Non-Invasive Zinc Protoporphyrin Screening Offers Opportunities for Secondary Prevention of Iron Deficiency in Blood Donors.

Background: Frequent blood donors are at high risk of developing iron deficiency. Currently, there is no potent screening during blood donation to detect iron deficient erythropoiesis (IDE) before anemia develops and deferral from donation is inevitable. Study Design and Methods: In addition to capillary and venous hemoglobin, the iron status of 99 frequent blood donors was assessed by various venous blood parameters and zinc protoporphyrin IX (ZnPP). ZnPP was determined by high-performance liquid chromatography (HPLC) and a new prototype fiber-optic device was employed for non-invasive measurements of ZnPP through the blood collection tubing (NI-tubing) and on lip tissue (NI-lip). We aimed to evaluate the feasibility and diagnostic value of the NI-tubing measurement for early detection of severe iron deficiency in blood donors. Results: NI-tubing and HPLC reference measurements of ZnPP showed narrow limits of agreement of 12.2 µmol ZnPP/mol heme and very high correlation (Spearman's Rho = 0.938). Using a cutoff of 65 µmol ZnPP/mol heme, NI-tubing measurements (n = 93) identified 100% of donors with iron deficiency anemia (IDA) and an additional 38% of donors with IDE. Accordingly, NI-tubing measurements would allow detection and selective protection of particularly vulnerable donors. Conclusion: NI-tubing measurements are an accurate and simple method to implement ZnPP determination into the routine blood donation process. ZnPP was able to identify the majority of subjects with IDE and IDA and might therefore be a valuable tool to provide qualified information to donors about dietary measures and adjustments of the donation interval and thereby help to prevent IDA and hemoglobin deferral in the future.

Screening for iron deficiency in surgical patients based on noninvasive zinc protoporphyrin measurements.

BACKGROUND: Approximately every third surgical patient is anemic. The most common form, iron deficiency anemia, results from persisting iron-deficient erythropoiesis (IDE). Zinc protoporphyrin (ZnPP) is a promising parameter for diagnosing IDE, hitherto requiring blood drawing and laboratory workup. STUDY DESIGN AND METHODS: Noninvasive ZnPP (ZnPP-NI) measurements are compared to ZnPP reference determination of the ZnPP/heme ratio by high-performance liquid chromatography (ZnPP-HPLC) and the analytical performance in detecting IDE is evaluated against traditional iron status parameters (ferritin, transferrin saturation [TSAT], soluble transferrin receptor-ferritin index [sTfR-F], soluble transferrin receptor [sTfR]), likewise measured in blood. The study was conducted at the University Hospitals of Frankfurt and Zurich. RESULTS: Limits of agreement between ZnPP-NI and ZnPP-HPLC measurements for 584 cardiac and noncardiac surgical patients equaled 19.7 µmol/mol heme (95% confidence interval, 18.0-21.3; acceptance criteria, 23.2 µmol/mol heme; absolute bias, 0 µmol/mol heme). Analytical performance for detecting IDE (inferred from area under the curve receiver operating characteristics) of parameters measured in blood was: ZnPP-HPLC (0.95), sTfR (0.92), sTfR-F (0.89), TSAT (0.87), and ferritin (0.67). Noninvasively measured ZnPP-NI yielded results of 0.90. CONCLUSION: ZnPP-NI appears well suited for an initial IDE screening, informing on the state of erythropoiesis at the point of care without blood drawing and laboratory analysis. Comparison with a multiparameter IDE test revealed that ZnPP-NI values of 40 µmol/mol heme or less allows exclusion of IDE, whereas for 65 µmol/mol heme or greater, IDE is very likely if other causes of increased values are excluded. In these cases (77% of our patients) ZnPP-NI may suffice for a diagnosis, while values in between require analyses of additional iron status parameters.

Non-invasive measurement of erythrocyte zinc protoporphyrin in children.

