Lymphocytes subsets reference values in childhood.

Immunophenotyping of blood lymphocyte subsets and activation markers is a basic tool in the diagnostic process of primary immunodeficiency diseases, its use becoming more and more widespread as the knowledge about these illnesses increases. However, the availability of reliable reference values, which need to be age-matched for the pediatric population, is a pre-requisite for the reliable interpretation of immunophenotyping data. Aim of this study is to analyze the lymphocyte subsets and activation markers distribution in children aged 0-18 years referring to the University Hospital of Padova and to create age-matched reference values expressed by percentiles, thus providing a valuable guideline for the interpretation of the immunophenotype.

I. Adrenal cortex and steroid 21-hydroxylase autoantibodies in adult patients with organ-specific autoimmune diseases: markers of low progression to clinical Addison's disease.

Adrenal cortex antibodies (ACA) were measured by immunofluorescence in 8840 adult patients with organ-specific autoimmune diseases without overt hypoadrenalism. Sixty-seven (0.8%) patients were ACA-positive, with the highest prevalence in those with premature ovarian failure (8.9%). Forty-eight ACA-positive and 20 ACA-negative individuals were enrolled into a prospective study. Antibodies to steroid 21-hydroxylases (21-OH), steroid 17 alpha-hydroxylase (17 alpha-OH) and cytochrome P450 side chain cleavage enzyme (P450scc) were measured by immunoprecipitation assay. Human leucocyte antigens D-related (HLA-DR) genotyping was also carried out and adrenal function assessed by ACTH test. On enrollment, 75% of ACA-positive patients had a normal adrenal function, while 25% revealed a subclinical hypoadrenalism. 21-OH antibodies were positive in 91% of ACA-positive sera. Eleven patients were positive for steroid-cell antibodies by immunofluorescence, and 9 revealed a positivity for antibodies to 17 alpha-OH and/or P450scc. During the prospective study, overt Addison's disease developed in 21% and subclinical hypoadrenalism in 29% of ACA-positive patients, while 50% maintained normal adrenal function. Progression to Addison's disease was more frequent in patients with subclinical hypoadrenalism, high titers of ACA and higher levels of 21-OH antibodies, complement-fixing ACA and HLA-DR3 status. All 20 persistently ACA-negative patients were also negative for antibodies to 21-OH, 17 alpha-OH, and P450scc, and all maintained normal adrenal function during follow-up. In conclusion, the detection of ACA/21-OH antibodies in adults is a marker of low progression toward clinical Addison's disease.

The TEST 1 automated system: a new method for measuring the erythrocyte sedimentation rate.

We evaluated performance of the TEST 1 (SIRE Analytical Systems, Udine, Italy), a fully automated analyzer for the measurement of the erythrocyte sedimentation rate (ESR). Intra-assay reproducibility was satisfactory for a wide range of ESR values, whereas there was a significant decrease in ESR data when the samples were stored at 4 degrees C for up to 24 hours. We compared TEST 1 with the Westergren ESR method approved by the International Council for Standardization in Haematology (ICSH) and with the Diesse Ves-Matic analyzer Linear regression analysis comparing the TEST 1 and the ICSH reference method yielded satisfactory correlations for K3 EDTA- and sodium citrate-anticoagulated samples. Bland-Altman analysis showed no evidence of a systematic bias between the TEST 1 and the reference method. A close correlation was found between the TEST 1 system and the Diesse Ves-Matic analyzer despite a significant positive systematic bias. Reference values for men and women were analyzed according to nonparametric statistics. The TEST 1 was easy to use, had a satisfactory operative practicability required minimal maintenance, and reduced contact with potential biohazards. This system enables the determination of ESR with any common standard-sized tube; the use of samples anticoagulated with K3 EDTA can widely reduce the workload in clinical laboratories.

Low frequency of autoantibodies to islet cell, glutamic acid decarboxylase, and second-islet antigen in patients with gestational diabetes mellitus: a follow-up study.

