Search

BACKGROUND : The objective was to apply statistical analysis to the false passes and fails that occur with the primary and secondary Hb-screening methods used at blood-donor sessions. STUDY DESIGN AND METHODS : Venous samples from 1513 potential donors who had undergone primary CuSO 4 screening using capillary blood (Hb cut-offs: women, 125 g/L; men, 135 g/L) were tested at the session by a secondary method (HemoCue; cut-offs: women, 120 g/L; men, 130 g/L) and again at the base laboratory using another system (Beckman Coulter General S system), which generated the “true” Hb value. RESULTS : False-pass and -fail rates for women and men, respectively, were 11.2 and 6.3 percent (women) and 5.2 and 1.8 percent (men) for CuSO 4 ; 1.9 and 3.7 percent (women) and 1.5 and 0.4 percent (men) for HemoCue; and 2.7 and 2.4 percent (women) and 1.8 and 0.2 percent (men) for a combined procedure that mimicked current practice of only testing CuSO 4 fails by HemoCue. CONCLUSION : CuSO 4 Hb screening gives large numbers of false passes, particularly in women. Using venous samples, the majority correctly pass at the lower HemoCue cut-offs. The current dual-testing policy appears convenient for donor sessions, but because small percentages of false passes and fails represent large numbers of donors, every effort should be made to improve the accuracy of Hb screening.

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.

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.

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.

IgA is best known in transfusion practice for its deficiency when anti-IgA antibodies cause severe anaphylactic reactions. Following the realization that IgA deficient products were needed on demand, blood donors were routinely screened, initially by latex agglutination inhibition and subsequently by hemagglutination inhibition using an Olympus PK-7200™ blood grouping machine. IgA deficiency (

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.

SummaryAccording to history, the population of the British Isles derives its genepool from a succession of invaders and immigrants. The settlement pattern of these invaders gave rise to a patchwork of genepools, shown in previous genetic surveys. Specimens from 1117 blood donors of regionally subdivided East Midlands (Derbyshire, Nottinghamshire and Leicestershire) were analysed for 18 conventional genetic systems (blood groups, serum proteins and red cell enzymes), according to place of residence. Significant differences exist among the five geographically defined sub-populations, and it is argued that these are derived from the historical settlement of continental European populations in the region, especially the Danes and the Vikings.

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.

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.

The identification of blood proteins in ancient bone is important (1) because detection of simple proteins can give information on species specificity and evolution, and (2) because the methods used provide a sound technical basis for investigating the survival of more informative proteins, such as HLA and blood groups, which would shed light on genetic profiles and disease predispositions of ancient populations. Our previous studies used a sensitive and specific enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and showed that albumin can be identified in human skeletal remains at least 4000 years old. However, the factors which affect survival and diagenesis of proteins within ancient bone remain largely unresolved. In the present studies, the results obtained after testing extracts of ancient bovine and human bone for albumin with the ELISA have been compared. The human extracts were prepared from intact (“closed”) vertebrae from formal burial sites. Well-preserved bovine material, on the other hand, could not easily be found and most of the bovine extracts had to be prepared from assemblages of bone which were “open” (i.e. the spongy interior was exposed), either because of butchering or weathering. Although albumin could be detected in both bovine and human bones which were several thousand years old, the survival pattern between the species was strikingly different (chi-square=11·7,P

Summary. 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 IgG 1 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.

HPS and PPHTN are unusual and challenging pulmonary manifestations of liver disease. We report two pediatric cases in association with heterotaxy polysplenia syndrome and congenital absence of the portal vein. Both patients were symptomatic and hemodynamically compromised and required aggressive medical therapy. One patient with PPHTN alone achieved a successful liver transplant. The second child presented with combined HPS and PPHTN and exhibited a different evolution of pulmonary vascular disease. These cases illustrate associations that must be entertained in the setting of heterotaxy syndrome, cyanosis, or pulmonary hypertension and how strategic medical combined with surgical management can provide a good outcome.

