Interobserver agreement on definition of the target volume in stereotactic radiotherapy for pancreatic adenocarcinoma using different imaging modalities.

PURPOSE: The aim of this study was to evaluate interobserver agreement (IOA) on target volume definition for pancreatic cancer (PACA) within the Radiosurgery and Stereotactic Radiotherapy Working Group of the German Society of Radiation Oncology (DEGRO) and to identify the influence of imaging modalities on the definition of the target volumes. METHODS: Two cases of locally advanced PACA and one local recurrence were selected from a large SBRT database. Delineation was based on either a planning 4D CT with or without (w/wo) IV contrast, w/wo PET/CT, and w/wo diagnostic MRI. Novel compared to other studies, a combination of four metrics was used to integrate several aspects of target volume segmentation: the Dice coefficient (DSC), the Hausdorff distance (HD), the probabilistic distance (PBD), and the volumetric similarity (VS). RESULTS: For all three GTVs, the median DSC was 0.75 (range 0.17-0.95), the median HD 15 (range 3.22-67.11) mm, the median PBD 0.33 (range 0.06-4.86), and the median VS was 0.88 (range 0.31-1). For ITVs and PTVs the results were similar. When comparing the imaging modalities for delineation, the best agreement for the GTV was achieved using PET/CT, and for the ITV and PTV using 4D PET/CT, in treatment position with abdominal compression. CONCLUSION: Overall, there was good GTV agreement (DSC). Combined metrics appeared to allow a more valid detection of interobserver variation. For SBRT, either 4D PET/CT or 3D PET/CT in treatment position with abdominal compression leads to better agreement and should be considered as a very useful imaging modality for the definition of treatment volumes in pancreatic SBRT. Contouring does not appear to be the weakest link in the treatment planning chain of SBRT for PACA.

Stereotactic radiosurgery and radiotherapy for resected brain metastases: current pattern of care in the Radiosurgery and Stereotactic Radiotherapy Working Group of the German Association for Radiation Oncology (DEGRO).

PURPOSE: Preoperative stereotactic radiosurgery (SRS) of brain metastases may achieve similar local control and better leptomeningeal control rates than postoperative fractionated stereotactic radiotherapy (FSRT) in patients treated with elective metastasectomy. To plan a multicentre trial of preoperative SRS compared with postoperative FSRT, a survey of experts was conducted to determine current practice. METHODS: A survey with 15 questions was distributed to the DEGRO Radiosurgery and Stereotactic Radiotherapy Working Group. Participants were asked under what circumstances they offered SRS, FSRT, partial and/or whole brain radiotherapy before or after resection of a brain metastasis, as well as the feasibility of preoperative stereotactic radiosurgery and neurosurgical resection within 6 days. RESULTS: Of 25 participants from 24 centres, 22 completed 100% of the questions. 24 respondents were radiation oncologists and 1 was a neurosurgeon. All 24 centres have one or more dedicated radiosurgery platform and all offer postoperative FSRT. Preoperative SRS is offered by 4/24 (16.7%) centres, and 9/24 (37.5%) sometimes recommend single-fraction postoperative SRS. Partial brain irradiation is offered by 8/24 (33.3%) centres and 12/24 (50%) occasionally recommend whole-brain irradiation. Two centres are participating in clinical trials of preoperative SRS. SRS techniques and fractionation varied between centres. CONCLUSION: All responding centres currently offer postoperative FSRT after brain metastasectomy. Approximately one third offer single-fraction postoperative SRS and four already perform preoperative SRS. With regard to potential co-investigators, 18 were identified for the PREOP­2 multicentre trial, which will randomise between preoperative SRS and postoperative FSRT.

Improving interinstitutional and intertechnology consistency of pulmonary SBRT by dose prescription to the mean internal target volume dose.

