LC-MS/MS in clinical chemistry: Did it live up to its promise?: Consideration from the Dutch EQAS organisation.

BACKGROUND: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the promise of improvements in sensitivity and specificity, better standardization with often non-commutable international standards, and better between-laboratory comparison. However, it remains unclear whether routine performance of the LC-MS/MS methods have met these expectations. METHOD: This study examined the EQAS results, from the Dutch SKML, of serum cortisol, testosterone, 25OH-vitaminD and cortisol in urine and saliva over 9 surveys (2020 to first half of 2021). RESULTS: The study found a significant increase in the number of compounds and in the number of results measured in the different matrices, with LC-MS/MS over a period of eleven years. In 2021, approximately 4000 LC-MS/MS results were submitted (serum: urine: saliva = 58:31:11%) compared to only 34 in 2010. When compared to the individual immunoassays, the LC-MS/MS based methods for serum cortisol, testosterone and 25OH-vitaminD showed comparable but also higher between-laboratory CVs in different samples of the surveys. For cortisol, testosterone and 25OH-vitaminD the median CV was 6.8%, 6.1% and 4.7% respectively for the LC-MS/MS compared to 3.9-8.0%,4.5-6.7%, and 7.5-18.3% for immunoassays. However, the bias and imprecision of the LC-MS/MS was better than that of the immunoassays. CONCLUSION: Despite the expectation that LC-MS/MS methods would result in smaller between-laboratory differences, as they are relatively matrix independent and better to standardize, the results of the SKML round robins do not reflect this for some analytes and may be in part explained by the fact that in most cases laboratory developed tests were used.

Reference ranges for analytes of thyroid function in children.

Promptly detecting pediatric thyroid dysfunction requires age-appropriate reference ranges for serum thyroid-stimulating hormone (TSH), serum free thyroxine (FT4), and serum free triiodothyronine (FT3). We sought to establish such ranges, employing the widely-used Immulite® 2000 automated immunoluminometric assays in a large population. We assayed the analytes according to manufacturer's instructions in serum samples from 359 male and 297 female university hospital patients, aged between newborn to 18 years, without evidence of thyroid or pituitary dysfunction. As data were not normally distributed, the reference ranges were assumed to lie between the 2.5th and 97.5th percentiles. Curves for age-related changes in the reference ranges were calculated using the linearity, median and skewness method. TSH, FT4, and FT3 reference ranges showed a wide spread immediately after birth, rapidly decreasing within the first 2 years of life. Reference range width was fairly stable after about age 4 years. However, from that time, the ranges' lower and upper limits steadily declined, essentially reaching (FT3) or approximating (TSH, FT4) healthy adult values by age 18 years. Age-specific reference ranges should be used when measuring TSH, FT4, and FT3 in children. During very early life, values of these analytes range widely, making it challenging to interpret measurements in infants, and, especially, newborns.

Heterophile antibodies rarely influence the measurement of thyroglobulin and thyroglobulin antibodies in differentiated thyroid cancer patients.

The aim of the study was to determine the impact of heterophile antibodies on the measurement of serum thyroglobulin (Tg), thyroglobulin recovery, and thyroglobulin antibody levels in differentiated thyroid carcinoma patients. We studied serum samples of 201 individual patients that were followed in our hospital for differentiated thyroid carcinoma and 52 control samples. Samples were split; half were treated by incubating the sample for 1 h in HAB-blocking tubes, the remainder was left untreated. Subsequently thyroglobulin and thyroglobulin antibody levels were measured in both the blocked and untreated samples. A difference between the two samples was considered significant if the blocked sample deviated from the untreated one by more than 2.77 times the standard deviation for the method. In the measurement of Tg, 2 patients showed a moderate, but significant lowering of Tg levels after blocking treatment, but not so great as to affect clinical management. None of the 52 controls showed heterophile antibody interference in thyroglobulin measurement. Neither in DTC patients, nor in controls was any possible heterophile antibody interference encountered. And in all thyroid carcinoma patients, and in all but one controls, no interference was found in the thyroglobulin antibody measurement. All in all a possible heterophile antibody interference was found in 3/759 tests (0.4%). We can assume that heterophile antibody interference is not a factor to be reckoned with in the daily practice of Tg measurement in the treatment and follow-up of differentiated thyroid carcinoma patients.