The aim of the study was to determine the frequency of patients with gestational diabetes mellitus (GDM) who have serological markers typical of autoimmune type 1 DM. The specific pancreatic markers, ICAs, glutamic decarboxylase (GADAbs), and second islet antigen (IA2Abs), were measured in 70 women with GDM during the pregnancy and after delivery. ICAs were measured by indirect immunofluorescence and GADAbs and IA2Abs were determined by a radiobinding assay with recombinant antigens. On entering the study, 1 of 70 (1.4%) patients was positive for both ICAs (80 JDF-U) and GADAbs (167 U/mL), while another (1.4%) was positive for ICAs (40 JDF-U). None of the patients was positive for IA2Abs. During follow-up, positivity was maintained unchanged in the two positive patients. Four previously negative patients had seroconversion: one for both ICAs (20 JDF-U) and GADAbs (49.3 U/mL) and the other three for GADAbs (1.8, 1.4, and 15.3 U/mL, respectively). The IA2Abs remained negative in all patients. Overall, during the observation period 6 of 70 (8.6%) patients had or developed autoantibodies against endocrine pancreas. During follow-up 15 patients developed clinical DM (10 type 2, 5 type 1) and 7 demonstrated impaired glucose tolerance (IGT) after OGTT. No correlations were demonstrated between the immunological patterns and the evolution in DM. In patients with GDM, the frequency of pancreatic autoantibodies varies during the pregnancy and after delivery, but a small subgroup of patients bearing these markers is identifiable. GDM is a complex syndrome, constituted by different types of diabetes mellitus where the autoimmune form is very rare.

Zinc metabolism and immune function in healthy elderly people.

Changes in immune function have been described in the elderly, and zinc is known to influence immune parameters. Zinc supplementation has, however, produced contradictory results. We evaluated zinc metabolism and immunity in 15 healthy non-institutionalised elderly people on a self-selected diet and compared the results with those obtained from 10 young healthy subjects. The elderly subjects had reduced serum zinc levels and their zinc absorption values were lower than in the young subjects. Circulating immune parameters were within the normal range, while delayed immune response was altered in 8 of the 15 elderly subjects. Zinc supplementation (25 mg/day) did not result in a significant improvement in dermal hypersensitivity responses although some effect was seen in 5 cases. It is concluded that zinc supplementation is unnecessary in elderly healthy subjects even if they present zinc metabolism alterations, but may be indicated in the presence of disease.

B lymphocytes in newborns.

B lymphocyte markers in 24 full term newborn infants were evaluated within four days after birth. It was demonstrated that the percentage of cells with surface immunoglobulins (SmIg) in the blood of these subjects was normal when the lymphocytes were stained with polyvalent as well as monospecific antisera. The percentage of cells bearing Fc receptors were found to be slightly, but not significantly, reduced. The ability of newborn lymphocytes to form rosettes with mouse erythrocytes was significantly reduced. The hypothesis that the B dependent system reaches complete maturity already at the first days after birth is discussed.

An immunological evaluation of type II diabetic patients with periodontal disease.

Peridontal disease is a frequent complication of diabetes, and diabetic subjects often exhibit decreased immune response with increased susceptibility to infection. We evaluated the possible relationship between immune response and periodontal disease in 40 type II diabetic patients, mean (+/- SD) age 59 +/- 8 years and mean disease duration 17 +/- 4 years, with good metabolic control (mean fasting plasma glucose, 10.5 +/- 3.8 mM/L, mean HbA1c 8.1 +/- 1.66%), and in 40 age and gender-matched controls. Interproximal alveolar bone loss (ABL), as the percentage of bone loss from the cement enamel junction (CEJ) to the apex, was measured with a modified Schei ruler at the deepest point on the mesial/distal surface of the teeth, except third molars, on a panoramic radiograph. Immunological evaluation involved study of NADPH neutrophil superoxide production, neutrophil chemotaxis, lymphocyte subpopulations, immunoglobulins and complement. Diabetic patients showed significant differences compared with controls regarding ABL (30.6 +/- 14.7% versus 17.6 +/- 4.3%; p < 0.0001) and the T-helper/T-suppressor ratio (2.3 +/- 1.0% versus 1.8 +/- 0.8%; p < 0.05). Other parameters of cell-mediated immunity and humoral immune response did not show any significant variations. No correlation between immunological and radiographic analysis parameters were found. Further studies are needed to verify the exact role played by immunological factors in type II diabetic patients with periodontal disease.