PURPOSE: Dose, fractionation, normalization and the dose profile inside the target volume vary substantially in pulmonary stereotactic body radiotherapy (SBRT) between different institutions and SBRT technologies. Published planning studies have shown large variations of the mean dose in planning target volume (PTV) and gross tumor volume (GTV) or internal target volume (ITV) when dose prescription is performed to the PTV covering isodose. This planning study investigated whether dose prescription to the mean dose of the ITV improves consistency in pulmonary SBRT dose distributions. MATERIALS AND METHODS: This was a multi-institutional planning study by the German Society of Radiation Oncology (DEGRO) working group Radiosurgery and Stereotactic Radiotherapy. CT images and structures of ITV, PTV and all relevant organs at risk (OAR) for two patients with early stage non-small cell lung cancer (NSCLC) were distributed to all participating institutions. Each institute created a treatment plan with the technique commonly used in the institute for lung SBRT. The specified dose fractionation was 3â€¯× 21.5 Gy normalized to the mean ITV dose. Additional dose objectives for target volumes and OAR were provided. RESULTS: In all, 52 plans from 25 institutions were included in this analysis: 8 robotic radiosurgery (RRS), 34 intensity-modulated (MOD), and 10 3D-conformal (3D) radiation therapy plans. The distribution of the mean dose in the PTV did not differ significantly between the two patients (median 56.9 Gy vs 56.6 Gy). There was only a small difference between the techniques, with RRS having the lowest mean PTV dose with a median of 55.9 Gy followed by MOD plans with 56.7 Gy and 3D plans with 57.4 Gy having the highest. For the different organs at risk no significant difference between the techniques could be found. CONCLUSIONS: This planning study pointed out that multiparameter dose prescription including normalization on the mean ITV dose in combination with detailed objectives for the PTV and ITV achieve consistent dose distributions for peripheral lung tumors in combination with an ITV concept between different delivery techniques and across institutions.

[Results of surgical and radiotherapeutic treatment of adenoid cystic carcinoma of the salivary glands]. / Resultate der operativen und strahlentherapeutischen Behandlung von adenoidzystischen Karzinomen der Speicheldrüsen.

Surgical treatment in patients with rare adenoid cystic carcinoma (ACC) of the salivary glands is considered to be the state of the art. With respect to an additional radiotherapy there are different approaches regarding the type of radiotherapy and timing. In this study the overall survival and recurrence-free survival in 52 individuals with salivary gland ACC who were treated at the University Hospital in Essen and received irradiation with fast neutrons and photons (mixed beam technique) either A) immediately following surgical treatment or B) only after the appearance of local recurrence were compared. Group A (n = 28, first diagnosis, FD September 1991-September 2009) received adjuvant radiotherapy immediately postoperative, group B (n = 24, FD June 1979-November 2001) underwent primarily surgical tumor resection according to the treatment regimen at that time and were irradiated only on the appearance of a local recurrence. In comparison to group B, patients in group A showed a lower recurrence rate and a significantly longer local relapse-free survival. Group B, however, showed a significantly higher overall survival. The frequency of distant metastasis occurred equally in both groups but the onset of distant metastasis was significantly earlier in group A. In general, overall survival was negatively influenced by distant metastasis. The local recurrence rate was very high after primary surgical treatment only. The immediate adjuvant high-linear energy transfer (LET) radiotherapy reduced the local recurrence rates. Irradiation after the appearance of a recurrence had a positive influence on overall survival. Overall, definitive high-LET radiotherapy in the mixed beam technique enabled high local control rates both primarily postoperative and also locoregional recurrences.

[Necrotizing esophagitis by stress-cardiomyopathy with right ventricular insufficiency]. / Nekrotisierende Ösophagitis bei Stresskardiomyopathie mit Rechtsherzversagen.

Acute necrotizing esophagitis ("black esophagus") is defined as complete necrosis of the esophageal mucosa, which typically affects the entire circumference. We report a case of a healthy 62-year-old woman, who became hemodynamically unstable due to stress cardiomyopathy with acute right heart failure. Transfusion-dependent anemia occurred 24 h later and an upper gastrointestinal endoscopy revealed a black discoloured mucosa of the distal esophagus. After hemodynamic stabilization and treatment with proton pump inhibitors and sucralfate, complete healing of the esophageal mucosa was achieved.

The impact of local control on overall survival after stereotactic body radiotherapy for liver and lung metastases from colorectal cancer: a combined analysis of 388 patients with 500 metastases.

