Anti-Nform antibody in hemodialysis patients.

The anti-Nform antibody is produced by dialysis patients following reuse of dialyzers sterilized with formaldehyde and it has been implicated as a cause of hemolytic anemia. Formaldehyde is one of the common disinfectants used for reprocessing capillary hemodialyzers. The safety of formaldehyde and the clinical significance of anti-Nform antibody need further evaluation. Amongst 45 patients practising dialyzer reuse, anti-Nform antibody was detected in 5 (11.1%), but not amongst 111 patients not reusing their dialyzer (p < 0.005). The presence of anti-Nform was not related to the sex, or duration of dialysis with positive anti-Nform antibody. Direct Coombs' test was positive amongst 80% of all tested patients with anti-Nform antibody, and in 38% of patients reusing dialyzers but without anti-Nform antibody. No tests of hemolysis (including direct Coombs' test) discriminated between anti-Nform antibody-positive and -negative patients, nor between anti-Nform antibody patients with and without overt hemolysis. The best diagnostic test for hemolysis in anti-Nform antibody-positive patients was hematocrit rise after cessation of dialyzer reuse. It appears that despite the induction of anti-Nform antibody, hemolysis is rarely a serious consequence of dialyzer reuse.

Recombinant human granulocyte colony-stimulating factor infusion and/or autologous peripheral blood stem cell transplantation as a rescue for sequential high-dose combination chemotherapy: a preliminary report.

BACKGROUND: Sequential cycles of combination chemotherapy with high-dose cyclophosphamide, etoposide and cisplatin (sHDCEP) can largely increase the total dose (TD) of drug delivered. If granulocyte colony-stimulating factor (G-CSF) and/or autologous peripheral blood stem cell (PBSC) rescue can shorten the duration of cytopenia between cycles of sHDCEP, the dose intensity (DI) can be increased as well. In order to explore the feasibility of delivering maximal TD and DI by administration of sHDCEP with G-CSF and/or PBSC rescue, this trial is undertaken to investigate the hematologic and nonhematologic toxicity observed with sHDCEP by G-CSF and/or PBSC rescue. METHODS: Patients with refractory malignancy and well preserved physiologic function for whom no available therapy is likely to cure or prolong the survival were eligible for the study. Each cycle of high-dose chemotherapy consisted of: cyclophosphamide 5,000 mg/m2, etoposide 1,500 mg/m2 and cisplatin 150 mg/m2. G-CSF and/or PBSC were administered alternatively after each cycle as rescue for myelosuppression. The next cycle was given to patient who showed response to the previous cycle after recovery from toxicity for a maximal of 4 cycles. RESULTS: Two cases of refractory malignancy with progressive disease were treated by sHDCEP for 7 cycles, including 4 cycles with G-CSF rescue, 2 cycle with PBSC rescue, and 1 cycle with G-CSF + PBSC rescue. In the 4 cycles rescued by G-CSF alone, we observed a slightly slower granulocyte and markedly prolonged platelet recovery in the subsequent cycle. By comparing the effect of G-CSF and/or PBSC rescue on hematologic recovery with the preceding cycle in the same patient, we found that G-CSF rescue provided faster granulocyte recovery than PBSC, but PBSC rescue provided faster platelet recovery than G-CSF. Rescue by larger number of PBSCs provided only faster platelet but not granulocyte recovery than rescue by adding G-CSF to a very small number of PBSCs. However, G-CSF plus the very small number of PBSCs provided shorter duration of both granulocytopenia and thrombocytopenia than rescue by G-CSF alone. The most common nonhematologic toxicity from sHDCEP included transient nausea, vomiting, diarrhea and mild impairment of liver function but we observed no significant or irreversible major organ damage. The side effect from PBSC collection was mild and toxicity from reinfusion of the thawed PBSCs was not obvious. Using G-CSF and/or PBSC rescue, sHDCEP was delivered repeatedly in no more than 4 weeks for the next-cycles except for patient 1 who had cycle 4 delayed because of prolonged platelet recovery by only G-CSF rescue in cycle 3. CONCLUSIONS: Our initial experience has shown that the nonhematologic toxicity from sHDCEP, G-CSF and PBSC rescue was well tolerated. Prolonged platelet recovery after sequential cycles of HDCEP by only G-CSF rescue delayed the next cycle of chemotherapy. Although the next cycle was delivered within 4 weeks by only PBSC rescue, concurrent infusion of G-CSF and larger number of PBSCs should provide the most rapid hematologic recovery. Sequential high-dose chemotherapy administered by this model is likely to provide the maximal delivery of TD and DI, and is worthy of further clinical trials.

Do the reticulocyte maturation fractions and bone marrow reticulocyte count further help the classification of anemias?