Complete analytical and diagnostic performances of the Abbott Cell Dyn 3500.

METHODS: The study reports the analytical and diagnostic performances of Cell Dyn 3500 (Abbott), an automated haematology analyser that provides the electrical impedance and the optical detection of total leukocyte and a five population leukocyte differential count. The evaluation of complete blood count and differential leukocyte count parameters was performed following the guidelines of the International Council for Standardization in Haematology and those of the National Committee for Clinical Laboratory Standards document H20-A. RESULTS: The CD 3500 demonstrated a good linearity, minimal carry-over, as well as acceptable levels of within and between-batch imprecision and stability. An excellent correlation between CD 3500 and the routine systems used in our laboratory (Technicon H*2 and Coulter STKS) was found for the major haematological indices, though agreement was not as good for MCHC. There was a good correlation between CD 3500 and manual reference differential method for neutrophils, lymphocytes, eosinophils and basophils, with the exception of monocytes. CONCLUSIONS: Regarding clinical sensitivity, the CD 3500 showed a high specificity to detect morphological abnormalities, but low sensitivity especially for the identification of immature granulocytes and nucleated red blood cells; if we overall evaluate any qualitative flags, we found an appreciable increase in sensitivity.

Evaluation of T-cell receptor CD3+ gamma delta in gestational diabetes mellitus.

Few studies have shown a significant increase of CD3+ T-cell receptor (TCR) gamma delta in the early phases of type 1 diabetes. We wished to determine if CD3+ TCR gamma delta is involved in the pathogenesis of gestational diabetes mellitus (GDM). We studied 29 GDM patients and 21 normal pregnant women. Lymphocyte subpopulations (CD3+ TCR alpha beta, CD3+ TCR gamma delta), islet cell antibodies (ICA), glutamic acid decarboxylase antibodies (GAD) and protein tyrosine phosphatase antibodies (IA2-Ab) were evaluated in all patients. The percentage of CD3+ TCR gamma delta was significantly higher in GDM women than in the control group (5.1 +/- 2.9% vs 3.7 +/- 1.7%; p < 0.05). No abnormalities of the other lymphocyte subpopulations were found. All subjects were negative for ICA; 2 GDM patients were positive for GAD, but no relationship was found between GAD positivity and CD3+ gamma delta levels in these 2 patients. Further follow-up studies of these patients are required to verify if the CD3+ TCR gamma delta receptor is a useful marker for diabetes development.

An immunological and genetic study of patients with gestational diabetes mellitus.

The aim of the study was to evaluate the frequency of islet cell (ICA) and insulin (IAA) antibodies and of HLA antigen typing in a group of subjects diagnosed with gestational diabetes mellitus (GDM) in a screening-diagnostic program during pregnancy. ICA, complement-fixing (CF) ICA and other autoantibodies, absolute number and percentage of lymphocyte subpopulations, and HLA antigens were evaluated in 68 women with GDM and compared with those of matched controls. ICA were found in 2 (2.9%) and IAA in 1 (1.5%). Both ICA-positive women had CF-ICA; one of them was receiving insulin therapy. while the other was on a special diet. No correlations were found between ICA and IAA, nor between IAA and insulin treatment. As far as lymphocyte subsets were concerned, we found a significant increase in the absolute number of total and activated (CD3+HLA-DR+) T lymphocytes and a significant increase in the absolute number and percentage of suppressor/cytotoxic T lymphocytes (CD8) and NK lymphocytes (CD57) in GDM patients compared with normal pregnant controls. Concerning frequency for HLA A, B, C, DR antigens in the GDM population, only Cw7 was found to be significantly increased and A10 significantly decreased in comparison with controls. Our study suggests that GDM is a heterogeneous disorder in which few patients present with the immunologic and genetic markers of type 1 diabetes.