Low bone mineral density in adult patients with moderate to severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease commonly treated with topical corticosteroids. The inflammatory nature of this disorder and the use of topical corticosteroids represent potential risk factors for bone loss. OBJECTIVES: The aim was to assess the prevalence of osteoporosis and osteopenia in adult patients with moderate to severe AD. In addition, the associations between topical/oral corticosteroid use and bone mineral density (BMD) and between disease activity and BMD were studied. PATIENTS AND METHODS: We studied 125 adult patients with moderate to severe AD. Using dual-energy X-ray absorptiometry, BMD was measured at lumbar spine and hips. The cumulative dose of topical and oral corticosteroids was calculated from pharmacy prescription records. Lifestyle parameters were collected by a questionnaire. Biochemical parameters of bone metabolism and disease activity [serum concentration of thymus and activation-regulated chemokine (TARC) levels] were also measured. RESULTS: Osteoporosis was documented in six patients (4.8%) and osteopenia in 41 patients (32.8%); 30.4% of the patients had a Z-score

Value of diagnostic radioiodine scintigraphy and thyroglobulin measurements after rhTSH injection.

UNLABELLED: Measurements of thyroglobulin (Tg) levels 72 h after administration of recombinant human thyrotropin (rhTSH) are recommended by the manufacturer in the follow-up of patients with differentiated thyroid carcinoma (DTC). In our department, Tg measurements are performed both 24 h and 72 h after administration of rhTSH, together with 72 h post rhTSH 131I whole body scintigraphy (WBS). The OBJECTIVE of this study is to compare the diagnostic usefulness of Tg measurements 24 and 72 h after rhTSH administration, and 131I WBS. PATIENTS AND METHODS: 181 patients were included who had been referred to our Nuclear Medicine Department for follow-up after 131I ablation of DTC. Tg measurements 24 h (Tg24) and 72 h (Tg72) after rhTSH, and 131I WBS, were done in all patients. The lower detection limit of Tg was 0,2 microg/l. RESULTS: 47 patients (26%) had detectable Tg levels: in 4/47 cases (8%) only Tg24 was detectable (always <1 microg/l), and in 6/47 cases (11%), only Tg72 was detectable. In 10/47 patients with detectable Tg-levels, Tg24 and Tg72 tested equally. In 27/47 cases, Tg24 was lower, and in 10/47 higher, than Tg72. Two patients with one or two positive Tg-test results also had a positive 131I WBS. In 8 patients (14%) only the 131I WBS was positive; an anatomical substrate for such a Tg-negative positive WBS was confirmed in only 2 patients. CONCLUSION: Tg-measurement 72 hours after rhTSH injection reveals all clinically relevant detectable Tg-levels. Diagnostic 131I scintigraphy may be omitted, even in high-risk patients.

Elevated plasma corticotrophin-releasing hormone levels in veterans with posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is associated with alterations in corticotrophin-releasing hormone (CRH) secretion. Plasma CRH levels, which are easily acquired, might serve as a predictor of hypothalamic CRH levels. Assessment of plasma CRH, adrenocorticotrophin hormone (ACTH), and cortisol levels in 31 veterans with PTSD, 30 traumatized veterans without PTSD matched on age, year, and region of deployment (traumacontrols), and 28 age-matched healthy controls (HCs) was carried out. Plasma CRH levels were higher in PTSD patients compared to both HCs (p=0.005) and traumacontrols (p=0.007). This led to our conclusion, that elevated plasma CRH levels are specifically related to PTSD and not to exposure to traumatic stress during deployment.

Enhanced cortisol suppression in response to dexamethasone administration in traumatized veterans with and without posttraumatic stress disorder.

BACKGROUND: While enhanced cortisol suppression in response to dexamethasone is one of the most consistent biological findings in posttraumatic stress disorder (PTSD), the relative contribution of trauma exposure to this finding remains unclear. METHODS: Assessment of diurnal salivary cortisol levels and 1600 h salivary cortisol before and after oral administration of 0.5mg dexamethasone in veterans with PTSD, veterans without PTSD (trauma controls) and healthy controls. Assessment of 1600 h plasma cortisol, ACTH and corticotrophin binding globulin (CBG) in response to dexamethasone in PTSD patients and trauma controls. RESULTS: Both PTSD patients and trauma controls demonstrated significantly more salivary cortisol suppression compared to healthy controls. Salivary cortisol, plasma cortisol and ACTH suppression as well as CBG levels did not differ between PTSD patients and trauma controls. PTSD patients showed a reduced awakening cortisol response (ACR) compared to healthy controls that correlated significantly with PTSD symptoms. No significant differences were observed in ACR between PTSD patients and trauma controls. CONCLUSIONS: These data suggest that enhanced cortisol suppression to dexamethasone is related to trauma exposure and not specifically to PTSD. The correlation between the ACR and PTSD severity suggests that a flattened ACR may be a result of clinical symptoms.

