Determination of reference intervals for 10 serum proteins measured by rate nephelometry, taking into consideration different sample groups and different distribution functions

Reference intervals were established for 10 serum proteins (IgA, IgG, IgM, transferrin, haptoglobin, complement C3, complement C4, alpha 1-acid-glycoprotein, alpha 1-antitrypsin, alpha 2-macroglobulin) measured by rate nephelometry. The reference individuals - 200 blood donors - were divided into 5 subgroups: men aged 19-39 and 40-60 years, women aged 19-39 and 40-60 years and women aged 19-48 years using oral contraceptives. Where possible, two or more subgroups were combined to give reference sample groups. Criteria for this procedure are given. The reference limits of the sample groups were estimated by parametric methods. Assuming that for a specified serum protein the type of distribution is the same in each subgroup, the data were standardized with estimated group specific parameter values and combined into one big sample. This permitted an improved determination of the underlying type of distribution. As a possible form of distribution we also considered the normal distribution truncated on the left side at c greater than or equal to 0. In some cases, after determination of an optimal c, this unusual distribution fitted the data significantly better than the generally used normal or log-normal distribution.     

Typing of genetic variants of alpha 1-antitrypsin from dried blood

The alpha 1-antitrypsin phenotype was determined from both plasma and dried blood in 35 children and adolescents who had or were suspected to have alpha 1-antitrypsin deficiency. A disc of paper with dried blood was eluted and analysed by flat bed electrofocusing on polyacrylamide gel. The gel was stained according to a silver-staining technique until the microheterogeneous bands appeared with adequate intensity. The alpha 1-antitrypsin patterns of plasma and dried blood were identical. The stability of the dried blood samples was 3 days at room temperature, up to 1 week at +4 degrees C, and up to 1 month at -20 degrees C. The alpha 1-antitrypsin screening method and the Pi-typing procedure described in this study may be combined in future screening studies using dried blood specimens obtained for the Guthrie test.     

Reference intervals for serum proteins: similarities and differences between adult Caucasian and Asian Indian males in Yorkshire, UK.

The aim of this study was to investigate similarities and differences in the distribution of serum concentrations of nine proteins in two racial groups (Caucasian and Asian Indian) of adult males living in the same geographical area (Leeds, Bradford, UK) for at least two generations. This is part of a larger study to determine the need for separating reference intervals for racial and ethnic groups worldwide. The distributions of concentrations for all proteins evaluated in the Indians fit In-Gaussian distributions, indicating probable homogeneity. However, for the Caucasians, the distributions for alpha1-antitrypsin and possibly haptoglobin were not In-Gaussian. In the former case, this is undoubtedly due to the number of Caucasians with lower-concentration phenotypes (Pi MS and MZ). Although haptoglobin differences may be due to genetic variants as well, this is not a complete explanation. In addition, the Indians have lower serum concentrations of orosomucoid (alpha1-acid glycoprotein), as has been reported by others. It is apparent that for some proteins, including alpha1-antitrypsin, orosomucoid, and possibly haptoglobin, the populations show differences that require the use of separate reference intervals. In addition to genetic influences, environmental differences cannot be ruled out as partial causes for some of the differences noted.     

Laser immunonephelometry reference intervals for eight serum proteins in healthy children.

Eight serum proteins were analyzed with the Behring nephelometer in samples from 479 healthy French children, ages three to 16 years. Girls had higher concentrations of IgM and albumin than boys had. Age appeared to be a main factor of variation for the proteins tested. Reference intervals are presented for IgG, IgA, IgM, albumin, transthyretin (prealbumin), retinol-binding protein, alpha 1-acid glycoprotein, and C-reactive protein. The significance of increased concentrations of C-reactive protein within a community is discussed.     