The survival of human proteins in blood stains on fragments of cloth buried in exposed soil was examined in a 15-month investigation carried out from September 1990 to December 1991. During this period there was a wide variety of weather conditions. Samples were exhumed at 4-weekly intervals for 16 weeks and finally at 65 weeks; extracts of the stains were tested for albumin and IgG using a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) performed with monoclonal antibodies. Human albumin survived well throughout the 15 months of study, but IgG could be detected only in the 4- and 8-week samples. The reactions for IgG were weaker than those for albumin, although the method's sensitivity (10 ng) was the same for each protein. Appropriate buried and non-buried control experiments were carried out using cloth, either unstained or stained with human blood or animal sera; there was no cross-reactivity between human and the other species investigated and soil did not affect the assay; under laboratory conditions, IgG and albumin survived equally well. The system's versatility was illustrated by using monoclonal anti-bovine-albumin to detect specific albumin in the extracts of buried cloth which had been stained with bovine serum. It was concluded that ELISA performed with monoclonal antibodies could be of great value in identifying blood stains for forensic purposes. The method has several advantages other than sensitivity and specificity: by using Terasaki plates, very small 5μl-aliquots of extract can be tested as opposed to the 50μl-samples required for conventional micro-ELISA plates; cross reactivity from environmental factors is avoided and, compared with radioimmunoassay and standard ELISA, the technique does not require complicated measuring equipment to provide a result. Decreased antigen survival with time and exposure suggests that it will be better to test for the more abundant blood proteins such as albumin. The system would be ideal for experimental forensic work, for example for examining the effects of different variables such as soil composition on protein survival.

The immunological identification of species-specific blood proteins in skeletal remains has an important role in the reconstruction of ancient dietary, ritual and domestic behaviour. However, which protein provides the most suitable target for such work has not been considered previously, and the present investigation was carried out on human bone to assess the relative merits of IgG and albumin. Extracts of bone from 31 individuals (from the English Civil War, medieval, Early Saxon, Roman, Iron Age and Bronze Age periods) were tested by an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Albumin was detected in 23 of the 31 skeletons, including those from the Iron and Bronze Ages, whereas IgG was identified in only one; this difference was very highly significant (P < 0.0005, χ2 30.0). The better detection rate for albumin was thought to be due to its higher original blood level, inherent differences in survival pattern being considered unlikely. Testing the same extract for both proteins in the same assay system ensured that any effects due to soil factors, burial conditions, physical integrity of the bone, chronological age, amount of original specimen, method of analysis and type of reagent were the same for each part of the study, thus permitting a valid comparison of antigen survival to be made. Control samples, including fresh and ancient animal bone extracts and human and animal sera, confirmed that the results were consistent and specific, with no cross-reactivity between human and animal material, and that as little as 10 ng of protein was detectable. In summary, the investigation compared the suitability of IgG and albumin for osteoarchaeological studies using a highly specific, sensitive and versatile ELISA; the results showed that albumin was a far better target molecule for such work and that it can survive in ancient bone for long periods of time.

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.

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. Immunohematology 1997;13:9–11.

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 haptoglobin0.1 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 haptoglobin0.1 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.

IgA is best known in transfusion practice for its deficiency when anti- IgA antibodies cause severe anaphylactic reactions. Following the realization that IgA deficient products were needed on demand, blood donors were routinely screened, initially by latex agglutination inhibition and subsequently by hemagglutination inhibition using an Olympus PK-7200 blood grouping machine. IgA deficiency (.0016 g/L) was found in 357 (with anti-IgA in 28%) of 301,310 donors, an incidence of 1 in 844. By screening new donors and directed call-up, group O, D- red blood cell (RBC) units are always in stock. During 1 year, the center supplied 79 units of RBCs and 64 units of fresh frozen plasma to a variety of patients with IgA deficiency, including three undergoing liver transplantation. The center also provides a reference service for IgA/anti-IgA status. The technique used (hemagglutination inhibition) has a sensitivity well below the threshold of standard quantitation methods. Samples were most commonly referred from departments investigating possible immunodeficiency and suspected transfusion reactions. Of 247 patients investigated, 122 had IgA deficiency, 43 with anti-IgA (of whom 5 had suffered a transfusion reaction). Donors and patients with anti- IgA were issued blood group cards warning that they should only receive IgA deficient products.

