Hemoglobin Contamination Research Articles

Analytical and clinical validation of a circulating tumor DNA–based assay for multicancer early detection.

10548 Background: The development of multi-cancer early detection (MCED) through a single blood test has emerged as a promising method for improving the efficiency of early cancer detection and benefiting population health. However, the lack of analytical validation and clinical evidence for their utility in diverse populations have prevented their use in clinical practice. To address these challenges, we conducted a comprehensive analytical and clinical validation for an MCED test, SPOT-MAS (Screening for the Presence of Tumor by DNA Methylation and Size). Methods: A prospective cohort study (ClinicalTrials.gov identifier: NCT05227261) was conducted at 13 major hospitals and one research institute in Vietnam. Our study recruited asymptomatic participants aged 40 years or older and followed up for 6 and 12 months. Analytical sensitivity of our assay was assessed by using our healthy and cancer-standard samples with known tumor fraction. The clinical performance was assessed by computing the positive predictive value (PPV), the negative predictive value (NPV) and accuracy in detecting tumor tissue-of-origin (TOO). Results: SPOT-MAS demonstrates the capability to detect at least 50% of cancer samples with a specificity of 98% when the samples have a tumor fraction of 0.049 (95% CI: 0.043-0.059). These results were consistently reproduced in both intra- and inter-batch analyses. Furthermore, our test maintains robustness even in the presence of hemoglobin contamination up to 500 mg/dL and genomic DNA contamination of up to 100%. Our analysis of 10,027 eligible participants demonstrated its ability to detect cancers in asymptomatic individuals with a positive predictive value of 58.14% (95%CI: 43.33- 71.62), an accuracy of 84.00% (95%CI: 65.35-93.60) in predicting tumor location and a negative predictive value of 99.92% (95%CI: 99.83-99.96). Conclusions: To our knowledge, this is the first and largest prospective validation study in Asia supporting the utility of SPOT-MAS as a multi-cancer blood test for early detection in a low- and middle-income country, where a nationwide cancer screening program is urgently needed but currently not available. Clinical trial information: NCT05227261 .

Novel ELISAs to measure total and phosphorylated tau in cerebrospinal fluid

Cerebrospinal fluid (CSF) total tau (t-tau) and tau protein phosphorylated at threonine 181 (p181tau) are established biomarkers for Alzheimer’s disease (AD). Herein, we measured t-tau and p181tau to evaluate novel enzyme-linked immunosorbent assays (ELISAs) using 72 CSF samples including from patients with AD with dementia (ADD) and various neurodegenerative diseases. Our assay system showed good correlations with widely used ELISA systems for t-tau and p181tau and showed that serum and hemoglobin contamination in CSF samples did not decrease sensitivity. Significant increases in both t-tau and p181tau levels were observed in ADD. These findings suggested that our ELISAs were reliable assays for CSF t-tau and p181tau similar to commonly used ELISAs.

Short-term effects of hemogram in healthy donors after peripheral blood stem cell collection

目的探讨连续外周血造血干细胞（PBSC）采集对异基因造血干细胞供者外周血细胞计数的影响以及供者对连续采集的耐受性。方法166名于2013至2014年在浙江大学医学院附属第一医院血液科捐献PBSC的亲缘异基因供者，男86名，女80名，中位年龄40.5（15.0~60.0）岁。动员方案为G-CSF 5~10 µg·kg−1·d−1皮下注射至采集结束。于PBSC动员前、采集前、采集1次及2次后检测供者外周血以及采集物中WBC、PLT、HGB。结果连续2次PBSC采集后供者外周血HGB中位数由145（94~181）g/L降至138（93~167）g/L（P<0.05），PLT中位数由231（105~490）×109/L降至95（39~210）× 109/L（P<0.05），WBC差异无统计学意义（P>0.05）。采集物中HGB与供者外周血HGB降幅呈低度正相关（r=0.297，P=0.017），采集物中PLT与供者外周血PLT降幅高度相关（r=0.719，P<0.001）。不同年龄组供者在PBSC采集后的HGB降幅两两比较差异无统计学意义（P≥0.05）。PLT差异无统计学意义（P>0.05）。不同体重指数供者在PBSC采集1次及2次后外周血HGB降幅差异均有统计学意义（P=0.003，P<0.001），体重指数<18.5 kg/m2的偏瘦组供者HGB下降最明显（P<0.05）。结论健康异基因造血干细胞供者在连续PBSC采集过程中HGB轻度下降，对于偏瘦供者应调整采集参数以保证安全。采集PBSC会使血小板减少的风险增加，减少的程度与年龄及体重指数无关，且均可耐受。

