Immunological Screening Tests Research Articles

Quantification of (9R)- and (9S)-hexahydrocannabinol (HHC) via GC-MS in serum/plasma samples from drivers suspected of cannabis consumption and immunological detection of HHC and related substances in serum, urine, and saliva.

The semisynthetic cannabinoid hexahydrocannabinol (HHC) is currently getting a lot of media attention because the legal status in many countries is not clearly specified. In this study, a GC-MS method for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) was extended to (9R)- and (9S)-HHC. The applicability was proven by serum/plasma samples from drivers suspected of cannabis consumption. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.15 and 0.25 ng/mL, respectively. Within-run imprecision was <6.5% and between-run imprecision was <10.0%. Inter-injection stability, processed sample stability (3 days), freeze-thaw stability (three cycles), and storage stability (1 week room temperature; 1 month 4°C, -20°C) could be proven. Both HHC diastereomers could be detected in 17 (5.3%) out of 321 analyzed samples from traffic controls in Western Saxony. The mean ratio between (9R)- and (9S)-HHC was 1.99 (CV = 14.6%). Quantification resulted in concentrations between <LLOQ and 35.35 ng/mL for (9R)-HHC and <LLOQ and 21.76 ng/mL for (9S)-HHC. Additionally, cross-reactivities of HHC, 11-hydroxy-hexahydrocannabinol (11-OH-HHC), 11-nor-9-carboxy-hexahydrocannabinol (HHC-COOH), hexahydrocannabiphorol (HHC-P), hexahydrocannabinol acetate (HHC-O), and tetrahydrocannabidiol (H4-CBD) were evaluated in five immunological screening tests for serum, urine, and saliva. Urine test strips and ELISA tests for the detection in serum seem to be beneficial to detect HHC consumption in comparison with saliva tests. HHC analogs and H4-CBD showed no cross-reactivity with any of the tests.

Rapid Immune Tests SARS-COV-2 – An Experience in Beira Baixa

Introduction: SARS-CoV-2 affects the epithelial cells of the respiratory tract, causing severe infections. Since it is a pathology with a high level of transmissibility, it becomes central to mass testing. In addition, there was also a need to monitor the epidemic through serological tests. Objective: Evaluate the presence of antibodies against SARS-CoV-2 in the community residing in Beira Baixa through immunological screening tests. Materials and methods: Analytical, cross-sectional, and observational study, whose sample consists of 206 individuals. Data collection took place between February and April 2021, in the laboratories of the Dr. Lopes Dias Higher School of Health. Verbal informed consent, a questionnaire to collect sociodemographic data and the serological test were applied. Results: Of the total number of participants, 15.5% admitted to having had COVID-19, of which 0.5% suspected they had been infected and 84% said they had never been infected. Regarding the presence of antibodies, 2.9% of the tests performed were positive for the presence of IgM's while 30.1% were positive for the presence of IgG's. Regarding vaccination, at the time of the investigation only 10.2% of the participants were vaccinated, of which 9.7% had IgG antibodies. Conclusion: Rapid serological tests can provide information about the presence of antibodies to SARS-CoV-2, thus being a very advantageous tool for immunity studies.

874 A NOT-SO-RARE COMPLICATION OF CONNECTIVE TISSUE DISEASE

Abstract Pulmonary hypertension is a not-so-rare complication of connective tissue diseases and is usually related with an inauspicious prognosis. We present the case of a 67-years-old woman hospitalized for progressive dyspnea. Two-dimensional echocardiography showed numerous features related to pulmonary hypertension (PH) with moderate Mitral Regurgitation. The right cardiac catheterization confirmed mixed PH (pre and post capillary) without chest angio-TC evidence of pulmonary thromboembolism. Thanks to optimized medical therapy (B-Blocker, ACE-i and diuretics), there was a significant clinical improvement documented at discharge. After 1 year, due to recurrence of dyspnea, she was admitted to our medical unit. Her clinical examination showed some of the characteristics of a connective tissue disease. Compared to previous echocardiography, a moderate to mild reduction of MR was documented. Chest CT scan revealed interstitial lung disease likely related to patient's underlying pathology. Immunological screening tests detected the presence of antinuclear (ANA) and anti-topoisomerase (ATA) antibodies. Right heart catheterization documented a worsening of PH with only pre-capillary component. On the basis of the above findings, a diagnosis of CREST syndrome complicated with PH was made, and the patient was started on oral combination of macitentan and riociguat. The frequency of this complication in connective tissue disease, the need for screening tests and different treatment approaches of pre and post capillary pulmonary hypertension is discussed.