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 of0.1 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 was0.1 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.

In some patients with autoimmune hemolytic anemia or hemolytic 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°C, 25°C, 30°C, and 37°C, and tested 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°C and 37°C than at 18°C or 25°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°C and at 37°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°C. However, when treatment was performed at 37°C, antigenicity 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°C or for not more than 30 minutes at 37°C. Immunohematology 1994;10:22.

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.

Two hundred and twenty-one patients with cold haemagglutinins of thermal amplitudeor = 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 levels0.1 g/l compared with Groups 1 and 2, respectively (P0.005 and P0.001). 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.

It was hypothesized that prenatal exposure to cocaine and other substances would be related to delayed expressive language development. Speech and language data were available for 458 6-year olds (204 were exposed to cocaine). No significant univariate or multivariate differences by cocaine exposure group were observed. Classification and regression tree modeling was then used to identify language variable composites predictive of cocaine exposure status. Meaningful cut points for two language measures were identified and validated. Children with a type token ratio of less than 0.42 and with fewer than 97 word types were classified into a low language group. Low language children (n = 57) were more likely to be cocaine exposed (63.1%), with cocaine-exposed children 2.4 times more likely to be in the low language group compared with control children after adjustment for covariates. Prenatal cigarette, but not alcohol exposure, was also significantly related to expressive language delays.

