Early Diagnosis Of Primary Immunodeficiencies Research Articles

Flow-cytometry as an auxiliary in the diagnosis of primary humoral immunodeficiencies.

Antibody deficiencies encompass a wide spectrum of pathologies and constitute approximately 50 % of primary immunodeficiencies; with cytometry, it is possible to evaluate the immune status rapidly, effectively and at low cost. To assess, by means of flow cytometry, the cells of patients with three types of primary humoral immunodeficiencies. Using flow cytometry, blood samples from patients and healthy subjects were analyzed with different monoclonal antibodies. Using various stains, a severe decrease in B lymphocytes was shown in patients with X-linked agammaglobulinemia, as well as a lack of CD154 expression in patients with hyper-immunoglobulin M syndrome, and heterogeneity of B lymphocyte subpopulations in patients with common variable immunodeficiency. Flow cytometry enables early diagnosis of primary immunodeficiencies with a high level of confidence and, in many cases, identification of the genes involved.

Evaluation of patients with primary immunodeficiency associated with Bacille Calmette-Guerin (BCG)-vaccine-derived complications

BackgroundBacille Calmette-Guerin (BCG) vaccination has a great impact on the prevention of severe complications of tuberculosis. However, in patients with primary immunodeficiencies (PID), it can lead to severe complications such as severe combined immunodeficiency, chronic granulomatous disease, and Mendelian susceptibility to mycobacterial disease. This study highlights the demographics, clinical complications and laboratory parameters among PID patients associated with BCG vaccination side effects. MethodsOne hundred and thirty-seven PID patients with BCGosis were evaluated in this study, based on the complications following BCG vaccination. ResultsThe mean age of the patients with BCG complications at the time of the first visit was five years. The within-group comparison of patients showed a highly significant incidence of pneumonia and hepatomegaly in severe combined immunodeficiency patients. Furthermore, the immunologic data showed an increase in the overall rates of lymphocytes such as CD3+, CD4+ and CD8 + T cells in Mendelian susceptibility to mycobacterial disease patients. The level of immunoglobulins has also increased in chronic granulomatous disease patients. ConclusionThe high rate of undiagnosed PIDs predisposes individuals to a high risk of severe side effects as a result of BCG vaccination, as well as infants that are less than one month of age. Therefore, there is a need for early screening and diagnosis of PIDs before exposing unknown PID status patients to BCG vaccination. The benefits of screening and early diagnosis of PID cannot be overemphasized, especially in patients with a previous family history of immunodeficiency.

Rapid electrophoretic recovery of DNA from dried blood spots.

Large-scale genetic screening of neonatal dried blood spots for episomal DNA has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of dried blood spots remains one of the most time consuming, costly, and labor-intensive parts of DNA analysis. In the present study, we developed and optimized a rapid methodology using only 50V and heat to extract episomal DNA from dried blood spots prepared from diagnostic cord blood samples. This electric field DNA extraction is the first methodology to use an electric field to extract episomal DNA from a dried blood spot. This 25-minute procedure has one of the lowest times for the extraction of episomal DNA found within the literature and this novel procedure not only negates the need for costly treatment and wash steps, but reduces the time of manual procedures by more than 30 min while retaining the 75-80% of the yield. Combined with real-time PCR, this novel method of electric field extraction not only provides an effective tool for the large scale genetic analysis of neonates, but a key step forward in the simplification and standardization of diagnostic testing.

Rapid Recovery of DNA from Dried Blood Spots: Use of a Novel Electrophoretic Method

The demand for rapid, effective point of care diagnostics has created a pressing need for new and improved methods to quantify DNA from patient samples. Dried blood spotting is a well‐established testing methodology that has gained increasing importance in in vitro diagnostics due to its low cost, low sampling requirements, and long‐term sample stability. In recent years, dried blood spots have been increasingly used for targeted genetic and metabolite profiling. Large‐scale genetic screening of neonatal dried blood spots has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of the dried blood spot remains the most time consuming, costly and labor‐intensive part of DNA analysis. Thus, the objective of this study was to develop and optimize a simple and rapid methodology to extract DNA from dried blood spots.In the present study, we created a novel methodology to extract DNA from a dried blood spot using only a lysis buffer (10mM KOH/30mM Tris buffer), 50V, and heat. Briefly, during the electric field extraction spots and lysis buffer were placed in an electroporation cuvette and DNA was extracted by applying 25–50 volts across the plates of the cuvette. The spot was then removed and DNA in the supernatant was analyzed using qPCR or fluorescent nucleic acid stains. This methodology was optimized by the addition of a 70°C heating step before the electric field extraction to better retrieve episomal DNA.This electric field DNA extraction is to our knowledge, the first methodology to use an electric field to extract episomal DNA from a dried blood spot. Results of this study show that this methodology produces significant recoveries of both genomic and episomal DNA (Ct (FAM) 33.0 (50V, 60s) to Ct (FAM) 35.0 (heating alone) p <0.01). Varying recoveries of episomal and genomic DNA were also seen at different voltage conditions suggesting that this methodology may be useful for the isolation and purification of episomal DNA (FAM (25V, 300s (no amplification) and 50V, 60s (Ct 33.0)). Furthermore, preliminary qualitative and spectroscopic analyses suggest that protein removal could be achieved using this dual voltage technique. The 25‐minute electric field extraction procedure has one of the lowest times for the extraction of DNA found within the literature. This optimized procedure negates the need for costly treatment and wash steps, simplifying DNA extraction considerably. Most importantly, the electric field extraction reduces the time of proprietary procedures by more than 30 minutes, while retaining 75–80% of the yield. Thus, the results show that this novel method of electric field extraction provides a rapid and effective protocol to remove episomal and genomic DNA from dried blood spots. This optimized electric field device not only simplifies bioanalytical techniques, but provides a significant step towards point of care diagnostics and should be expanded to other sample types.Support or Funding InformationThe authors thank PerkinElmer for providing the funding for this study.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diagnostic biologique des déficits immunitaires primitifs