Synthetic thymic fraction 5: effects of high dose administration on circulating lymphocytes in patients.

The synthetic pentapeptide (Arg-Lys-Asp-Val-Tyr; TP-5) corresponding to the active site of the hormone thymopoietin, was given at the dose of 300 mg/m2/day (1 day), higher than the usually administered, to a group of 27 immunodepressed patients in order to determine the tolerability and the immunomodulatory activity. The examination of a series of hematological parameters including counts of differential clustering of lymphocytes by cytofluorimetric analysis was performed 24 hr and 48 hr after treatment, and repeated at different intervals up to 14 days after treatment. TP-5 caused a significant increase of circulating lymphocytes and particularly of CD3+CD4+ and CD3+CD8+ subtypes, peaking at 48 hr and maintaining the increased values up to the last examination on day 14 from treatment. A faster increase (zenith at 24 hr) was observed for CD4+ cells, in comparison with CD8+ cells (zenith at 48 hr). The number of patients that increased total lymphocytes or lymphocyte subset after treatment ranged between 52.6 (CD4+ cells) and 69.2% (NK cells), whereas about 7.7% (NK cells) to 36.9% (CD4+ cells) remained unchanged and a smaller amount of 10.5% (CD4+ and CD8+ cells) or 23.1% (NK cells) showed a decrease greater than 10% of their respective basal value. No significant relationship between responders and non-responders can be found on the basis of previous treatments, cancer type, sex or age.

[Analytical evaluation of an automated hematologic analyzer: Cell Dyn 3500]. / Valutazione analitica di un analizzatore ematologico automatico: il Cell Dyn 3500.

In this study we evaluated the analytical performances of the Cell Dyn 3500, an automated hematology analyzer that provides the electronic and optical detection of leukocyte count and the complete cell counts in samples of whole blood. The protocol included evaluation of complete blood count and differential leukocyte count parameters, using the ICSH and the NCCLS H20-A protocols. Technical performances with regards, to linearity, carry over, precision and stability were quite well acceptable; the accuracy showed a good agreement between Cell Dyn 3500 and instruments used in our laboratory for the major hematologic indices and a good correlation for neutrophils, lymphocytes and eosinophils compared with the manual count.

A study on lymphocyte subpopulations in diabetic pregnant women and their newborn.

We studied the lymphocyte subpopulations in 14 pregnant women with type 1 insulin-dependent diabetes mellitus (IDDM), mean age (+/- SD) 30 +/- 4 years, mean disease duration 12 +/- 5 years, in 14 with gestational diabetes mellitus (GDM) (mean age 33 +/- 6 years) and 21 matched healthy pregnant controls (C), when the subjects delivered, and in their newborn. The GDM and IDDM mothers show a significant increase (p < 0.05) of lymphocytes in comparison with C (GDM 1.83 +/- 0.5 x 10(9)/l, IDDM 1.6 +/- 0.68 x 10(9)/l, C 1.06 +/- 0.45 x 10(9)/l lymphocytes), which has repercussion on lymphocyte subpopulations absolute values. The newborn of GDM mothers have an increase of lymphocytes T-activated (0.6 +/- 0.3 vs 0.3 +/- 0.2%; p < 0.05) and a reduction of NK lymphocytes than C (8.9 +/- 9 vs 15.5 +/- 7.6%; p < 0.05). The newborn of IDDM mothers show a significant reduction of NK lymphocytes than C (10 +/- 6 vs 15.5 +/- 7.6%; p < 0.05). So in newborn of diabetic mothers there is a deficit of natural immunity at birth which has to be evaluated by follow-up study.