Pediatric reference intervals for lymphocyte vitamin C (ascorbic acid)

ObjectiveTo establish pediatric reference intervals for lymphocyte vitamin C.Design and methodsThis was a prospective study of 194 well children aged 0–7 years old of mixed ethnicity who had blood drawn for the purpose of this study. Blood was collected during elective surgery under general anesthesia and lymphocytes isolated and stored as frozen ascorbic acid lymphocyte lysates for later HPLC analysis by previously described methodology. Reference intervals were established according to the Clinical and Laboratory Standards Institute (CLSI) and the International Federation of Clinical Chemistry (IFCC) guidelines (C28-A3). Horn–Pesce robust method was used to estimate the 95% confidence interval and 95% reference interval.ResultsReference intervals were independent of age or gender and shown to be 12.9–52.8 μg/10⁸ cells (lymphocytes).ConclusionWe have defined pediatric reference ranges for lymphocyte vitamin C in healthy, fasted children at a relevant age group (0–7 years). The new reference interval can now be used to more reliably explore possible implications of variation of vitamin C levels on bleeding and other clinical signs.     

Age- and sex-specific reference intervals for blood copper, zinc, calcium, magnesium, iron, lead, and cadmium in infants and children

ObjectivesReference intervals for clinically important elements in infants and children are rarely reported, despite their importance for accurate clinical decision-making. The exploration of such reference intervals is essential.Design and MethodsSeven elements, including copper (Cu), zinc (Zn), calcium (Ca), magnesium (Mg), iron (Fe), lead (Pb), and cadmium (Cd), were analyzed on BOHUI 5100 and 2100 analyzers using blood samples from 4044 healthy infants and children.ResultsAge- and sex-specific reference intervals were established for Cu, Zn, Ca, Mg, Fe, Pb, and Cd.ConclusionsEstablished reference intervals for Cu, Zn, Ca, Mg and Fe can provide important guidance for the reasonable supplementation of trace elements and other essential elements in infants and children. Reference intervals for Pb and Cd can play a role in the surveillance and diagnosis of environmental overexposure.     

Glycoproteins of pleural effusions (author's transl)]

Using single radial immunodiffusion, ten glycoproteins from non purulent pleural fluids have been estimated in different diseases. For five proteins (prealbumin, ceruloplasmin, alpha2HS-glycoprotein, transferrin, beta2-glycoprotein 1) the results have been found not to correlate with the causal disease. However for orosomucoid, alpha1-antitrypsin, haptoglobin, alpha2-macroglobulin and hemopexin, there was good correlation between proteins levels and aetiology. The glycoprotein concentration was low in mechanical effusions from cirrhosis and chronic cardiac failure. It was high in inflammatory, post-embolism and particularly neoplastic effusions. A raised orosomucoid level occurred as the most characteristic of cancer states especially when associated with a parallel increase of the four other glycoproteins. A simultaneously elevated level of these five pleural glycoproteins seems to be a good and significant biological sign for neoplastic effusion diagnosis.     

Reference distributions for the positive acute phase proteins, alpha1-acid glycoprotein (orosomucoid), alpha1-antitrypsin, and haptoglobin: a comparison of a large cohort to the world's literature

Limiting bedside use of positive acute phase protein measurements (alpha1-acid glycoprotein (orosomucoid), alpha1-antitrypsin, and haptoglobin) has been the lack of satisfactory methods for quantifying serum levels and a credible reference material. Great strides have been made in the last few years. The remaining barrier to more relevant and cost-effective use of serum protein data for diagnosis and prognosis is the availability of reliable reference intervals from birth to old age for both males and females. Sixty publications reporting reference intervals have been identified which meet the criteria used in our prior two studies, and these have been analyzed statistically. Previous small studies of these individual proteins agree on average, over their constrained age ranges, with our life-long reference ranges. This meta-analysis provides support for our reference ranges and places them in the perspective of previous publications.     

Alpha1-antitrypsin phenotypes in chronic active liver disease and primary biliary cirrhosis.

Alpha1-antitrypsin concentrations and phenotypes were determined in groups of patients with chronic active liver disease and primary biliary cirrhosis. The concentrations of alpha1-antitrypsin were above normal values in both groups; the patients with primary biliary cirrhosis had higher concentrations than those with chronic active liver disease. The prevalence of common phenotypes in these two groups did not differ from that in a sample of healthy blood donors from this institution of from a large Norwegian sample. We interpret our data as disputing the view that alpha1-antitrypsin phenotypes, other than Z. significantly predispose adults to hepatic cirrhosis.     