Early diagnosis of primary immunodeficiencies (PID) is critical to improve patients care and to prevent complications. Clinical and biological signs of PID include infections, but also cancers and autoimmune diseases. First-line biological tests - such as blood count, immunoglobulins quantitation and post-vaccines serologies – could be helpful to make the diagnosis. More specialized tests, such as lymphocytes phenotyping and IgG subclasses quantitation, should be done as second-line tests. All these blood exams have to be rigorously interpreted, notably regarding patient’s age and infectious agents.

Frequency of Mycobacterium bovis and mycobacteria in primary immunodeficiencies.

Susceptibility to mycobacterial diseases is observed in some primary immunodeficiency diseases. In this study, we aimed to evaluate mycobacterial infections in primary immunodeficiency diseases. Patients under follow-up by Ege University Pediatric Immunology Department for severe combined and combined immunodeficiencies, interleukin 12/ interferon gamma receptor deficiency, nuclear factor kappa-beta essential modulator deficiency and chronic granulomatosis disease were evaluated retrospectively in terms of the frequency and characteristics of mycobacterial infections using a questionnaire form for demographic properties, clinical features and laboratory tests. A diagnosis of mycobacterial infection was made clinically in a total of 25 patients including five (11.3%) of 45 patients who had severe combined immune deficiency, 12 (52.3%) of 21 patients who had chronic granulomatous disease, four patients (100%) who had interferon gamma receptor 2 partical deficiency, two patients (100%) who had interleukin 12 receptor beta 1 deficiency and one patient (100%) who had nuclear factor kapa-beta essential modulator deficiency. Mycobacterium strain could be typed in 14 (33%) of these 25 patients including Mycobacterium bovis, Mycobacterium chelonea, Mycobacterium elephantis, Mycobacterium fortuitum, and Mycobacterium tuberculosis. All patients were treated with anti-tuberculosis therapy. Thirty-six percent of these 25 patients underwent hematopoietic stem cell transplantation. Eight patients (five before, three after transplantation) died. Non-tuberculosis mycobacteria including mainly Mycobacterium bovis were observed with a higher rate compared to Mycobacterium tuberculosis in primary immunodeficiencies, especially in those affecting the interleukin 12/interferon gamma pathway. Early diagnosis of primary immunodeficiencies with neonatal screening program and preventing administration of the Bacille Calmette-Guerin vaccine in these patients is important.

Survival of the fetus: fetal B and T cell receptor repertoire development.

A mature and diverse T and B cell receptor repertoire is a prerequisite for immunocompetence. In light of its increased susceptibility to infection, the human fetus has long been considered deficient in this regard. However, data accumulated since the 1990s and in earnest in the past couple of years paints a more complicated picture. As we describe in this review, mechanisms responsible for generating a diverse receptor repertoire, such as somatic recombination, class switch recombination, and somatic hypermutation, are all operational to surprising extents in the growing fetus. The composition of the fetal repertoire differs from that of adults, with preferential usage of certain variable (V), diversity (D), and joining (J) gene segments and a shorter complementarity determining (CDR3) region, primarily due to decreased terminal deoxynucleotidyl transferase (TdT) expression. Both T and B cell receptor repertoires are extremely diverse by the end of the second trimester, and in the case of T cells, are capable of responding to an invading pathogen with in utero clonal expansion. Thus, it would appear as though the T and B cell receptor repertoires are not a hindrance towards immunocompetence of the newborn. Our improved understanding of fetal receptor repertoire development is already bearing fruit in the early diagnosis of primary immunodeficiencies (PID) and may help clarify the pathogenesis of congenital infections, recurrent abortions, and autoimmune disorders in the near future.

