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.

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.

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

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.

Radiosensitive SCID patients with Artemis gene mutations show a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint in bone marrow.

Severe combined immunodeficiency disease (SCID) can be immunologically classified by the absence or presence of T, B, and natural killer (NK) cells. About 30% of T(-)B(-)NK(+) SCID patients carry mutations in the recombination activating genes (RAG). Some T(-)B(-)NK(+) SCID patients without RAG gene mutations are sensitive to ionizing radiation, and several of these radiosensitive (RS) SCID patients were recently shown to have large deletions or truncation mutations in the Artemis gene, implying a role for Artemis in DNA double-strand break (dsb) repair.

XLA patients with BTK splice-site mutations produce low levels of wild-type BTK transcripts.

X-linked agammaglobulinemia is caused by mutations in the BTK gene, which result in a precursor B-cell differentiation arrest in the bone marrow and the absence of or strongly reduced B lymphocytes in blood. We identified a patient with a mild clinical phenotype, low numbers of B lymphocytes, and a splice-site mutation in the BTK gene. The precursor B-cell compartment in the bone marrow of this patient was almost identical to that in healthy children.

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 proteins with partial recombination activity.

Composition of precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared with healthy children.

X-linked agammaglobulinemia (XLA) is characterized by a severe B-cell deficiency, resulting from a differentiation arrest in the bone marrow (BM). Because XLA is clinically and immunologically heterogeneous, we investigated whether the B-cell differentiation arrest in BM of XLA patients is heterogeneous as well. First, we analyzed BM samples from 19 healthy children by flow cytometry.

Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus.

Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a "single-cell" model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Igkappa gene (IGK) rearrangements, followed by IGK deletions and Iglambda gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified.

Neonatal blood lymphocyte subpopulations: a different perspective when using absolute counts.

We compared the absolute counts of lymphocyte subpopulations in 15 neonates, and 9 adults using the whole lysed blood technique with 15 different triple immunostainings. To obtain accurate absolute lymphocyte counts in neonatal cord blood samples, the flow cytometric 'lympho-gate' was corrected for the erythroid cell contamination by normoblasts and unlysed erythrocytes. In contrast to earlier studies where relative frequencies were reported, we found that the major difference between neonatal and adult lymphocyte subpopulations concerned the much larger pool of naive 'untriggered' cells in neonates, standby for participation in primary immune responses.

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

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.

Analysing the developing lymphocyte system of neonates and infants.

Unlabelled: Technical developments in immunophenotyping and function testing have greatly facilitated studies on the developing lymphocyte system in the past decade and contributed to a better interpretation of the data obtained in these studies. This is important for the correct interpretation of data obtained in paediatric patients with possible immunological diseases. The age-related differences in lymphocyte subpopulations and function imply that the available adult reference values cannot be used in children.

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.

Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vkappa gene segments (Vkappa-Kde rearrangements) or to the heptamer recombination signal sequence in the Jkappa-Ckappa intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias.

Detection of minimal residual disease in acute leukemia patients.

Diagnostic techniques, routinely used in clinical practice for monitoring acute leukemia patients, are able to detect only 1-5% of malignant cells. At present, two main techniques are being introduced for detection of minimal residual disease (MRD) in leukemia, namely immunological marker analysis and the polymerase chain reaction (PCR) technique with general sensitivity of 10(-4)-10(-5). Immunological marker analysis allows detection of unusual and aberrant immunophenotypes, and is usually performed by flow cytometry.