BACKGROUND: Reliable screening for iron deficiency (ID) has required a blood sample and cost-intensive laboratory measurements. A novel method to non-invasively measure erythrocyte zinc protoporphyrin (ZnPP), an established marker for ID, is evaluated in children. METHODS: ZnPP was determined non-invasively by fluorescence measurements on the wet vermillion of the lower lip in 99 hospitalized children aged 9 months to 5 years. For comparison, conventional ID parameters and ZnPP were determined from blood samples. RESULTS: The non-invasively measured ZnPP values had limits of agreement (LoA) of 14 µmol ZnPP/mol heme (95% confidence interval: 9-20) compared to fluorescence measurements directly in blood. Repeated high-performance liquid chromatography reference determinations had comparable LoA of 14 µmol ZnPP/mol heme (9-17). Non-invasive ZnPP measurements had sensitivity and specificity of 67% (39-88%) and 97% (91-99%), and negative and positive predictive value of 94% (90-97%) and 80% (55-93%), for detecting ID as defined by the soluble transferrin receptor (sTfR). In groups with more severe ID as defined by serum ferritin and sTfR, higher ZnPP values were found, with the highest ZnPP values for the group with ID anemia. CONCLUSION: Non-invasive ZnPP measurements are reliably feasible in children. The simple and fast method has the potential to enable wide-spread screening for ID.

Falsely low values of oxygen saturation measured by pulse oximetry in a boy treated with Chinese herb tea.

An 8-year-old boy suffering from progressive glioblastoma was scheduled for neurosurgery. Prior to induction of anaesthesia pulse oximetry measured 64 % saturation of oxygen (SpO2). Arterial blood gas analysis revealed normal oxygen saturation and normal oxygen partial pressure. After having ruled out technical problems of pulse oximetry the neurosurgical procedure was halted. Meticulous examination of the child's history and medication did not explain a possible interaction of drugs with pulse oximetry. A Chinese herb tea had been given to the child, but was then stopped on the day of admission. The surgical procedure took place the next day without any complications. During the subsequent inpatient stay, repeated blood gas analyses showed normal oxygenation, but pulse oximetry measured initially SpO2 values of 64 %, gradually increasing over 7 days up to 91 % by the time of discharge from hospital. Blood samples were taken and analysed. Absorption spectroscopy from the patient's blood showed an uncommon absorption maximum at 684 nm besides the normal maxima. The normalisation of SpO2 values after stopping Chinese herb tea administration leads to the conclusion that one of its ingredients caused the distorted pulse oximetry measurement.

Comparison of an accelerated weighted fluorescence Monte Carlo simulation method with reference methods in multi-layered turbid media.

Monte Carlo (MC) simulations are frequently used to simulate the radial distribution of remitted fluorescence light from tissue surfaces upon pencil beam excitation to gather information about influences of different tissue parameters. Here, the "weighted direct emission method" (WDEM) is proposed, which uses a weighted MC simulation approach for both excitation and fluorescence photons, and is compared to four other methods in terms of accuracy and speed, and using a broad range of tissue-relevant optical parameters. The WDEM is 5.2× faster on average than a fixed weight MC approach while still preserving its accuracy. Additional gain of speed can be achieved by implementing it on graphics processing units.

The 1,318-nm diode laser supported partial nephrectomy in laparoscopic and open surgery: preliminary results of a prospective feasibility study.