P416 A NOT–SO–RARE COMPLICATION OF CONNECTIVE TISSUE DISEASE

Abstract Pulmonary hypertension is a not–so–rare complication of connective tissue diseases and is usually related with an inauspicious prognosis.We present the case of a 67–years–old woman hospitalized for progressive dyspnea. Two–dimensional echocardiography showed numerous features related to pulmonary hypertension (PH) with moderate Mitral Regurgitation. The right cardiac catheterization confirmed mixed PH (pre and post capillary) without chest angio–TC evidence of pulmonary thromboembolism. Thanks to optimized medical therapy (B–Blocker, ACE–i and diuretics), there was a significant clinical improvement documented at discharge. After 1 year, due to recurrence of dyspnea, she was admitted to our medical unit. Her clinical examination showed some of the characteristics of a connective tissue disease. Compared to previous echocardiography, a moderate to mild reduction of MR was documented. Chest CT scan revealed interstitial lung disease likely related to patient’s underlying pathology. Immunological screening tests detected the presence of antinuclear (ANA) and anti–topoisomerase (ATA) antibodies. Right heart catheterization documented a worsening of PH with only pre–capillary component. On the basis of the above findings, a diagnosis of CREST syndrome complicated with PH was made, and the patient was started on oral combination of macitentan and riociguat. The frequency of this complication in connective tissue disease, the need for screening tests and different treatment approaches of pre and post capillary pulmonary hypertension is discussed.

The Critical Role of Prenatal Genetic Study in Prevention of Primary Immunodeficiency in High-risk Families: The Largest Report of 107 Cases.

This study aims to investigate the role of prenatal diagnosis (PND) in Iranian couples with a previous history of primary immunodeficiency disorders (PIDD) in their family. All referred couples with a family history of PIDD and a tendency for PND were included in this project. Based on gestational age, chorionic villus sampling(CVS) was performed to analyze the molecular defect of the fetus according to the previous gene defect of the affected case in the family. Postnatal confirmation was performed by immunological screening tests. In a total of 100 cases, CVS was not evaluated in 19 patients due to unwillingness (n=5), late prenatal referral (n=7), miscarriage before CVS (n=3), and female fetus with x-linked diseases in previous children (n=4). In the remaining 81 patients, heterozygous and homozygous mutations were found in 33 and 23 cases, respectively. The hemizygous mutation was obtained in 6 and no pathogenic mutations were found in 19 individuals. Postnatal evaluations revealed that a total of 65 babies were healthy, 32 fetuses were aborted (3 cases before CVS, 2 spontaneous abortions of a healthy and as affected fetus in the CVS subgroup, and 27 cases were aborted due to therapeutic causes). One fetus from the heterozygous subgroup was spontaneously aborted with severe combined immunodeficiency (SCID) and one fetus from the homozygous subgroup that was supposed to be healthy was affected by the autosomal dominant-chronic granulomatous disease (AR-CGD). The diagnostic error was 1.2%. PND is highly recommended in families with a history of PID in their previous child to prevent an affected baby being born and to reduce the government, family, and personal burden of these diseases.

Identification of a Cyclospora cayetanensis Oocyst Antigens and Their Validity in the Detection of Immunogenic Patterns of Cyclosporiasis Patients.

The diagnosis of cyclosporiasis is currently based on the microscopic detection of oocysts, which may provide invalid results. The availability of simple, objective immunological screening tests would facilitate epidemiological studies of cyclosporiasis. Therefore, the present study aimed to identify the antigens of Cyclospora cayetanensis oocysts and their validity in serodiagnosis. According to parasitological and molecular diagnoses, three study groups were specified. Group (G) I included 30 patients with cyclosporiasis, GII included 12 patients with other parasitic infections, and GIII included 16 healthy subjects. SDS-PAGE was used to analyse C. cayetanensis antigens, and the validity of western blotting and enzyme-linked immunosorbent assays (ELISAs) was then assessed amongst the sera of all study groups. The C. cayetanensis antigenic profile showed eight characteristic bands with molecular weights ranging from 14 to 175kDa. Western blot analysis of sera revealed 93.3% (28/30 of GI) and 92.8% (26/28 of GII and III) sensitivity and specificity, respectively, dividing the patients in GI into four subgroups. The most frequent diagnostic bands (71.4% of GI sera) showed weights of 26-28kDa, followed by 71kDa (53.6%). ELISA sensitivity was 90% (27/30), and specificity was 78.6%. Validation showed perfect agreement between the PCR and western blot results, and ELISA presented substantial agreement with both the PCR and western blot results. Our findings suggest the existence of high immunogenic diversity in C. cayetanensis and indicate that the 26-28kDa immunogenic groups may potentially be used as a diagnostic marker of cyclosporiasis. Due to the high validity of ELISA, it might be the test of choice for the routine serodiagnosis of cyclosporiasis.

