Volume 43, Issue 1

Inflammation is an important indicator of tissue injury. In the acute form, there is usually accumulation of fluids and plasma components in the affected tissues. Platelet activation and the appearance in blood of abnormally increased numbers of polymorphonucleocytes, lymphocytes, plasma cells and macrophages usually occur. Infectious disorders such as sepsis, meningitis, respiratory infection, urinary tract infection, viral infection, and bacterial infection usually induce an inflammatory response. Chronic inflammation is often associated with diabetes mellitus, acute myocardial infarction, coronary artery disease, kidney diseases, and certain auto-immune disorders, such as rheumatoid arthritis, organ failures and other disorders with an inflammatory component or etiology. The disorder may occur before inflammation is apparent. Markers of inflammation such as C-reactive protein (CRP) and urinary trypsin inhibitors have changed our appraisal of acute events such as myocardial infarction; the infarct may be a response to acute infection and (or) inflammation. We describe here the pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi). It is an important anti-inflammatory substance that is present in urine, blood and all organs. We also describe the anti-inflammatory agent bikunin, a selective inhibitor of serine proteases. The latter are important in modulating inflammatory events and even shutting them down.

Apolipoprotein H (apoH) is considered to be a necessary cofactor for the binding of certain antiphospholipid antibodies to anionic phospholipids. Some apoH-dependent antiphospholipid antibodies also exert lupus anticoagulant (LA) activity, which seems to depend on antiphospholipid antibody epitope specificity. The aim of this study was to evaluate whether the presence of less frequent apoH alleles may induce structural or conformational changes in these “LA-dependent” regions that may initiate more frequent autoimmune responses in subjects. We selected patients with confirmed LA activity and none or low titers of anticardiolipin antibodies that had been sent to the laboratory for routine antiphospholipid antibody determination. Many of them had some clinical manifestation of antiphospholipid syndrome. Antibodies to apoH were determined with a commercially available anticardiolipin/apoH ELISA kit. ApoH protein polymorphism (apoH phenotype) was demonstrated by isoelectric focusing and immunoblotting. Our results showed that 47/74 (63.5%) of our selected LA-positive patients also had elevated apoH-dependent antiphospholipid antibody titers. These results point to two subgroups of patients according to the LA potency of apoH-dependent antibodies. A strong positive correlation (non-linear or linear) for apoH-dependent antibody titers and LA activity was observed in both subgroups of patients. In this study, we did not find significant differences in the distribution of apoH phenotypes among control subjects and patients with apoH-dependent/LA-positive auto- antibodies.

As B-lymphocytes play an important role in innate and adaptive immunity, we aimed to examine the effects of CpG oligodeoxynucleotides (ODNs) on purified tonsil-originating CD19+ B-cells, representing mucosal B-cells. We screened various K-type ODNs, reactive with human B-cells, and tested for the production of immunoglobulins in vitro. Using one CpG-ODN, DSP30, we observed that it could upregulate not only Toll-like receptor 9 (TLR9) mRNA expression in activated B-cells, but also the early expression of CD69 followed by the sequential expression of CD80, CD86 and the nuclear factor (NF)-κB pathway. Furthermore, mRNA expression of certain B-cell-derived cytokines was influenced by exposure to DSP30, with a strong upregulation of interleukin 6 (IL-6) and downregulation of IL1-β. Stimulation of B-cells, co-stimulated with IL-2, IL-10 and soluble CD40 ligand (sCD40L) with different CpG-ODNs, had differing effects on the terminal differentiation in vitro of B-cells into immunoglobulin-secreting cells. TLR9 is involved in innate immunity and the recognition of bound CpG DNA from invading bacterial pathogens. As tonsillar B-cells are mucosal-type B-lymphocytes, this study suggests that CpG-ODNs show promise as mucosal adjuvants in modulating the local production of immunoglobulins of certain classes and subclasses, a crucial issue in vaccine perspectives.