Correction of peripheral blood mononuclear cell cytosolic protein for hemoglobin contamination

Pharmacodynamic (PD) analysis requires accurate and precise quantification of enzyme activity targeted by anticancer agents in surrogate cells like peripheral blood mononuclear cells (PBMCs). Enzyme activity is normally reported per mass unit of protein input. However, high and fluctuating hemoglobin (Hb) contamination strongly influences the protein content of PBMC cytosolic lysate. We present the development and validation of a spectrophotometrical Hb quantification method to correct for this contamination. The applicability of Hb correction was demonstrated by determination of the dihydropyrimidine dehydrogenase enzyme activity in PBMC cytosolic lysates.

Variable oxygen affinity among terrestrial and diving vertebrates

From being the first protein to have its three‐dimensional structure determined to being sequenced for hundreds of species, myoglobin (Mb) has had a distinguished history as a research subject. Although Mb is one of the most studied proteins, surprisingly little is known of its oxygen affinity at physiologically significant temperature or the variability of this oxygen affinity between species. In this study we compared the oxygen affinity (P50) of skeletal muscle Mb from six terrestrial and ten diving vertebrates. After extracting Mb from muscle samples and removing hemoglobin contamination, oxygen affinity was determined using a TCS Hemox Blood Analyzer. We found Mb P50 ranging from 2.5 to 5.3 mmHg at 37°C. Modeling of oxygen transport in muscle tissue will be necessary to determine if this twofold difference in oxygen affinity has significant implications in oxygen transport under various physiological conditions. Variants in Mb structure and function could be adaptive for species that regularly experience muscle hypoxia such as diving vertebrates.

Exfoliated Buccal Mucosa Cells as a Source of DNA to Study Oxidative Stress

The extent of oxidative DNA damage is considered a biomarker of carcinogenic process and could be investigated in population studies using easily obtained cells. The oxidized DNA base adduct 8-hydroxy-2-deoxyguanosine (8-OHdG) released by enzymatic hydrolysis of DNA is commonly assayed by high performance liquid chromatography with electrochemical detection. It is expressed as a ratio of 8-OHdG to unoxidized deoxyguanosine. We modified and improved this method, determined the optimal time for harvesting buccal mucosa cells (BMC), assessed whether they mirror peripheral circulating blood cell DNA damage, and compared the anticoagulants, heparin, and EDTA for consistency in measurement of leukocyte 8-OHdG. Thirty-one healthy participants, randomized into two groups, donated BMC and blood samples. Samples were collected at baseline and either 3 or 7 days after baseline. Results showed no correlation between 8-OHdG/deoxyguanosine ratios in BMC and peripheral blood leukocytes at any time point regardless of harvest time. BMC had much higher oxidative DNA damage, but displayed a 25.6% reduction in the oxidized DNA adduct level (P < 0.04) at 3 days after baseline. Leukocytes collected in heparin and EDTA had similar 8OHdG/deoxyguanosine ratios; however, EDTA was preferred, as it produced a clean nuclear pellet without hemoglobin contamination, and the results were less variable. This improved assay shows within subject stability over time in both leukocyte and BMC DNA damage, increasing the probability that small intervention differences can be detected in healthy subjects. Buccal cells provide an accessible pool of epithelial cells that represents higher levels of DNA damage than circulating leukocytes.