Advances in molecular biology can be applied to forensic practice with spectacular success. However, the extraction of DNA from scarce or badly degraded substances remains a significant problem and an ability to overcome it remains crucial because the success of all DNA analyses depend on a successful initial extraction. Extraction methods must solve problems associated with low quantities of DNA, DNA degradation, polymerase chain reaction (PCR) inhibitors and failure to solubilize or separate DNA from the substrate. These problems are compounded by the relative lack of knowledge concerning DNA degradation in different forensic environments. The extraction method must be suitable for the starting material. Fresh material with abundant DNA, such as blood, soft tissue, or saliva, presents no difficulties and DNA suitable for PCR analysis can be extracted easily. In mixed substrates, for instance vaginal swabs from rape victims, male and female DNA can be separated by differential lysis procedures. Putrefied material with abundant DNA, such as blood, soft tissues, and entomological and botanical material, has plenty of DNA, but PCR inhibitors may be present and extraction methods must be chosen that select DNA but not the inhibitors. Clean material with scarce DNA, such as stains (blood, saliva, sperm), swabs (vaginal and from bitemarks, fingernails and stains on bodies), fingernails, hair, desquamated cells (skin, dandruff), and certain body liquids (urine, plasma), has a paucity of cellular material and hence low quantities of DNA. The prerequisite here is to reduce the loss of DNA, for example by introducing more specific methods of extraction and reducing the number of steps in the procedure. In the case of dirty material with scarce DNA, such as sperm, saliva and blood stains, nails and feces, not only are there low quantities of DNA, but there are also large amounts of organic and inorganic contamination. The problems faced by any extraction method are to recuperate small quantities of DNA as well as eliminating inhibitors of PCR. Formalin-fixed and paraffin-embedded tissues and stained smears on slides present particular problems associated with damage to DNA due to the processing of the materials. Nevertheless, DNA can be successfully extracted from these substrates. The greatest challenge in forensic DNA analysis is extraction from calcified tissues (bone and teeth). These substrates have obviously undergone considerable degradation and are often contaminated with soil. In addition, DNA undergoes binding interactions with the hydroxyapatite matrix which chemically stabilizes DNA; this aids its survival, but makes it difficult to extract. An essential part of the extraction process is therefore to release DNA from this binding. Before DNA can be isolated from a substrate, several important steps must be taken. These involve lysing the cells to release the DNA and digesting protein so that as much DNA as possible is solubilized. Many (if not most) techniques – and there are many variations – involve the use of proteinase K (PK) to digest protein and a detergent (often sodium dodecyl sulfate (SDS)) to lyse the cell membranes. The extraction procedures themselves can be divided into two categories: nonaffinity methods where all extraneous material is removed leaving DNA in solution, and affinity methods whereby the DNA is targeted directly and removed from the solution. Each category has many variations and on occasions they can be combined. The main nonaffinity techniques are phenol/chloroform, salt precipitation and chelex. The phenol/chloroform method is the best known and has an excellent forensic record; it is based on removing protein, thus purifying the nucleic acids which are extracted in aqueous solution; the disadvantages are that it does not remove nonprotein contaminants, several steps are involved in the procedure, and the reagents are toxic. The salt precipitation method is based on salting-out by dehydration and precipitation with a saturated salt solution. It is an excellent method when abundant clean samples are available and is quicker, cheaper and less toxic than the phenol/chloroform procedure. The disadvantages are that it does not eliminate all PCR inhibitors and considerable amounts of protein may be retained. With chelation (e.g. chelex), the active reagent binds to inorganic substances and clears the extract of certain PCR inhibitors, particularly metal ions; an alkaline pH disrupts cell membranes leaving DNA in solution. The method is simple, rapid and does not involve multiple tube transfers, though it does not have a particularly selective action on protein. The novelty of affinity methods is that they actively select DNA by either specific or nonspecific reactions. The main nonspecific affinity method is the glass–milk or silica gel procedure. Very small glass beads are combined with isothiocyanate which itself is bound to guanidium. The positively charged amine groups on the guanidium link with the negatively charged phosphate groups on DNA. Once bound to the glass beads, the DNA can be repeatedly washed and cleansed of all other material before being eluted and subjected to PCR analysis. This is currently the method of choice for difficult substrates; the disadvantage is that when a great deal of contaminant is present, it might sterically hinder the adsorption process of DNA on to the glass substrate. The only specific affinity method yet published uses a murine anti-dsDNA monoclonal antibody of IgG2A subclass bound to paramagnetic beads via anti-murine IgG. The antibody-coated beads react specifically with DNA and can easily be washed free of contaminants. The method is extremely specific and sensitive, positive PCR results being obtainable from as little as 0.005 ng per microliter of DNA. In addition, the bead–anti-DNA–DNA complex can be put directly into the PCR. As yet this method has not been fully tested on all forensic substrates, but initial results are very promising. Commercial DNA extraction kits, for example Dynal DNA direct™, QIAamp®, Prep-A-Gene® and Scotlab Geneclean®, are becoming increasingly available and some provide protocols for forensic substrates. Most of these kits are based on an affinity mechanism. In practice they seem to give reliable results. In conclusion, DNA extraction methods in forensic analysis seem to be going two ways. For substrates with fresh abundant DNA, extraction presents no problems and any method would suffice; it would seem logical to choose one that uses nontoxic reagents and is inexpensive. The most interesting evolution in extraction techniques is with those dealing with difficult substrates where DNA is scarce or tightly bound and non-DNA contamination is abundant. Recent work suggests that the future in these situations lies with affinity methods and the development of immunoaffinity seems to point the way for future research. Such studies should be combined with investigations into the interaction between DNA and forensic environments so that extraction procedures can be devised for particular circumstances, for only with proper extraction procedures will subsequent DNA analysis be successful.

Although many studies have examined the development of children of male alcoholics, few studies have considered substance use by the female caregiver. This study evaluated the relationship between substance use by female caregivers and factors that affect the child-rearing environment.A total of 480 inner-city African-American women were recruited during pregnancy for a longitudinal study of the effects of prenatal alcohol use and substance use by caregivers on the development of their children. All women were screened for alcohol consumption at their first prenatal visit to a large urban maternity hospital. Those who averaged seven or more drinks per week (0.5 oz absolute alcohol per day) at the time of conception were invited to participate in the study, as was a 5% random sample of lighter drinkers and abstainers. At the 7.5-year follow-up assessment, the quantity and frequency of alcohol consumption and drug use, as well as several measures of the child-rearing environment, were assessed for 231 of the caregivers.Current alcohol use was uncorrelated with standard demographic factors, such as socioeconomic status, but was related to poorer family functioning, lower quality of parental intellectual stimulation, and higher levels of domestic violence. There were independent effects of illicit drug use on family environment, domestic violence, and caregiver depression. History of drinking during pregnancy, however, was not related to the current child-rearing environment. Poorer parental functioning generally was found only among the caregivers who currently drank both heavily (six or more drinks/occasion) and frequently (three or more days/week). After controlling for lifetime alcohol problems, current drinking still predicted a less cohesive and organized family environment and higher levels of domestic violence.Current heavy, frequent drinking in this relatively homogenous, economically disadvantaged sample was unrelated to demographics and seemed to have an important negative impact on the quality of the child-rearing environment, whereas drinking during pregnancy was unrelated to the current child-rearing environment.