Clinical and Genetic Study of X-linked Agammaglobulinemia Patients (The Benefit of Early Diagnosis).

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by genetic defects in the Bruton tyrosine kinase (Btk) gene. XLA is characterized as an antibody deficiency by recurrent bacterial infections, the absence of peripheral B cells, and profound reductions in all immunoglobulin isotypes. This study aims to report the clinical and genetic features of five Iranian patients with XLA. Five male cases with recurrent bacterial infection entered this study based on clinical evaluation and Immunological screening tests. The levels of T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) were also measured in dried blood spot (DBS) samples. Sanger sequencing was applied to PCR products of DNA samples of the patients for genetic studies. All patients were from unrelated families with a mean age of 6.7 years (2.5-11) at the time of diagnosis with 4.8 mean years of delay in diagnosis. The most frequent clinical manifestations were recurrent respiratory infections and arthritis. In these patients, five previously reported mutations were found including four mutations (p.Q496X, p.Q497X, p.R520X, and p.R641H) in the Kinase domain besides one mutation (p.L37P) in the pleckstrin homology (PH) domain. Evaluations of KREC and TREC level in patients' DBS showed low-to-undetectable copies of KREC (0-2 copies/3.2mm DBS) with normal copies of TREC. As patients with XLA have complete immunoglobulin defects and develop severe and recurrent infections, early diagnosis would be beneficial for the improvement of their quality of life. The study results may provide valuable information for the diagnosis, genetic counseling and prenatal diagnosis for the patients and their family members and emphasize performing KREC as an early diagnostic test in patients with XLA.

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine, expressing the outer membrane protein OMP19 of Brucella species

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.

Detection of Paralytic Shellfish Toxins in Mussels and Oysters Using the Qualitative Neogen Lateral-Flow Immunoassay: An Interlaboratory Study.

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

Cat-scratch disease with hepatosplenic lesions in two brothers

A one-year-old boy was hospitalized after 6 days of high fever. Antimicrobials were administered for a suspected lower respiratory tract infection, but the fever persisted. On day 23, a diffusion-weighted magnetic resonance imaging (MRI) revealed multiple cystic lesions in the liver and spleen (Fig. 1). Fig. 1 In case 1, diffusion-weighted magnetic resonance imaging showed multiple cystic lesions in the liver and spleen. About 3 years later, his 2-year-old younger brother was hospitalized after 12 days of fever. Both brothers had been in contact with the same cat. Abdominal ultrasonography showed hepatosplenomegaly and multiple cystic lesions in the spleen. Furthermore, MRI showed multifocal small diffuse cystic liver lesions (Fig. 2). Fig. 2 In case 2, magnetic resonance imaging showed multifocal small diffuse cystic liver lesions. Suspecting cat-scratch disease (CSD), azithromycin and rifampin were administered in both cases; the fever gradually abated. In the first case, the Bartonella henselae IgM antibody titer was 1:160; in both cases, the IgG antibody titer was >1:1024, confirming CSD. Immunological screening tests showed no abnormal findings. We could not find any skin lesions showing injury by the cat. CSD is caused by the Gram-negative bacterium B. henselae, which causes zoonotic infections, primarily through domestic cats, particularly kittens [1]. Although localized necrotizing lymphadenitis is the most typical presentation, some cases display atypical symptoms such as Parinaud's syndrome, encephalitis and, as in these cases, fever with hepatic and splenic involvement. It is also important in the differential diagnosis of fevers of unknown origin in children. B. henselae colonizes about 10% of cats and is transmitted by cat fleas. There have been some cases of multiple infections in the same household [2], [3], [4]. These cases suggest that multiple family members may be infected with CSD from the same pet over several years.