The predictive power of serum κ/λ ratios on initial presentation of immunoglobulin G (IgG) or IgA monoclonal component was studied to differentiate between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. The retrospective study involved 145 patients clinically diagnosed with monoclonal gammopathy of undetermined significance or multiple myeloma, who had serum M-protein IgG <35g/L or IgA <20g/L at M-protein detection. Serum light chains κ and λ were measured by fixed-time nephelometry. Test performance indices, predictive values and likelihood ratios were calculated according to the Weissler recommendation. MM patients were considered as diseased and MGUS patients as non-diseased in order to estimate the performance characteristics of serum κ/λ ratios. There was a statistically significant difference in κ/λ ratios distribution between both groups of patients, in both M-protein κ-type (Mann-Whitney U=168, p<0.001) and in M-protein λ-type (Mann-Whitney U=143, p<0.001). Negative likelihood ratios at threshold levels of 0.6 and 4.2 were 2.17- and 3.32-fold greater, respectively, than positive likelihood ratios, so that the predictive power of a serum κ/λ ratio within these limits is better in ruling out (negative predictive power) than ruling in disease (positive predictive power). The post-test characteristics of a serum κ/λ ratio interval between 0.6 and 4.2 in discriminating MGUS from MM in our geographic population were: sensitivity 0.96 (0.93–0.99 95%CI); specificity 0.70 (0.63–0.77); positive predictive value 0.68 (0.64–0.73); negative predictive value 0.96 (0.94–0.99); likelihood ratios (+)LR 3.23 (2.68–4.04); and (−)LR 17.16 (11.00–63.00). Thus, serum M-protein with a κ/λ ratio between 0.6 and 4.2 increases the posterior probability of MGUS from 0.60 to 0.96 in asymptomatic patients, for whom only monitoring may be suggested when the serum κ/λ ratio is within these limits.

The soluble transferrin receptor (sTfR) distinguishes iron deficiency anemia from other types of anemia. Refractory iron deficiency anemia is often the onset symptom in malabsorption-induced celiac disease. We evaluated whether sTfR levels distinguish celiac disease-associated iron deficiency anemia from iron deficiency anemia of other origin. To this aim we measured sTfR and ferritin levels and their ratio (the sTfR/ferritin index) and other hematological parameters in 42 anemic children (20 with and 22 without celiac disease) vs. 22 non-anemic children with celiac disease and 31 healthy controls (age range 4–12years). Hemoglobin parameters, mean cell volume, and serum iron and ferritin levels were decreased to a similar extent in the anemic patients (celiac and non-celiac). The sTfR level in non-anemic celiac patients was similar to that of normal controls (1.7±0.35mg/L), whereas it was significantly increased in non-celiac and celiac anemic patients (2.2±0.5mg/L, p<0.05 and 2.7±1.2mg/L, p<0.001, respectively). The sTfR/ferritin index was also increased more in the anemic celiac patients (mean 4.4, range 1.5–12.0) than in anemic non-celiac children (mean 2.6, range 1.4–4.0) compared with non-anemic children (mean 1.2, range 0.7–2.0). Differences were more pronounced when ferritin was <5ng/mL. Thus, the sTfR/ferritin index may be a predictive measure in discriminating anemic patients with celiac disease from those without celiac disease.

The aim of the study was to assess the effect of cardiothoracic surgery on the dynamics of plasminogen, D-dimers and plasminogen activator inhibitor (PAI-I) during the first 24h after surgery. The study included 14 patients operated with (on-pump) and 14 without (off-pump) the use of extracorporeal circulation (ECC). Blood sampling was carried out on induction of anesthesia (timepoint 1), on introduction of heparin (point 2) and protamine (point 3), at the end of surgery (point 4), and the next morning (point 5). Relative to point 1, the utilization of plasminogen at point 2 was 24% and 17% in the on-pump and off-pump groups, respectively (p=0.001 both). Increased D-dimer concentration from the baseline was more pronounced in the on-pump group (p=0.001). At point 5, D-dimer concentrations were comparable in both groups and different from baseline levels. PAI-I activity showed within-group differences from baseline at point 5 in the off-pump group (p=0.001), and at points 3 and 5 in the on-pump group (p=0.002 and 0.001, respectively). At point 5, the activity of PAI-I was comparable in both groups, yielding p=0.001 vs. baseline. Fibrinolysis was more pronounced and more dynamic in the on-pump group due to activation of the systemic inflammatory response induced by the use of ECC. In the off-pump group, fibrinolysis was a normal physiological response to the surgical procedure.

Hematological and biochemical tests, including white blood cell count (WBC), C-reactive protein (CRP) and other acute-phase reactants, have been used in the diagnosis of acute appendicitis. However, there is controversy among physicians about the value of this practice in children. The objective of our study was to evaluate serum amyloid A protein (SAA) levels in children with confirmed acute appendicitis and to compare the sensitivity and specificity of this marker of inflammation with those for WBC and CRP. A prospective cohort study of 60 children admitted with abdominal pain to rule out appendicitis was used in the study. Of these, 42 underwent surgery, while 18 children who had spontaneous amelioration within 24h of admission were not operated on and served as controls. WBC and serum SAA and CRP levels were obtained preoperatively. Serum concentrations of the analytes were determined with particle-enhanced immunonephelometric methods. Patients with acute appendicitis had WBC, SAA and CRP levels higher than those of the control group (p<0.001). There was no appendicitis patient with a normal SAA value, while 21.4% of the patients had CRP values within the normal range. The performance of each test was measured by receiver-operating characteristic curves. Area under the curve (AUC) values were 0.849 for WBC, 0.868 for CRP and 0.964 for SAA. The sensitivity and specificity of these methods were 76% and 75% for WBC>10.0×10 9 /L, 62% and 94% for CRP>10mg/L and 86% and 83% for SAA >45.0 mg/L, respectively. Circulating SAA levels have better discriminatory value than WBC or CRP in the assessment of acute appendicitis in children. Thus, this test appears to be of higher value than the current standards of care in the diagnosis of this condition.