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.

In situations where badly burnt fragments of bone are found, identification of their human or non-human origin may be impossible by gross morphology alone and other techniques have to be employed. In order to determine whether histological methods were redundant and should be superseded by biomolecular analyses, small fragments of artificially burnt bone (human and non-human) were examined by quantitative and standard light microscopy, and the findings compared with newer biomolecular analyses based on identifying specific human albumin by ELISA and amplifying human mitochondrial DNA by PCR. For quantitative microscopy, reference data were first created using burnt bones from 15 human and 20 common domestic and farm animals. Measured osteon and Haversian canal parameters were analysed using multivariate statistical methods. Highly significant differences were found between values for human and non-human bone, and a canonical discriminant function equation was derived, giving a predicted correct classification of 79%. For the main study, samples of cortical bone were taken from three fresh cadavers, six human skeletons and ten freshly slaughtered animals and burnt by exposure to temperatures ranging from 800 to 1200 degrees C; charred fragments of human cortical bone from two forensic cases were also tested. Quantitative microscopy and canonical discriminant function gave the correct origin of every sample. Standard microscopy falsely assigned burnt bone from one human skeleton and one forensic case to a non-human source, but otherwise gave correct results. Human albumin was identified in five individuals, including one of the forensic cases, but mitochondrial DNA could not be amplified from any of the human bone. No false positive test results were seen with either biomolecular method; and human albumin and mitochondrial DNA were correctly identified in all unburnt control specimens. It was concluded that histological methods were not redundant and that quantitative microscopy provided an accurate and consistent means of determining the human or non-human origin of burnt bone and was more reliable than standard microscopy or the newer immunological and DNA techniques tested here.

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.

The purpose of this survey was to describe the job setting, professional activities, and satisfaction of maternal-fetal medicine (MFM) subspecialists and to compare these with previous surveys. A questionnaire was mailed to members of the Society of Perinatal Obstetricians (SPO). The results were compared to a similar survey in 1986. Of 1,352 members, 58% responded. In comparison to 1986, there was an increase in percentage of women (25%); a decrease in university hospital-based faculty (55%); and an increase in community hospital-based salaried physicians (23%) and private practice (15%). The distribution of professional time changes with an increase in clinical care, a decrease in teaching and research. The annual number of procedures increased for ultrasound and genetics and decreased for gynecologic procedures. Since 1986, there have been significant changes among MFM subspecialists in job setting, allocation of professional time, and number and types of procedures. Job satisfaction remains high.

Oxidant stress has been implicated as playing a role in the pathogenesis of cholestatic liver injury. The objective of this study was to determine whether the xanthine oxidase/xanthine dehydrogenase enzyme system was involved in this oxidant stress. Adult Sprague-Dawley rats were treated with the xanthine oxidase inhibitor, oxypurinol, and randomized to bile duct ligation or sham surgery; vehicle-treated, sham-operated rats served as controls. After 5 d of bile duct ligation, serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total and direct bilirubin concentrations were significantly elevated, and increased lipid peroxidation of hepatic mitochondria and microsomes was present. Treatment with oxypurinol reduced the aspartate aminotransferase, alanine aminotransferase, and bilirubin values by 26-47% but did not alter the increased lipid peroxidation of mitochondria and microsomes. Serum vitamin E:total lipids ratio was also reduced in both bile duct-ligated groups, consistent with oxidant injury. These data show that inhibition of xanthine oxidase reduces biochemical evidence of hepatocellular injury during bile duct ligation without affecting oxidant damage to intracellular hepatocyte organelles. Thus, in this model a component of cholestatic injury appears to have been caused by oxidant stress from a source outside of the hepatocyte.