BACKGROUND: The aim of this analysis was to model the effect of local control (LC) on overall survival (OS) in patients treated with stereotactic body radiotherapy (SBRT) for liver or lung metastases from colorectal cancer. METHODS: The analysis is based on pooled data from two retrospective SBRT databases for pulmonary and hepatic metastases from 27 centers from Germany and Switzerland. Only patients with metastases from colorectal cancer were considered to avoid histology as a confounding factor. An illness-death model was employed to model the relationship between LC and OS. RESULTS: Three hundred eighty-eight patients with 500 metastatic lesions (lung n = 209, liver n = 291) were included and analyzed. Median follow-up time for local recurrence assessment was 12.1 months. Ninety-nine patients with 112 lesions experienced local failure. Seventy-one of these patients died after local failure. Median survival time was 27.9 months in all patients and 25.4 months versus 30.6 months in patients with and without local failure after SBRT. The baseline risk of death after local failure exceeds the baseline risk of death without local failure at 10 months indicating better survival with LC. CONCLUSION: In CRC patients with lung or liver metastases, our findings suggest improved long-term OS by achieving metastatic disease control using SBRT in patients with a projected OS estimate of > 12 months.

Stereotactic body radiotherapy (SBRT) for multiple pulmonary oligometastases: Analysis of number and timing of repeat SBRT as impact factors on treatment safety and efficacy.

BACKGROUND: Stereotactic body radiotherapy (SBRT) for oligometastatic disease is characterized by an excellent safety profile; however, experiences are mostly based on treatment of one single metastasis. It was the aim of this study to evaluate safety and efficacy of SBRT for multiple pulmonary metastases. PATIENTS AND METHODS: This study is based on a retrospective database of the DEGRO stereotactic working group, consisting of 637 patients with 858 treatments. Cox regression and logistic regression were used to analyze the association between the number of SBRT treatments or the number and the timing of repeat SBRT courses with overall survival (OS) and the risk of early death. RESULTS: Out of 637 patients, 145 patients were treated for multiple pulmonary metastases; 88 patients received all SBRT treatments within one month whereas 57 patients were treated with repeat SBRT separated by at least one month. Median OS for the total patient population was 23.5 months and OS was not significantly influenced by the overall number of SBRT treatments or the number and timing of repeat SBRT courses. The risk of early death within 3 and 6 months was not increased in patients treated with multiple SBRT treatments, and no grade 4 or grade 5 toxicity was observed in these patients. CONCLUSIONS: In appropriately selected patients, synchronous SBRT for multiple pulmonary oligometastases and repeat SBRT may have a comparable safety and efficacy profile compared to SBRT for one single oligometastasis.

Nomogram based overall survival prediction in stereotactic body radiotherapy for oligo-metastatic lung disease.

BACKGROUND: Radical local treatment of pulmonary metastases is practiced with increasing frequency due to acknowledgment and better understanding of oligo-metastatic disease. This study aimed to develop a nomogram predicting overall survival (OS) after stereotactic body radiotherapy (SBRT) for pulmonary metastases. PATIENTS AND METHODS: A multi-institutional database of 670 patients treated with SBRT for pulmonary metastases was used as training cohort. Cox regression analysis with bidirectional variable elimination was performed to identify factors to be included into the nomogram model to predict 2-year OS. The calibration rate of the nomogram was assessed by plotting the actual Kaplan-Meier 2-year OS against the nomogram predicted survival. The nomogram was externally validated using two separate monocentric databases of 145 and 92 patients treated with SBRT for pulmonary metastases. RESULTS: The median follow up of the trainings cohort was 14.3months, the 2-year and 5-year OS was 52.6% and 23.7%, respectively. Karnofsky performance index, type of the primary tumor, control of the primary tumor, maximum diameter of the largest treated metastasis and number of metastases (1 versus >1) were significant prognostic factors in the Cox model (all p<0.05). The calculated concordance-index for the nomogram was 0.73 (concordance indexes of all prognostic factors between 0.54 and 0.6). Based on the nomogram the training cohort was divided into 4 groups and 2-year OS ranged between 24.2% and 76.1% (predicted OS between 30.2% and 78.4%). The nomogram discriminated between risk groups in the two validation cohorts (concordance index 0.68 and 0.67). CONCLUSIONS: A nomogram for prediction of OS after SBRT for pulmonary metastases was generated and externally validated. This tool might be helpful for interdisciplinary discussion and evaluation of local and systemic treatment options in the oligo-metastatic setting. KEY MESSAGE: A nomogram for prediction of overall survival after stereotactic body radiotherapy (SBRT) for pulmonary metastases was developed and externally validated. This tool might be helpful for interdisciplinary discussion and evaluation of local and systemic treatment options in the oligo-metastatic setting.