Warm ischemia (WI) and bleeding during laparoscopic partial nephrectomy (LPN) place technical constraints on surgeons. Our aim was to develop a safe and effective laser-assisted LPN-technique without the need for WI. In this study, a diode laser-emitting light at a wavelength of 1,318 nm at output powers between 45 and 70 W in continuous-wave mode was used. Light was coupled into a flexible 600-µm bare fiber to be transported to the tissues. After dry lab experience, 13 patients (six males, seven females) underwent five open and eight laparoscopic/retroperitoneoscopic partial nephrectomies. Postoperative renal function and serum C-reactive protein (CRP) were monitored and coagulation depth and effects on resection margins (RR) were evaluated. Demographic, clinical, and follow-up data are presented. Mean operative time was 116.5 min (range 60-175 min) with mean blood loss of 238 ml (range 50-600 ml). Laser light application took a maximum of 17 min. All patients had a favorable outcome. The locations of the treated tumors (eight left and five right) were central (two), upper pole (two), lower pole (three) and middle kidney parenchyma (six anterior, two posterior, and five peripheral). Mean tumor size was 3.3 cm (range 1.8-5 cm). Two WI (19 and 24 min) were needed. Immediate postoperative serum creatinine and CRP were elevated within 0.1 to 0.6 mg/dl (mean 0.18) and 2.1-10 mg/dl (mean 6.24), respectively. Coagulation depth ranged from <1 to 2 mm without an effect on histopathological evaluation of tumors or RR. One patient had positive RR. During follow-up (2-6 months), one patient developed an A-V fistula that needed embolization. This prospective in-vivo feasibility study showed that the diode laser is a safe and promising device for LPN. Its advantages are minimal gas formation, good hemostasis, and minimal parenchymal damage. Oncological safety appears to be warranted by the use of a diode laser.

A second-derivate fitting algorithm for the quantification of free hemoglobin in human plasma.

BACKGROUND: Assessment of hemolysis in vivo is becoming increasingly relevant in critical care. Current methods (Harboe, 1959) for quantifying the free hemoglobin (fHb) content produce unsatisfactory results in case of hyperbilirubinemia, a frequent condition in patients at risk for intravascular hemolysis. METHODS: A novel evaluation method based on second-derivative fitting to quantify fHb content was developed. The method uses spectrophotometric data from 350 to 650 nm recorded with standard instruments as input. To evaluate the power of the new method, plasma of patients and non-icteric plasma of healthy volunteers were spiked with fHb concentrations up to 2000 mg/L and compared to methods described in the literature by Harboe, Noe and Fairbanks. All measurements were done in compliance with the bioanalytical method validation protocol from the European Medicines Agency. RESULTS: Both the second-derivative fitting algorithm as well as the methods of Harboe, Noe and Fairbanks quantified fHb accurately in non-icteric samples, with inaccuracy and imprecision below 10%. For icteric specimen, false high results were obtained with the established formulas for fHb concentrations below 700 mg/L. In contrast, no interference was found with the second-derivate fitting method for bilirubin concentrations up to 465 µmol/L. The lower limits of quantifications for the second-derivative fitting algorithm were specified in agreement with the EMA guideline with 25 mg/L fHb for both non-icteric and icteric specimens. CONCLUSIONS: A user-friendly, computer-based algorithm is reported that allows the accurate quantification of fHb concentrations in the presence of high bilirubin concentrations. The new method allows for uniform sample preparation with only a single dilution step and can be readily implemented in any laboratory on standard spectrophotometers using the provided supplementary Microsoft Excel macro.

Rapid spectrophotometric quantification of urinary porphyrins and porphobilinogen as screening tool for attacks of acute porphyria.

Autosomal-dominant acute porphyria, a group of rare diseases, can lead to life-threatening neurovisceral attacks. No efficient screening test is available today. Elevated urinary porphobilinogen in addition to elevated porphyrins is highly specific for an attack of acute porphyria. This study proposes and evaluates a custom-made device, algorithm, and methods for a two-step quantification of urinary porphyrins and porphobilinogen. The first step is oxidation of the nonfluorescent porphyrinogens and subsequent fluorescence-spectroscopic determination of total urinary porphyrins (TUP) using second derivative spectral fitting. Photo-oxidation is compared with chemical oxidation methods. The second step is the quantification of porphobilinogen in case of elevated TUP. Heat-induced conversion products of porphobilinogen, namely uroporphyrin and porphobilin, are quantified by fluorescence and absorption spectroscopy. Results show that the preferred method combination is TUP quantification (lower limit of quantification: 0.2 µmol / L) after photo-oxidation with subsequent absorption-spectroscopic determination of porphobilin after heating for indirect quantification of porphobilinogen (quantification range: 0 to 20 mg / L). Urinary porphobilinogen and porphyrins of one acute porphyria patient were quantified with <10 % deviation from an external reference determination. The spectrophotometric approach requires only minimal sample processing and yields a result within 15 min, thus closing the screening gap for acute porphyria.