Intracavitary deposits on Essure® hysteroscopic sterilization devices: A case report.

OBJECTIVE: To study the composition of intracavitary deposits on Essure® hysteroscopic sterilization devices. DESIGN: Case report. SETTING: Reproductive Medicine and Gynecology department of a University Hospital. PATIENTS: A 39 years old patient presenting with a request for surgical removal of Essure® sterilization devices. Diagnostic hysteroscopy showed a crystal like white deposit attached to one of the devices. INTERVENTION: Diagnostic hysteroscopy and surgical removal of Essure® devices was performed. The deposits were collected and infrared spectroscopy analysis was performed. MAIN OUTCOME MEASURE: Chemical composition of the deposits attached to the device. RESULTS: Infrared spectroscopy of the material showed patterns conclusive with calcite (calcium carbonate, CaCO3). CONCLUSION: Until now, it is not clear if there is a relationship between reported complaints and formation of calcite deposits on Essure®. CAPSULE: Infrared spectroscopy of deposits on Essure® devices showed a pattern conclusive with calcite. The relationship between reported complaints and the formation of calcite deposits on Essure® remains unclear.

A potential role for the mast cell in the pathogenesis of idiopathic osteoporosis in men.

Osteoporosis is increasingly being recognized in men. Secondary causes are often implicated, but the mechanism of bone loss remains unclear in about a third of patients. The mast cell is a complex cell that stores a number of factors known to affect bone metabolism. Patients with systemic mastocytosis often demonstrate osteoporosis and bone marrow mast cells may be increased in osteoporotic postmenopausal women. We address the possible role of the mast cell in the pathophysiology of male osteoporosis by studying the relationship between bone marrow infiltration with mast cells and the 24 h urine excretion of N-methylhistamine, and the severity of osteoporosis in 48 consecutive men with idiopathic osteoporosis (bone mineral density Z score of <-1 and/or at least one prevalent vertebral fracture). Secondary causes for osteoporosis were excluded and none of the patients had systemic manifestations of enhanced mast cell activity. A widely variable number of morphologically normal mast cells were counted in toluidine blue-stained sections from 42 of 46 evaluable bone marrow biopsies. In 4 of the 42 biopsies (9%), clusters of abnormal mast cells were identified. These four patients were the only ones who also demonstrated increased 24 h urine excretion of N-methylhistamine. There was a significant positive relationship between mast cell number and the 24 h urine excretion of N-methylhistamine reflecting mast cell activity (p = 0.0001), and this latter measurement correlated negatively with bone mineral density (BMD) at the lumbar spine (p < 0.001). We identified clinically important bone marrow cell infiltration with pathologic mast cells in the absence of systemic manifestations of mast cell hyperactivity as an additional secondary cause for osteoporosis in some 9% of men with idiopathic osteoporosis, and found urinary excretion of N-methylhistamine to be above the upper limit of the normal laboratory reference range diagnostic for this cause of secondary osteoporosis. The more continuous spectrum in the relationship between mast cell activity and BMD supports a potential role for this cell in the pathogenesis of idiopathic male osteoporosis.

Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind, placebo-controlled study.

Cisplatin-induced toxicities are mainly caused by the formation of free radicals, leading to oxidative organ damage. Plasma concentrations of antioxidants decrease significantly during cisplatin chemotherapy for cancer. Forty-eight cancer patients treated with cisplatin-based chemotherapy were randomised in a double-blind manner to receive either supplementation with vitamin C, vitamin E and selenium dissolved in a beverage or to receive a placebo beverage. Primary outcome measures were the amount of nephrotoxicity and ototoxicity induced by cisplatin. No significant differences were found between the two study groups with respect to these primary outcome measures. However, patients who achieved the highest plasma concentrations of the three antioxidant micronutrients had significantly less loss of high-tone hearing. In addition, significant correlations were found between the reduced/oxidised vitamin C ratio and malondialdehyde (MDA), markers of oxidative stress, and cisplatin-induced ototoxicity and nephrotoxicity. The lack of protection against cisplatin-induced toxicities in patients in the intervention arm may be related to poor compliance and/or inadequate supplementation. Supplementation with a higher dose (intensity) and in combination with other antioxidants should be investigated further.