[Human papillomavirus and squamous cell cancer of the head and neck region : Prognostic, therapeutic and prophylactic implications]. / Humane Papillomviren bei Plattenepithelkarzinomen der Kopf- und Halsregion : Relevanz für Prognose, Therapie und Prophylaxe.

Human papilloma viruses (HPV) are responsible for approximately half of all oropharyngeal squamous cell carcinomas (OPSCC) and incidence rates of HPV-associated OPSCC continue to increase substantially. The defined viral carcinogenesis permits development of specific diagnostic, therapeutic, and prophylactic approaches. Laboratory identification of HPV-associated OPSCC may be achieved by p16(INK4a) immunohistochemistry combined with HPV DNA detection by polymerase chain reaction (PCR) using tumor tissue. Patients with HPV-associated OPSCC have a relatively good prognosis; therefore, the HPV status plays an important role in patient guidance. Due to the relatively favorable prognosis, ongoing studies are evaluating whether less rigorous therapy for HPV-positive patients results in equally good cure rates. The criteria for patient selection are, however, still uncertain. Particularly markers for detection of HPV-positive patients with a high risk of treatment failure are lacking. Besides tumor stage and comorbidities, distinct genomic, epigenetic, and immunologic alterations are prognostically relevant for HPV-associated OPSCC, and might be of predictive value. Furthermore, the characteristic molecular alterations suggest the possibility of novel vigilant and specific therapy approaches. These may be inhibitors of the phosphatidylinositol 3­kinase (PI3K) pathway, which is frequently activated in HPV-associated OPSCC, and immunotherapeutic methods, e. g., therapeutic vaccination. Although prophylactic HPV vaccinations may also prevent development of HPV-associated OPSCC, foreseeable effects on OPSCC incidence will be low, given the low vaccination rates in Germany. This highlights the fact that interdisciplinary research networks should enhance the necessary activities related to HPV-associated OPSCC.

The influence of the local effect model parameters on the prediction of the tumor control probability for prostate cancer.

In carbon ion beams, biological effects vary along the ion track; hence, to quantify them, specific radiobiological models are needed. One of them, the local effect model (LEM), in particular version I (LEM I), is implemented in treatment planning systems (TPS) clinically used in European particle therapy centers. From the physical properties of the specific ion radiation, the LEM calculates the survival probabilities of the cell or tissue type under study, provided that some determinant input parameters are initially defined. Mathematical models can be used to predict, for instance, the tumor control probability (TCP), and then evaluate treatment outcomes. This work studies the influence of the LEM I input parameters on the TCP predictions in the specific case of prostate cancer. Several published input parameters and their combinations were tested. Their influence on the dose distributions calculated for a water phantom and for a patient geometry was evaluated using the TPS TRiP98. Changing input parameters induced clinically significant modifications of the mean dose (up to a factor of 3.5), spatial dose distribution, and TCP predictions (up to factor of 2.6 for D50). TCP predictions were found to be more sensitive to the parameter threshold dose (Dt) than to the biological parameters α and ß. Additionally, an analytical expression was derived for correlating α, ß and Dt, and this has emphasized the importance of [Formula: see text]. The improvement of radiobiological models for particle TPS will only be achieved when more patient outcome data with well-defined patient groups, fractionation schemes and well-defined end-points are available.

Dosimetric consequences of pencil beam width variations in scanned beam particle therapy.

Scanned ion beam delivery enables the highest degree of target dose conformation attainable in external beam radiotherapy. Nominal pencil beam widths (spot sizes) are recorded during treatment planning system commissioning. Due to changes in the beam-line optics, the actual spot sizes may differ from these commissioning values, leading to differences between planned and delivered dose. The purpose of this study was to analyse the dosimetric consequences of spot size variations in particle therapy treatment plans. For 12 patients with skull base tumours and 12 patients with prostate carcinoma, scanned-beam carbon ion and proton treatment plans were prepared and recomputed simulating spot size changes of (1) ±10% to simulate the typical magnitude of fluctuations, (2) ±25% representing the worst-case scenario and (3) ±50% as a part of a risk analysis in case of fault conditions. The primary effect of the spot size variation was a dose deterioration affecting the target edge: loss of target coverage and broadening of the lateral penumbra (increased spot size) or overdosage and contraction of the lateral penumbra (reduced spot size). For changes ≤25%, the resulting planning target volume mean 95%-isodose line coverage (CI-95%) deterioration was ranging from negligible to moderate. In some cases changes in the dose to adjoining critical structures were observed.

