, immune-complexes, and nonspecific adsorption. There was no association between positive ELDAT results and the presence of active hemolysis. The ELDAT was far more sensitive than the agglutination tests for detecting RBC-bound C3b and also for C3d if the monoclonal reagent was employed.

An 80-year-old female presented with melena and anemia due to bleeding from a benign gastric ulcer. Her blood group was O, D+. The serum contained anti-B and a weak anti-A (titer 2 at 18 degrees C). She was inadvertently transfused with approximately 3.5 units of group A red blood cells with no initial ill effects. One week later, the anti- A titer increased to 8 and the direct antiglobulin test (DAT) was weakly positive (IgG and C3d). The next day, intravascular hemolysis became evident. The DAT was still weakly positive and the serum contained a weak cold autoagglutinin, which did not correlate with the severity of the hemolysis. A Donath-Landsteiner test was performed and found to be strongly positive. The antibody showed P specificity, confirming a diagnosis of paroxysmal cold hemoglobinuria (PCH). Exchange transfusion was followed by rapid recovery even though the Donath-Landsteiner test remained positive for at least a month. The patient was well when last seen 11 months after presentation. It was thought that the original low titer of anti-A reflected compromised immune homeostasis in an elderly patient and that stimulation by incompatible blood in those circumstances resulted in a delayed hemolytic transfusion reaction that triggered, exacerbated, or was accompanied by an autoimmune response manifesting as PCH.

Autoimmune hemolytic anemia, in which the direct antiglobulin test (DAT) is negative or weakly positive, may be due to low-affinity autoantibodies. We describe two such cases. An 8-year-old male presented with weight loss, jaundice, a hemoglobin of 33 g/L, reticulocytes of 306 x 10(9)/L, and haptoglobin of < 0.1 g/L. The DAT was negative. After washing the red blood cells (RBCs) with saline at 4 degrees C, the DAT was positive for IgG and an eluate contained an IgG3 autoantibody, thus confirming a diagnosis of autoimmune hemolytic anemia (AIHA). Red cell transfusions and corticosteroids were given with eventual complete recovery. A 73-year-old male had a hemoglobin of 89 g/L and haptoglobin of < 0.1 g/L. The DAT was initially negative but was positive for IgG using cold-washed (4 degrees C) RBCs; it was also positive with unwashed cells in the DiaMed system and an eluate contained IgG1 autoantibody. AIHA was therefore confirmed and prednisolone started but continued hemolysis necessitated splenectomy before full recovery occurred. Although RBCs may be strongly sensitized with low-affinity autoantibodies in vivo, the IgG is easily removed when RBCs are washed at room temperature for a DAT. The DiaMed system that uses unwashed RBCs overcomes this problem, but cold washing the RBCs at 4 degrees C must be used when preparing eluates.

Factors governing the functional activity of red cell autoantibodies are poorly defined. Here we report the presence of qualitative differences in the glycosylation of IgG autoantibodies which affect in vitro interactions with Fc gamma RIII. The following antibodies were affinity-purified by adsorption and elution from normal red cells: IgG eluted from the red cells of 27 haemolysing or non-haemolysing patients, anti-D in sera from 11 pregnant women, and IgG1 and IgG3 human monoclonal anti-D. Monoclonal antibodies with differing levels of agalactosyl IgG were produced by culturing cell lines at high or low cell density. The % IgG with oligosaccharides lacking terminal galactose residues (agalactosyl IgG) of antibodies was designated as low, medium or high according to their reactivity with a monoclonal antibody to terminal N-acetylglucosamine. Fc gamma RIII-mediated functional activity was assessed by measuring the K-cell-mediated lysis of red cells in eluates diluted to achieve comparable levels of red cells sensitization. All eluates containing allo-anti-D were lytic (range 74-100%). In contrast, lysis by autoantibodies varied from 0 to 100%; 11/13 eluates from red cells of haemolysing patients promoted > 5% lysis compared to 2/7 eluates from red cells of non-haemolysing patients (P < 0.02). The ability of autoantibodies to promote K-cell-mediated red cell lysis correlated inversely with their level of agalactosyl IgG (r = -0.56, P < 0.01, n = 23). Further, monoclonal anti-D antibodies with very low levels of agalactosyl IgG were comparatively more lytic than the same antibodies containing more agalactosyl IgG. Analysis of the ratio of kappa:lambda light chains suggested that autoantibodies from 6/19 patients were monoclonal or oligoclonal in nature. The data indicate that IgG red cell autoantibodies from different patients are functionally heterogenous, and that this may be due, at least in part, to qualitative differences in the Fc region glycosylation reflected by differences in the proportion of agalactosyl IgG. This heterogeneity is consistent with the clonally-restricted nature of the autoantibodies in some patients.