Insights into the etiology of page hypertension - a model of hypertensive heart disease and diastolic dysfunction

Background: Page (renal wrap) hypertension is an established model of experimental hypertension and produces hypertensive heart disease characterized by ventricular hypertrophy and fibrosis as well as diastolic dysfunction. Known vasoconstrictor systems (renin-angiotensin-aldosterone system, endothelin and catecholamines) are not activated in this model. The etiology of Page hypertension is unclear. We explored two possible mechanisms of hypertension in this model: one; role of sodium (Na) retention during the evolution of hypertension and two; production of a novel vasoconstrictor substance by the wrapped kidney. Methods: Two groups of mongrel dogs (sham, n = 6 and renal wrap, n = 6; age 1–2 years) were studied. 24-hour urine collections for Na were performed for one week before and four weeks after the wrap or sham surgery. The kidneys were harvested and extracts from cortex and medulla were tested for vasoconstrictor and vasodilatory properties in vitro using rat aortic strips in organ chamber experiments. Strips were pretreated with L-NMMA to control for any hemoglobin contamination of extracts. Seperate aortic strips were exposed to extracts after pre-constriction with norepinephrine to assess potential differential vasodilatory effects of extracts. Results: Blood pressure (see figure) in the renal wrapped animals was significantly higher than in the sham operated group. Renal function (serum creatinine), plasma renin activity and aldosterone levels were not significantlty different between the two groups (2-way ANOVA, p > 0.05). Urinary Na excretion over time was not significantly different between the two groups (see figure) (2-way ANOVA, p > 0.05). Both cortical and medullary renal extracts produced vasoconstriction in the aortic strips but there was no significant difference in the degree of vasoconstriction induced by extracts from wrapped or sham operated kidneys. While pre-constricted aortic strips showed relaxation with cortical extracts, medullary extracts produced further constriction. Again, the extent of constriction or relaxation produced by the renal tissue extracts was not significantly different between the sham operated and wrapped kidneys. Conclusions: Sodium retention does not contribute to evolution of hypertension following renal wrapping. No evidence exists for presence of novel active vasoconstrictor substance in wrapped kidneys. However, the renal production of a non-vasoactive precursor factor that is activated peripherally cannot be excluded. The etiology of Page hypertension remains unclear.

Correlates of IL-10 and IL-12 concentrations in cervical secretions.

Interindividual variations in host immune responses to HPV infection are thought to be important determinants of viral persistence and progression to cervical intraepithelial neoplasia and cancer. However, few studies have measured local immune markers at the site of infection (e.g., the cervical mucosa). We sought to determine biologic correlates of IL-10 and IL-12 concentrations in cervical secretions. Cervical secretions were passively collected using a WeckCel sponge from 247 women participating in a natural history study of human papillomavirus infection as part of an immunologic ancillary study. IL-10 and IL-12 concentrations were determined using standard ELISA assays. In general, IL-10 and IL-12 levels were significantly intercorrelated (Pearson's correlation coefficient = 0.6) but had somewhat different determinants. Significant increases (P < 0.05) in IL-10 concentrations were observed for nonovulatory phases of the menstrual cycle, postmenopausal status, recent use of oral contraceptives (OC), low secretion volume, macrolevels of heme contamination, and high vaginal pH. Increasing IL-10 levels were also observed among smokers, women with increasing numbers of lifetime sex partners, and women who report having less frequent sex (less than once per week), however, these results were not statistically significant. Significantly higher IL-12 concentrations were observed among recent OC users, women with low secretion volume, and women with a high vaginal pH. There was a non-statistically significant observation of increasing IL-12 levels among nonsmokers, women with increasing numbers of lifetime and recent pregnancies, and increasing levels of heme contamination. We failed to observe a significant association between HPV and IL-10 or IL-12 levels in this crosssectional sample. Future analyses of cervical cytokine levels and HPV infection should control for the inherent variation of local cytokine levels due to hormonal influences, hemoglobin contamination, pH, and cervical secretion volume differences.

Improved Myoglobin Saturation Measurement Made by Partial Least-Squares Analysis of Optical Reflectance Spectra

Accurate myoglobin oxygen saturation determination from tissue using optical spectral analysis has been limited by overlap in spectral features between myoglobin and hemoglobin. Partial least-squares analysis of reflectance spectra has been successfully used to predict myoglobin oxygen saturation from blood perfused hearts, although uncertainty remains as to how much error in the myoglobin saturation estimate occurs due to hemoglobin contamination. This study was undertaken to quantify the error due to hemoglobin in the partial least-squares estimate of myoglobin saturation. Calibration spectra were developed by mathematical addition of individually acquired in vitro reflectance spectra from various absorbing species in scattering media. The isolated perfused guinea pig heart model was used to allow for switching between crystalloid and red blood cell perfusion in a beating heart. Inclusion of cytochrome c and cytochrome oxidase in a calibration set containing hemoglobin and myoglobin improved the accuracy of the partial least-squares analysis of myoglobin saturation. Both the Mahalanobis distance test and the residual ratio test demonstrated improved representation of spectra obtained from the heart using a calibration set that included both cytochromes. A 4.3% error in myoglobin oxygen saturation measurements was determined.