Administration of parenteral nutrition to the small infant has decreased morbidity but is associated with the development of cholestasis and liver dysfunction in a high proportion of infants. In this review, contributing factors of the etiopathogenesis of parenteral nutrition-associated liver disease will be outlined, forming the basis for a unifying theory of its pathogenesis. It is proposed that oxidant stress and stimulation of hepatic Kupffer cells by bacterial cell wall products absorbed from the injured intestine are major factors leading to cholestasis and liver injury during prolonged parenteral nutrition. Improved outcome in patients has been related to the early introduction of feedings. New proposed therapeutic modalities have included antibiotics and probiotics to prevent bacterial overgrowth of the small intestine, enterally-administered ursodeoxycholic acid and intravenous cholecystokinin. Improved understanding of the fundamental mechanisms producing liver injury and fibrosis during parenteral nutrition will lead to new preventative and treatment measures in the future.

Considerable variation in offspring outcome is observed after intrauterine alcohol exposure. The underlying mechanism may include genetic diversity in the enzymes responsible for alcohol metabolism. Of the known genetic polymorphisms, differences at the alcohol dehydrogenase-2 locus (ADH2) are likely most critical because the resulting enzymes are30-fold different in their kinetic constants. To test whether differences in maternal or offspring ADH2 genotype are determinants of risk for alcohol-related birth defects, maternal-infant pairs (n = 243) were enrolled on the basis of maternal alcohol intake during pregnancy and maternal ADH2 genotype. Infant outcome was measured using the Bayley Scales of Infant Development Mental Index (MDI) at 12 months of age. Drinking during pregnancy was associated with lower MDI scores but only in the offspring of mothers without an ADH2*3 allele (P.01, analysis of variance, post hoc). The offspring of drinking women with at least one ADH2*3 allele had MDI scores similar to those of nondrinking women of either ADH2 genotype. Lower MDI scores were associated with the three-way interaction among increasing alcohol intake and maternal and offspring absence of the ADH2*3 allele (P.01, multiple linear regression). We suggest that the protection afforded by this allele is secondary to its encoding of the high-Km/high-Vmax ADH beta3 isoenzyme, which would provide more efficient alcohol metabolism at high blood alcohol concentrations. These observations are supportive of alcohol, rather than acetaldehyde, being the more important proximate teratogen and are the first observations of a specific genetic explanation for susceptibility differences to alcohol-related birth defects.

Extraction of amplifiable DNA-from degraded human material in the forensic context remains a problem, and maximization of yield and elimination of inhibitors of the Polymerase Chain Reaction (PCR) are important issues which rarely feature in comparative studies. The present work used PCR amplification of three DNA sequences (HLA DPB1, amelogenin and mitochondrial) to assess the efficiency of three methods for extracting DNA (sodium acetate, magnetic beads and glass-milk) from 32 skeletal samples and 25 blood stains up to 43 years old. The results, analyzed using multivariate statistics, confirmed that the extraction method was crucial to the subsequent detection of amplification products; the glass-milk protocol performed better than sodium acetate, which was better than magnetic beads. Successful amplification also depended on gene sequence, multiple copy mitochondrial sequences performing best; however, with the singly copy sequences, the longer HLA DPB1 (327 bp) being detected just as often as the shorter amelogenin (106/112 bp). Amplification products were obtained more frequently from blood stains than bone, perhaps reflecting differences inherent in the material, and from younger compared with older specimens, though plateauing seemed to occur after 10 years. PCR inhibitors were more frequent in sodium acetate extracts.