In order to study red cell autoantibodies with ABO specificity, the records of 4668 patients seen over 32 years were examined. Five group A patients had high thermal amplitude cold agglutinins showing anti-A, -A1 or -AI specificity; none had ever received blood or blood products. They presented as chronic cold haemagglutinin disease, autoimmune haemolytic anaemia or were discovered during investigation of pregnancy, carcinomatosis or anaemia. In vivo haemolysis was evident in all but the antenatal case; only the patient with autoimmune haemolytic anaemia required treatment, responding well to steroids. ABO-specific autoantibodies thus appear similar in reactions and clinical manifestations to autoantibodies in general.

Aims: To examine a large series of patients in whom both red cell autoantibodies and carcinoma are present; and to determine whether this rare occurrence is a true association or a chance event., Methods: The laboratory records of 160 patients (76 men, 84 women; mean age 68 years) with erythrocyte autoantibodies and confirmed carcinoma were examined for site of tumour origin and clinical and immunohematological findings. To test whether the concomitant occurrence of autoantibodies and carcinoma was fortuitous, data on total population and carcinoma incidence were included in a chi 2 analysis., Results: The association was significant (chi 2 = 97.5, p < 0.0005); erythrocyte autoantibodies and carcinoma were found together 12-13 times more often than expected from their relative frequencies. Autoantibodies occurred with a variety of carcinomas, particularly those of breast, lung, colon, rectum, and prostate; this largely reflected tumour incidence. Adenocarcinoma, squamous, anaplastic, and transitional cell types were all represented. Warm, cold, and mixed autoantibodies were not associated with particular tumour sites or histology. Eighty six patients had haemolysis of varying severity, 37 had metastatic disease, and 28 died within a few months of presentation., Conclusions: The presence of erythrocyte autoantibodies and carcinoma in the same patient is a true association and probably reflects a fundamental disturbance in immune homeostasis. It tends to occur with a large tumour mass and metastatic disease, and generally indicates a poor prognosis.

In some patients with autoimmune hemolytic anemia or bemolytic disease of the newborn, the red cells are so heavily coated with immunoglobulin that phenotyping cannot be carried out unless the antibody is removed without destroying the red cell antigens. Studies were performed initially to determine the optimum conditions for removal of immunoglobulin from red blood cells (RBCs) using chloroquine. Group O, R1r RBCs were coated with serial dilutions of anti-D; aliquots were incubated in chloroquine diphosphate (CDP) solution (200 g/L, pH 5.0) at 18 degrees C, 25 degrees C, 30 degrees C, and 37 degrees C, and test- ed by the antiglobulin technique at intervals of 30 minutes for up to 2 hours, the results being expressed as titration scores. These studies showed that antibody removal was much more efficient at 30 degrees C and 37 degrees C than at 18 degrees C or 25 degrees C. A further series of experiments was then carried out to assess the effect of chloroquine on red cell antigenicity. A 5 percent suspension of RBCs heterozygous for c, D, and E antigens, and for Kell, Duffy, and Kidd antigens, was incubated at 30 degrees C and at 37 degrees C in chloroquine solution. Aliquots were removed at 30-minute intervals for up to 2 hours, tested with serial dilutions of the appropriate antisera, and titration scores obtained. The antigens were well preserved after two hours of chloroquine treatment at 30 degrees C. However, when treatment was performed at 37 degrees C, aitigenicity had markedly deteriorated by 60 minutes, although the antigens were still reasonably well preserved (except for Jkb) at 30 minutes. It is therefore recommended that treatment with chloroquine solution prior to typing RBCs heavily coated with antibody should be carried out for 90 minutes at 30 degrees C or for not more than 30 minutes at 37 degrees C.