This study employed customer feedback surveys to assess the quality of the laboratory services provided by an accredited clinical microbiology laboratory. The purpose of the study was to classify external failures and determine the quantity of such errors, and to establish how much time was spent processing and correcting these errors, together with the costs arising. The study indicates that the quality of laboratory test results can be regarded as good based on the accuracy of the result. Customer needs were also well taken into account in practice because of the good quality of the service. However, improvements are required in providing customers with test results without delay. The cost of processing and correcting such errors was negligible. The use of a customer feedback system allows systematic monitoring of external failures. In the accredited laboratory under study, it is unnecessary to monitor continually the costs and working time involved in processing and correcting external failures. The most essential point is to monitor the effects of the quality measures taken to reduce the number of failures.

The aim of this study was to investigate whether or not there is an increase in serum total and free prostate specific antigen levels (t-PSA, f-PSA) in patients with and without benign prostatic hyperplasia (BPH) undergoing extracorporeal circulation during cardiovascular bypass. The study included a total of 50 men. Of these, 35 patients underwent elective coronary artery bypass grafting with extracorporeal circulation [with (n=20, group I) and without (n=15, group II) BPH]. Another 15 patients underwent renal or ureteral surgery (group III) and served as a control group. Serum t-PSA and f-PSA levels were measured before surgery and 3h and 3days after surgery. All patients underwent urethral catheterization 24h before surgery. In groups I and II, patients had an increase in t-PSA 3h after surgery compared to baseline values (p=0.0001 and p=0.011, respectively). Also, 3days after surgery, mean t-PSA levels were higher than baseline levels (p=0.004) in group I. Serum t-PSA levels were higher at 3h than at day 3 in groups I and II (p=0.003 and p=0.02, respectively). Mean serum f-PSA levels obtained 3h after surgery were increased in both groups I and II when compared to baseline values (p=0.0001 and p=0.001, respectively). There was no significant difference between f-PSA values before and 3days after surgery in all groups. In the control group, there was no significant increase in either serum t-PSA or f-PSA levels obtained at different times. There was a modest increase in the percentage of free prostate specific antigen (% f-PSA) 3h after the operation in group II (p=0.025); the values returned to baseline within 3days. It was suggested that t-PSA and f-PSA serum levels increase due to extracorporeal cardiopulmonary circulation as a consequence of ischemic damage to the prostate. In patients with BPH, this rise may be higher than in those without BPH, because BPH patients generally have larger prostate volumes that may be more vulnerable to ischemic damage. Because f-PSA has a shorter half-life, postsurgical levels of f-PSA may not show this ischemic damage to the prostate during the late postoperative period and f-PSA appears to be cleared more rapidly than t-PSA.

Ongoing demands on laboratory performance require optimization of processes. An obvious way to achieve this is to reduce manual labor in favor of automated methods. We describe the validation of an automated quantitative urine human chorionic gonadotropin (hCG) analysis on the Roche Modular E170 analyzer to replace the manual qualitative pregnancy test in urine. At urine hCG concentrations of 476, 45 and 11U/L, we found inter-assay variation of 4.3%, 4.3% and 6.8% and average intra-assay variation of 3.0%, 2.6% and 3.0%, respectively. The analytical detection limit was 0.7U/L. We did not detect any loss (due to degradation or adsorption) during a storage period of 5days at 4°C or at −20°C. Recoveries of hCG in urine of a pregnant woman diluted with urine of a pre-menopausal non-pregnant woman (concentration range between 6 and 800mU/L) were between 93% and 112% (y=0.997x−3.843, r 2 =0.999). Diluting a serum sample (hCG 42,000U/L) with urine (negative for hCG) up to 8000-fold yielded a completely linear hCG response, indicating that the assay was not affected by the urine matrix. In a correlation study with 60 urine samples (of which 10 were of male origin), we did not find any discrepancies between results for the manual pregnancy test and the hCG test on the Roche Modular E170 (using a cutoff value of 50U/L).

