Flow cytometric assessment of leukocyte kinetics for the monitoring of tissue damage

Leukocyte populations quickly respond to tissue damage, but most leukocyte kinetic studies are not based on multiparameter flow cytometry. We systematically investigated several blood leukocyte populations after controlled tissue damage. 48 patients were assigned to either an anterior or posterolateral total hip arthroplasty. Peripheral blood was collected pre-operatively and at 2 h, 24 h, 48 h, 2 and 6 weeks postoperatively and assessed by flow cytometry for absolute counts of multiple leukocyte populations using standardized EuroFlow protocols. Absolute counts of leukocyte subsets differed significantly between consecutive time points. Neutrophils increased instantly after surgery, while most leukocyte subsets initially decreased, followed by increasing cell counts until 48 h. At two weeks all leukocyte counts were restored to pre-operative counts. Immune cell kinetics upon acute tissue damage exhibit reproducible patterns, which differ between the leukocyte subsets and with “opposite kinetics” among monocyte subsets. Flow cytometric leukocyte monitoring can be used to minimally invasively monitor tissue damage.This was supported by Stichting Anna Fonds/NOREF (Dutch Orthopedic Research and Education Fund) and the Erasmus MC Medical research grant (grant no. DRP337224)

Clinical heterogeneity can hamper the diagnosis of patients with ZAP70 deficiency

One of the severe combined immunodeficiencies (SCIDs), which is caused by a genetic defect in the signal transduction pathways involved in T-cell activation, is the ZAP70 deficiency. Mutations in ZAP70 lead to both abnormal thymic development and defective T-cell receptor (TCR) signaling of peripheral T-cells. In contrast to the lymphopenia in most SCID patients, ZAP70-deficient patients have lymphocytosis, despite the selective absence of CD8+ T-cells. The clinical presentation is usually before 2 years of age with typical findings of SCID. Here, we present three new ZAP70-deficient patients who vary in their clinical presentation. One of the ZAP70-deficient patients presented as a classical SCID, the second patient presented as a healthy looking wheezy infant, whereas the third patient came to clinical attention for the eczematous skin lesions simulating atopic dermatitis with eosinophilia and elevated immunoglobulin E (IgE), similar to the Omenn syndrome. This study illustrates that awareness of the clinical heterogeneity of ZAP70 deficiency is of utmost importance for making a fast and accurate diagnosis, which will contribute to the improvement of the adequate treatment of this severe immunodeficiency

DataSheet_1_Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood.zip

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual “expert-based”) gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.Peer reviewe

The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins

The protein products of the recombination activating genes (RAG1 and RAG2) initiate the formation of immunoglobulin (Ig) and T-cell receptors, which are essential for B- and T-cell development, respectively. Mutations in the RAG genes result in severe combined immunodeficiency disease (SCID), generally characterized by the absence of mature B and T lymphocytes, but presence of natural killer (NK) cells. Biochemically, mutations in the RAG genes result either in nonfunctional proteins or in protein

Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood.

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings

Longitudinal survey of lymphocyte subpopulations in the first year of life.

Contains fulltext : 87145.pdf (publisher's version ) (Closed access)Age-matched reference values for lymphocyte subpopulations are generally obtained via cross-sectional studies, whereas patients are followed longitudinally. We performed a detailed longitudinal analysis of the changes in lymphocyte subpopulations in a group of 11 healthy infants followed from birth up to 1 y of age, with special attention for early developmental markers, markers of maturation, and markers of activation. We found that T and B lymphocytes increased at 1 and 6 wk of age, respectively. In contrast, NK cells showed a sharp decline directly after birth, suggesting that they are more important during pregnancy than thereafter. CD45RA+--mainly CD4+--naive T lymphocytes were high at birth, and increased further during the first year of life; they form a large expanding pool of cells, ready for participation in primary immune responses. The absolute counts of CD45RO+ memory T lymphocytes were similar in infants and adults, albeit with a lower level of expression of CD45RO on infant T lymphocytes. Almost all infant T lymphocytes expressed CD38 throughout the first year of life. The abundant expression of CD38 on an infant's T lymphocytes might be related to a greater metabolic need of the large population of naive untriggered cells that are continually involved in primary immune responses during the first year of life. The high B lymphocyte counts in infants mainly concerned CD38+ B lymphocytes throughout the first year of life. Also, the relative frequencies of CD1c+ and CD5+ B lymphocytes were higher throughout the first year of life than in adults. Therefore, CD1c, CD5, and CD38 could be markers of untriggered B lymphocytes. In conclusion, our longitudinal survey of T and B lymphocytes, NK cells, and their subpopulations during the first year of life helps to complete the picture of lymphocyte development in infants. This information contributes to the correct interpretation of data from infants with possible immune disorders.1 april 200

Two patients with complete defects in interferon gamma receptor-dependent signaling.

Contains fulltext : 52114.pdf (publisher's version ) (Closed access)Unusual susceptibility to mycobacterial infections can be caused by deleterious mutations in genes that encode the interferon-gamma receptor 1 chain. Such mutations hamper the activation of macrophages by a type 1 immune response and result in enhanced survival of intracellular pathogens. We here report two patients with unusual mycobacterial infections, both diagnosed with homozygous deleterious interferon-gamma receptor 1 gene mutations. Patient 1 became ill after Bacillus Calmette-Guerin vaccination at the age of 9 months and died at the age of 18 months. She carried a homozygous C71Y mutation in the extracellular part of the mature interferon-gamma receptor 1 protein, resulting in the lack of detectable protein expression and absence of interferon-gamma dependent signaling. Patient 2 became ill at the age of 3 years, is still alive at 19 years of age, and has suffered from five successive infection episodes with atypical mycobacteria. A homozygous splice-site mutation in intron 3 was identified, resulting in the deletion of exon 3 at the mRNA level and consequently a truncated interferon-gamma receptor 1 protein with absence of the transmembrane domain. Protein expression and interferon-gamma dependent signaling were not detectable