Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

Diffuse mesangial sclerosis in a PDSS2 mutation-induced coenzyme Q10 deficiency.

BACKGROUND: A 7-month-old male infant was admitted because he was suffering from nephrotic syndrome, along with encephalomyopathy, hypertrophic cardiomyopathy, clinically suspected deafness and retinitis pigmentosa, and an elevated serum lactate level. METHODS: Coenzyme Q10 supplementation was started because of the clinical suspicion of primary CoQ10 deficiency. Despite intensive efforts, he passed away 4 weeks after admission. RESULTS: The results of genetic tests, available postmortem, explored two hitherto undescribed mutations in the PDSS2 gene. Both were located within the polyprenyl synthetase domain. Clinical exome sequencing revealed a heterozygous missense mutation in exon 3, and our in-house joint-analysis algorithm detected a heterozygous large 2923-bp deletion that affected the 5 prime end of exon 8. Other causative defects in the CoQ10 and infantile nephrosis-related genes examined were not found. A postmortem histological, immunohistochemical, and electron microscopic evaluation of the glomeruli revealed collapsing-sclerosing lesions consistent with diffuse mesangial sclerosis. The extrarenal alterations included hypertrophic cardiomyopathy and diffuse alveolar damage. A histological evaluation of the central nervous system and skeletal muscles did not demonstrate any obvious abnormality. CONCLUSIONS: Until now, the clinical features and the mutational status of 6 patients with a PDSS2 gene defect have been reported in the English literature. Here, we describe for the first time detailed kidney morphology features in a patient with nephrotic syndrome carrying mutations in the PDSS2 gene.

[Maternal and neonatal vitamin B12 deficiency detected by expanded newborn screening]. / Anyai és újszülöttkori B12-vitamin-hiány felismerése kiterjesztett újszülöttkori szuréssel.

INTRODUCTION: Infant vitamin B12 deficiency can manifest as a severe neurodegenerative disorder and is usually caused by maternal deficiency due to vegetarian diet or pernicious anaemia. Its early recognition and treatment can prevent potentially serious and irreversible neurologic damage. Biochemically, vitamin B12 deficiency leads to an accumulation of methylmalonic acid, homocysteine, and propionylcarnitine. Expanded newborn screening using tandem mass spectrometry may identify neonatal and maternal vitamin B12 deficiency by measurement of propionylcarnitine and other metabolites in the dried blood spot sample of newborns. AIM: To summarize our experiences gained by screening for vitamin B12 deficiency. METHOD: Clinical and laboratory data of vitamin B12-deficient infants diagnosed in Szeged Screening Centre were retrospectively analysed. RESULTS: In Hungary, expanded newborn screening was introduced in 2007. Since then approximately 395 000 newborns were screened in our centre and among them, we identified four newborns with vitamin B12 deficiency based on their screening results. In three cases an elevated propionylcarnitine level and in the fourth one a low methionine level were indicative of vitamin B12 deficiency. We also detected an additional vitamin B12-deficient infant with neurological symptoms at 4 months of age, after a normal newborn screening, because of elevated urinary methylmalonic acid concentration. Vitamin B12 deficiency was secondary to maternal autoimmune pernicious anaemia in all the five infants. As a result of the recognized cases the incidence of infant vitamin B12 deficiency in the East-Hungarian region was 1.26/100 000 births, but the real frequency may be higher. Conslusions: Optimizing the cut off values of current screening parameters and measuring of methylmalonic acid and/or homocysteine in the dried blood spot, as a second tier test, can improve recognition rate of vitamin B12 deficiency. Orv Hetil. 2017; 158(48): 1909-1918.

Metabolites of Aliphatic Alcohols Detected in Alcoholic Beverages Inhibit Phagocytosis.

AIMS: The aim of our study was to measure granulocyte and monocyte phagocytosis following treatment of cells with some metabolites of aliphatic alcohols alone and in combination with acetaldehyde. METHODS: The cells were separated from human peripheral blood prior to determination of phagocytosis of opsonized zymosan particles by granulocytes and monocytes treated individually with metabolites of aliphatic alcohols including formaldehyde, 1-propanal, acetone, 1-butanal, and 2-butanone and in combination with acetaldehyde. RESULTS: The findings revealed that metabolites of aliphatic alcohols inhibited phagocytosis by granulocytes and monocytes in a concentration-dependent manner and when combined with acetaldehyde, they caused a further decrease in phagocytic activity. CONCLUSION: Due to their additive effects, it is possible that, in combination with acetaldehyde, metabolites of aliphatic alcohols may inhibit phagocytosis at physiologically realistic concentrations in episodic heavy drinkers, thereby contributing to their increased susceptibility to infectious diseases.

Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes.

A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-induced immunosuppression in heavy drinkers, thereby increasing susceptibility to infectious diseases. However, the aliphatic alcohols contained in alcoholic beverages might also impair immune cell function, thereby contributing to a further decrease in microbicidal activity. Previous research has shown that aliphatic alcohols inhibit phagocytosis by granulocytes but their effect on human monocytes has not been studied. This is important as they play a crucial role in engulfment and killing of pathogenic microorganisms and a decrease in their phagocytic activity could lead to impaired antimicrobial defence in heavy drinkers. The aim of this study was to measure monocyte phagocytosis following their treatment with those aliphatic alcohols detected in alcoholic beverages. Monocytes were separated from human peripheral blood and phagocytosis of opsonized zymosan particles by monocytes treated with ethanol and aliphatic alcohols individually and in combination was determined. It was shown that these alcohols could suppress the phagocytic activity of monocytes in a concentration-dependent manner and when combined with ethanol, they caused a further decrease in phagocytosis. Due to their additive effects, it is possible that they may inhibit phagocytosis in a clinically meaningful way in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

Cost Utility Analysis of Percutaneous Adhesiolysis in Managing Pain of Post-lumbar Surgery Syndrome and Lumbar Central Spinal Stenosis.

BACKGROUND: The increase in the number of interventions for the management of chronic pain and associated escalation of healthcare costs has captured the attention of health policymakers, in no small part due to the lack of documentation of efficacy, cost-effectiveness, or cost utility analysis. A recent cost utility analysis of caudal epidural injections in managing chronic low back pain of various pathologies showed a high cost utility with improvement in quality of life years, competitive with various other modalities of treatments. However, there are no analyses derived from high-quality controlled studies related to the cost utility of percutaneous adhesiolysis in the treatment of post-lumbar surgery syndrome or lumbar central spinal stenosis. STUDY DESIGN: This analysis is based on 2 previously published controlled studies. OBJECTIVE: To assess the cost utility of percutaneous adhesiolysis procedures in managing chronic low back and lower extremity pain secondary to post-lumbar surgery syndrome and lumbar central spinal stenosis. SETTING: A private, specialty referral interventional pain management center in the United States. METHODS: Two controlled studies were conducted assessing the clinical effectiveness of percutaneous adhesiolysis for post-lumbar surgery syndrome and lumbar central spinal stenosis in an interventional pain management setting utilizing contemporary interventional pain management practices. A cost utility analysis was performed with direct payment data for a total of 130 patients in treatment groups over a 2-year period. Various outcome measures were included with significant improvement, defined as at least 50% improvement with reduction in pain and disability status. RESULTS: The results of 2 controlled studies of low back pain with 60 and 70 patients and a 2-year follow-up with the actual reimbursement data showed cost utility for 1 year of quality-adjusted life year (QALY) of USD $2,652 for post-lumbar surgery syndrome and USD $2,649 for lumbar central spinal stenosis. The results of this assessment show that the cost utility of managing chronic, intractable low back pain with percutaneous adhesiolysis at a QALY that is similar or lower in price than medical therapy only, physical therapy, manipulation, spinal cord stimulation, and surgery. LIMITATIONS: The limitations of this cost utility analysis are that it is a single-center evaluation, with the inclusion of costs of adhesiolysis procedures in an ambulatory surgery center and physician visits, rather than all related costs including drug therapy and costs of disability in multiple settings. CONCLUSION: This cost utility analysis of percutaneous adhesiolysis in the treatment of post-lumbar surgery syndrome and lumbar central spinal stenosis shows the clinical effectiveness and cost utility of these procedures at USD $2,650 per one year of QALY when performed in an ambulatory surgery center.

[ParC-10 cells for modelling parotid gland tissue reorganization]. / Par-C10 sejtek a parotis szöveti szervezodésének modellezésére.