Accurate estimation of dose distributions inside an eye irradiated with 106Ru plaques.

BACKGROUND: Irradiation of intraocular tumors requires dedicated techniques, such as brachytherapy with (106)Ru plaques. The currently available treatment planning system relies on the assumption that the eye is a homogeneous water sphere and on simplified radiation transport physics. However, accurate dose distributions and their assessment demand better models for both the eye and the physics. METHODS: The Monte Carlo code PENELOPE, conveniently adapted to simulate the beta decay of (106)Ru over (106)Rh into (106)Pd, was used to simulate radiation transport based on a computerized tomography scan of a patient's eye. A detailed geometrical description of two plaques (models CCA and CCB) from the manufacturer BEBIG was embedded in the computerized tomography scan. RESULTS: The simulations were firstly validated by comparison with experimental results in a water phantom. Dose maps were computed for three plaque locations on the eyeball. From these maps, isodose curves and cumulative dose-volume histograms in the eye and for the structures at risk were assessed. For example, it was observed that a 4-mm anterior displacement with respect to a posterior placement of a CCA plaque for treating a posterior tumor would reduce from 40 to 0% the volume of the optic disc receiving more than 80 Gy. Such a small difference in anatomical position leads to a change in the dose that is crucial for side effects, especially with respect to visual acuity. The radiation oncologist has to bring these large changes in absorbed dose in the structures at risk to the attention of the surgeon, especially when the plaque has to be positioned close to relevant tissues. CONCLUSION: The detailed geometry of an eye plaque in computerized and segmented tomography of a realistic patient phantom was simulated accurately. Dose-volume histograms for relevant anatomical structures of the eye and the orbit were obtained with unprecedented accuracy. This represents an important step toward an optimized brachytherapy treatment of ocular tumors.

Long survival of primary diffuse leptomeningeal gliomatosis following radiotherapy and temozolomide: case report and literature review.

OBJECTIVE: Primary diffuse leptomeningeal gliomatosis (PDLG) is a rare neoplasm with a short survival time of a few months. There is currently no standardized therapeutic approach for PDLG. - MATERIAL AND METHODS: We report on a 53-year-old male patient who presented with epileptic seizures, gait disturbance, paraparesis and sensory deficits in the dermatomes T8-10. - RESULTS: Magnetic resonance imaging (MRI) revealing numerous spinal and cranial gadolinium-enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with WHO grade III astrocytic cells. Consecutively, the patient underwent craniospinal radiotherapy (30Gy) and 11 sequential cycles of temozolomide. This regimen led to partial tumor regression. Thirteen months later, spinal MRI revealed tumor progression. Second-line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration. The patient died twenty-two months after diagnosis, being the longest survival time described thus far with respect to PDLG consisting of astrocytic tumor cells. - CONCLUSIONS: Radiochemotherapy including temozolomide, as established standard therapy for brain malignant astrocytomas, might be valid as a basic therapeutic strategy for this PDLG subtype.

Cardiac side effects of conventional and particle radiotherapy in cancer patients.

Radiation therapy, which has become an integral part of modern treatment strategies for many types of cancer in recent decades, is associated with the risk of long-term adverse effects. Particularly in the case of patients with a good prognosis as a result of these modern treatment modalities, avoiding therapy-related side effects plays an increasingly important role. Of these side effects, cardiac complications are particularly relevant since they not only adversely affect quality of life but can also be potentially life-threatening. This review summarizes and critically analyzes the available evidence on the impact of ionizing irradiation on the cardiovascular system. The paper presents an overview of the current knowledge on epidemiological evidence, clinical presentation and risk factors of radiation-induced cardiovascular disease. Additionally, mechanisms of development of this type of complication and strategies for screening, treatment, and avoiding cardiac complications are discussed.

Continuous glucose monitoring reveals associations of glucose levels with QT interval length.