Column chromatographic characterization of complex formation of pro-IGF-II isoforms with acid labile subunit and IGF-binding proteins associated with non-islet cell tumour induced hypoglycaemia.

OBJECTIVE AND DESIGN: Non-islet cell tumour induced hypoglycaemia (NICTH) is a paraneoplastic phenomenon that is associated with the formation of several isoforms of pro-insulin like growth factor 2 (pro-IGF-II), or so called "big" IGF-II. Disturbance of ternary complex formation by big IGF-II is assumed to be a crucial early event in the pathogenic cascade of hypoglycaemia. By size-exclusion chromatography, we investigated complex formation by adding different naturally occurring isoforms of pro-IGF-II to pooled normal adult serum. Results were compared with the analysis of the serum from a patient with NICTH. RESULTS: Gel filtration experiments with the serum of a patient with NICTH demonstrated that ternary complex formation was severely compromised. The various forms of pro-IGF-II did not induce a shift of IGF-binding protein 3 (IGFBP-3) from 150kD towards smaller binary complexes in the normal adult serum, suggesting that they did not interfere with the interaction between the acid labile subunit and IGFBP-3. Instead, unglycosylated recombinant pro-IGF-II[1-104] was capable of forming a 150kD complex. In contrast, predominantly glycosylated and unglycosylated pro-IGF-II[1-87] eluted in the free unbound form. We showed that mature IGF-II and isoforms of pro-IGF-II were able to phosphorylate the IGF-I receptors of MC7 cells, albeit to a markedly lesser extent than IGF-I. When the patient's serum was tested in this system, the IGF-I receptor phosphorylation activity was considerably less than that in sera from age matched healthy individuals. CONCLUSION: We postulate that, alongside the presence of big IGF-II in the circulation, additional steps are required to stimulate the release of IGF-II and pro-IGF-II isoforms from IGFBPs in vivo. These factors may be proteases, that are present in the local environment of the tumour and in insulin-sensitive tissues.

Hereditary persistence of alpha-fetoprotein (HPAF P): review of the literature.

Alpha-fetoprotein (AFP) serum levels are raised in several clinical conditions, ranging from non-pathological conditions to malignancies. Hereditary persistence of alpha-fetoprotein (HPAFP) is a rare benign disorder with elevated AFP levels. HPAFP is described as a benign autosomal dominantly inherited condition which is not associated with any clinical disability or additional symptoms. In the past 28 years, only 19 families have been described; due to this unfamiliarity with HPAFP, elevated AFP levels are never attributed to HPAFP. However, undiagnosed HPAFP can result in inappropriate and unnecessary treatment decisions. Therefore, HPAFP should be taken into consideration in patients with unexplained elevated AFP levels, and especially in patients with urological disorders.

Heterophilic antibodies may be a cause of falsely low total IGF1 levels.

BACKGROUND: A low serum total IGF1 is considered as a diagnostic indicator of GH deficiency (GHD) in the presence of hypopituitarism. Introduction of IRMA and chemiluminescent immunometric assay (CLIA) IGF1 immunoassays has introduced endogenous antibodies as a new source of interference. In general, this goes unnoticed and might lead to unnecessary diagnostic and therapeutic interventions. CASE: A 56-year-old man was referred with a decline in physical performance, unexplained osteopenia, and weight loss of 3 kg over the past 8 months. Although clinical signs and symptoms were unremarkable, laboratory results pointed to secondary hypothyroidism and secondary hypogonadism. In addition, the serum total IGF1 level (CLIA; Siemens Medical Solutions Diagnostics) was in the low normal range. Two GH stimulation tests were performed, but these tests did not support the diagnosis GHD. Moreover, IGF1 bioactivity measured by the kinase receptor activation assay was normal. Interference of heterophilic antibodies was considered. After pretreatment with specific heterophilic blocking tubes that contain blocking reagents to eliminate heterophilic antibodies, serum-free thyroxine, testosterone, and IGF1 levels turned out to be normal. CONCLUSION: To the best of our knowledge, we here describe the first case in the literature of a patient with low serum total IGF1 levels due to interference from heterophilic antibodies in the used IGF1 immunoassay. When confronted with low-IGF1 levels that do not fit the clinical picture, interference of heterophilic antibodies should be considered in the differential diagnosis.

