Darci Block - Academia.edu (original) (raw)

Papers by Darci Block

Journal of Inorganic Biochemistry, 2016

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this repor... more The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pK a values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wildtype; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the wild-type protein (68 ± 3 mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process was one to three days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.

American Journal of Clinical Pathology

The guideline-recommended lipid panel for cardiovascular disease (CVD) risk assessment measures t... more The guideline-recommended lipid panel for cardiovascular disease (CVD) risk assessment measures total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and calculated low-density lipoprotein (LDL) cholesterol. Measured cholesterol in subfractions of HDL and LDL purportedly improve CVD risk prediction. Homogenous enzymatic methods are now available for measurement of the cholesterol within small dense LDL (sLDL), small dense HDL (HDL3), and triglyceride-rich lipoproteins (TRL). For meaningful interpretation of these measurements, an understanding of the potential sources and extent of result variability is needed. The smallest difference between serial measurements within a patient that likely reflects a change in clinical status is called the reference change value (RCV). Biological variability and reference change values (RCV) are well-characterized for basic lipids but there is limited information for sLDL, HDL3 or TRL. The objective of this study was to dete...

The Bone & Joint Journal

Aims The aim of this study was to determine the diagnostic accuracy of α defensin (AD) lateral fl... more Aims The aim of this study was to determine the diagnostic accuracy of α defensin (AD) lateral flow assay (LFA) and enzyme-linked immunosorbent assay (ELISA) tests for periprosthetic joint infection (PJI) in comparison to conventional synovial white blood cell (WBC) count and polymorphonuclear neutrophil percentage (PMN%) analysis. Methods Patients undergoing joint aspiration for evaluation of pain after total knee arthroplasty (TKA) or total hip arthroplasty (THA) were considered for inclusion. Synovial fluids from 99 patients (25 THA and 74 TKA) were analyzed by WBC count and PMN% analysis, AD LFA, and AD ELISA. WBC and PMN% cutoffs of ≥ 1,700 cells/mm3 and ≥ 65% for TKA and ≥ 3,000 cells/mm3 and ≥ 80% for THA were used, respectively. A panel of three physicians, all with expertise in orthopaedic infections and who were blinded to the results of AD tests, independently reviewed patient data to diagnose subjects as with or without PJI. Consensus PJI classification was used as the r...

Journal of Medical Internet Research

Background COVID-19 is caused by the SARS-CoV-2 virus and has strikingly heterogeneous clinical m... more Background COVID-19 is caused by the SARS-CoV-2 virus and has strikingly heterogeneous clinical manifestations, with most individuals contracting mild disease but a substantial minority experiencing fulminant cardiopulmonary symptoms or death. The clinical covariates and the laboratory tests performed on a patient provide robust statistics to guide clinical treatment. Deep learning approaches on a data set of this nature enable patient stratification and provide methods to guide clinical treatment. Objective Here, we report on the development and prospective validation of a state-of-the-art machine learning model to provide mortality prediction shortly after confirmation of SARS-CoV-2 infection in the Mayo Clinic patient population. Methods We retrospectively constructed one of the largest reported and most geographically diverse laboratory information system and electronic health record of COVID-19 data sets in the published literature, which included 11,807 patients residing in 41...

