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A woman, aged 66, fulfilled all the usual criteria of dermatitis herpetiformis. Subsequently, she developed circulating IgA and IgM basement membrane zone antibodies, a labile erythrocyte sedimentation rate (ESR), and the clinical picture changed to one of bullous pemphigoid. Her labile ESR was obviously caused by a factor related to the erythrocytes. Direct Coombs test was positive.
Here we are presenting the case of a 70-years-old woman who has hepatic cirrhosis anti-HCV and insulin-dependent diabetes mellitus, without relevant epidemiologic ascendants or previous transfusions and HBV, HIV negatives. On admission to our hospital she showed signs of autoimmune hemolytic anaemia (AHA) which was confirmed by positive direct Coombs test and an improvement in blood test after corticoid treatment. Having discarded other possible causes such as drugs infectious diseases or essential mixed cryoglobulinemia (CME), we put forward the possible association between AHA and infection by HCV, where AHA was an extrahepatic immunological manifestation of HCV. This fact has never been brought to light in previous medical literature.
BACKGROUND AND OBJECTIVE: Severe anemia is the outstanding problem in approximately 50 percent of patients with myelofibrosis with myeloid metaplasia (MMM). The present trial was based on the considerations that abnormal immune responses are frequently associated with MMM and that cyclosporin A (Cy-A) has proven to be effective in improving anemia in autoimmune disorders. The aim of this study was to evaluate the effect of Cy-A on anemia of MMM. DESIGN AND METHODS: We studied 10 patients with MMM and severe anemia who were not responsive to corticosteroids. Eight of them showed evidence of immune defects (direct or indirect Coombs' test, antinuclear or antimitochondrial antibodies, circulating immune complexes). Cy-A was delivered orally in two refracted doses of 5 mg per kilogram bw every day and the serum level of the drug was maintained between 100 and 200 ng/mL for at least 6 months. Clinical effects were measured by calculating a normalized transfusional need (NTN), and response was defined as about a 30% reduction in the initial transfusion requirement. Hematologic parameters, s-Epo, s-TfR, s-IL2R and lymphocyte flow cytometric analysis were also evaluated. The results were analyzed with the Student's t-test. RESULTS: Only 6 patients completed the entire 6 months of planned therapy. Three of these responded, with one no longer needing transfusions. A high CD4/CD8 ratio was predictive of response (mean value 4.7 +/- 3.5 in responders versus 0.9 +/- 0.4 in non-responders, p = 0.06). INTERPRETATION AND CONCLUSIONS: An immunomediated mechanism negatively affects erythropoiesis in MMM. Cy-A may be effective for patients with severe refractory anemia in this disease.
INTRODUCTION: In the foetus in utero predominates a considerable percentage of immunologically so-called naive lymphocytes of the T and B series. The objective of the presented work was to assess by means of flow cytometry and labelled monoclonal antibodies quantitative changes in cell sub-populations of the foetal immune system before the foetus is altered by severe heamolysis as a result of Rh(D) isoimmunization. METHOD: The authors obtained by intrauterine puncture of the umbilical cord peripheral blood from 10 foetuses with isoimmunization during the 23rd-35th week of gestation, confirmed by the direct Coombs test and with a mean haematocrit value of 30.8% +/- 8.02. These findings were compared with values in the peripheral blood stream in a control group of 35 foetuses during the 18th-39th week of pregnancy with normal intrauterine development (haematocrit 34.2% +/- 5.87). The authors assessed the haemogram and CD signs in the lymphocyte population. RESULTS: In the peripheral bloodstream of foetuses with erythrocyte isoimmunization the authors did not detect, as compared with the control group (p > 0.01), a lower haemoglobin level (10.6 +/- 2.77 g/dl vs. 11.9 +/- 2.03 g/dl) a lower haematocrit (30.8% +/- 8.02 vs. 34.2% +/- 5.87) and fewer leucocytes 5.1 +/- 2.39 x 10(9)/l versus 6.97 +/- 3.29 x 10(9)/l. In foetuses with Rh(D) isoimmunization the authors found a higher percentage of T(CD3+) lymphocytes (79.0% +/- 11.23 vs. 73.7% +/- 12.79, but did not prove an increase of activated T lymphocytes (%DR+ from CD3+) (1.0% +/- 0.52 vs. 1.3 +/- 0.58). The percentage ratio of T helper cells (CD+) was higher than in the control group (61.0% +/- 10.25 vs. 56.1% +/- 12.45). In foetuses with Rh(D) isoimmunization there was no difference in the ratio of CD8 positive cells (24.1% +/- 8.23 vs. 23.6% +/- 7.26). Suppressor T cells (CD8+CD11b+) were fewer (4.1% +/- 1.24 vs. 7.5 +/- 11.23) than in the control group. The number of NK cells in foetuses with Rh(D) isoimmunization was 5.1% +/- 3.26 vs. 6.9% +/- 3.86, in isoimmunized foetuses there is a higher ratio of so-called naive T helper cells Th1 (CD4+ CD45 RA+) 49.3% +/- 12.71 vs. 43.0% +/- 12.88. When assessing naive B1 cells (CD19+CD5+), the authors did not find a difference between the two groups (10.4% +/- 6.20 vs. 9.06% +/- 12.1). The ratio of CD4: CD8 in the group with isoimmunization was higher than in the control group (3.1 +/- 1.39 vs. 2.5 +/- 1.13). CONCLUSION: In isoimmunized foetuses in the initial stages of haemolysis no detactable immune response with significant changes in the lymphocyte sub-populations was found.