Chronic anemia due to mitomycin C is drug dose-dependent, normocytic, progressive, related to erythropoietin levels and quantitatively predictable: implications for radiochemotherapy.

Mitomycin C (MC) is used as therapy against solid tumors, also combined with other chemotherapeutic agents or radiotherapy. It may cause acute, subacute, or chronic anemia capable of modifying the results of chemo- and radiotherapy. Erythropoietin may be lowered by cancer itself or because of chemoradiotherapy. There are few studies investigating the relationship between erythropoietin and chronic anemia.We prospectively analyzed the chronic anemia and erythropoietin in 38 patients with solid cancer. Patients were 40 to 82 years of age. MC was randomly given every 3 weeks as a single drug at 10 or 20 mg/m². When myelotoxicity occurred the next therapy cycle was delayed until recovery. RBC indices, hemolysis, erythropoietin, liver and kidney function were studied. MC cycles were 136 (3.6 ± 1.4 per pt), 32 being delayed because of myelotoxicity.Hematocrit, hemoglobin and RBC were inversely related to the cumulative dose (r = 0.70 to 0.86; p 0.03 to 0.01) of MC. Other tests remained stable. Anemia occurred almost twofold earlier in the 20 mg/m² group (p=0.049). basal erythropoietin, already lower than in age and sex watched 81 non cancerous subjects (p<0.001), decreased during MC therapy (p<0.01). For each given MC mg/m² a 0.0372 Hb mg/dl reduction occurred. Chronic anemia due to MC is accompanied by erythropoietin reduction. These results can help in designing chemoradiotherapy.

Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197: implications for pancreatic cancer gene therapy.

Vectors combining the heat shock proteins (HSPs) promoter with the catalytic subunit A of the diphtheria toxin (DTA) or its variants, cross-reacting material (CRM) 176 and 197, were engineered to investigate the effect of bacterial toxins on pancreatic cancer (PC) cells. Three heat-inducible enhanced green fluorescent protein (eGFP)-expression vectors were obtained: V1 (91% homology to HSPA6), V2 (five heat shock elements upstream the minimal HSPA6 promoter) and V3 (V1 and V2 combined). The highest eGFP transcription and translation levels were found in V3 transfected PC cells. The V3 promoter was used to control DTA, CRM176 and CRM197 expression, treatment response being investigated in four PC cell lines. DTAwt or CRM176 transfected cell growth was completely arrested after heat shock. CRM197 toxin presumed to be inactive, caused mild distress at 37 degrees C and induced a 25-50% reduction in cell growth after heat shock. Preliminary in vivo findings showed that heat treatment arrests tumor growth in DTA197 stably transfected PSN1 cells. In conclusion, the efficient HSP promoter identified in this study may be extremely useful in controlling the transcription of toxins such as CRM197, which have lethal dose-related effects, and may thus be a promising tool in PC gene therapy in vivo.

Lymphocyte subsets and cytokines in women with gestational diabetes mellitus and their newborn.

This study aimed to identify potential immunological markers for predicting type 1 diabetes in patients with gestational diabetes mellitus (GDM) and any immunological impairment in their newborn. In 62 GDM patients and 74 women with normal glucose tolerance (NGT), and their babies, we assessed total lymphocytes, T lymphocyte subsets CD3 and CD8 expressing T cell receptor (TCR) alpha/beta or gamma/delta, CD16 and CD19, pancreatic autoantibodies and cytokines (IL-5, IL-2, soluble receptor IL-2). At delivery, umbilical cord blood samples were taken for lymphocyte subpopulations and cytokine measurements. GDM mothers had higher levels of total lymphocytes, CD8 expressing TCR gamma/delta, and lower levels of CD3 expressing TCR alpha/beta than NGT controls. Insulin-treated GDM mothers had lower CD4 and CD4/CD8 ratios, and higher CD8 and IL-5 than diet-treated GDM or controls. Five women were positive for pancreatic autoantibodies, with lower CD4 (p<0.01) and CD4/CD8 ratios (p<0.05), and higher CD8 (p<0.03) and CD19 than GDM and control mothers negative for autoantibodies. GDM newborn had higher CD8 gamma/delta and lower CD16 than NGT babies. There were no significant differences in TNF-alpha concentrations in the cord blood obtained from the GDM and NGT newborn. In conclusion, GDM women and their newborn have lymphocyte subset impairments, which are more important in patients positive for autoantibodies and/or treated with insulin.