BACKGROUND: Reticulocyte count plays a major role in anemic evaluation. The conventional method done by the manual supravital staining cannot subclassify the group of less than 2% of corrected reticulocyte count. The newly-developed flow cytometer provides different maturation fractions by measuring its fluorescent intensity. The reticulocytes are believed to shift to the circulation from the bone marrow earlier in more severe anemia. Therefore, the purpose of this study is to evaluate the role of reticulocyte maturation fractions and bone marrow reticulocyte in anemia classification. METHODS: By using a fully automated counter, the roles of the reticulocyte with maturation and their shifting from bone marrow were evaluated in anemias. Different groups of subjects (243 in total) including aplastic, nutritional, and infiltrative anemias and anemia due to excess destruction and blood loss were studied. Each subject had bone marrow examination for morphologic diagnosis and reticulocyte evaluation. RESULTS: Both the absolute count and the maturation fractions of reticulocytes showed significant difference among marrow infiltration, aplastic anemia, and hemolytic anemia. Both the absolute reticulocyte count and less mature fractions were lowest in aplastic group. The marrow reticulocyte counts and shift ratio to circulating blood added little benefit in the classification of anemias. CONCLUSIONS: The automated reticulocyte count with maturation fractions helps classify anemias, particularly for those with low reticulocyte count by the manual method.

[A rapid and simple method for direct genotype determination of ABO blood group].

We report the use of an allele-specific polymerase chain reaction (ASPCR) format together with low melting temperature agarose gel electrophoresis which allows rapid identification of the six major genotypes of the ABO blood group. Four sequence specific primer sets, each specific for a different set of ABO alleles, were used. Twenty individuals, whose ABO genotypes were previously determined by serological and family analysis, were typed with this new approach. A 100% correlation between serology and the ASPCR was found. This method is rapid, simple, and reproducible. Potential applications include identification of ABO subgroups and variants, paternity testing, as well as forensic science.

Red cells, HLA and platelet antibody formation in patients with multiple transfusions.

Totally 436 Chinese patients having received multiple transfusions of red cells and platelets on more than three occasions were screened for red cell antibodies. Twenty-six (6%) of them were positive. Anti-E, -Mia, and -c were the common alloantibodies. Nine patients were immunized during the period of regular transfusions, with a newly immunized rate of 2% (9/436). Among 436 patients, 387 were screened for HLA antibodies by lymphocytotoxicity test (LCT). The overall positive rate was 35%. Most of the antibodies identified were against the high-frequency HLA antigens in the Chinese population. About 10% of the LCT-positive cases reverted to negative state during the follow-up period. After chloroquine stripping of the target platelets, mixed passive hemagglutination assay was used to detect platelet antibodies other than HLA antibody. Fifty-eight of 161 cases (36%) were positive for platelet antibodies, but half of them disappeared within 1 month. Nineteen of the 58 patients had sepsis and 2 had jaundice. These findings suggested that HLA and platelet antibodies are common among Chinese, though their clinical significance and the role in platelet damage are doubtful.

Incidence of post-transfusion hepatitis before and after screening for hepatitis C virus antibody.

To evaluate the effectiveness of screening test for antibody to hepatitis C virus (anti-HCV), the incidence of acute post-transfusion HCV infection in patients who underwent cardiovascular surgery and received blood transfusion was studied. All patients were followed prospectively with serum biochemistry tests and viral hepatitis markers before and periodically for at least 6 months after cardiovascular surgery. None of them had history of liver disease and none tested positive for anti-HCV prior to blood transfusion. Before blood donors were screened for anti-HCV with a second-generation HCV diagnostic kit, 28 (12.4%) of 226 patients or 0.49% of 5,690 unit transfusion had seroconverted to anti-HCV during a 6-month follow-up. The incidence of post-transfusion hepatitis (PTH) C in 91 patients who had received 1-12 units transfusion was significantly lower than in 135 patients who had received more than 12 units transfusion (6.6 vs. 16.3%, p < 0.05). However, none of the 87 transfused patients, since anti-HCV screening in July 1992, developed PTH C (p < 0.05). The result demonstrates that screening for anti-HCV by a more sensitive second-generation HCV diagnostic assay may protect the patients studied from PTH C. It further provides a firm argument for the necessity of a nation-wide blood donor screening.

HLA antigen and antibody association in platelet-alloimmunized patients.