The Use Of Rituximab For Preventing Delayed Haemolytic Transfusion Reaction In Highly Immunized Patients With Sickle Cell Disease

IntroductionTransfusion plays a major role in the management of sickle cell disease (SCD). Delayed haemolytic transfusion reaction (DHTR) is frequent in SCD patients and may have a lethal outcome. DHTR is characterized by recurrence of clinical symptoms related to SCD with a marked haemoglobin (Hb) drop, rapid disappearance of HbA, within 5 to 15 days after transfusion. DHTR is mainly caused by a secondary immune response to an allo-antigen (Ag) on transfused RBCs, but some cases are described without significant antibodies (Abs) or detectable Abs. The increased rate of alloAbs in SCD patients is the result of blood groups polymorphisms between recipients (mainly of Afro-Caribbean's origin) and donors (Caucasians). Inhibition of a primary or secondary immune response to blood group antigens is therefore a major goal for these patients. B cell depletion therapy with anti-CD20 is commonly used to treat auto-antibodies mediated diseases. Thus, one can speculate that rituximab could be helpful in preventing alloimmunization in SCD patients. Here, we report 8 cases of SCD patients previously allo-immunized, in whom a new transfusion was indicated, and treated with rituximab in order to prevent further immunization and DHTR. Patients and rituximab protocolPatients treated with rituximab were already highly immunized (high responders). All but one had a previous history of life threatening DHTR characterized by severe clinical vaso-occlusive crisis (VOC), acute chest syndrome (ACS) and/or multi organ failure (MOF) and a marked decrease of Hb compared to immediate post-transfusion level, ranging from 2 to 6 g/dl. Treatment with rituximab was given according 2 different regimens depending on the patient's condition as follows: 1) at a fixed dose of 1,000 mg 2 weeks apart, 1 month and 2 weeks before the procedure in case of a planned surgery requiring a transfusion or 2) a single infusion of 1,000 mg in acute situations of life-threatening VOC with ACS and/or MOF. In all cases, a premedication by low dose of methyprednisolone (10 mg) was administered to limit the risk of secondary VOC. All patients were transfused with leucodepleted RBCs units with matched phenotype for the known antibodies and the most immunogenic blood groups (Rh/Kell/Duffy/Kidd/MNS). OutcomeFollowing rituximab and after transfusion, 4 patients had a non eventful clinical course. Four patients presented a mild DHTR, with a drop of Hb ranging from 2 to 3 g/dl from baseline. Among them, 2 had mild clinical symptoms of intravascular hemolysis and/or exacerbation of VOC, but none of them had an ACS or a MOF, or needed new admission in intensive care unit. In all patients, the post transfusional immunological screening tests remained identical to the pre transfusion screening test without any newly detectable allo-Abs. None of the patients experienced a severe adverse event related to rituximab. ConclusionThis retrospective analysis of 8 cases from the French referral centre for SCD suggests that rituximab may at least prevent the occurrence of newly formed antibodies in highly immunized patients and potentially minimize the risk of severe DHTR This study confirms that the mechanisms of DHTR are complex in SCD, and does not rely only on the classical conflict between red blood cell Ag and Abs. Thus, rituximab may be considered when a new transfusion seems inevitable in SCD patients with a previous history of DHTR linked to immunization. Disclosures:Off Label Use: administration of rituximab in a off-label setting.

Which Amphetamine-Type Stimulants Can Be Detected by Oral Fluid Immunoassays?

Introduction: The use of oral fluid for monitoring drug consumption on roads has many advantages over conventional biological fluids; therefore, several immunoassays have been developed for this purpose. In this work, the ability of 3 commercial immunoassays to detect amphetamine-type stimulants (ATSs) in oral fluid was assessed. In addition, it was reviewed the main controlled ATSs available worldwide, as well as the oral fluid immunological screening tests that have been used for identifying ATSs in drivers. Materials and Methods: The analytical specificity of amphetamine direct enzyme-linked immunosorbent assay (ELISA), methamphetamine direct ELISA (Immunalysis Corporation), and Oral-View saliva multidrug of abuse test (Alfa Scientific Designs) was evaluated using ATS-spiked oral fluid. Legislation and published articles that report the use of immunological screening tests to detect ATS consumption in conductors were reviewed, including the kit's technical information, project reports, police and drug databases. Results and Discussion: Even at high concentrations, the tested assays were not able to detect methylphenidate, fenproporex, or diethylpropion, controlled ATSs legally marketed in many countries. Conclusions: This evidences the need to develop new kits that enable one to control the misuse of prescription ATSs on roads through oral fluid immunoassays.