Non-invasive detection of iron deficiency by fluorescence measurement of erythrocyte zinc protoporphyrin in the lip.

Worldwide, more individuals have iron deficiency than any other health problem. Most of those affected are unaware of their lack of iron, in part because detection of iron deficiency has required a blood sample. Here we report a non-invasive method to optically measure an established indicator of iron status, red blood cell zinc protoporphyrin, in the microcirculation of the lower lip. An optical fibre probe is used to illuminate the lip and acquire fluorescence emission spectra in ∼1 min. Dual-wavelength excitation with spectral fitting is used to distinguish the faint zinc protoporphyrin fluorescence from the much greater tissue background fluorescence, providing immediate results. In 56 women, 35 of whom were iron-deficient, the sensitivity and specificity of optical non-invasive detection of iron deficiency were 97% and 90%, respectively. This fluorescence method potentially provides a rapid, easy to use means for point-of-care screening for iron deficiency in resource-limited settings lacking laboratory infrastructure.

Medical laser application: translation into the clinics.

Medical laser applications based on widespread research and development is a very dynamic and increasingly popular field from an ecological as well as an economic point of view. Conferences and personal communication are necessary to identify specific requests and potential unmet needs in this multi- and interdisciplinary discipline. Precise gathering of all information on innovative, new, or renewed techniques is necessary to design medical devices for introduction into clinical applications and finally to become established for routine treatment or diagnosis. Five examples of successfully addressed clinical requests are described to show the long-term endurance in developing light-based innovative clinical concepts and devices. Starting from laboratory medicine, a noninvasive approach to detect signals related to iron deficiency is shown. Based upon photosensitization, fluorescence-guided resection had been discovered, opening the door for photodynamic approaches for the treatment of brain cancer. Thermal laser application in the nasal cavity obtained clinical acceptance by the introduction of new laser wavelengths in clinical consciousness. Varicose veins can be treated by innovative endoluminal treatment methods, thus reducing side effects and saving time. Techniques and developments are presented with potential for diagnosis and treatment to improve the clinical situation for the benefit of the patient.

In-vitro investigations on laser-induced smoke generation mimicking the laparoscopic laser surgery purposes.

Intraoperative smoke-generation limits the quality of vision during laparoscopic/endoscopic laser-assisted surgeries. The current study aimed at the evaluation of factors affecting this phenomenon. As a first step, a suitable experimental setup and a test tissue model were established for this investigation. The experimental setup is composed of a specific sample container, a laser therapy component suitable for the ablation of model tissue at different treatment wavelengths (λ = 980 nm, 1350 nm, 1470 nm), a suction unit providing continuous smoke extraction, and a detection unit for smoke quantification via detection of light (λ = 633 nm) scattered from smoke particles. The ablation rate (AR) was calculated by dividing the ablated volume by the ablation time (60 sec). The laser-induced scattering signal intensity of the smoke (SI) was determined from time-charts of the signal intensity as a measure for vision, in addition a delay-time tdelay could be derived defining the onset of SI after the laser was switched on. The ratio SI/AR is used as a measure for smoke generation in relation to the ablation rate. Additionally the light transmission of the tissue samples was used to estimate their optical properties. In this set-up, smoke generation using λ = 980 nm as ablation laser wavelength was detected after a delay-time tdelay = (121.6 ± 24.8) sec which is significantly longer compared to the wavelengths λ = 1350 nm with tdelay = (89.8 ± 19.3) sec and λ = 1470 nm with tdelay = (24.7 ± 5.4) sec. Thus, the delay Experimental set-up consisting of sample container, laser therapy component, suction unit and scattered-light detection compartment. time is wavelength-dependent. The SI/AR ratio was significantly different (p < 0.001) for 1470 nm irradiation compared to 980 nm irradiation [SI/AR(1470) = (11.8 ± 2.6) · 10(3) vs. SI/AR(980) = (8.6 ± 2.0) · 10(3) ]. The ablation crater for 980 nm irradiation was comparable with 1470 nm irradiation, but the coagulation rim was thicker in the 980 nm case. In conclusion, it could be shown experimentally that smoke-generation depends on the wavelength used for laser ablation.

