It's About Everything: Validation and Optimization of 96-Well Tray Flow Cytometry Crossmatch.

Flow cytometric crossmatch (FCXM) is widely used in many centers as part of pretransplant risk assessment to detect donor-specific anti-HLA antibodies. The limited number of crossmatches that can be performed during on-call work-up for deceased donors within reasonable time remains the main obstacle to accommodating the majority of highly sensitized listed patients to be tested by the standard tube FCXM method. This limitation often directs the organs to nonsensitized patients and deprives highly sensitized patients who could be compatible if their sera were included in the crossmatch test. The goal of this study is to optimize a 96-well tray FCXM protocol that allows more sera to be crossmatched without prolonging the overall procedural time while maintaining quality and sensitivity of the assay. The new method was validated against use of the standard tube method and included total of 63 crossmatches performed simultaneously by both methods using 20 donors' cells with patients' sera, pooled positive controls tested on different dilutions, and commercial negative control. In the new protocol we modify various assay parameters including tube platform, incubation time, amount of reagent antibody cocktail, and cell volumes. An overall concordance of 98% was achieved with the protocols with slight improvement in sensitivity (2 negative B-cell reactions converted to positive in presence of weak donor-specific antibodies and mild T-cell reactivity could be picked up at 1:80 diluted positive control by the tray method only). The median channel fluorescence values of the 2 methods were essentially equivalent for both T and B crossmatches (r2 of 0.98 and 0.97, respectively). In conclusion, 96-well tray assay has the potential to increase the probability of highly sensitized patients receiving transplants by allowing increased number of crossmatches to be performed with significant reduction in turnaround time and assay cost. Furthermore, the enhanced sensitivity of the assay will provide more accurate information about sensitization status and strength of donor-specific antibodies to treating physicians, allowing them to choose the best therapeutic option and to provide better patient care.

Tolerance to Noninherited Maternal Antigens Allows Successful Second Kidney Transplantation: A Case Report.

The 2-way traffic of cells between mother and fetus may introduce noninherited maternal HLA antigens (NIMAs) from the mother to her fetus and expose her to the inherited paternal antigens presented on fetal cells. While contact of maternal immune system with paternal antigens leads to sensitization and development of anti-paternal HLA antibodies, exposure of the fetus to NIMAs can lead to tolerance because of immaturity of the fetal immune system. This state of tolerance is associated with better graft survival from NIMA-mismatched siblings. There are few studies on the effect of tolerance to NIMAs in renal transplant recipients with emphasis on first kidney graft. This reported case combines both effects (sensitization and tolerance) of the same NIMA in a patient who failed the first graft of maternal origin yet demonstrated persistence tolerance toward repeated NIMA mismatches presented on the second sibling donor. To our knowledge, this is the first report of combined effects of the same NIMA encountered by a single patient.

Evaluation of PCR, culture and serology for the diagnosis of acute human brucellosis.

BACKGROUND: The diagnosis of brucellosis is frequently difficult to establish. This is not only because clinically, the disease can mimic any infectious and noninfectious disease, but also because the established diagnostic methods are not always successful. In this study, we have tried to evaluate PCR techniques in the diagnosis of brucellosis in comparison to conventional techniques. PATIENTS AND METHODS: Fifty peripheral blood samples from the following groups were collected: patients with brucellosis (17); patients with febrile illnesses due to factors other than brucella etiology (19); symptomatic occupationally exposed persons (9); and healthy volunteers (5). The last three groups were considered controls. Among the 17 Brucella samples, only 14 were obtained before treatment was begun. The samples were tested by serology, using the standard tube agglutination method (STA), blood culture using Bactec machines, and PCR using primer pair to amplify a 223-bp region within a gene coding for a 31-kD Brucella antigen. Diagnosis of brucellosis was based on compatible clinical picture in addition to positive blood culture and/or positive serology. RESULTS: Of the 17 blood samples from patients with brucellosis, eight were culture positive for Brucella species, and all showed high titer antibrucella antibodies. Only 14 of them were positive by PCR, and these were the samples submitted before initiation of therapy, representing 100% sensitivity. Among the 33 controls, blood culture was negative for Brucella in all of them, while one sample showed high-titer antibrucella antibodies. The latter was from the febrile illnesses group. PCR-based assay was able to detect four bands in the controls, all of which were from the occupationally exposed asymptomatic group. CONCLUSION: In view of the several advantages of PCR over the conventional methods for the diagnosis of brucellosis, such as speed, safety, high sensitivity and specificity, the technique might be considered for laboratory diagnosis of brucellosis. However, for the evaluation of asymptomatic highly exposed persons, PCR might be considered complementary to the traditional methods and followed up by serology and/or culture.

Primary antibody deficiency in Arabs: first report from eastern Saudi Arabia.

Epidemiological studies have shown wide geographical and racial variations in the prevalence and pattern of immunodeficiency diseases. As there is no national registry, very little is known of the prevalence and nature of humoral immunodeficiency in the Arabian peninsula. We report here for the first time the analysis of serum immunoglobulin (Ig) levels in 2000 consecutive patients (age, 1-80 years). They were seen over a period of 6 years and were referred to us from six district hospitals for suspected immunodeficiency, autoimmunity, allergy, or immunoglobulin dyscrasia. Forty-six were found to be immunodeficient, in whom at least one of the Ig class was low; 15 had secondary immunodeficiency. The remaining 31 cases, representing 1.5% of the population studied (giving a prevalence of 1550/100,000 hospital registered patients), were categorized into four primary humoral immunodeficiency groups: these included, in order of frequency, (i) selective IgA deficiency (45%; 700/100,000) (ii) common variable immunodeficiency (CVID) (29%; 450/100,000), (iii) agammaglobulinemia (16%; 250/100,000), and (iv) selective IgG deficiency (10%; 150/100,000). Compared with similar hospital-based surveys in the west the prevalence of humoral immunodeficiency seems to be higher in Arabs; this in part may be related to race and higher rate of consanguinity. Most patients with IgA deficiency had either infection, atopy or autoimmunity. Compared with some other races, agammaglobulinemia (X- and non-X-linked) seems to be more prevalent.

Common variable immunodeficiency with CD4+ T lymphocytopenia and overproduction of soluble IL-2 receptor associated with Turner's syndrome and dorsal kyphoscoliosis.

An unusual combination of common variable immunodeficiency (CVID) and Turner's syndrome in a Saudi woman aged 20 years is presented. In addition to panhypogammaglobulinaemia, the patient had CD4+ T lymphocytopenia; however, there was evidence of in vivo activation of T cells and overproduction of soluble interleukin 2 receptor in culture supernate. Mantoux test was positive, but lymphoblastic response to non-specific mitogen was impaired. Immunogenetically the patient was HLA-DR3 positive and karyotypically she was a mosaic (45XO/46XX) with ring X chromosome (46Xr(X)). The presence of severe kyphoscoliosis was possibly related to ring X chromosome. This case highlights the grave consequences of the delayed diagnosis of immunodeficiency and emphasises the heterogeneous nature of CVID.