Basics of primary immunodeficiencies

Primary immunodeficiencies (PID) are a heterogeneous group of inherited disorders, the etiology are the defects in the development or function of the immune system. The principal PID manifestations are the infections in early age, malignancy and diseases of immune dysregulation as autoimmunity and allergy. PIDs are genetics disorders and most of them are inherited as autosomal recessive, also this group of diseases is more prevalent in males and in childhood. The antibody immunodeficiency is the PID more common in adults. The more frequent disorders are the infections in the respiratory tract, abscesses, candidiasis, diarrhea, BCGosis etc. Initial approach included a complete blood count and quantification of immunoglobulins. The delay in diagnosis could be explained due to a perception that the recurrent infections are normal process or think that they are exclusively of childhood. The early diagnosis of PID by primary care physicians is important to opportune treatment and better prognosis.

Autoinflammatory diseases: An immunologist’s view

Autoinflammatory syndromes (AISs) are a heterogeneous group of genetically determined diseases, whose basis is the dysregulated mechanisms of inflammation. Despite the fact that traditional primary immunodeficiencies and AISs have an external dissimilarity, there are many parallels, such as hypodiagnosis, multisystem pattern of lesion, chronic inflammation-induced complications, possible medical therapy using cytokine inhibitors, and cardinal cure by stem cell transplantation and gene therapy. There are more than 25 different AISs, their common manifestations are fever episodes accompanied by laboratory inflammatory activity, polymorphous eruption, lymphoproliferative syndrome, and injury to different organs. Genetic analysis plays an important role in the diagnosis in traditional AISs and primary immunodeficiencies. This is particularly relevant to AISs that are not infrequently very closely allied phenotypically. Moreover, it is important that the detection of a causative genetic defect frequently determines man-agement tactics for patients. For AIS treatment, there is a spectrum of agents modulating these or those inflammatory components. However, there is a problem of resistance to standard therapy as before. Pathogenetic therapy for AISs is lifetime, expensive, and commonly responsible for serious adverse reactions, which worsens quality of life in patients. Patients with AISs, like those with other primary immunodeficiencies, need a multidisciplinary approach with the participation of various specialists. These patients should be followed up in specialized centers that perform treatments according to the international algorithms. Since the early diagnosis of primary immunodeficiencies and AISs is a key to their successful treatment; primary care physician’s awareness of these rare diseases is of the most importance.

Treatment of Primary Immunodeficiency with Human Gammaglobulin

Primary immunodeficiencies are characterized by recurrent or prolonged infections associated with growth retardation, infections by specific microorganisms or by low virulence germs, inappropriate response to the antibiotics used, a high risk of complications and hospitalization and severe vaccine complications. The evaluation of immunological status is essential for the diagnosis of these diseases. Immunoglobulin replacement therapy is the best option for most antibody deficiencies and for some diseases that do not belong to this group, like hyper IgM syndrome, immunodeficiency with thymoma and severe combined immunodeficiency. This therapy is a safe procedure that induces dramatic positive changes in the clinical outcome of patients who carry antibody defects. An early diagnosis of primary immunodeficiencies is essential so that therapeutic measures may be taken quickly, such as the use of immunoglobulin when properly indicated, reducing the risks of death and complications

The impact of physician education and public awareness on early diagnosis of primary immunodeficiencies

Thank you Hans for that kind and gracious introduction. We are deeply honored to have been asked by Bibi Day to speak at the opening session of the first Robert A. Good Immunology Symposium. It is with deep humility that we come before such an illustrious faculty and assembled delegates. There are no words to describe how moved we are that this session is dedicated to our son, Jeffrey. What a great honor. We first met Dr. Good in 1975 when our son Jeffrey was 5 years old. He had already been diagnosed with Hypogammaglobulinenemia. He was treated superbly by his doctors, many of whom are here with us today. We asked Jeffrey’s doctors to reach out to the greatest experts, anywhere in the world, on this disease; especially experts on the specific B cell complexities that affected our son. In our quest for answers, we eventually reached Dr. Joshua Lederberg who, at the time, was President of Rockefeller University. To our surprise, he told us that the greatest expert, anywhere in the world on Primary Immunodeficiencies, was not half way around the globe but, in fact, was only two city blocks from our home. Of course he was talking about Dr. Robert A. Good, President of the Sloan Kettering Cancer Institute. Dr. Good took our call, met with Jeffrey, and monitored Jeffrey’s progress over many years. Doctor and patient got along famously. Both of them were constantly filled with energy and enthusiasm. Dr. Good gave Fred and I the confidence to share a normal life with our son. Jeffrey did well in school, played sports, traveled, enjoyed summer camp, and even had a girlfriend. But repeated episodes of high fever, multiple hospitalizations, and eventually serious bouts of pneumonia were too much for Jeffrey to overcome and he lost his courageous battle at the age of 15. Throughout those years, we never met another family and Jeffrey never met another child facing this condition. We were sure that he was the only one. A few months after we lost Jeffrey, with the urging of family and friends to do something to remember his life, we