Quantifying the Hydrological Effect of Permitted Water Abstractions across Spatial Scales.

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.

Influence of hydrological regime and land cover on traits and potential export capacity of adult aquatic insects from river channels.

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.

Recruitment of Anguilla spp. glass eels in the Waikato River, New Zealand. Evidence of declining migrations?

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.

An enzyme-linked direct antiglobulin test for assessing erythrocyte bound immunoglobulins.

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.

Erythrocyte autoantibodies, subclasses of IgG and autoimmune haemolysis.

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.

Erythrocyte autoantibodies, autoimmune haemolysis, and carcinoma.

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.

Erythrocyte autoantibodies, autoimmune haemolysis, and myelodysplastic syndromes.

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 direct antiglobulin tests in patients with autoimmune haemolysis.

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.

Small quantities of erythrocyte bound immunoglobulins and autoimmune haemolysis.

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.

Paroxysmal cold hemoglobinuria and the elusive Donath-Landsteiner antibody.

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.

Autoimmune hemolytic anemia due to IgA class autoantibodies.

Autoimmune hemolytic anemia due to warm-reacting autoantibodies solely of the IgA class is very rare, and only five cases were identified among 5,177 patients referred during 13.5 years. All were females (ages 21-69 years) and all presented with idiopathic "Coombs negative" autoimmune hemolytic anemia, a diagnosis that was confirmed using monospecific anti-human IgA reagents. Red cell-bound IgG was reduced, but in two patients IgM was initially increased, an occurrence that was thought to reflect the developing autoimmune response. The autoantibodies had high affinity for red cells with very little free antibody detectable in the serum; in two instances Rh specificity was evident. Hemolysis was severe in four patients. Two of them had intravascular hemolysis, one of whom also had marked dyserythropoiesis and a transiently positive Ham's test. Although IgA autoantibodies caused hemolysis predominantly through immune adherence, on occasions they also seemed to be able to induce complement activation, possibly via the alternative pathway. Prednisolone was the mainstay of treatment, and was occasionally augmented with azathioprine and intravenous immunoglobulin. Blood transfusion was required in two patients, both of whom eventually required splenectomy that resulted in full remission. The one patient with mild hemolysis recovered without treatment.

An improved method for removal of red cell-bound immunoglobulin using chloroquine solution.

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.

Direct Coombs test-negative autoimmune hemolytic anemia and low-affinity IgG class antibodies.

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.

Measurement of red blood cell-bound C3b and C3d using an enzyme-linked direct antiglobulin test.

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.

Immune hemolytic anemia due to diclofenac.

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.

Autoimmune hemolytic anemia caused by warm-reacting IgM-class antibodies.

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.

Delayed hemolytic transfusion reaction and paroxysmal cold hemoglobinuria: an unusual association.

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.

IgG2 class red cell antibodies and autoimmune haemolysis.

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.