Dual-wavelength excitation for fluorescence-based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood.

Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron-restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual-wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual-wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (rs = 0.943, p < 0.0001; range 37-689 µmol ZnPP/mol heme, 84-1238 nmol/L) and for PPIX (rs = 0.959, p < 0.0001; range 42-4212 µmol PPIX/mol heme, 93-5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single-wavelength excitation and with commercial front-face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual-wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX.

Bandwidth-variable tunable optical filter unit for illumination and spectral imaging systems using thin-film optical band-pass filters.

An optical filter unit is demonstrated, which uses two successively arranged tunable thin-film optical band-pass filters and allows for simultaneous adjustment of the central wavelength in the spectral range 522-555 nm and of the spectral bandwidth in the range 3-16 nm with a wavelength switching time of 8 ms∕nm. Different spectral filter combinations can cover the complete visible spectral range. The transmitted intensity was found to decrease only linearly with the spectral bandwidth for bandwidths >6 nm, allowing a high maximum transmission efficiency of >75%. The image of a fiber bundle was spectrally filtered and analyzed in terms of position-dependency of the transmitted bandwidth and central wavelength.

Ex vivo comparison of the tissue effects of six laser wavelengths for potential use in laser supported partial nephrectomy.

Laparoscopic/robotic partial nephrectomy (LPN) is increasingly considered for small renal tumors (RT). This demands new compatible surgical tools for RT-resection, such as lasers, to optimize cutting and coagulation. This work aims to characterize ex vivo handling requirements for six medically approved laser devices emitting different light wavelengths (940, 1064, 1318, 1470, 1940, and 2010 nm) amenable for LPN. Incisions were made by laser fibers driven by a computer-controlled stepping motor allowing precise linear movement with a preset velocity at a fixed fiber-tip distance to tissue. Optical parameters were measured on 200 µm tissue slices. Cutting quality depended on power output, fiber velocity and fiber-tip distance to tissue. Contact manner is suitable for cutting while a noncontact manner (5 mm distance) induces coagulation. Ablation threshold differs for each wavelength. Ablation depth is proportional to power output (within limit) while axial and superficial coagulation remains mostly constant. Increased fiber velocity compromises the coagulation quality. Optical parameters of porcine kidney tissue demonstrate that renal absorption coefficient follows water absorption in the 2 µm region while for other spectral regions (900 to 1500 and 1 µm) the tissue effects are influenced by other chromophores and scattering. Tissue color changes demonstrate dependencies on irradiance, scan velocity, and wavelength. Current results clearly demonstrate that surgeons considering laser-assisted RT excisions should be aware of the mentioned technical parameters (power output, fiber velocity and fiber-tip tissue-distance) rather than wavelength only.

Photobleaching-based method to individualize irradiation time during interstitial 5-aminolevulinic acid photodynamic therapy.

Interstitial photodynamic therapy (iPDT) is being investigated for the treatment of high-grade human brain malignancies. In recent clinical studies, fluorescence monitoring during iPDT of glioblastoma multiforme has revealed patient-specific accumulation of photosensitizer (aminolevulinic acid (ALA) induced protoporphyrin IX, PpIX) and its photobleaching kinetics. As photosensitizer degradation, also referred to as photobleaching, and tissue damage are caused by the same underlying processes, the photobleaching kinetics might provide a tool for real-time treatment supervision. Here, we show with computer simulations that varying optical properties have a strong influence on the irradiation time required to fully bleach the photosensitizer. We propose a method to potentially determine the time point during iPDT, when the photosensitizer within the target volume has been largely photobleached. Simulations show that it is possible to determine this time point by continuously monitoring the ratio of the fluorescence intensities at two time points during irradiation. We show that this method works for a large range of optical properties, different photobleaching rates and varying inter-fibre distances. In conclusion, the relative fluorescence method offers the potential to individualize irradiation times to consume the photosensitizer within the target tissue during iPDT.