American Journal of Clinical Pathology

American Journal of Clinical Pathology

The Journal of Applied Laboratory Medicine: An AACC Publication

Clinical Biochemistry

Lactate, white blood cell (WBC) and neutrophil count, procalcitonin and immature granulocyte (IG)... more Lactate, white blood cell (WBC) and neutrophil count, procalcitonin and immature granulocyte (IG) count were compared for the prediction of sepsis, and severe sepsis or septic shock, in patients presenting to the emergency department (ED). We prospectively enrolled 501 ED patients with a sepsis panel ordered for suspicion of sepsis. WBC, neutrophil, and IG counts were measured on a Sysmex XT-2000i analyzer. Lactate was measured by i-STAT, and procalcitonin by Brahms Kryptor. We classified patients as having sepsis using a simplification of the 1992 consensus conference sepsis definitions. Patients with sepsis were further classified as having severe sepsis or septic shock using established criteria. Univariate receiver operating characteristic (ROC) analysis was performed to determine odds ratio (OR), area under the ROC curve (AUC), and sensitivity/specificity at optimal cut-off for prediction of sepsis (vs. no sepsis), and prediction of severe sepsis or septic shock (vs. no sepsis). There were 267 patients without sepsis; and 234 with sepsis, including 35 patients with severe sepsis or septic shock. Lactate had the highest OR (1.44, 95th% CI 1.20-1.73) for the prediction of sepsis; while WBC, neutrophil count and percent (neutrophil/WBC) had OR>1.00 (p<0.05). All biomarkers had AUC<0.70 and sensitivity and specificity <70% at the optimal cut-off. Initial lactate was the best biomarker for predicting severe sepsis or septic shock, with an odds ratio (95th% CI) of 2.70 (2.02-3.61) and AUC 0.89 (0.82-0.96). Traditional biomarkers (lactate, WBC, neutrophil count, procalcitonin, IG) have limited utility in the prediction of sepsis.

American Journal of Clinical Pathology, 2017

Clinical biochemistry, Jan 25, 2016

Measuring lipoprotein-associated phospholipase A2 (Lp-PLA2) activity can aid in identifying indiv... more Measuring lipoprotein-associated phospholipase A2 (Lp-PLA2) activity can aid in identifying individuals at higher risk of coronary heart disease. However, the biological variation of Lp-PLA2 activity and corresponding reference change value (RCV) is unknown which limits interpretation of results. In this study we aim to define the intra- and inter-individual variability of Lp-PLA2 activity in a healthy reference population. A total of 24 healthy individuals (22-47years of age) were prospectively collected at several time points: daily for five days (after overnight fast), daily for three days (while non-fasting), weekly for four weeks (after overnight fast), and monthly for 6months (after overnight fast). Intra-individual and inter-individual variability was determined. The index of individuality (IoI) and reference change value (RCV) were calculated for each time period. Variability in Lp-PLA2 activity was not different in fasting versus non-fasting states and also did not change i...

Clinical Chemistry, 2015

A 65-year-old woman with congestive heart failure presented for a mitral valve replacement. She e... more A 65-year-old woman with congestive heart failure presented for a mitral valve replacement. She experienced excessive bleeding and thrombosis during surgery, with subsequent hemolytic anemia. Serum samples were abnormally dark (Fig. 1). The hemolysis and icteric indices were not increased above the laboratory's threshold for electrolyte measurement; however, the lipemia index was. Upon visual inspection, the sample was not milky or turbid in appearance. QUESTIONS 1. What is the most likely cause of the dark color observed in the patient sample? 2. What factors can influence the measurement of lipemic index? 3. What laboratory tests might identify the cause of the dark color? How should samples with abnormal color be handled to verify that the color is not interfering with analytical methods? The answers are on the next page.

MLO: medical laboratory observer, 2013

Journal of Biological Chemistry, 2006

3 The abbreviations used are: heme, iron protoporphyrin IX irrespective of oxidation state, HO, h... more 3 The abbreviations used are: heme, iron protoporphyrin IX irrespective of oxidation state, HO, heme oxygenase; pa-HO, Pseudomonas aeruginosa heme oxygenase; rR, resonance Raman; 5-c, five-coordinate; 6-c, six-coordinate; HS, high spin; LS, low spin; MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.

Clinical Biochemistry, 2013

The performance of three point of care methods for pleural fluid pH analysis was compared to a cu... more The performance of three point of care methods for pleural fluid pH analysis was compared to a currently validated blood gas analyzer. An ABL 725 (Radiometer America, Westlake, OH) was used as the reference method to evaluate three cartridge-based assays: ABL 90 FLEX (Radiometer), and i-STAT 1 (Abbott Point of Care, Abbott Park, IL) CG4+ and G3+ cartridges for pleural fluid pH analysis. Pooled residual pleural fluid samples and quality control material were analyzed to determine intra- and inter-assay precision. Method comparison was performed with spiked (n=40) and clinically-ordered (n=10) pleural fluid samples across the analytical measuring range. All methods demonstrated inter-assay CVs<0.1% at pH values of 7.1 and 7.6, and intra-assay CVs<0.3% at pH values of 7.2 and 7.7. Bland-Altman plots demonstrated clinically significant bias between ABL 725 and each cartridge-based method only at pH>7.6. For samples with pH<7.6 mean bias vs. ABL 725 was -0.01 for ABL 90 FLEX and 0.03 for i-STAT 1 CG4+ and G3+ cartridges. Clinical concordance using a decision limit of pH7.2 was 96-98% for the three methods. Analytical and clinical performance of the three cartridge-based methods was comparable to a validated blood gas analyzer for pleural fluid pH analysis. Cartridge-based pH methods offer the advantage of easier troubleshooting for clots and clogs as they use disposable electrodes. However cartridge-based methods are not currently FDA-approved for pleural fluid samples, such that additional validation would be required for this specimen type.