The frequencies of HLA antigens were compared between 55 patients with and 38 patients without lymphocytotoxic antibodies formation after long-term platelet transfusions. Only HLA-B60 and B75 were found to manifest significant difference between these two groups. Patients with HLA homozygosity had a higher incidence of alloimmunization. Although most of the platelet alloantibodies were against HLA antigens of high frequency, the HLA-antibodies were induced at a rate different from the frequency of their corresponding antigens. The antibodies against the first and second HLA loci are of similar frequencies. In conclusion, the patients with HLA homozygous alleles have a higher incidence of platelet alloimmunization, and the antibody of certain specificities has higher rate of occurrence. These findings may be helpful in platelet-donor selection.

Resistance to erythropoietin: immunohemolytic anemia induced by residual formaldehyde in dialyzers.

Because of inadequate renal synthesis of erythropoietin, the anemia associated with chronic renal failure has been treated successfully in most patients on hemodialysis with recombinant human erythropoietin. Hemolysis due to anti-Nform antibody in dialysis patients with the reused dialyzer may be one of the factors that cause refractoriness to erythropoietin therapy. Patients who do not respond to erythropoietin administration should be screened for anti-Nform antibody.

HLA antibodies in multiple transfused patients.

Totally 86 patients with multiple transfusions were followed up regularly for more than six months. Their sera were screened for HLA antibodies every two to four weeks. Subsequently 28 (33%) of them developed HLA antibodies. The specificities of identified antibodies were anti-A2, All, B16 and B60. However, most of the antibodies were either multiple or undetermined. The panel reactive activity (PRA) was correlated with the amount of blood components transfused. We concluded that about one third of the patients will develop HLA antibodies after multiple transfusions and the reactivity of the antibodies is correlated to the amount of blood components transfused.

Evaluation of anti-HTLV-1 in physical-checkup individuals and blood donors in Taiwan.

The gelatin particle agglutination (GPA) test specific for antibody detection of human T-cell lymphotropic virus type 1 (HTLV-1) was used to screen 500 blood donors and 5000 physical-checkup individuals at Veterans General Hospital-Taipei. The positive rate of physical-checkup individuals and the blood donors was 0.18% (9/5000) and 0% (0/5000) respectively. Among the 9 GPA positive specimens, eight were confirmed to be positive by western blot analysis and a prevalence rate of 0.16% (8/5000). Seven of the nine GPA positive samples were also positive by indirect fluorescent antibody test and two of them had indeterminate results. Since GPA is less expensive, relatively simple and convenient, we recommend that GPA could be used as screening test for HTLV-1 infection of blood donors, followed by western blot method as a confirmatory test in blood bank.

Imported kala-azar: a case report.

A 77-year-old man returned from Honan Province, mainland China, and developed intermittent fever and loss of body weight. On physical examination there was evidence of chest infection but no lymphadenopathy or hepatosplenomegaly. Laboratory data suggested anemia, thrombocytopenia and polyclonal gammopathy. The diagnosis of Leishmaniasis was finally established by bone marrow aspiration which disclosed Leishman-Donovan bodies. Unfortunately, the patient expired soon after the diagnosis was made and a partial autopsy was performed. Kala-azar is a rare disease in Taiwan. However, it should be suspected in those patients who have visited the endemic areas, even though the clinical manifestations are atypical.

Detection of antibodies to human immunodeficiency virus (HIV-1) with the particle agglutination test.

This study was designed to evaluate the sensitivity and specificity of the particle agglutination test (PA) for antibody detection and to determine whether it is possible to use this test as a screening routine for anti-human immunodeficiency virus type I antibodies instead of the enzyme linked immunosorbent assay (ELISA). Serum samples were collected from 5,142 subjects and tested with the above two tests. A total of 83 samples were observed to be ELISA-positive. However, only 36 were found to be seropositive by the PA test. These samples were then confirmed by the Western blot analysis and 28 were positive for HIV-1, 33 were negative and 22 gave indeterminate results. The most common indeterminate band was p15/17 and the p24 band came next. The sensitivity and specificity of the PA test were 100% and 99.9%. The results indicate that the PA test is not only a convenient technique but also has a high sensitivity and specificity. It can be used as a primary screening test for antibody against HIV-1 instead of the ELISA.

HLA antibodies--the cause of platelet alloimmunization in Chinese.

Lymphocytotoxicity test (LCT) and platelet suspension immunofluorescence test (PSIFT) were used together to screen platelet-associated antibodies in patients who received long-term platelet transfusion. Twenty-four of 53 patients (45.3%) were immunized subsequently. Since the concordance of LCT and PSIFT was 100%, most of the platelet associated antibodies were of HLA specificity, and platelet specific antibody alone (in absence of HLA) was not detected. The identified antibodies were anti-A2, A11, A24, B5, B46, B57, B60, and B62. The majority of them were against the high frequency HLA antigens in the Chinese population. The development of antibody could not be correlated with the number of platelet-donors exposed, the time interval after the initiation of platelet transfusion, or the percentage of reactive lymphocytotoxic panels. HLA antibody was the major factor in causing platelet alloimmunization in the Chinese patients. However, some other unknown factors should be looked for. In addition, ABO incompatibility did not affect the posttransfusional increment while the platelet was compatible with LCT crossmatching.