INTRODUCTION: Acute infections mononucleosis is the most common clinical manifestation of primary Epstein-Barr virus (EBV) infection occurring during adolescence. It is a benign lymphoproliferative, usually self-limiting disease. Complications are relatively rare, but they may occur, especially hematological. Most common are autoimmune hematolytic anemia and thrombocytopenia, and they respond to corticoid therapy. Deuteration of white blood cells is rather rare, whereas mild neutropenia is a normal finding during the course of acute disease. On the other hand, agranulocytosis is extremely rate, and almost every case has been reported in the literature. Filgrastim--the recombinant human granulocyte colony-stimulating factor (G-CSF) stimulates the activation, proliferation and maturation of progenitor granulocyte cells. This drug is usually applied in treatment of iatrogenic neutropenia, during chemotherapy of malignancies and in some idiopathic and cyclic neutopenias. CASE REPORT: A female patient, 18 years of age, has been hospitalized at the Clinic of Infectious Diseases in Novi Sad on two occasions. First because of severe acute infectious mononucleosis with acute hepatitis and jaundice 10 days after onset of symptoms. Physical examination revealed severe intoxication, dehydration, icteric skin, mucosis and massive hepatosplenomegaly. The diagnosis was confirmed by ELISA IgM, EBV VCA positive and ELISA IgG EBV VCA and IgG EBVNA negative results. The patient was discharged from hospital after 24 days without complaints and with normal physical and laboratory findings. For several days she felt well, but gradually severe fatigue and malaise occurred and she became febrile again. That was the reason why she was hospitalized again, two weeks later. This time she was febrile, extremely intoxicated with general lymphadenopathy, catarrhal gingivostomatitis and massive splenomegaly. The first laboratory findings showed severe neutropenia (absolute count of granulocytes was 0.156 x 10/l, with only 12% segmented neutrophils). Mild anemia--3.05 x 10/l was also registered, while the platelet count was normal. Other biochemical analyses were normal, the Coombs' test negative, while the serological response was also normal. Bone marrow puncture was performed and normocellular bone marrow was registered, somewhere hypercellular due to hyperplasia of granulocyte progenitor cells from promyelocytes to normal maturated cells. Anemia showed megaloblastoid proliferation, while megakaryocytes were normal. High doses of corticosteroids were applied (dexamethasone 160 mg daily) and filgrastim 5 micrograms every other day. From the very beginning of therapy the patient felt better, whereas granulocytes responded with elevation as soon as 48 hours after initiation of therapy. On the sixth day the treatment was stopped because the level of granulocytes was normal and the patient has completely recovered. She was discharged from hospital 4 weeks later with mild meteorism, but normal physical and laboratory findings and mild splenomegaly registered only by ultrasonography. DISCUSSION: During the last 10 years only several cases of severe leukopenia with acute infectious mononucleosis had been reported in literature. In all cases it was associated with some other hematological complications and it occurred in young adults without previously registered immunodeficiency. We have no knowledge about application of filgrastim in treatment of EBV-induced agranulocytosis, but the International Association for Studying Agranulocytosis and Aplastic Anemia reported that in 4% of patients Epstein-Barr virus can cause agranulocytosis even a year after the occurrence of acute disease.
An 88-year-old woman was admitted with generalized lymphadenopathy, anemia, and thrombocytopenia. On admission, a peripheral blood examination showed a red blood cell count of 146 x 10(6)/microliter, a hemoglobin concentration of 6.9 g/dl, and a platelet count of 5.0 x 10(4)/microliter. Blood examination detected polyclonal hypergammaglobulinemia; the results of the direct/indirect Coombs' test were positive; and an elevated cold agglutinin titer and high platelet associated IgG (PA-IgG) level indicated the existence of autoantibodies. Serum cytokine measurements disclosed an elevated level of interleukin-6 (IL-6). Immunoblastic lymphadenopathy-like T cell lymphoma was diagnosed on the basis of lymph node biopsy specimens. VP-16 and steroid therapy alleviated the patient's lymphadenopathy, anemia, thrombocytopenia, and hypergammaglobulinemia. These findings suggest that tumor cells with a T cell phenotype produced IL-6 in large quantities, thus provoking B-cell and plasmacytic histologic changes and humoral disease manifestations, including hypergammaglobulinemia.