Biochemistry, 2008

To my loving and patient wife Dawn, who without, none of this would have been possible. She is my... more To my loving and patient wife Dawn, who without, none of this would have been possible. She is my biggest supporter and fan, and has kept me on the path. And to my son, Clinton Roland, and daughter, Lillian Riley, who have given me strength and purpose to complete this project.

Critical Reviews in Clinical Laboratory Sciences, 2013

Requests for testing various analytes in serous fluids (e.g. pleural, peritoneal, pericardial eff... more Requests for testing various analytes in serous fluids (e.g. pleural, peritoneal, pericardial effusions) are submitted daily to clinical laboratories. Testing of these fluids deviates from assay manufacturers' specifications, as most laboratory assays are optimized for testing blood or urine specimens. These requests add a burden to clinical laboratories, which need to validate assay performance characteristics in these fluids to exclude matrix interferences (given the different composition of body fluids) while maintaining regulatory compliance. Body fluid testing for a number of analytes has been reported in the literature; however, understanding the clinical utility of these analytes is critical because laboratories must address the analytic and clinical validation requirements, while educating clinicians on proper test utilization. In this article, we review the published data to evaluate the clinical utility of testing for numerous analytes in body fluid specimens. We also highlight the pre-analytic and analytic variables that need to be considered when reviewing published studies in body fluid testing. Finally, we provide guidance on how published studies might (or might not) guide interpretation of test results in today's clinical laboratories.

Journal of Inorganic Biochemistry, 2016

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this repor... more The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pK a values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wildtype; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the wild-type protein (68 ± 3 mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process was one to three days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.

American Journal of Clinical Pathology

The guideline-recommended lipid panel for cardiovascular disease (CVD) risk assessment measures t... more The guideline-recommended lipid panel for cardiovascular disease (CVD) risk assessment measures total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and calculated low-density lipoprotein (LDL) cholesterol. Measured cholesterol in subfractions of HDL and LDL purportedly improve CVD risk prediction. Homogenous enzymatic methods are now available for measurement of the cholesterol within small dense LDL (sLDL), small dense HDL (HDL3), and triglyceride-rich lipoproteins (TRL). For meaningful interpretation of these measurements, an understanding of the potential sources and extent of result variability is needed. The smallest difference between serial measurements within a patient that likely reflects a change in clinical status is called the reference change value (RCV). Biological variability and reference change values (RCV) are well-characterized for basic lipids but there is limited information for sLDL, HDL3 or TRL. The objective of this study was to dete...

The Bone & Joint Journal

Aims The aim of this study was to determine the diagnostic accuracy of α defensin (AD) lateral fl... more Aims The aim of this study was to determine the diagnostic accuracy of α defensin (AD) lateral flow assay (LFA) and enzyme-linked immunosorbent assay (ELISA) tests for periprosthetic joint infection (PJI) in comparison to conventional synovial white blood cell (WBC) count and polymorphonuclear neutrophil percentage (PMN%) analysis. Methods Patients undergoing joint aspiration for evaluation of pain after total knee arthroplasty (TKA) or total hip arthroplasty (THA) were considered for inclusion. Synovial fluids from 99 patients (25 THA and 74 TKA) were analyzed by WBC count and PMN% analysis, AD LFA, and AD ELISA. WBC and PMN% cutoffs of ≥ 1,700 cells/mm3 and ≥ 65% for TKA and ≥ 3,000 cells/mm3 and ≥ 80% for THA were used, respectively. A panel of three physicians, all with expertise in orthopaedic infections and who were blinded to the results of AD tests, independently reviewed patient data to diagnose subjects as with or without PJI. Consensus PJI classification was used as the r...

