Study of the naturally occurring radionuclide Beryllium-7 (Be-7) in Hong Kong.

Beryllium-7 (Be-7) is one of the naturally occurring radionuclides being monitored under the Global Atmosphere Watch Programme of the World Meteorological Organization. Be-7 mainly originates from cosmic rays. It can be used as a tracer to facilitate understanding of the atmospheric vertical transport by observing its spatial and temporal distribution characteristics. The Hong Kong Observatory has been operating an environmental radiation monitoring programme for decades, and long record of Be-7 activity concentration data in airborne particulate samples are available to characterize the behaviour of Be-7 in the lower atmosphere in Hong Kong. In this study, Be-7 activity concentration data of airborne particulates collected at three locations in Hong Kong from 1993 to 2020 are examined. Temporal variations are analyzed. In particular, the long-term monthly average Be-7 activity concentrations are found to be most sensitive to precipitation. The relevant data analysis, as well as key factors affecting the Be-7 activity concentrations in the lower atmosphere in Hong Kong, will be described.

A young woman with hypogonadism, hypertension and hypokalaemia.

We report a case of a 16 years old girl who presented sequentially with primary amenorrhoea, hypertension and hypokalaemia. Eight years later, she was finally diagnosed with 17alpha-hydroxylase deficiency congenital adrenal hyperplasia. Previous antihypertensive medications were stopped. Hydrocortisone alone successfully maintained normotension and normokalaemia.

A young woman with hypogonadism, hypertension and hypokalaemia

We report a case of a 16 years old girl who presented sequentially with primary amenorrhoea, hypertension and hypokalaemia. Eight years later, she was finally diagnosed with 17alpha-hydroxylase deficiency congenital adrenal hyperplasia. Previous antihypertensive medications were stopped. Hydrocortisone alone successfully maintained normotension and normokalaemia.

Cytokine release in febrile non-haemolytic red cell transfusion reactions.

BACKGROUND AND OBJECTIVES: The aim of this study was to elucidate the role and identity of cytokines involved in febrile non-haemolytic red cell transfusion reactions (FNHTRs). MATERIALS AND METHODS: Eighty-one patients experiencing transfusion reactions after receiving packed red blood cells (RBCs) were divided into three groups, as follows, based on the reaction experienced: FNHTRs (n = 60); chills without fever (n = 8); and allergic reaction with urticaria (n = 13). The concentrations of interleukin (IL)-1beta, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha were measured in the packed transfused unit and patients' plasma by using enzyme immunoassays. Wilcoxon's matched-pairs signed test was used to compare the difference in cytokine levels in patients' plasma before and after transfusion. The Kruskal-Wallis test was used first, followed by the Mann-Whitney test, to compare the pretransfusion cytokine levels in patients' plasma between groups and to compare the cytokine levels in packed RBCs transfused to each group of patients. RESULTS: The age of the implicated packed RBC was 11.5 +/- 5.7 days. Significant increases were observed in IL-6 (P < 0.001) and IL-8 (P < 0.001) patients' plasma levels, but not in IL-1beta or TNF-alpha levels, in those patients exhibiting FNHTR. No changes were observed in the patients' plasma samples of the other groups. Cytokine levels in the RBC concentrate supernatants were not appreciably elevated. CONCLUSIONS: Transfusion of packed RBCs may significantly increase intravascular levels of IL-6 and IL-8 in patients with FNHTRs.

Influence of gamma irradiation and storage on apheresis platelets.