Low basal serum cortisol in patients with severe atopic dermatitis: potent topical corticosteroids wrongfully accused.

BACKGROUND: Topical corticosteroids are used extensively to treat inflammatory skin disorders including atopic dermatitis (AD). Several studies have described temporary reversible suppression of hypothalamic-pituitary-adrenal function. However, sound evidence of permanent disturbance of adrenal gland function is lacking. OBJECTIVES: To relate basal cortisol levels to prior use of topical corticosteroids and disease activity in patients with moderate to severe AD and to investigate the effect on basal serum cortisol levels of topical corticosteroid treatment during hospitalization. METHODS: Two groups of patients with AD were evaluated: 25 inpatients with severe AD who required hospitalization (group 1) and 28 outpatients with moderate to severe AD (group 2). In group 1, morning basal serum cortisol levels were measured twice, at admission and at discharge; in group 2, morning basal serum cortisol levels were measured once. Use of topical corticosteroids in the 3 months prior to the cortisol measurement was recorded and disease activity was monitored using the Six Area, Six Sign Atopic Dermatitis (SASSAD) score and serum thymus and activation-regulated chemokine (TARC) levels. RESULTS: On admission, basal cortisol levels in group 1 were significantly (P < 0.001) decreased in 80% of the patients. In group 2, the basal cortisol levels were normal in all but three patients. Comparing the two groups, group 1 on admission had a significantly lower cortisol level than that of group 2 (P < 0.001). Disease activity in group 1 on admission was significantly higher than that of group 2 (P < 0.001). There was no difference in use of topical corticosteroids in the 3 months before cortisol measurement. At discharge in group 1 there was a significant increase (P < 0.0001) of basal cortisol levels and a significant (P < 0.001) decrease in disease activity reflected by the decrease in serum TARC levels and SASSAD score. CONCLUSIONS: Disease activity, rather than the use of topical corticosteroids, is responsible for the low basal cortisol values in patients with severe AD.

Detection of circulating Tg-mRNA in the follow-up of papillary and follicular thyroid cancer: how useful is it?

To investigate the usefulness of thyroglobulin mRNA (Tg-mRNA) detection in peripheral blood in the follow-up of papillary and follicular (differentiated) thyroid cancer, a literature study was performed. Both evidence for and evidence against the usefulness of Tg-mRNA detection were found. Also, evidence for the expression of Tg-mRNA in cells other than normal or neoplastic thyroid follicular cells was found. It is concluded that currently Tg-mRNA detection is not a useful tool in the follow-up of differentiated thyroid carcinoma, but that the concept of using RT-PCR measurements during follow-up still warrants further research.

Non-protein bound iron release during chemotherapy in cancer patients.

Non-protein bound iron (NPBI) is able to catalyse oxidative reactions, causing damage to vital structures. Adverse effects induced by cisplatin seem, in part, to be mediated by free radicals. In the present study, we have measured plasma NPBI, various other iron parameters and antioxidants in 28 cancer patients undergoing cisplatin-based chemotherapy at various time points before and during chemotherapy. No NPBI was present prior to therapy, but within 1-4 days following the first administration of chemotherapy, mean NPBI rose significantly to 10.6+/-6.6 micromol/l (range, 0.6-21.3 micromol/l) in 18 (64.3%) of the 28 patients measured. The rise in NPBI was accompanied by a significant rise in total plasma iron and ferritin and a marked decrease in the latent iron-binding capacity. Concomitantly, plasma vitamins C and E decreased significantly, indicating consumption of antioxidants. Similar observations were also made during the fourth chemotherapy cycle. The increase in NPBI preceded and correlated significantly with chemotherapy toxicity, such as a decrease in leucocyte count and haemoglobin, with a transient rise in various liver enzymes and with known cisplatin-related toxicity, i.e. the loss of renal and hearing function. In conclusion, cisplatin chemotherapy induces oxidative damage which rapidly leads to release of iron from intracellular proteins and the appearance of NPBI. Bone marrow, red blood cells, liver and kidney seem to be a likely source of NPBI. The observed high levels of NPBI may be a major causative determinant in chemotherapy-induced toxicity.