Hair follicle: a reliable source of recipient origin after allogeneic hematopoietic stem cell transplantation.

Blood, buccal swab and hair follicles are among the most commonly used sources for forensic science, parentage testing and personal identification. A total of 29 patients who have had a sustained engraftment from 15 months to 21.5 years after allogeneic hematopoietic stem cell transplantation (HSCT) without rejection, relapse or chronic GVHD involving oral mucosa were enrolled for a chimerism study. PCR-amplified short tandem repeat analyses were conducted per patient every 3 months for at least three consecutive times. The results for blood were all donor type except one who had a mixed chimerism, 14.5 years after receiving a transplant for lymphoma. As for buccal swab, mixed chimerism ranging from 10 to 96% donor origin was noted for 28 recipients except the one who had mixed chimerism of blood and retained total recipient type. In contrast, hair follicles were 100% recipient type for the entire group. It is concluded that the hair follicle is devoid of adult stem cell plasticity and may serve as a reliable source of recipient's origin when pre-transplant DNA fingerprinting or reference DNA is not available for people who have successfully received allogeneic HSCT while in need of a personal identification.

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.

Novel polymorphisms in exons 6 and 7 of A/B alleles detected by polymerase chain reaction-single strand conformation polymorphism.

BACKGROUND AND OBJECTIVES: The ABO blood group system is the most important blood group system in transfusion medicine. In addition to the major A, B and O alleles, many rare alleles have been defined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and analysis by PCR sequence specific primers (SSP) are commonly conducted for genotyping but have the limitation of being unable to detect unknown substitution(s) in amplified DNA fragments, whereas PCR-single strand conformation polymorphism (SSCP) can be used for both. MATERIALS AND METHODS: Three-hundred unrelated blood donors of the AB phenotype were enrolled. Four pairs of primers were designed to constitute two sets of multiplex PCRs: this amplifies four fragments spanning the entire exon 6 and its immediate flanking regions, nucleotides 432-1065, as well as the 3' untranslated region of exon 7 of the ABO gene. The SSCP electrophoresis was carried out on a 12.5% polyacrylamide gel in a GenePhor electrophoresis unit. For those with unexpected banding patterns, SSCP analyses were performed in duplicate and samples were cloned and sequenced for exons 6 and 7. RESULTS: Seven samples were noted to have six variant alleles, of which five have not been previously reported in the literature. Of these five novel variants, four were derived from the B allele, while the other derived from the A allele. CONCLUSIONS: By using PCR-SSCP, five novel A/B alleles were found.

Confirmation of a recombinant allele B*5603 and a hypothetical reciprocal hybrid.

From its DNA sequence, B*5603 is thought to be a product of gene conversion. We present here serological evidence of such an event and further speculate on a possible reciprocal hybrid yet to be identified. In addition, we report the allelic frequency of B*5603 in the Taiwanese population and its association with A*1101, Cw*01 and DRB1*1201.

[Blood group phenotyping and their application in Taiwan].

During the 1950's and 60's as new blood group systems were identified, antigen distribution studies were performed in Europe and North America among Caucasians and American Blacks. However, to date only limited studies have been performed in Africa and Asia. Because of lack of knowledge of the antigen distribution of most other than ABO and Rho (D) blood group systems within these areas and among the people of non-Caucasian races, questions of testing needs and problems have occurred. In recent years, three big matters have been encountered off and on in blood banking in Taiwan. First, multiple-transfusion recipients develop so many alloantibodies that finding compatible donors becomes a difficult task. Second, since bone marrow transplantation technology is being instituted in many teaching hospitals, it is a task of blood banks to monitor the antigen changes of other blood group systems (including of Rh system other than D) before and after transplantation. Third, more than enough disputed paternity cases that can not be resolved by simple ABO testing. Therefore, blood banks should be staffed with suitable backgrounds to cope with the procedures needed for analysing all blood group antigens. In order to resolve all the problems effectively, we ran the tests for blood group antigens other than ABO and D in our blood bank from 1984 to 1986. A total of 31 sets of antisera were used to identify the specificity of 13 blood group system antigens of the Chinese population ranging from 99 to 2257. Based on the datum obtained, we found a significant difference between Chinese and Caucasians in the distribution of eight blood group systems (Rhesus, MNSs, Kell, Duffy, Kidd, P, Lutheran and Colton). The antigen frequency of Fya and s are 99.74, 99.91 respectively. The results are higher than those of the Caucasian population. On the other hand, Fyb and S are 9.22 and 6.56, much lower than those in Caucasians. We found no K, Lua and Cob antigens among the Chinese. We conclude that this study is a significant contribution to the knowledge of blood group antigen systems and antigen distribution, and also will benefit this population in many areas of medical care.

Alloantibodies other than anti-D in pregnant women.

Alloantibodies other than ant-D in two primigravidas have been reported. There was no history of exposure to risk factors. One of these primigravida developed anti-E and anti-c of IgM specificity and another developed anti-Jkb of IgG specificity. No evidence of hemolytic disease of the newborn was noted in both cases. From the family studies of both cases, the maternal alloantibodies seem to have been induced by the fetal red cell antigens. Although alloantibodies can occur in primigravidas, the incidence is very low. It seems unnecessary to do routine prenatal antibody screening in our population.

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.

Blood group phenotypes in Taiwan.

This report describes a study of 31 red cell antigens in 13 blood group systems tested over a period of 3 years in the Chinese population of Taiwan. The study provides evidence that major differences exist between Taiwanese and whites or blacks in five blood group systems: Rh, MNSs, Duffy, P, and Xg.

Determination of sibship by PCR-amplified short tandem repeat analysis in Taiwan.

BACKGROUND: Sibship determination for any two persons whose parents have died is one of the most fundamental issues of personal identification, second only to those of a parent-child relationship. STUDY DESIGN AND METHODS: By automated fluorescence analysis of a PCR-amplified short tandem repeat (STR) system in conjunction with capillary electrophoresis, a panel of up to 15 polymorphic, autosomal, unlinked STR loci was used to investigate sibship index (SI) values in a cohort of 126 true sibling pairs. These SI values were then compared with those of 126 random pairs. RESULTS: The 15-loci STR panel provides a cumulative power of exclusion of 0. 9999997. Of the 126 random pairs, 124 (98.4%) had cumulative sibship indices (CSIs) of <1.0, and none had a CSI of >3.0 (median, 0.0101; range, 0.0000003-2.5376). In contrast, 107 (85%) of the 126 sibling pairs had a CSI of >100 (median, 5,579.9853; range, 0.0747-9,406,829, 249.8461). However, five pairs (4%) of the sibling group had a CSI of <3.0. True sibship was confirmed for this particular group by additional paternity testing and mitochondrial DNA sequence analysis. Among a total of 1890 observations (15 loci x 126 pairs), two alleles per locus were shared 760 times (40.21%) (mean, 6.03 loci; range, 1-10) in the sibling group, but only 192 times (10.16%) in the random group (mean, 1.52 loci; range, 1-5) (p<0.001). No alleles were shared 696 times (36.83%) in the unrelated pairs, as compared to 176 times (9.31%) in the sibling group (p<0.001). A polarized distribution was not noted in the sharing of single alleles in either the random or the sibling group: 1002 observations (53.02%) and 954 observations (50.48%), respectively. CONCLUSION: Highly polymorphic STR analysis can be discriminative in most sibship determinations, and the sharing of two alleles per locus is most informative in indicating sibship. Complementary mitochondrial DNA sequence analysis is mandatory in a few cases to exclude or establish true sibship when CSIs are equivocal and neither parent is available.

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.

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.

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.

[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.

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.