Journal of Medical Internet Research

Background COVID-19 is caused by the SARS-CoV-2 virus and has strikingly heterogeneous clinical m... more Background COVID-19 is caused by the SARS-CoV-2 virus and has strikingly heterogeneous clinical manifestations, with most individuals contracting mild disease but a substantial minority experiencing fulminant cardiopulmonary symptoms or death. The clinical covariates and the laboratory tests performed on a patient provide robust statistics to guide clinical treatment. Deep learning approaches on a data set of this nature enable patient stratification and provide methods to guide clinical treatment. Objective Here, we report on the development and prospective validation of a state-of-the-art machine learning model to provide mortality prediction shortly after confirmation of SARS-CoV-2 infection in the Mayo Clinic patient population. Methods We retrospectively constructed one of the largest reported and most geographically diverse laboratory information system and electronic health record of COVID-19 data sets in the published literature, which included 11,807 patients residing in 41...

American Journal of Clinical Pathology

American Journal of Clinical Pathology

The Journal of Applied Laboratory Medicine: An AACC Publication

Clinical Biochemistry

Lactate, white blood cell (WBC) and neutrophil count, procalcitonin and immature granulocyte (IG)... more Lactate, white blood cell (WBC) and neutrophil count, procalcitonin and immature granulocyte (IG) count were compared for the prediction of sepsis, and severe sepsis or septic shock, in patients presenting to the emergency department (ED). We prospectively enrolled 501 ED patients with a sepsis panel ordered for suspicion of sepsis. WBC, neutrophil, and IG counts were measured on a Sysmex XT-2000i analyzer. Lactate was measured by i-STAT, and procalcitonin by Brahms Kryptor. We classified patients as having sepsis using a simplification of the 1992 consensus conference sepsis definitions. Patients with sepsis were further classified as having severe sepsis or septic shock using established criteria. Univariate receiver operating characteristic (ROC) analysis was performed to determine odds ratio (OR), area under the ROC curve (AUC), and sensitivity/specificity at optimal cut-off for prediction of sepsis (vs. no sepsis), and prediction of severe sepsis or septic shock (vs. no sepsis). There were 267 patients without sepsis; and 234 with sepsis, including 35 patients with severe sepsis or septic shock. Lactate had the highest OR (1.44, 95th% CI 1.20-1.73) for the prediction of sepsis; while WBC, neutrophil count and percent (neutrophil/WBC) had OR>1.00 (p<0.05). All biomarkers had AUC<0.70 and sensitivity and specificity <70% at the optimal cut-off. Initial lactate was the best biomarker for predicting severe sepsis or septic shock, with an odds ratio (95th% CI) of 2.70 (2.02-3.61) and AUC 0.89 (0.82-0.96). Traditional biomarkers (lactate, WBC, neutrophil count, procalcitonin, IG) have limited utility in the prediction of sepsis.

American Journal of Clinical Pathology, 2017

Clinical biochemistry, Jan 25, 2016

Measuring lipoprotein-associated phospholipase A2 (Lp-PLA2) activity can aid in identifying indiv... more Measuring lipoprotein-associated phospholipase A2 (Lp-PLA2) activity can aid in identifying individuals at higher risk of coronary heart disease. However, the biological variation of Lp-PLA2 activity and corresponding reference change value (RCV) is unknown which limits interpretation of results. In this study we aim to define the intra- and inter-individual variability of Lp-PLA2 activity in a healthy reference population. A total of 24 healthy individuals (22-47years of age) were prospectively collected at several time points: daily for five days (after overnight fast), daily for three days (while non-fasting), weekly for four weeks (after overnight fast), and monthly for 6months (after overnight fast). Intra-individual and inter-individual variability was determined. The index of individuality (IoI) and reference change value (RCV) were calculated for each time period. Variability in Lp-PLA2 activity was not different in fasting versus non-fasting states and also did not change i...