Salivary gland hypofunction, which may occur in head and neck cancers following therapeutic irradiation or in Sjogren's syndrome, drastically impair the patient's quality of life. Conventional treatments do not provide a satisfactory solution to the problem, therefore it is becoming increasingly urgent to develop completely new management approaches in particular, the challenge of restoring the function of acini. Many biologically based interventions studied, thus "reprogramming" with gene therapy of survivor ducts or regeneration potential of progenitor cells in the salivary gland. Our research group has been working on several models, which have shown that by using appropriate media containing extracellular proteins (e.g. BME, basal membrane extract) can be achieved acinar differentiation. A significant proportion of in vitro models of salivary gland are submandibular of origin, which however is different from the development and function of parotid. Our research group aimed to model the potential treatment options for salivary gland hypofunction, the carrier or bioactive molecules directed differentiation, as well as the potential of gene therapy on rat parotid-derived cell line (Par-C10). In our experiments, we have studied the morphological changes of Par-C10 cells cultured on permeable polyester membrane, or in three-dimensional cultures, using varying concentrations of BME. In addition, we have tested the use of recombinant adenovirus vectors that could modify Par-C10 cells and make them useful in gene therapy models. Our data suggest that Par-C10 cell line is suitable for modelling parotid gland tissue organization and may also serve as a useful gene therapy model system.

Cervical Nerve Root Block Using a Curved Blunt Needle and Posterior Approach.

BACKGROUND: Cervical transforaminal epidural steroid injections have become less popular due to the risk of catastrophic complications they pose. However, cervical nerve root blocks are useful for surgical planning in patients with cervical radicular pain syndromes. OBJECTIVES: Our aim was to find a method of performing cervical selective nerve root blocks that removed the risk of catastrophic complications. STUDY DESIGN: Retrospective case review. SETTING: Academic multidisciplinary spine center. METHODS: Among patients, 50 consecutive cases were retrospectively reviewed for immediate pain scores and follow-up results. In the intervention, a posterior approach using a curved blunt needle was employed for cervical selective nerve root blocks to minimize the risk of arterial injection. To measure the outcomes, we used quantitative pain severity scores and qualitative responses. RESULTS: This technique detailed in this study has a high immediate analgesic effect that can be used for diagnostic purposes. It is not known if this technique has prognostic value with respect to surgery. The prolonged response rate is about 50%, which is in line with other techniques. LIMITATIONS: This study had no control group. CONCLUSION(S): Cervical selective nerve root blocks using a curved blunt needle and a posterior approach are effective in selectively identifying nerves that cause clinical symptoms. This technique minimizes the risk of arterial or spinal cord impingement and therefore may be safer than transforaminal selective nerve root blocks.

Aliphatic alcohol contaminants of illegally produced spirits inhibit phagocytosis by human granulocytes.

CONTEXT: Unregulated production of spirits in many countries leads to products containing appreciable levels of aliphatic alcohols (AAs) and is the main source of human exposure to these substances worldwide. Previous studies have confirmed that alcohol abuse can lead to ethanol-induced immunosuppression and thereby increased susceptibility to infectious diseases. Granulocytes, as professional phagocytic cells, play a crucial role in engulfment and killing of pathogenic microorganisms. Thus, a decrease in their phagocytic activity has been invoked as a factor in the impaired antimicrobial defense observed in alcoholics. However, AAs consumed as contaminants of illicit spirits may also influence phagocytosis, thereby contributing to a further decrease in microbicidal activity but, so far, this has not been studied. OBJECTIVE: Therefore, the aim of this study was to measure granulocyte phagocytosis following treatment of granulocytes with those higher alcohols found in illegal spirits. MATERIALS AND METHODS: Granulocytes were isolated from human peripheral blood. Then phagocytosis of opsonized zymosan particles by granulocytes treated with AAs individually and in combination was determined. RESULTS: These alcohols inhibited phagocytosis in a concentration-dependent manner and at lower concentrations when combined than when tested individually. DISCUSSION AND CONCLUSION: Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit phagocytosis in a clinically meaningful way in episodic heavy drinkers.

The MILD procedure.

We are following with great interest the increasing generally favorable impressions of the long-term results of the MILD (minimally invasive lumbar decompression) procedure for treating spinal stenosis due to hypertrophied ligamentum flavum (LF). We are also influenced by the cautionary surgical observations and opinions of Tumialan et al and publications about the lack of efficacy or placebo effect. The impression indeed has been virtual safety of the MILD procedure, but Tumialan et al describe some major complications resulting from the procedure. An algorithm for clinical use is needed.

Fulminant Cervical Epidural Hematomas: Why Do They Happen, How Can We Minimize Their Occurrence, and What Can We Do When They Do Occur? A Perspective.