BACKGROUND: The objective of the study was to examine the interrelationships and clinical significance of IgA red cell antibodies in the autoimmune response. STUDY DESIGN AND METHODS: The records of 5235 patients referred to an immunohematology center over a 14-year period were critically examined for patients who had IgA autoantibodies, defined as elutable IgA immunoglobulins that would rebind to normal cells. RESULTS: One hundred twenty-four patients (61 male) aged 6 to 98 years had warm-reacting IgA autoantibodies. In 75 individuals, these were idiopathic; neoplasms were the most common associated conditions in the individuals with secondary IgA autoantibodies. IgA was the only immunoglobulin present in 6 patients; all others also had IgG and/or IgM coating their cells, and 102 individuals also had increased amounts of cell-bound complement. In a comparison by chi-square test of populations with haptoglobins of < 0.1 g per L, IgA was shown to act synergistically with IgG in producing hemolysis (p < 0.01). CONCLUSION: Autoimmune hemolysis due to IgA antibodies alone in rare, with red cell destruction occurring through mechanisms similar to those for IgG. Most commonly, IgA acts synergistically with other immunoglobulins (usually IgG) and complement; the hemolysis may be severe. Whether IgA autoantibodies alone can activate complement remains controversial, but increasing evidence suggests that they can, possibly via the alternative pathway, and that this activation may result in intravascular hemolysis.

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.

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 of0.1 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 of0.1 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.

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.

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 was0.1 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.

Regular, heavy alcohol intake results in transferrin that is deficient in carbohydrate moieties. Carbohydrate-deficient transferrin (CDT) has been used as a biologic marker of heavy alcohol exposure in nonpregnant humans. There have been no reports of CDT levels in pregnancy. Our objective was to determine maternal and cord blood levels of CDT. Parturients were recruited at delivery based on graded representative alcohol consumption, from abstainers to heavy drinkers, as determined by screeners skilled at eliciting drug and alcohol histories. Maternal and cord blood serum samples were obtained at delivery. A double antibody radioimmunoassay was used to determine CDT in each sample. There were 83 paired specimens analyzed by paired t tests and stepwise regression analysis. Cord blood CDT units/liter (44.0 +/- 29.5) were significantly (P0.0001) higher than maternal (18.4 +/- 7.0). Maternal and cord CDT did not correlate with race, perinatal risk score, gestational age at delivery, birth weight, Apgar scores, or reported alcohol intake. Maternal CDT levels had a significant negative correlation with cigarette smoking. Cord blood CDT levels are significantly higher than maternal. While regular, heavy alcohol consumption by adults results in serum transferrin deficient in carbohydrate moieties, the reason for elevated fetal CDT is unknown.

To examine the relationship between maternal methadone exposure and neonatal head circumference and abstinence syndrome, we examined the records of 172 opiate-addicted gravidas enrolled in a methadone maintenance program in an urban hospital over a 2-year period. Higher doses of methadone in the third trimester were associated with increased head circumference reflecting both increased gestational duration and improved overall growth. Neonatal withdrawal was positively correlated with gestational age at delivery and race, with nonblack infants exhibiting higher neonatal abstinence scores than blacks following adjustment for maternal dose and gestational age at delivery. Selection of optimal methadone dosage is a complex problem in which the favorable neurobehavioral outcome associated with increased growth and gestational age must be weighed against the risks associated with more severe neonatal withdrawal. Our findings of improved overall fetal growth and gestational duration associated with higher methadone doses suggest that more liberal methadone dosing in pregnancy may improve long-term neonatal outcome.

Traditional Gaussian approaches to biochemical screening make calculations very cumbersome. We have sought to develop a logistic regression mathematical model for the calculation of risk for trisomy 21 in multiple marker screening that would be more "user friendly." Biochemical screening data from 15,444 patients with confirmed normal outcomes and 28 with trisomy 21 were analyzed by logistic regression analysis (LRA) to produce a direct mathematical approach to risk calculation that combines the improvements in sensitivity and specificity already demonstrated by us with extreme value cumulative function surface fittings, but which can be extended for other exploratory variables. LRA yields a 71.4% sensitivity with a 95% specificity which is comparable to our previously published discriminant aneuploidy detection (DADs) approach; both are an improvement over the commonly used Gaussian approach of risk assessment. In our study population, LRA performed as well as DADs; both performed better than the Gaussian method, and LRA is a simpler approach.