A 37-year old patient, HIV-1-infected, consulted for progressive weakness and dyspnea on exertion, increasing over three months. Complete blood count showed pancytopenia, while the bone marrow revealed severe hypoplasia. Other investigations, including serology for CMV, vitamin levels, Coombs test, gastroscopy and colonoscopy were non contributing. A diagnosis of zidovudine-induced medullary aplasia was made; the clinical course was favourable after this drug was replaced by triple therapy. The authors discuss the differential diagnosis of cytopenias in the patient infected by HIV and they suggest a diagnostic approach.
In the routine laboratory for hematology conflicting results may be obtained for the red blood cell parameters with the Coulter Counter Model S. These parameters2) are: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). When the values of the MCHC are above 36 g/dl something must be wrong with the blood sample of the patient. One of the reasons can be agglutination e.g. by cold agglutinins. The blood sample should be reanalysed before and after heating for 1 hour at 37 degrees C. If the values change: cold agglutinins are present; if no change occurs paraproteins, or other disturbing factors, such as bilirubin or high leucocyte levels, will be found. MCH values may also be high in some cases e.g. if the red blood cells are coated with antibodies (Coombs test positive) or after ingestion of medicines like Azathioprine. These examples show that it is possible in some cases to correlate immunological findings with the red blood cell parameters. In addition to the results with the Coulter Counter Model S, some observations on the Hemalog (Technicon) are also presented.
Brucellosis which is an important public health problem is a zoonotic disease that causes economic loss, and seen all over the world as well as in our country. The aim of this study was to compare the results of Rose Bengal test, standard tube agglutination test (STA), Coombs tube agglutination test, Rivanol tube agglutination test and ELISA (IgA, IgG and IgM) method, in patients who were suspected to have brucellosis. Blood and serum samples collected from 77 patients were included to the study. Blood cultures were performed from all of the patients and Brucella spp were isolated in 35 (45.4%) of them. At least one of the serological tests for Brucella was found positive in 62 samples (80.5%). When culture positivity accepted as the gold standard, the sensitivities of the tests were calculated as follows; 100% for Rose Bengal, 94.3% for STA, 97.1% for ELISA IgG, 94.3 for ELISA IgA and 71.4 for ELISA IgM. No prezone phenomenon was observed in the samples. Brucella Coombs tube agglutination test did not increase the chance of diagnosis of brucellosis, unlike the results of previous studies. We found that Rose Bengal and STA tests were still efficient methods for brucellosis serodiagnosis. The results of rivanol tube agglutination test were similar to STA test, and this test can be used especially in patients with brucellosis relapse. ELISA test detecting IgA and IgG antibody titers together found to be effective methods increasing the chance of brucellosis diagnosis, and could be helpful for follow up of brucellosis.
A panel of eight tests was used to study 200 cases of acute brucellosis, 200 patients negative for brucella as a control group and 200 apparently healthy individuals as a second control group. The best diagnostic test was the rose Bengal test using an imported reagent (BioMérieux, France) and 2 local reagents. This test was improved from being a screening test to be a titrable one. The best two tests used together were the tube agglutination test with Coomb-like test. The indirect fluorescent antibody test had no advantages over the use of other tests. The 2-mercaptoethanol test and C-reactive protein test were useful in checking the brucellosis activity. Normal numbers of E-rosette forming cells and inefficient neutrophils in phagocytosis were found in peripheral blood during acute brucellosis.
The direct antiglobulin test (DAT) was performed on 243 subjects in Kenya. A high incidence of positive DAT was found in children with malaria of whom 70% had RBCs coated with IgG, C3 and C4 either separately or together. Only 12% of paediatric patients with conditions other than malaria had a positive DAT. Most positive DATs in association with malaria occurred in children between 18 months and five years of age. There was a lower incidence of positive DAT in Kikuyu children from an area of low malarial transmission than in children from other tribal groups who reside in areas of high malaria endemicity. A high rate of positive DAT was also found in patients with visceral leishmaniasis. Interestingly there was an association of IgG and C4 but without C3 on red cells of five patients with visceral leishmaniasis and two schoolchildren from an area endemic for visceral leishmaniasis. This combination was not found on other patients.