BACKGROUND: Epidural hematomas after appropriately performed cervicothoracic interlaminar epidural injections have been associated with the rapid onset of neurological symptoms and devastating outcomes, despite prompt identification and treatment. Anticoagulation issues were initially felt to be the problem, but the occurrence of fulminant hematomas in patients without coagulation forced a reassessment of the causes and responses to this problem. OBJECTIVES: To evaluate why fulminant epidural hematomas occur after cervicothoracic epidural injections, with a literature review to survey knowledge about them in the surgical literature, and to offer comments as to what the interventional pain physician can do to minimize their occurrence. STUDY DESIGN: A perspective piece with a literature review. SETTINGS: Interventional pain management practices. METHODS: A perspective on the issue of fulminant cervical hematomas and an associated literature review. RESULTS: Anatomical studies show that there are no meaningful arteries in the posterior epidural spaces which would explain hematomas. There is a dense posterior intravertebral epidural venous plexus at C1 and also at C6-C7 extending caudally to the upper thoracic region. A venous origin has been questioned because venous pressure was felt to be too low to explain the bleeding. The surgical literature, going back 80 years, contains numerous reports of engorged epidural veins causing radiculopathy and myelopathy. These engorged veins can occur in the presence or absence of spinal pathology. There is no known means of reliably identifying these engorged veins; they have been mistaken for disc protrusions. At least one report documents massive bleeding from these veins. Studies done on a feline model of cervical stenosis suggest that the epidural pressure can reach arterial levels. LIMITATIONS: No direct documentation of arterialized posterior intravertebral epidural venous pressures has been made. While anatomical anomalies and degeneration contribute to epidural scarring, we do not have a full understanding as to the cause of arterialization of veins, particularly in younger patients with no obvious intraspinal pathology. CONCLUSION: Fulminant cervicothoracic epidural hematomas after an epidural injection appear to arise from the unintentional and unavoidable puncture of arterialized veins with sharp needles. A technique to open a path out from the foramen so that the blood can escape is described. Alternatively, providers should consider injecting more cephalad, between C2-C3 and C6-C7 in the cervical spine, or an alternative procedure, such as a selective nerve root injection. A cervical transforaminal approach should only be attempted with a blunt needle, which cannot enter an artery. Should symptoms occur, cervical flexion rotation maneuvers should be implemented while awaiting prompt transfer to a facility where an appropriate diagnosis and treatment can be provided. KEY WORDS: Cervical epidural hematoma, cervical epidural injection, posterior intravertebral venous plexus, arterialized epidural veins, pressurized epidural veins.

A Morphometric Study Analyzing the Anterior Epidural Space Volume Throughout Childhood.

BACKGROUND: Following disc herniations, fragments migrate into the anterior epidural space within the lumbar spine. Although the volume of this area has been previously described in the adult population, the volume is relatively unknown within children. OBJECTIVES: Investigate the relative volume in the lumbar anterior epidural space within the growing spine by using imaging studies. STUDY DESIGN: Retrospective chart review. SETTING: University Medical Center in Lubbock Texas. A teaching hospital affiliated with Texas Tech University Health Sciences Center. METHODS: We conducted a retrospective review of the charts of pediatric patients seen at our institution from 2018 through 2020. Charts chosen for our investigation contained computed tomography imaging of the lumber spine, showing no deformities. Thirty patients were stratified equally among 3 age groups, 2-5 years old, 10-12 years old, and 16-18 years old. The anterior epidural space was measured in each patient 3 times using the previously reported method used by Teske et al (1). Results were compared with a combination of analysis of variance (ANOVA) and single tail paired t test. RESULTS: There was a statistically significant difference in the anterior epidural space size among age groups at all levels of the lumbar spine. When comparing only 2 groups together, the younger age group had anterior epidural space sizes significantly smaller than the other age group for all levels of the lumbar spine. The 10-12 age group had a significantly smaller space in the anterior epidural space than the 16-18-year olds only at the level of L2, L4, and L5 (P = 0.048,0.039, and 0.031, respectively). Within the 16-18-year age group, the anterior epidural space was significantly different between L4 and L3 and L2 and L3 (P < 0.001 and P = 0.019, respectively). LIMITATIONS: Our study is limited by its retrospective nature and the sample size of the patient groups. Furthermore, the use of computed tomography imaging and not making physical measurements limits our accuracy. CONCLUSION: The volume of the anterior epidural space is smaller in the pediatric population than the adult population. The inability of herniated discs to fit within the epidural space in children and adolescents could potentially be the cause of the increased failure of conservative treatment for pediatric lumbar disc herniations.

Identification of potential interferents of methylmalonic acid: A previously unrecognized pitfall in clinical diagnostics and newborn screening.