BACKGROUND AND PURPOSE: Gamma irradiation of platelet concentrates to prevent graft-versus-host disease may inactivate contaminated lymphocytes and subsequently inhibit the synthesis of cytokines in the apheresis platelets during storage. We investigated the influence of irradiation and storage on apheresis platelets collected with the COBE Spectra or Fenwal CS-3000 Plus systems. METHODS: Eleven units of apheresis platelets were collected with a COBE Spectra cell separator and another 11 units with a Fenwal CS-3000 Plus system. Each unit of apheresis platelets was divided into two equal parts: one was irradiated with 3000 cGy directly after blood donation, and the other served as a control. Cell counts, platelet activation marker CD62 antigen, blood gas values, and supernatant concentrations of K+, Na+, lactate, glucose, interleukin-1 beta (IL-1 beta), IL-8, and tumor necrosis factor-alpha (TNF-alpha) were determined in paired samples on the day of collection (day 0) and after 5 days of storage (day 5). RESULTS: No significant differences in white cell counts or TNF-alpha concentrations were noted between the irradiated and control platelets on day 0 or day 5, whereas the mean proportion of platelets expressing CD62P (22.65% vs 25%, p = 0.014) and the mean IL-1 beta (45.55 pg/mL vs 52.75 pg/mL, p = 0.004) and IL-8 concentrations (10.68 pg/mL vs 13.07 pg/mL, p = 0.015) were significantly lower in irradiated than control platelets on day 5. The 5-day storage significantly increased the mean proportion of platelets expressing CD62P (25.00% vs 15.02%, p = 0.008), mean PO2 (116.34 mm Hg vs 98.07 mm Hg, p = 0.002), and mean concentrations of K+ (3.30 mmol/L vs 3.06 mmol/L, p < 0.001), lactate (15.12 mmol/L vs 3.23 mmol/L, p < 0.001), IL-1 beta (52.75 pg/mL vs 29.73 pg/mL, p = 0.001), and IL-8 (13.07 pg/mL vs 3.62 pg/mL, p < 0.001). Five-day storage also significantly decreased white cell count (0.18 x 10(8) vs 0.74 x 10(8), p < 0.001), PCO2 (19.38 mm Hg vs 50.51 mm Hg, p < 0.001), and concentrations of HCO3- (10.36 mmol/L vs 21.34 mmol/L, p < 0.001) and glucose (193.37 mg/dL vs 309.18 mg/dL, p < 0.001). Platelet counts and concentrations of IL-1 beta, IL-8, and TNF-alpha on day 0 did not differ significantly between control apheresis platelets collected with the Fenwal CS-3000 Plus and those collected with COBE Spectra. The mean white cell count (1.29 x 10(8) vs 0.19 x 10(8), p = 0.002) and the proportion of platelets expressing CD62P (24.71% vs 7.09%, p < 0.001) on day 0, however, were significantly higher in the platelets collected with the Fenwal CS3000-Plus than in those collected with the COBE Spectra. CONCLUSIONS: Gamma irradiation of apheresis platelets inhibits the expression of platelet CD62P and the secretion of IL-1 beta and IL-8 after 5 days' storage.

Allogeneic peripheral blood stem cell transplantation and early detection of donor engraftment by polymerase chain reaction.

Patients with hematologic malignancy or severe aplastic anemia after myeloablative chemo- and radiotherapy were given granulocyte colony-stimulating factor (G-CSF)-mobilized, cryopreserved allogeneic peripheral blood stem cells (PBSCs) from 15 healthy donors who were either human leukocyte antigen (HLA)-matched siblings (n = 13) or haploidentical offspring (2). Polymerase chain reaction-amplified short tandem repeat genotyping was used for early confirmation of donor engraftment after PBSC transplantation (PBSCT). A standard cyclosporine A/methotrexate combination was used to prevent acute graft-versus-host disease (GVHD). All donors, including one in the third trimester of pregnancy, tolerated G-CSF administration and 3-day PBSC harvesting procedures well. Engraftment was prompt for all patients; it was verified using a panel of 12 human polymorphic short tandem repeat loci from bone marrow as early as 7 days posttransplantation. This status was maintained until relapse, when mixed chimerism was detected using the polymerase chain reaction. A minimum resurgence of recipient cells to 1% of the population was required to detect chimerism. The median times to recovery of the absolute neutrophil count to greater than 0.5 x 10(9)/L and the sustained platelet count to greater than 20 x 10(9)/L without transfusion were 10 and 12 days after PBSCT, respectively. Six patients experienced acute GVHD, Grade I in two patients and Grade II in four, including two HLA-haploidentical recipients. Chronic GVHD was noticed in three of the 11 patients who were followed for at least 100 days after PBSCT. Ten patients were still alive at the latest follow-up and have been disease free for a median of 278 days (range 60-671). Five patients died from causes other than graft failure: three from leukemia relapse and two from transplant-related complications. The results confirm that G-CSF can be safely administered to healthy donors and that engraftment after allogeneic PBSCT is fast and durable. Complete chimerism can be detected early by genomic analysis. PBSCT may offer an alternative to bone marrow transplantation.