Wunderlich Syndrome during antiplatelet drug therapy

Spontaneous hemorrhage in the kidney, also known as Wunderlich Syndrome, is a rare clinical problem. The most common cause of spontaneous renal hemorrhage is tumor. Other causes are rupture of a renal cyst, vasculitis, hydronephrosis, preeclampsia, and kidney infections. A 67-year-old man was admitted complaining left flank colic pain. A contrast CT scan showed the presence of a subcapsular hematoma of the left kidney extending from the upper to the lower pole. He had no history of trauma and immunological screening tests for vasculitis were normal. His current therapy included acetylsalicylic acid (100 mg/daily) and lisinopril (20 mg/ daily). The patient was hospitalized for 4 days and his circulatory state remained stable. Nine months later an ultrasound examination showed complete resolution of the hematoma. One year later the patient was admitted again because of a spontaneous right calf hematoma and a thoroughly investigation of his coagulation pattern was carried out. Laboratory finding revealed a platelet defect, as the number of adenine nucleotides and other marker related to platelet activation were increased: ADP 1 mcM lack 2 masculine wave, ADP 2 mcM lack 2 masculine wave, Adrenalina 5 mcM lack 2 masculine wave, Adrenalina 10 mcM lack 2 masculine wave. Platelet activation markers: Gp53 in lysosomal membrane 0.48 Dpar (0 - 0.26). Our case describes the recurrence of spontaneous hemorrhages (perirenal and intramuscular hematoma) as a result of an underlying platelet aggregation defect worsened by administration of acetylsalicylic acid. In patients on antiplatelet treatment with a history of excessive bleeding a thorough investigation of coagulation status appears beneficial.

Screening for drugs of abuse in hair with ion spray LC–MS–MS

Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high. The aim of this study was to develop an LC–MS–MS method for the simultaneous analysis of several drugs of abuse in human hair as an alternative to immunological screening tests. In 75 randomly selected autopsy cases, hair was analyzed in addition to the usual specimens of blood and urine. The method included nicotine, cotinine, morphine, codeine, 6-acetylmorphine, ethylmorphine, amphetamine, methamphetamine, MDA, MDMA, benzoylecgonine, cocaine, 7-aminoflunitrazepam and diazepam. The LC–MS–MS analysis was performed on a SCIEX API 2000 MS–MS instrument equipped with an electrospray interface. To 20–50 mg of hair, 0.5 ml of mobile phase A (acetonitril:methanol:20 mM formate buffer, pH 3.0 (10:10:80)) and 25 μl of internal standard were added and the sample was incubated in a water bath at 37 °C during 18 h. Using a threshold of 20 ng/sample, equivalent to 1 ng/mg if 20 mg hair is used, 26 positive results were found in 16 cases. Three of the 26 positive detections could not be confirmed by GC–MS. Two of the cases were not previously known as drug users. Of the 59 negative cases, only one case had a positive blood sample showing 0.01 and 0.07 μg/g femoral blood of 6-acetylmorphine and morphine, respectively. This might indicate drug abstinence resulting in decreased tolerance or even a “first time” use of heroin resulting in death. We conclude that the use of hair analysis in postmortem cases can reveal both unknown drug use, as well as confirm a period of drug abstinence prior to an acute fatal overdose. The proposed LC–MS–MS method showed high sensitivity, was very easy to perform and seemed appropriate for screening purposes.

Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus

An RT-PCR method was developed that amplified genetic material from the 5′ end of the S protein gene of both transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), but discriminated between the two by the size of the product generated. A number of restriction endonuclease enzymes were assessed for recognition of the amplicons so produced. The assay was shown to detect viral RNA from all of the 26 different TGEV and PRCV isolates examined, covering a period from 1946 to 1996. Detection of TGEV in clinical specimens was possible using a spin column method to extract RNA and sensitivity was compared to virus isolation and antigen detection ELISA. The method could provide a means of confirming positive results from immunological screening tests such as FAT and ELISA, reducing the need for virus isolation and convalescent serology.

Lymphoproliferative response to mitogen (ConA) after treatment of rats with gentamicin and doxycyclin: relevance to human therapeutics.

Interaction of xenobiotics with the immune system may result in undesirable effects such as immunosuppression. In the future, it is probable that immunological screening tests may be required for safety evaluation of new drug candidates as an adjunct to more traditional toxicity testing. The lymphoproliferative response to mitogens has been proposed as a screening test. This test has been set up and validated in mice, but will probably have to be done in rats, the species of choice for drug safety studies. This test has been adapted to rats, using both in vivo and in vitro treatments, and used to validate drugs, including doxycyclin and gentamicin (with cyclosporin A and hydroxycortisone as controls) at the dose levels used in toxicology studies. Discrepant results obtained with doxycyclin and hydroxycortisone (effect in vitro but not in vivo) suggest that further validation studies are necessary in order to assess the reliability of this test.