Length of sedimentation reaction in undiluted blood (erythrocyte sedimentation rate): variations with sex and age and reference limits.

Although the length of sedimentation reaction in blood (LSRB) (commonly, but improperly called erythrocyte sedimentation rate, ESR) has long been used in clinical laboratories because it is simple and low-cost, its sensitivity and specificity are unsatisfactory. Usually, the values are reported using the Westergren method with sodium citrate-anticoagulated specimens. We used a new procedure, the TEST1, which measures the length of sedimentation reaction in undiluted K3EDTA anticoagulated blood samples following ICSH (International Committee for Standardization in Haematology) recommendations. Samples obtained from 840 reference individuals (430 females and 410 males, mean age 44 and 46.5 years respectively, range 1 to 90 years) were utilised to estimate the reference limits. The subjects, classified by sex, were subdivided into four statistically different age groups to determine the reference limits (2.5th and 97.5th percentiles). Sex difference was statistically significant in two age groups, from 14 to 50 (p < 0.0001) and from 50 to 70 years (p < 0.01). We did not observe significant sex difference within the age bracket from 1 to 14 years and from 70 to 90 years. In both sexes LSRB values increased with age, in significant correlation with fibrinogen concentration (p < 0.0001), and became significantly higher in subjects older than 70 compared to all the younger subjects (p<0.01 in females and p < 0.02 in males). Thus, we defined adequate reference ranges in elderly.

[Properties of human lymphocytes binding to mouse red blood cells]. / osservazioni sulla proprieta' dei linfociti umani di legare le emazie di topo.

Certain properties of the bond between lymphocytes and mouse red blood cells were studied. The turnover of the receptor on the lymphocytes of twelve healthy subjects was evaluated. After two hours of incubation in a medium devoid of additives the receptor is released and its resynthesis is completed in twenty-four hours. In experiments employing proteolytic enzymes inhibition of rosette formation is observed, whereas neuraminidase, under the same experimental conditions, significantly increases the number of rosette forming cells. The percentage of mouse rosette forming cells in nineteen patients affected with chronic lymphatic leukaemia was then evaluated (17 with type B and 2 with type T). On the basis of these cases and other recently published data, an ontogenetic explanation for the appearance of this receptor is proposed.

B lymphocytes in sarcoidosis.

Twenty-six patients with active and inactive sarcoidosis were studied. Surface immunofluorescence techniques with polyvalent and monospecific antisera after resynthesis of intrinsic Ig, were employed to determine the number of B cells. The peroxidase positive cells were not included in the count. Percentage and absolute number of lymphocyte forming rosettes with mouse erythrocytes were also determined. Compared to normal controls the numbers of SmIg positive cells were practically unchanged. Moderate decrease in the absolute number of mouse rosette forming cells was found in active disease. Our results will be correlated with the staging of the disease.

Effect of high-dose steroid therapy on T-cell subpopulations. A longitudinal study in MS patients.

Lymphocyte subpopulations, T cell activation antigens, and serum levels of interleukin 2 (IL-2) and soluble IL-2 receptor (sIL2R), were studied in relapsing-remitting MS (RR-MS) patients before and after high-dose steroid therapy. Prior to therapy, a minority of patients showed increased HLA-DR antigen expression, and an increased number of CD16+ and CD19+ cells. Steroid treatment induced a significant increase in HLA-DR and CD19 expression, a significant reduction in CD16+, CD57+, and CD8+ CD57+ cells, and a slight, non-significant, decrease in IL-2 and sIL-2R levels and CD25 expression on CD4+ T lymphocytes.