Dextran interference in biuret-type assays of total serum proteins was investigated in a Belgian National External Quality Assurance Survey with 256 participants. In vitro supplementation of therapeutic (10% Gentran 70 ® ) dextran concentrations showed a broadly varying (from 0 to 20%) negative interference. The analytical interference was found to depend on both the sodium hydroxide and tartrate concentrations in the reagent formulation. The dry chemistry biuret method was not affected by the dextran interference. In a number of cases, the effects observed may be of clinical importance. Both clinicians and laboratory staff should be aware of the persistence of this analytical problem.

Insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P) have been proposed as indirect biomarkers for the detection of the misuse of recombinant human growth hormone in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays was carried out. For total IGF-I, two radioimmunoassay (RIA) kits (IGF-I/RIA1, Nichols Institute Diagnostics and IGF-I/RIA2, Mediagnost) and one enzyme-linked immunosorbent assay (ELISA) (R&D) were evaluated. For P-III-P, two RIA kits (P-III-P/RIA3, Cis-bioInternational and P-III-P/RIA4, Orion Diagnostica) were studied. The intra-laboratory precision and accuracy values for all IGF-I assays were better than 15%. The IGF-I/ELISA showed the lowest limit of quantification (LOQ) and its calibration curve covered the range of concentrations found in human serum samples. Higher agreement between laboratory results was obtained for IGF-I/ELISA and IGF-I/RIA1. Low inter-technique correlation was obtained for the three assays; the only comparable results were obtained between IGF-I/ELISA and IGF-I/RIA1. For P-III-P, intra-laboratory precision and accuracy values better than 15% were obtained for both assays in almost all cases. The calibration curve for P-III-P/RIA4 covered the range of concentrations of serum samples, while 30% of the values for P-III-P/RIA3 were below the calibration sample with the lowest concentration. Inter-laboratory correlation was also higher for P-III-P/RIA4. In summary, ELISA and RIA4 were the most suitable assays for measurement of IGF-I and P-III-P, respectively, in serum samples. However, the validation studies carried out show the need for harmonization of immunoassay parameters to improve the reproducibility and comparability of results between different laboratories and in different studies.

Serum sex hormone-binding globulin (SHBG) regulates the cellular bioavailability of SHBG-bound steroid hormones. Since variations in SHBG levels may affect the concentration of free, i.e., biologically active testosterone in serum, SHBG levels are commonly measured as a supplement to total testosterone determination. The recently developed electrochemiluminescence Elecsys ® SHBG immunoassay was evaluated analytically on a Modular E170 (Roche Diagnostics, Mannheim, Germany) immunoanalyzer. Major differences in SHBG concentrations have been described among the commercially available methods; we therefore compared the new method with an established SHBG immunoradiometric assay (IRMA) in 99 routine serum samples. To provide reference values to clinicians, SHBG concentration was measured by Elecsys ® in 304 serum samples from healthy volunteers and several relevant clinical subgroups. The within-run and total imprecision coefficients of variation were ≤2.9% and ≤3.3%, respectively. Functional sensitivity was at least 0.74nmol/L. Recoveries after dilution of high-concentration samples in low-titer human serum or in assay diluent were within the range of 85–110%. The Elecsys ® SHBG assay correlated well (r=0.98) with the SHBG immunoradiometric assay, but values were higher for the Elecsys ® assay (Passing Bablok regression analysis: slope 1.14, intercept +2.5). In healthy subjects and clinical subgroups, we confirmed the differences in SHBG values reported in the literature. The Elecsys ® SHBG immunoassay provides precision and reliability in combination with reduced turnaround time.

Although the establishment of medical laboratory institutions was a continuous process that matured only after the 16th century, several attempts had already been made to attain a diagnosis by investigating bodily excrement. In the first part of our work, published in a previous issue of this journal, we presented data on urine, sperm, menses and stools. In this paper we present data on sputum, vomit, blood, sweat, and autopsies, thus completing the list of human materials used for laboratory examinations. All the data used are extracted from codices of Late Antiquity and Byzantium and translated by us. We did not study medical texts from the other great ancestors of Western medicine, namely Arabic and Jewish writings. From the texts cited, it is apparent that the lack of technological means was no obstacle for the doctor to create an “examinational” mind, i.e., to try to correlate the macroscopic findings in the excrement with the pathophysiological mechanism that induced them, solely with the use of the senses. This not only applies to the examination of urine, as is commonly assumed, but also to many other excrements of the upper and lower orifices of the body, as well as the human body as a whole.