The serological response of heifers to injection with Brucella abortus strain 19 vaccine and simultaneous administration of levamisole hydrochloride were compared with those of heifers inoculated with Brucella abortus strain 19 vaccine alone. The Rose Bengal, serum agglutination, complement fixation and Coombs' antibovine globulin tests were used to measure the humoral responses for a period of sixteen weeks after vaccination. The results indicated that antibody titres in the heifers injected with levamisole and the vaccine were moderately elevated. It was suggested that the heightened antibody titres were possibly mediated through helper T-cells and macrophages. The significance of using levamisole as an adjuvant with Brucella abortus strain 19 vaccine in cattle is briefly discussed.
In a previous paper (Brodie, 1977) the value of the Widal test in the diagnosis of typhoid fever was shown to be limited. Evaluation of possible alternative tests showed that: (1) the sensitivity of the anti-human globulin (Coombs') test appeared greater than that of the agglutination test but the length of time (48 h) before results were available rendered it of little value in rapid diagnosis: (2) the complement fixation test offered no particular help towards diagnosis; (3) immunized and non-immunized typhoid fever patients developed fimbrial antibodies, as also did immunized healthy individuals. In this latter group, however, those immunized with alcoholized TAB vaccine had higher antibody titres to fimbrial antigen than those immunized with heat-killed phenolized vaccine.
A 51-year old woman who had type-C chronic hepatitis developed autoimmune hemolytic anemia after 4 weeks treatment with a total 48 million interferon-beta, 6 million 8 times. Indirect Coomb's test turned from negative to positive through IFN therapy and a complication of asymptomatic primary biliary cirrhosis was confirmed by histologic findings of the biopsied liver which was performed during IFN therapy and a presence of anti-mitochondrial antibody. After stopping IFN therapy and adding prednisolone anemia improved in getting negative indirect Coombs test but remaining positive direct Coombs test.
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The diagnosis of autoimmune haemolytic anaemia (AIHA) requires the establishment of haemolysis and demonstration of autoantibodies against red cells. Most laboratories use the conventional Coomb's test for the demonstration of the autoantibodies. However, in approximately 2-6% of the patients who present with the clinical and haematological features of AIHA, the direct agglutination test is negative on repeated testing. Attempts are therefore being made to identify a test which could be more sensitive than the conventional test, yet retaining the simplicity and cost effectiveness of the test. In the present study, the efficacy of the newly developed gel card test has been compared with the conventional Coomb's test for detection of autoantibodies in 50 cases clinically suspected to have haemolytic anemia. The gel card picked up the antibodies in all the cases detected to be positive by the conventional test. In addition, the gel card also picked up 5 tests which were negative by the conventional method. The sensitivity and specificity of the gel card Direct Coomb's test (DCT) as compared to the conventional tube test for DCT was found to be 100% and 95.1% respectively. The Indirect Coomb's test (ICT) was 100% sensitive and 92.5% specific. In view of the high sensitivity and specificity and the simplicity of the procedure, this test may be effectively used for diagnosis of AIHA.
On 90 samples of amniotic fluid coming from 54 cases of Rh-isoimmunization submitted to amniocentesis one or more times, the following tests were done: 1.) Coombs indirect test; 2) immunoelectrophoresis; 3) quantitative determination of immunoglobulins. The results were related to the degree of immunization as determined by Coombs indirect test on the mothers' serum and spectrophotometric curve of the amniotic fluid. For reasons of comparison, quantitative determination of immunoglobulins were performed on samples of amniotic fluid from 39 pregnant non-immunized patients. It was found that there was a direct relationship between antibody titers in the mothers' serum and those in the amniotic fluid, with the latter values always being inferior. Moreover, it was found that the level of IgG in the amniotic fluid and the degree of maternal immunization were proportional, with higher titers in the more severe cases. In addition to the routine tests, the determination of anti-Rh antibodies and titers of IgG in amniotic fluid can be useful in further evaluating degree of immunization in Rh-incompatibility. Furthermore, small quantities of IgA were found in amniotic fluid: these IgA were probably of secretory type (SIgA) and of amniotic origin, and therefore independent of any active immunization. Finally, all determinations of IgM were negative.
We report the first results of a prospective study concerning antibody screening in transfusion recipients. We compared the standard tube test (Liss Coombs) with two commercial tests for the detection of red cell IgG antibodies: (1) a column/agglutination test and (2) a microplate/solid-phase system. The results of the study demonstrate several significant advantages of the two methods as compared with the standard tube test. The new methods, especially the microplate test, are superior in determining red cell antibodies which are relevant to transfusion. The two methods are more sensitive and, when automated, more efficient and safer as compared with the standard tube test.