Leucocyte-associated plasma proteins. Association of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, transferrin and haptoglobin with human lymphocytes, monocytes, granulocytes and a promyelocytic leukaemic cell line (HL-60)

The distribution of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, haptoglobin and transferrin, including their electrophoretic heterogeneous variants, was studied in isolated lymphocytes, monocytes and granulocytes and in a human promyelocytic cell line (HL-60) by crossed immunoelectrophoresis. Prealbumin, albumin and transferrin were present in lymphocytes, monocytes and granulocytes, whereas the cellular variants of orosomucoid and haptoglobin were present only in granulocytes. alpha 1-Antitrypsin was present in four electrophoretic variants which were differently distributed among the various cell types. Synthesis of alpha 1-antitrypsin by monocytes, granulocytes and HL-60 cells was demonstrated by 14C-leucine incorporation. The six plasma proteins could not be removed from intact cells by incubation with the respective antibodies at 0 degrees C, or iodinated by lactoperoxidase catalysed iodination at 23 degrees C. They were, however, readily solubilized by freeze-lysis of the cells, suggesting an intracellular localization. Compared to their plasma counterparts none of the proteins differed in their hydrophobic properties but the carbohydrate residues of orosomucoid, alpha 1-antitrypsin and haptoglobin were different. The pattern of disappearance of the proteins from the cells during incubation suggested that the localization of albumin and transferrin in relation to the cells differed from that of the other proteins.     

Biochemical modifications in a case of analbuminemia (author's transl)]

A new case of analbuminemia is described for a six month old child of Algerian origin. The serum albumin concentration was 64 mg/l and its immunochemical action was identical to that of normal albumin. The system reacted by an increase of the synthesis of globulins. For the subject, the alpha1-antitrypsin, ceruloplasmin, haptoglobin, alpha2-macroglobulin, transferrin and immunoglobulins M contents were three times higher than the standard figures. However, it was possible to show that the presence of free bilirubin independent from proteins could be detected at a concentration of 17 mumol/l.     

Identification and quantification of serum proteins secreted into the normal human jejunum

The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates. Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions. The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins and further 10-15% due to the remaining serum proteins. It is suggested that the serum proteins pass through the epithelium by a transcellular mechanism.     

Role of bioantioxidants depression and deficiency of protease inhibitor alpha 1-antitrypsin in mechanisms activating free radical oxidation and proteolysis in chronic pancreatitis

AIM: To elucidate the reason and mechanisms of neutralization of protease inhibitors and antioxidants by proteolytic enzymes and oxidants. MATERIALS AND METHODS: The trial included 92 patients with exacerbation of chronic pancreatitis. 47 of them had chronic recurrent pancreatitis (CRP), 45 patients had chronic fibrozing pancreatitis (CFP). Measurements were made of blood catalase and ceruloplasmin (according to P. Hubl, R. Breschneider and O. Houchin, respectively), alpha1-antitrypsin (by Reiderman), schiff bases (by B. Fletcher et al.), dienic conjugates (by Z. Placer), serum acid phosphatase (by Bodansky), acid phosphatase of polynuclear cells (by R. Nartsissiv), NBT-test was made according to B. Park. RESULTS: Exacerbation of CRP was associated with enhancement of free radical lipid peroxidation (FPOL), release of proteolytic and lysosomal enzymes from acinar cells, a fall in catalase level. Catalase depression depends on the level of blood lysosomal enzymes and partially on FPOL activity. In CFP moderate activity of FPOL and trypsin is associated with normal levels of lysosomal enzymes and catalase. In both pancreatitis forms, alpha1-antitrypsin levels are low. This lowering is primary and unrelated with inflammatory process in the pancreas. A trypsin rise in both forms depends on lowering of alpha1-antitrypsin which via trypsin inhibits formation of lysosomal enzymes in polynuclear cells. Inability of protease inhibitor to block proteolytic (lysosomal) enzymes manifests in initial intraacinar activation of trypsin from trypsinogen, in inflammatory focus under polynuclear cells release of lysosomal enzymes and in proteolytic enzymes release from the affected acinar structures. CONCLUSION: Lack of alpha1-antitrypsin--protease inhibitor--and depression of antioxidant catalase are main and intermediate elements in activation of mechanisms of proteolytic aggression and FPOL.     