A convenient method of DNA extraction from blood anticoagulated with EDTA.

Surplus blood often remains after routine clinical tests using EDTA-anticoagulated samples. To use this blood for DNA analysis, we isolated white cells by adherence to polyethylene terephthalate fibers, which could be stored for several weeks transported by mail after methanol fixation. DNA yield was sufficient and correlated with white cell count. Extracted DNA was free of hemoglobin contamination and durable to polymerase chain reaction and enzyme digestion, which yielded products visualized as well-separated bands on electrophoresis. We found our method to be practical for the routine clinical laboratory.

The state of ferriprotoporphyrin IX in malaria pigment.

To evaluate the state of ferriprotoporphyrin IX (FP) in malaria pigment, mouse erythrocytes infected with Plasmodium berghei NYU-2 parasites were lysed by hypotonic shock, and hemoglobin and other soluble material were removed by extensive washing. The amount of FP recovered in the insoluble pellet was 2.1 mumol/ml of packed infected erythrocytes, of which approximately 1% was attributable to hemoglobin contamination. This crude preparation then was digested with a nonspecific protease from Streptomyces griseus and extracted with chloroform/methanol. The residue of insoluble dark brown material had the spectral and solubility properties characteristic of the FP of malaria pigment, and various different preparations contained from 82 to 99% of FP by weight. By elemental analysis, highly purified preparations contained no chlorine and had an oxygen content consistent with 1 mol of hydroxyl/mol of FP (oxygen content: calculated, 12.6%; found, 12.5%). In comparison to hematin purchased from Sigma, which had a measured oxygen content of 14.7%, the low oxygen form of hematin purified from malaria pigment was remarkably less soluble in ethanol, 3% sodium bicarbonate, and chloroform.

Kinetics of T 4 and T 3 binding to plasma sites in salmonid teleost fish

The relative affinities and maximum capacities of the classes of l-thyroxine (T 4)- and 3,5,3′-triiodo- l-thyronine (T 3)-binding sites in plasma of three species of salmonid teleost fish were determined by saturation analysis on miniature Sephadex G-25 columns at 20–21° and kinetic data were analyzed by the multiligand, multisite LIGAND computer program. In plasma of rainbow trout ( Salmo gairdneri) homologous ligand displacement indicated that both thyroid hormones (TH) bound to a minimum of two classes of saturable sites and at least one nonsaturable site. For T 4 ( n = 13) the relative site affinities were 0.61 ± 0.08 (SEM) × 10 7 M −1 and 0.86 ± 0.11 × 10 5 M −1 and the site capacities were 8.3 ± 1.16 × 10 −7 M and 5.15 ± 1.34 × 10 −5 M, respectively; for T 3 ( n = 14) the relative site affinities were 1.8 ± 0.16 × 10 7 M −1 and 1.7 ± 0.17 × 10 5 M −1 and the site capacities were 7.8 ± 1.3 × 10 −7 M and 11.5 ± 5.29 × 10 −5 M, respectively. The greater affinities of T 3 than T 4 for plasma binding sites would explain the lower proportion of free T 3 than free T 4 in trout plasma. Two-site models with comparable values for TH-binding parameters were determined for brook trout ( Salvelinus fontinalis) and arctic charr ( Salvelinus alpinus). The TH-binding parameters were uninfluenced by severe hemoglobin contamination of plasma, bleeding of fish 24 hr previously, or 2 weeks starvation. Heterologous ligand displacement (T 4 displaced by T 3 or T 3 displaced by T 4) on rainbow trout plasma suggested (i) two low-affinity, high-capacity sites, one binding predominantly T 4 and one binding predominantly T 3; (ii) a high-affinity, low-capacity site binding T 3 exclusively; and (iii) a high-affinity, low-capacity site binding T 4 predominantly but also binding T 3 with low affinity.