Polymorphisms of twelve short tandem repeat loci in a Taiwanese population and their application in parentage testing.

With the advancement of techniques in molecular biology, rapid, sensitive, and reliable methods of DNA typing for parentage testing have become available. In this study, we evaluated the usefulness of multiplex polymerase chain reaction (PCR) with 12 unlinked short tandem repeat (STR) loci for paternity testing in Taiwan. The genetic informativeness of this test was then compared with that of conventional human leukocyte antigen (HLA) analysis in 167 parentage studies. The 12 STR loci alone provided a cumulative power of exclusion of up to 0.9998. Paternity was excluded in 59 (35.3%) cases, including 40 of 112 paternity trios and 19 of 55 paternity duos. In the 40 trios in which paternity was excluded, a mean of 6 (range, 3-9) incompatible STR markers were in the 19 duos in which paternity was excluded, a mean of 4 (range, 1-8) incompatible STR markers were noted. In the 72 trios in which the alleged paternity could not be excluded, the mean probabilities of paternity (PP) were 90.6863% with HLA testing alone, 99.9847% with STR analysis alone, and 99.9972% with combined HLA and STR analysis. In the 36 duos in which the alleged paternity could not be excluded, the mean PPs were 81.4768% with HLA testing alone, 99.6124% with STR analysis alone, and 99.9145% with combined HLA and STR analysis. These results suggest that STR analysis is very powerful when used alone for paternity trio testing and when combined with conventional serologic HLA typing for duo parentage testing in the Taiwan population.

Factors affecting progenitor cell yields using three tandem leukaphereses in previously treated malignancies.

BACKGROUND: Mobilized peripheral blood progenitor cells (PBPCs) have increasingly been used to replace autologous bone marrow to allow faster hematopoietic reconstitution after myeloablative therapy in various malignancies. There is a paucity of data concerning factors that affect the total yield of three tandem leukaphereses. METHODS: Factors affecting the yield of PBPCs were analyzed in a series of 121 consecutive patients including 36 with non-Hodgkin's lymphoma, two with Hodgkin's disease, four with multiple myeloma, 44 with acute leukemia, 20 with breast cancer and 15 with other solid tumors. PBPCs were mobilized using granulocyte-colony-stimulating factor (G-CSF) alone (group I, n = 15), or after conventional-dose (group II, n = 70) or high-dose (group III, n = 36) chemotherapy followed by G-CSF. The total yield of three tandem PBPC collections for each patient was assessed by the number of mononuclear cells (MNCs), CD34+ cells and colony-forming units of granulocyte macrophages (CFU-GM). The factors evaluated included age, sex, diagnosis, history of marrow involvement, previous radiotherapy, the number of prior chemotherapy cycles and mobilization method. The two -sample t-test and logistic regression analysis were performed for univariate and multivariate analysis, respectively. RESULTS: With univariate analysis, a diagnosis of acute leukemia, positive history of bone marrow involvement, more chemotherapy cycles and mobilization with high-dose chemotherapy adversely affected the yields of CD34+ cells. By multivariate analysis, Group II had higher yields of MNCs (p = 0.039), CFU-GM (p = 0.002) and CD34+ cells (p = 0.011) than Group III. Fewer cycles of prior chemotherapy is the common favorable factor for the yields of both CD34+ cells (p = 0.016) and CFU-GM (p = 0.017). CONCLUSIONS: The number of prior chemotherapy cycles adversely affects progenitor cell yield. Conventional-dose chemotherapy followed by G-CSF seems to be the mobilization methods of choice for heavily pretreated cancer patients with limited bone marrow reserve. PBPCs should be harvested early, when the tumor burden is less, to avoid cumulative marrow toxicity from chemotherapy.

Transfusion of donor peripheral blood buffy coat cells as effective treatment for relapsed acute leukemia after transplantation of allogeneic bone marrow or peripheral blood stem cells from the same donor.