Quantitative immunological determination of 12 plasma proteins excreted in human urine collected before and after exercise

Urine was collected from 6 healthy male adults at rest and from 20 male adults after a marathon race (25 miles). The concentrated urines were quantitatively analyzed, by single radial immunodiffusion, for their content in 12 different plasma proteins: tryptophan-rich prealbumin, albumin, alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, ceruloplasmin, haptoglobin, Gc-globulin, transferrin, hemopexin, beta(2)-glycoprotein I, gammaA-globulin, and gammaG-globulin.Albumin, gammaA-globulin, and gammaG-globulin represent the major part of the plasma proteins detected in normal urine excreted by humans at rest (12, 0.5, and 2.5 mg respectively, out of a total excretion of 17.5 mg of plasma proteins per 24 hr). The other plasma proteins were excreted at a lower rate (< 0.4 mg/24 hr). The relative content of tryptophan-rich prealbumin, alpha(1)-antitrypsin, Gc-globulin, transferrin, and gammaG-globulin was lower in normal urine than in normal serum, whereas that of alpha(1)-acid glycoprotein, beta(2)-glycoprotein I, and gammaA-globulin was higher. The ratio of gammaG-globulin to gammaA-globulin was 4.9:1. When plotted on a logarithmic scale, no direct relationship between the molecular weight of a protein and the value of its renal clearance could be observed.Strenuous exercise increased (up to 50-fold) the excretion of plasma proteins which represent 82% of the total proteins found in urine, instead of 57% in urine collected from humans at rest. There was particularly a significant rise of tryptophan-rich albumin, albumin, alpha(1)-acid glycoprotein, transferrin, gammaA-globulin, and gammaG-globulin (0.26, 127, 11.8, 3.3, 1.2, and 2.0 mug respectively, out of a total excretion of 167 mug of plasma proteins per min). The ratio of gammaG-globulin to gammaA-globulin was 16:1. After exercise, the renal clearance of proteins increased from 2 to 40 times, but, as for the urine of normal subjects at rest, no direct relationship between molecular weight and renal clearance could be observed.     

Serum proteins in diseases of the liver.

In 9 groups of liver diseases, 11 serum protein parameters were studied. Prealbumin, retinol-binding protein, albumin, alpha-lipoprotein and Normotest (NT) were usually highly intercorrelated and separated best among groups with different types of functional impairment. Haptoglobin, C3 and total iron-binding capacity were probably also sometimes reduced by restricted protein synthesis but appeared to be more easily affected by other factors, alpha1-antitrypsin was often increased in different liver diseases, but a pattern compatible with the "acute-phase reaction" was regularly present only in hepatic tumors. Bile retention was often accompanied by relatively high levels of ceruloplasmin, C3, and alpha-lipoprotein and by high NT values.     

Catabolic rate of alpha1-antitrypsin of Pi type M and Z in man.

1. Human alpha1-antitrypsin was isolated from three Pi M and two Pi Z subjects without alteration of its microheterogeneity. The purified proteins were labelled with either 125I or 131I by a lactoperoxidase method. 2. The disappearance rate of two types of alpha1-antitrypsin were studied after simultaneous injection of labelled M-protein and Z-protein into Pi M subjects. 3. The ratio of extravascular to plasma pools of alpha1-antitrypsin ranged between 1-2 and 1-6 with no difference between M- and Z-protein. The mean fractional catabolic rates of M-protein and Z-protein were respectively 0-26 and 0-40 per day. 4. The difference in catabolic rate of Z- and of M-protein is too small to explain why the alpha1-antitrypsin content of the blood in Pi ZZ subjects is only 15% of that normally found in Pi MM subjects. The low alpha1-antitrypsin in Pi ZZ subjects appears mainly to be due to a low rate of biosynthesis.     

Reference individuals, blood collection, treatment of samples and descriptive data from the questionnaire in the Nordic Reference Interval Project 2000

The rules for recruitment of reference individuals, inclusion and preparation of individuals, blood collection, treatment of samples (and control materials) and analysis at the 102 medical laboratories attending the Nordic Reference Interval Project (NORIP) are given as well as the rules for central exclusion of reference individuals. The individuals (18-91-year-olds) should be evenly distributed on age and gender groups. The 3002 reference individuals who contributed at least one reference value to the finally suggested reference intervals were characterized using the information in the questionnaire. Gender, age and country are the main entries in the tables. Other variables in the cross-tables or figure are height, weight, body mass index, ethnic origin, heredity for diabetes, chronic disease, oestrogens or oral contraceptives, other medication, hard physical activity, previous blood donations, smoking habits, use of alcohol, hours since last meal and time of blood collection (hour, day of week, month, year). The Danes had the highest alcohol consumption and the Icelanders had the highest body mass index. The information in this article may interest potential users of the Nordic Reference Interval Project bio-bank and database (NOBIDA) in which serum, Li-heparin plasma and EDTA buffy coat from the mentioned individuals are stored below -80 degrees C.     