Preparative affinity electrophoresis: Application to human erythrocyte carbonic anhydrase

A modification of affinity electrophoresis for preparative purposes is described. This method has been applied to the purification of human erythrocyte carbonic anhydrases B and C. During conventional affinity chromatography some hemoglobin contamination occurs. By introduction of an electrophoretic purification step after the immobilization of carbonic anhydrase to the affinity gel, the hemoglobin impurity is reduced about eight and two times in the preparations of the B and C enzymes, respectively, compared to the enzymes purified by affinity chromatography.

Purification of fluorescein-labeled specific anti-hemoglobin antibody using cross-linked immunoabsorbent

Fluorescein-labeled monospecific anti-hemoglobin antibodies are useful tools for studying hemoglobin expression and somatic point mutation. The yield of monospecific antibodies is extremely low and the purification of these antibodies using hemoglobin immunoabsorbents is further complicated by the fact that hemoglobin ‘leaks’ out from the immunoabsorbent during elution of the antibody with acid medium contaminating the eluted antibody. Two improvements are reported in this paper for the purification of fluorescein-labeled goat anti-mouse hemoglobin antibody specific for DBA/2J hemoglobin that does not react with C57BL/6J mouse hemoglobin: (1) The entire gamma globulin fraction of anti-hemoglobin antiserum was labeled with the fluorochrome before proceeding for the absorption and purification steps (in contrast to the conventional methods where the antibody is first purified followed by the fluorochrome labeling). This modification was expected to yield only the functionally active labeled antibody preparation devoid of molecules that may have undergone denaturation during the labeling procedure. Also, this modification eliminated the need of carrying out the labeling procedure with a small amount of the precious purified antibody thus decreasing the antibody loss. (2) Instead of conventional immunoabsorbents (hemoglobin coupled to CNBr-Sepharose), immunoabsorbents cross-linked with glutaraldehyde were used. The eluted antibody was free of any hemoglobin contamination. The antibody thus purified was found to specifically react with fixed red blood cells from DBA/2J mice but not cells from C57BL/6J mice. The method described in this paper should be of general applicability in the preparation of monospecific antibodies.

A simple method to remove hemoglobin from antiserum.

Hemoglobin contamination in antiserum occurs frequently in immunohematology. The hemoglobin obscures the ring test for the observation of antigen-antibody reaction. A simple method to remove hemoglobin from antiserum is described in this paper. An appropriate amount of CM-Sephadex was added to antiserum containing hemoglobin and the mixture was packed into a cellulose tube, followed by dialysing against 0.01 M monobasic sodium phosphate solution (pH approx. 6). Equilibrating with stirring, the hemoglobin was completely adsorbed with CM-Sephadex so that hemoglobin-free antiserum was prepared without dilution of the antiserum. This method is useful for immunohematology.

The occurrence of vitamin A in biological membranes

1. 1. Rats maintained on a low vitamin A intake were intraperitoneally injected with 50.7 μg [11,12- 3H 2]retinyl acetate. 15 h later, the animals were killed and liver and kidney plasma membranes, kidney endoplasmic reticulum and erythrocyte ghosts were prepared. 2. 2. The labeled vitamin A content of the membrane preparations varied consistently between 0.003–0.005 μg retinol/mg N, while the level in the homogenate varied from 0.001–0.009 μg retinol/mg N. The labeled vitamin A content of the erythrocyte ghosts was low (max. 0.0001 μg retinol/mg N) probably due to heavy hemoglobin contamination. 3. 3. The radioactive vitamin A compounds were strongly bound to the membranes: n- hexane removed only 7% of the radioactive compounds, whereas chloroform-methanol (2:1, v/v) extraction of the membrane preparations removed 90% of the radioactive compounds. 4. 4. Thin-layer chromatography of the chloroform-methanol (2:1, v/v) extract of the kidney endoplasmic reticulum, in three different solvent systems, revealed that retinol and retinoic acid were the dominant forms of vitamin A. This was confirmed by combined column and thin-layer chromatography (using a fourth solvent system) of the chloroform-methanol (2:1, v/v) extract of endoplasmic reticulum.