BACKGROUND: The transfusion of same-donor peripheral blood buffy coat (PBBC) cells to chronic myelocytic leukemia patients in relapse after bone marrow transplantation has been increasingly used as an effective antileukemic therapy. A graft-versus-leukemia effect mediated by immunocompetent donor T cells underlies its success. In acute leukemia, however, the effect of this adoptive cellular immunotherapy has not been established, and the results are generally poor. STUDY DESIGN AND METHODS: Five patients, three with acute lymphoblastic leukemia and two with acute myelocytic leukemia, who relapsed within 6 months after allogeneic marrow transplantation were enrolled in a nonrandomized pilot study to receive donor PBBC cell transfusions either before or after undergoing cytoreductive chemotherapy. ABO genotyping and polymerase chain reaction amplification of human tetrameric short tandem repeats DNA typing were used to test for marrow chimerism. RESULTS: Two acute lymphoblastic leukemia patients-both of whom underwent chemotherapy before PBBC cell transfusions, experienced a complete remission, and developed acute and then chronic, extensive graft-versus-host disease-have been leukemia-free for 9 and 7 months, respectively. Repeated molecular studies of their marrow as early as 2 weeks to 8 months after treatment confirmed that the marrow was of donor origin. The other three patients, who chose not to undergo chemotherapy before PBBC cell transfusions, failed to achieve remission and died 14, 16, and 30 days, respectively, after leukemia relapse. CONCLUSION: Adoptive cellular immunotherapy may be effective for acute lymphoblastic leukemia patients in relapse after bone marrow transplantation if chemotherapy is administered before PBBC cell transfusions are initiated.

Allogeneic peripheral blood progenitor cell transplant after bone marrow graft failure: report of a case.

A 31-year-old woman diagnosed with acute myelocytic leukemia received an allogeneic peripheral blood progenitor cell (PBPC) transplant one month after a previous bone marrow graft failed. PBPCs were mobilized with granulocyte-colony-stimulating factor and collected by apheresis. T-cell depletion was not performed and no further chemo- or radiotherapy was given for the second transplant. Engraftment was prompt, with the peripheral blood leukocyte count rising dramatically to 2,400/microL, six days after completion of PBPC transplant. The platelet count reached 36,000/microL on the eighth day and was self-sustained thereafter. Both blood grouping and bone marrow karyotyping confirmed donor origin of the engraftment. At the time of writing, the patient has been disease-free for over 200 days without any complications of acute or chronic graft-versus-host disease.

Varicella zoster virus infection after allogeneic or autologous hemopoietic stem cell transplantation.

A retrospective study was carried out in 161 patients who underwent allogeneic or autologous hemopoietic stem cell transplants. The aim was to determine the frequency, outcome and risk-factors associated with varicella zoster virus (VZV) infection. Post-transplant VZV infection occurred in 29 patients. The median onset of infection was 6.5 months post-transplant, with 82% of cases occurring within the first year. Localized herpes zoster was seen in 27 patients, one patient had varicella, and one patient had simultaneous presentation of both herpes zoster and varicella. No cutaneous or visceral dissemination was noted in the series. Each patient was treated with intravenous acyclovir. Mild complications with postherpetic neuralgia were reported by three patients. There were no deaths from VZV infection. Two risk factors noted to be associated with VZV infection were the presence of graft-versus-host disease in allogeneic transplants and leukemia as the underlying disease in autologous transplants. The overall incidence of post-transplant VZV infection in the present series was relatively low compared with that of other reports involving either allogeneic or autologous bone marrow transplantation.

[Hemolytic disease of the newborn caused by maternal anti-Di(a): a case report in Taiwan].