OBJECTIVES: Determination of methylmalonic acid (MMA) from dried blood spots (DBS) is commonly performed in clinical diagnostics and newborn screening for propionic acidemia (PA) and methylmalonic acidemia. Isobaric compounds of MMA having the same mass can affect diagnostic reliability and quantitative results, which represents a previously unrecognized pitfall in clinical assays for MMA. We set out to identify interfering substances of MMA in DBS, serum and urine samples from confirmed patients with PA and methylmalonic acidemia. METHODS: Techniques included quadrupole time-of-flight high-resolution mass spectrometry (QTOF HR-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC) and tandem mass spectrometry (MS/MS). RESULTS: The five isobaric metabolites detected in DBS, serum and urine from PA and methylmalonic acidemia patients were confirmed as 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-hydroxyisovalerate, 3-hydroxyvalerate and succinate using a series of experiments. An additional unknown substance with low abundance remained unidentified. CONCLUSIONS: The presented results facilitate the diagnostic and quantitative reliability of the MMA determination in clinical assays. Isobaric species should be investigated in assays for MMA to eliminate possible interference in a wide range of conditions including PA, methylmalonic acidemia, a vitamin B12 deficiency, ketosis and lactic acidosis.

The story of the Texas Pain Society: formation and function of a regional pain society.

The idea of forming a Texas Pain Society came to the Founders in 1987 due to disparity and deficiencies in the practice of pain management in the United States and, in particular, the State of Texas. The Founders considered very carefully the implication of forming such a society. They diligently mapped out the mission and goals of the Texas Pain Society in those early formative years. This report is the history of Texas Pain Society as the activities unfolded from 1989 to 2011. The reader may question why there is a need to tell such a story. We believe strongly that, with disparities of standards of practice in pain medicine and poor recognition of advances in pain management, this scenario is quite common in many states and countries. The practitioners of pain management in these regions certainly must have considered getting together and forming a consensus on the standards of practice in their communities. This historical report of the Texas Pain Society provides the relevant information necessary and the efforts to be made for a society's mission to achieve its goals and have an ongoing impact in its own region. We hope that we have shed some light on a process for the formation of a regional pain society such as ours.

Vitamin B12 (Cobalamin): Its Fate from Ingestion to Metabolism with Particular Emphasis on Diagnostic Approaches of Acquired Neonatal/Infantile Deficiency Detected by Newborn Screening.

Acquired vitamin B12 (vB12) deficiency (vB12D) of newborns is relatively frequent as compared with the incidence of inherited diseases included in newborn screening (NBS) of different countries across the globe. Infants may present signs of vB12D before 6 months of age with anemia and/or neurologic symptoms when not diagnosed in asymptomatic state. The possibility of identifying vitamin deficient mothers after their pregnancy during the breastfeeding period could be an additional benefit of the newborn screening. Vitamin supplementation is widely available and easy to administer. However, in many laboratories, vB12D is not included in the national screening program. Optimized screening requires either second-tier testing or analysis of new urine and blood samples combined with multiple clinical and laboratory follow ups. Our scope was to review the physiologic fate of vB12 and the pathobiochemical consequences of vB12D in the human body. Particular emphasis was put on the latest approaches for diagnosis and treatment of vB12D in NBS.

Comparison of different preparation techniques of dried blood spot quality controls in newborn screening for congenital adrenal hyperplasia.

In newborn screening, samples suspected for congenital adrenal hyperplasia (CAH), a potentially lethal inborn error of steroid biosynthesis, need to be confirmed using liquid chromatography-tandem mass spectrometry. Daily quality controls (QCs) for the 2nd-tier CAH assay are not commercially available and are therefore generally prepared within the laboratory. For the first time, we aimed to compare five different QC preparation approaches used in routine diagnostics for CAH on the concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone in dried blood spots. The techniques from Prep1 to Prep5 were tested at two analyte concentrations by spiking aliquots of a steroid-depleted blood, derived from washed erythrocyte suspension and steroid-depleted serum. The preparation processes differed in the sequence of the preparation steps and whether freeze-thaw cycles were used to facilitate blood homogeneity. The five types of dried blood spot QCs were assayed and quantitated in duplicate on five different days using a single calibration row per day. Inter-assay variations less than 15% and concentrations within ±15% of the nominal values were considered acceptable. Results obtained by means of the four dried blood spot QC preparation techniques (Prep1, Prep2, Prep4 and Prep5) were statistically similar and remained within the ±15% ranges in terms of both reproducibility and nominal values. However, concentration results for Prep3 (spiking prior to three freeze-thaw cycles) were significantly lower than the nominal values in this setting, with differences exceeding the ±15% range in many cases despite acceptable inter-assay variations. These findings have implications for the in-house preparation of QC samples in laboratory developed tests for CAH, including 2nd-tier assays in newborn screening.