Clinical Chemistry, 2015

A 65-year-old woman with congestive heart failure presented for a mitral valve replacement. She e... more A 65-year-old woman with congestive heart failure presented for a mitral valve replacement. She experienced excessive bleeding and thrombosis during surgery, with subsequent hemolytic anemia. Serum samples were abnormally dark (Fig. 1). The hemolysis and icteric indices were not increased above the laboratory's threshold for electrolyte measurement; however, the lipemia index was. Upon visual inspection, the sample was not milky or turbid in appearance. QUESTIONS 1. What is the most likely cause of the dark color observed in the patient sample? 2. What factors can influence the measurement of lipemic index? 3. What laboratory tests might identify the cause of the dark color? How should samples with abnormal color be handled to verify that the color is not interfering with analytical methods? The answers are on the next page.

MLO: medical laboratory observer, 2013

Journal of Biological Chemistry, 2006

3 The abbreviations used are: heme, iron protoporphyrin IX irrespective of oxidation state, HO, h... more 3 The abbreviations used are: heme, iron protoporphyrin IX irrespective of oxidation state, HO, heme oxygenase; pa-HO, Pseudomonas aeruginosa heme oxygenase; rR, resonance Raman; 5-c, five-coordinate; 6-c, six-coordinate; HS, high spin; LS, low spin; MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.

Clinical Biochemistry, 2013

The performance of three point of care methods for pleural fluid pH analysis was compared to a cu... more The performance of three point of care methods for pleural fluid pH analysis was compared to a currently validated blood gas analyzer. An ABL 725 (Radiometer America, Westlake, OH) was used as the reference method to evaluate three cartridge-based assays: ABL 90 FLEX (Radiometer), and i-STAT 1 (Abbott Point of Care, Abbott Park, IL) CG4+ and G3+ cartridges for pleural fluid pH analysis. Pooled residual pleural fluid samples and quality control material were analyzed to determine intra- and inter-assay precision. Method comparison was performed with spiked (n=40) and clinically-ordered (n=10) pleural fluid samples across the analytical measuring range. All methods demonstrated inter-assay CVs<0.1% at pH values of 7.1 and 7.6, and intra-assay CVs<0.3% at pH values of 7.2 and 7.7. Bland-Altman plots demonstrated clinically significant bias between ABL 725 and each cartridge-based method only at pH>7.6. For samples with pH<7.6 mean bias vs. ABL 725 was -0.01 for ABL 90 FLEX and 0.03 for i-STAT 1 CG4+ and G3+ cartridges. Clinical concordance using a decision limit of pH7.2 was 96-98% for the three methods. Analytical and clinical performance of the three cartridge-based methods was comparable to a validated blood gas analyzer for pleural fluid pH analysis. Cartridge-based pH methods offer the advantage of easier troubleshooting for clots and clogs as they use disposable electrodes. However cartridge-based methods are not currently FDA-approved for pleural fluid samples, such that additional validation would be required for this specimen type.

Biochemistry, 2008

To my loving and patient wife Dawn, who without, none of this would have been possible. She is my... more To my loving and patient wife Dawn, who without, none of this would have been possible. She is my biggest supporter and fan, and has kept me on the path. And to my son, Clinton Roland, and daughter, Lillian Riley, who have given me strength and purpose to complete this project.

Critical Reviews in Clinical Laboratory Sciences, 2013

Requests for testing various analytes in serous fluids (e.g. pleural, peritoneal, pericardial eff... more Requests for testing various analytes in serous fluids (e.g. pleural, peritoneal, pericardial effusions) are submitted daily to clinical laboratories. Testing of these fluids deviates from assay manufacturers' specifications, as most laboratory assays are optimized for testing blood or urine specimens. These requests add a burden to clinical laboratories, which need to validate assay performance characteristics in these fluids to exclude matrix interferences (given the different composition of body fluids) while maintaining regulatory compliance. Body fluid testing for a number of analytes has been reported in the literature; however, understanding the clinical utility of these analytes is critical because laboratories must address the analytic and clinical validation requirements, while educating clinicians on proper test utilization. In this article, we review the published data to evaluate the clinical utility of testing for numerous analytes in body fluid specimens. We also highlight the pre-analytic and analytic variables that need to be considered when reviewing published studies in body fluid testing. Finally, we provide guidance on how published studies might (or might not) guide interpretation of test results in today's clinical laboratories.