BACKGROUND: QTc interval lengthening during hypoglycemia is discussed as a mechanism linked to sudden death in diabetes patients and the so-called "dead in bed syndrome." Previous research reported a high interindividual variability in the glucose-QTc association. The present study aimed at deriving parameters for direction and strength of the glucose-QTc association on the patient level using combined Holter electrocardiogram (ECG) and continuous glucose monitoring. METHODS: Twenty type 1 diabetes patients were studied: mean (SD, range) age, 43.6 (10.8, 22-65) years; gender male (n [%]), 10 (50.0%); mean (SD) hemoglobin A1C, 8.5% (1.0%); and impaired hypoglycemia awareness (n [%]), six (30.0%). Continuous interstitial glucose monitoring and Holter ECG monitoring were performed for 48 h. Hierarchical (mixed) regression modeling was used to account for the structure of the data. RESULTS: Glucose levels during nighttime were negatively associated with QTc interval length if the data structure was accounted for (b [SE] = -0.76 [0.17], P = 0.000). Exploratory regression analysis revealed hypoglycemia awareness as the only predictor of the individual strength of the glucose-QTc association, with the impaired awareness group showing less evidence for an association of low glucose with QTc lengthening. CONCLUSIONS: Mixed regression allows for deriving parameters for the glucose-QTc association on the patient level. Consistent with previous studies, we found a large interindividual variability in the glucose-QTc association. The finding on impaired hypoglycemia awareness patients has to be interpreted with caution but provides some support for the role of sympathetic activation for the QTc-glucose link.

Comparison between PENELOPE and electron Monte Carlo simulations of electron fields used in the treatment of conjunctival lymphoma.

For the treatment of conjunctival lymphoma in the early stages, external beam radiotherapy offers a curative approach. Such treatment requires the use of highly conformed small radiation beams. The beam size is so small that even advanced treatment planning systems have difficulties in calculating dose distributions. One possible approach for optimizing the treatment technique and later performing treatment planning is by means of full Monte Carlo (MC) simulations. In this paper, we compare experimental absorbed dose profiles obtained with a collimator used at the University Hospital Essen, with MC simulations done with the general-purpose radiation transport code PENELOPE. The collimator is also simulated with the hybrid MC code electron Monte Carlo (eMC) implemented in the commercial treatment planning system Eclipse (Varian). The results obtained with PENELOPE have a maximum difference with experimental data of 2.3%, whereas the eMC code differs systematically from the experimental data about 7% in the penumbra tails. We also show that PENELOPE simulations are able to obtain absorbed dose maps with an equivalent statistical uncertainty to the one found with eMC in similar CPU times.

Comparison of adjoint and forward Monte Carlo based treatment planning for a realistic head phantom.

BNCT treatment planning can be improved by having the adjoint technique available in the Monte Carlo transport code. In adjoint MC, the simulated particles travel backwards instead of 'forward'. By speeding up the calculations, more beam positions can be investigated and thus a better plan can be composed. In a realistic head phantom with 10 disseminated lesions in the brain, the adjoint method is more favourable than the forward calculations whenever larger beam diameters are applied.

Boron neutron capture therapy for an explanted organ: the logistical challenges.

Single liver metastases of colorectal cancer can be cured by surgery; disseminated liver metastases are incurable. A research group in Pavia, Italy, used BNCT as an experimental method to irradiate in curative intention the explanted liver of patients suffering from disseminated hepatic metastases. The situation in Pavia, where a reactor with a specially adapted thermal column and the hospital are close by, is unique. For the purpose of the present study, it was necessary to investigate how the Pavia experience can be repeated with transplantation centers located at distance from a reactor. Some basic investigations of the logistics of such a procedure are reported.

Extension of the calibration curve for the PGRA facility in Petten.

At the boron neutron capture therapy (BNCT) facility in Petten, the Netherlands, (10)B concentrations in biological materials are measured with the prompt gamma ray analyses facility that is calibrated using certified (10)B solutions ranging from 0 to 210 ppm. For this study, newly certified (10)B solutions ranging up to 1972 ppm are added. MCNP simulations of the setup range to 5000 ppm. A second order polynomial (as already used) will fit (10)B-concentrations less than 300 ppm. Above 300 ppm a fitted third order polynomial is needed to describe the calibration curve accurately.