Antigen expression of frozen platelets.

Platelet antigens of platelet samples from 36 donors, frozen for different intervals, were evaluated by the platelet suspension immunofluorescence test (PSIFT). A, B, PLA1(HPA-1a) and various HLA antigens were tested by their corresponding antisera. The antigen could be detected in almost all the samples after one month of freezing. After 3 and 6 months, the platelet antigens could only be detected in 29.2% and 3.7% of the samples, respectively. There was no difference in decay of antigen expression among A, B, PLA1 and HLA antigens. When compared with the freshly prepared platelets, frozen platelets presented stronger antigen expression after 2 to 4 weeks of storage. This may suggest that the frozen platelets could be used for platelet crossmatching procedures without loss of their antigenicity within one month.

Serological identification of HLA-B13 subtypes.

A serological approach is used to confirm subtypes of HLA-B13 originally observed by one-dimensional isoelectric focusing (1D-IEF). Sixty anti-B13 alloantibody sera were screened against Chinese panel cells. Two clusters of sera showing distinct reactive patterns were identified. One is a shorter reactive pattern than the other. Using these serological reaction patterns, the B13 antigen can be divided into two subtypes, B13.1 and B13.2, in the Chinese population. These serological subtypes appear to correlate well with the 1D-IEF patterns of B13 subtypes. The serological subtyping is also in agreement with the differences in nucleotide sequence previously determined to exist in B13 antigen subtypes. Family studies show that both B13.1 and B13.2 segregate as HLA-B locus alleles. Gene frequencies for B13.1 and B13.2 were 0.0676 and 0.0612, respectively, in our study population of 337 southern Han Chinese.

Neonatal alloimmune thrombocytopenia due to HLA-A2 antibody.

A male, full-term baby with thrombocytopenia was born by a G3P2A1 mother who was not associated with autoimmune disease. Platelet antibody screening was positive by using lymphocytotoxicity test, platelet suspension immunofluorescence test and solid-phase red cell adherence test. The identified HLA antibody was of A2 specificity. It was confirmed by testing the mother's and the baby's sera against the lymphocytes and platelets of 10 HLA-A2-positive donors. The possibility of platelet-specific antibody as the cause of neonatal alloimmune thrombocytopenia was ruled out by testing against platelets of 10 HLA-A2-negative donors and the known platelet-specific antigens utilizing immobilized, purified platelet glycoprotein as targets. The mother's serum reacted strongly with both the father's and the baby's platelets and lymphocytes. This neonatal thrombocytopenia was most likely due to the maternal HLA antibody, which was induced by her antecedent gestations.

HIV, HBV and HCV seropositivity in hemophiliacs.

Eleven cases of severe type hemophiliacs who had received long-term factor VIII injections were tested for the serological markers of human immunodeficiency virus (HIV), hepatitis B virus and hepatitis C virus (HCV). The period of factor VIII concentrate injections ranged from 2 to 32 years. The seropositive rates of HIV and HCV were 9/11(82%) and 11/11(100%), respectively. The seropositive rate of hepatitis B surface antigen was only 1/11(9%), while the seropositive rates of antibody to hepatitis B core antigen and antibody to hepatitis B surface antigen were 9/11(82%) and 7/11(64%), respectively, Although the patients had no symptoms related to acquired immunodeficiency syndrome, they were noted to have inverted helper/suppressor T-lymphocyte ratio, suggesting that hemophiliacs with long-term factor VIII injections have a high incidence of HIV and HCV infection, with immunological aberration.

[The property of filamentous hemagglutinin of Bordetella pertussis].

For the purpose to clarifying the biologically active substance of B. pertussis, we prepared the filamentous hemagglutinin (FHA) from culture supernatant of the strain Tohama phase I and purified it through chromatography columns of hydroxylapatite, fetuin-Sepharose 4B and Sepharose CL 6B. There are several bands appeared in the polyacrylamide gel after SDS-PAGE, especially between 98 kD and 210 kD. The amount of 210 kD component is not proportional to hemagglutination (HA) activity of FHA among five different lots. Irons et al. reported that FHA preparation degraded the 220 kD and 210 kD polypeptides during the storage would be reduced the specific HA activity. Our result pointed out that the HA activity of purified Foffdid not relate to 210 kD component only. For the quality control of FHA purity in the B. pertussis acellular vaccine, we suggest that it would be necessary to test both HA titer and SDS-PAGE results.