The first case of hemolytic disease of the newborn (HDN) possibly caused by anti-Di(a) in a Chinese infant in Taiwan is reported. The mother had two pregnancies before but no history of blood transfusion. Her first male infant was normal, but her second full-term male one developed mild jaundice soon after birth, and the total bilirubin level was 12.1 mg/dL, 18.3 mg/dL, 23.6 mg/dL at 24 hours, 48 hours, and 72 hours of age, respectively. Total bilirubin was 9.1 mg/dL on the eighth day after receiving phototherapy and compatible blood exchange transfusion. The infant recovered uneventfully. The immunohematological study revealed that the mother was group AB, Rh (D)+; Di(a - b+), the father was group O, Rh (D)+; Di(a + b+), the infant boy and his 2-year-old brother were group B, Rh(D)+; Di(a + b+). The direct antiglobulin test (DAT) on the infant red cells was positive (4+ with polyspecific AHG; 4+ with anti-IgG). The maternal serum and infant's eluate from red blood cells showed negative reactions in routine antibody detection tests, but they contained alloantibody reacting against the Di(a+) cells by the manual polybrene test (MP) and indirect antiglobulin test (IAT) in AHG phase. The anti-Di(a) titers in the mother's serum was MP 1:256 and AHG 1:256, and in the infant's eluate was MP 1:128 and AHC 1:64 against Di(a + b+) cells. Based on the above results we conclude that the jaundice in this newborn baby was caused by maternal anti-Di(a) which was most likely induced by previous pregnancy. In conclusion, Diego blood group is a system of high value in anthropology because it accounts for the Mongoloid origin of American Indians, Japanese and Chinese. Anti-Di(a) may cause HDN, as in our case of HDN due to maternal anti-Di(a) in a Chinese infant. But in Europe and America, where practically all people are Di(a - b+) phenotypes, the system seems of no interest in parental studies as well as in blood transfusions. Owing to the Di(a) antigen is of higher incidence in Chinese population, we suggest that the Diego system should be involved in routine compatibility testing or antibody identification problems in parental studies and in blood transfusions in Taiwan.

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.

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.

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.

Purification and characterization of human macrophage-derived granulomonopoietic enhancing factor (GM-EF).

A granulomonopoietic enhancing factor (GM-EF) capable of promoting the effect of colony-stimulating factors (CSFs) on myeloid progenitor cells has been purified to homogeneity from serum-free medium conditioned by fully mature human macrophages. GM-EF was a glycoprotein with an apparent molecular weight of 74 kd and an isoelectric point of 5.2-5.3. The purified protein was heat stable (75 degrees C for 30 min) and was sensitive to treatment with trypsin, papain, and bacterial protease but not to neuraminidase. The activity of GM-EF could be effectively neutralized by GM-EF-specific antiserum, and no antigenic cross-reactivity was observed using antisera against interleukin (IL)-1, IL-4, and IL-6. These results suggest that GM-EF is a unique cytokine that is different biochemically and antigenically from other hematopoietic enhancing factors such as IL-1, IL-4, and IL-6.

Bone marrow transplantation for severe aplastic anemia--a study of twenty-one Chinese patients in Taiwan.

A total of 21 multiply transfused patients with severe aplastic anemia (SAA) were treated with bone marrow transplantation between March 1985 and September 1990: 20 allogeneic and one syngeneic transplants. A positive response in mixed lymphocyte culture (MLC) was also noted in 7 allogeneic recipients. Pregraft conditioning included high-dose cyclophosphamide (CY) 200 mg/kg over 4 consecutive days, followed by 300 cGy total-body irradiation the day before BMT. Seventeen patients older than 14 years received additional donor buffy-coat cells infusion for 5 days posttransplant. A combination of methotrexate and cyclosporine was used for prophylaxis of graft-versus-host disease. Seventeen patients were alive with a functional graft, and Kaplan-Meier product limit estimates showed a 80.95% probability of survival at 67.7 months. There were 4 deaths: two died of primary graft failure, one from secondary rejection, and the other from chronic GVHD-related complications. Acute GVHD, grade I was noted in only one patient (5.6%). In contrast, chronic GVHD was observed in 10 out of 18 (55.6%) evaluable patients. Venoocclusive liver disease and interstitial pneumonitis were not diagnosed. Our findings indicate that the combination of CY/TBI/BC is well tolerated and results in a low incidence of graft failure/rejection in multiply transfused Chinese patients who received transplants for SAA. The MTX/CsA combination was confirmed as being remarkable in reducing the incidence and severity of acute GVHD. For patients with SAA under the age of 40, with an HLA-identical sibling, we highly recommend BMT as the treatment of choice.

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.