Monocyte monolayer assays (MMA) were performed using red cells (RBCs) from 159 patients with a positive direct antiglobulin test and monocytes from healthy individuals. The results of the MMA reflected, to a great extent, in vivo haemolysis; 56/63 patients with a positive result, but only 10/96 with a negative one, had evidence of in vivo RBC destruction. Positive MMAs were observed with autoantibodies of all types: warm (42 cases), cold (14 cases), mixed (seven cases), but never in patients with only C3d on their RBCs and no detectable autoantibodies. With warm autoantibodies, the MMA results depended, in part, on the level of IgG sensitization; additional C3d coating acted synergistically to lower the amount of IgG necessary for monocyte interaction to occur. The presence of IgG3 as well as IgG1 on the RBCs was usually associated with a high MMA result; most of these cases, however, also exhibited larger amounts of cell-bound IgG. When the MMA results were analysed in respect of the various types of autoantibodies, differences in the phagocytosis:adherence ratio were observed; in patients with warm autoantibodies, there was a preponderance of phagocytosis, whereas in those with cold and mixed types, adherent RBCs predominated or were found alone.

The subclass pattern of red cell bound IgG autoantibody was studied in 304 patients on 426 occasions. Subclass interrelationships with time, other cell bound immunoglobulins (IgM, IgA), amount of bound IgG and serum haptoglobin levels were investigated using population proportions; because of the multiple statistical tests, P less than 0.01 was required for significance. IgG1 was most common, being found in 98% of cases and as the sole subclass in 64%; multiple subclasses occurred in 34.5%. The IgG subclass pattern possibly changed with time (P less than 0.02, greater than 0.01), populations being compared at 6 and 12 months. There was a highly significant and important correlation between multiple IgG subclasses and multiple immunoglobulin coating; in our further studies, this necessitated the use of samples where only cell bound IgG was increased. Multiple IgG subclasses strongly correlated with larger amounts of cell bound IgG, groups with greater than 2 and less than 1 OD units by the enzyme-linked direct antiglobulin test being compared (approximately greater than 800 and less than 400 molecules IgG per red cell respectively). Multiple subclasses (P less than 0.05, greater than 0.01), but not IgG3, were possibly associated with low haptoglobin levels; significance was reached, however, if the multiple immunoglobulin effect was ignored. IgG subclass interrelationships are clearly complex and require strictly defined populations for their study.

Immunohaematological investigations were carried out in 46 patients with erythrocyte autoantibodies associated with myelodysplastic syndromes. Eight patients had refractory anaemia, 17 refractory anaemia with ring sideroblasts, 11 refractory anaemia with excess of blasts, four chronic myelomonocytic leukaemia, five refractory anaemia with excess of blasts in transformation and one could not be classified. Standard agglutination direct antiglobulin tests showed that the red cells were most often coated with IgG and C3d, though increased amounts of IgM or IgA were also found in 15 of 35 cases (43%) when the more sensitive enzyme linked method was used. The IgG antibodies were predominantly of IgG1 subclass. Clinically important autoimmune haemolysis occurred in 15 patients, and was of "warm", "cold," and "mixed" types in seven, four, and four cases, respectively: it is important to recognise its presence in view of the good response to treatment. The increased incidence of erythrocyte autoantibodies in myelodysplastic syndromes is thought to be one manifestation of disturbed immune homeostasis.