Determination of reference intervals for 13 plasma proteins based on IFCC international reference preparation (CRM470) and NCCLS proposed guideline (C28-P, 1992): Trial to select reference individuals by results of screening tests and application of maximal likelihood method

By using a new international reference preparation for the plasma proteins (CRM470) prepared by IFCC, we launched a project to determine reference intervals for 13 plasma proteins (immunoglobulins, compliment components, transferrin, α₁ antitrypsin, α₁acid glycoprotein, haptoglobin, α₂macroglobulin, ceruloplasmin, albumin, transthyretin) in accordance with the guideline proposed by NCCLS. As reference individuals, 999 subjects were first selected through medical examination and a health-check questionnaire. However, there were many who had “abnormal” values in some of the 25 screening tests results measured simultaneously (serum enzymes, lipids, blood counts, etc.). Therefore, we adopted a criterion that those individuals who had “abnormal” values (beyond 99% confidence intervals) in the 25 tests would be excluded. The exclusion resulted in appreciable narrowing of the distributions in all 13 plasma proteins. This implied that “apparent” normality of major screening tests results may be used as a criterion to select truly relevant reference individuals. A parametric approach using a modified Box-Cox power transformation formula and the maximal likelihood estimation led to almost perfect normalization of the reference distributions on all the items tested. Since the nonparametric method proposed by the NCCLS guideline gave less reproducible results in our simulation study, the parametric method appears to be a method of choice for the calculation. © 1996 Wiley-Liss, Inc.     

Age and gender specific pediatric reference intervals for aldolase, amylase, ceruloplasmin, creatine kinase, pancreatic amylase, prealbumin, and uric acid

BackgroundReference intervals can vary based on age and gender. Proper partitioning is necessary to classify health status in different age groups.MethodsSeven analytes; aldolase, amylase, ceruloplasmin, creatine kinase, pancreatic amylase, prealbumin and uric acid; were assayed on Roche Modular P analyzers using serum samples from 1765 children (867 females and 898 males; age range, 6 months to 17 y). Subjects 6 months up to 7 y were undergoing minor surgical procedures. Children 7 to 17 y were apparently healthy. Subjects with significant medical history or who were taking any medications were excluded.ResultsSeparate reference intervals for boys and girls were required for 33% of the groups. Aldolase showed gender variation in the 6–8, 12–14, and 15–17 y. Amylase was the only analyte that showed no significant gender differences within any age group. Both ceruloplasmin and uric acid had significant differences between the 12–14 and 15–17 y groups. Creatine kinase exhibited statistically significant gender differences in all age groups with the exception of 6–8 y.ConclusionWe verified that when establishing pediatric reference intervals, partitioning by age and gender is frequently necessary.     

Cationic proteins of human granulocytes. V. Interaction with plasma protease inhibitors.

Several cationic proteins of human granulocytes possess chymotrypsin-like and bactericidal activities. The heat-labile chymotrypsin-like activity is inhibited by serum, owing to complex formation with alpha2-macroglobulin and alpha1-antitrypsin. The molar affinity of the cationic proteins for alpha2-macroglobulin is much higher than that for alpha1-antitrypsin. The results indicate that the molar combining ratios are 1:1 for cationic protein to alpha1-antitrypsin and 2:1 for cationic protein to alpha2-macroglobulin. The proteolytic activity against fibrinogen and casein is inhibited by both alpha2-macroglobulin and alpha1-antitrypsin, whereas the activity against small molecular synthetic substrates is inhibited by alpha1-antitrypsin but not alpha2-macroglobulin. The heat-stable bactericidal action of the cationic proteins against Staphylococcus was also inhibited by serum, probably owing to complex formation with alpha2-macroglobulin and alpha1-antitrypsin.