Enzyme linked and radioimmune direct antiglobulin tests (DAGTs) were used to assess red cell bound IgG, IgA, and IgM in 585 patients referred to an immunohaematology reference centre. One hundred and fifty eight patients with less than or equal to 200 mol IgG and small amounts of IgA and IgM coating their red cells were studied in detail. The presence of autoimmune haemolysis was determined from the clinical, haematological, and biochemical findings; it occurred in at least 25% of the 158 patients, the degree varying widely. There was a highly significant association between small increases in cell bound immunoglobulins and the presence of autoimmune haemolysis. Immunoglobulins of IgG, IgA, and IgM classes could produce autoimmune haemolysis when the classical agglutination DAGTs were negative; the IgA and IgM were usually found in association with IgG. The haemolytic effect was enhanced by the presence of complement and combinations of immunoglobulin classes on the red cells.

The provision of blood for transfusing patients whose sera contain red cell autoantibodies requires considerable expertise. Over 8 years, 3888 samples from 2149 patients were examined; the varying clinical presentation necessitated a flexible investigative approach. The autoantibodies showed evidence of blood group specificity in 706 patients (32.9%), usually within the Rh system for warm reacting antibodies, whereas cold antibodies were mostly anti-I. Concomitant alloantibodies were detected by noting varying reaction strengths during antibody investigation and compatibility testing, and by absorption techniques using autologous or selected allogenous red cells. Alloantibodies were found in 294 patients (13.7%); the most frequent were anti-E and anti-K. Compatibility tests were performed on SAG-M donor blood of suitable ABO group, similar Rh genotype, Kell negative and lacking antigens to any alloantibodies detected. All units of blood were incompatible by at least one technique and were issued as 'not compatible but considered suitable'. A total of 7052 units was issued for 1685 patients; no haemolytic reactions were reported. It was concluded that blood can be safely given to patients with autoantibodies, even in serologically complex cases, providing adequate investigations are carried out.

Enzyme linked and agglutination direct antiglobulin tests were carried out on blood samples from 219 patients suspected of having autoimmune haemolysis. The enzyme linked tests were more sensitive: they could detect the small amounts of IgG, IgA, and IgM which are normally present on red cells and showed increased amounts of cell bound immunoglobulins in patients with Coombs test negative autoimmune haemolysis. Many patients had immunoglobulins of more than one class bound to their red cells; considering the degree of haemolysis in individual patients, it appeared that the different immunoglobulin classes acted synergistically in effecting red cell destruction, even in amounts too small to be detected by the agglutination tests. In patients with cold reacting autoantibodies and complement coating of the red cells active haemolysis was found (with one exception) where IgM was detected on the cells by the enzyme linked method. Elution studies indicated that immunoglobulins detected just by the enzyme linked techniques were red cell antibodies. Both enzyme linked and agglutination tests were negative in 66 patients: 61 of these had no evidence of haemolysis, and in the other five the haemolysis was not autoimmune in origin.

An enzyme-linked direct antiglobulin test (DAGT) for assessing erythrocyte-bound IgG, IgM and IgA is described. The test is carried out in microtitre plates using heavy chain-specific, alkaline phosphatase-linked, goat anti-human globulin reagents with p-nitrophenyl phosphate as substrate. Results are expressed in optical density (OD) units per 3.6 X 10(7) red cells. The method is reproducible, with coefficients of variation of 0.056, 0.093 and 0.087 for IgG, IgM and IgA respectively. The linear relationship between the amount of red cell-bound antibody and the OD reading for each immunoglobulin class shows that the method is suitable for quantitative studies. Healthy individuals were found to have small amounts of immunoglobulin bound to their red cells with mean values of 0.251, 0.087 and 0.128 OD units per 3.6 X 10(7) red cells for IgG, IgM and IgA respectively; there was no difference between male and female subjects. In the clinical situation, the enzyme-linked DAGT was considered to show significantly increased amounts of cell-bound immunoglobulin when the results were more than three standard deviations above the mean and the quantitative results permitted an accurate assessment of the progress and response to treatment of patients with autoimmune haemolysis.