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This Month In J Lab Clin Med
Copyright © 2005 by Mosby, Inc.
VOLUME 145
JUNE 2005
NUMBER 6
THIS MONTH IN J Lab Clin Med Issue Highlights for June 2005
CRP— consistency in measurement C-reactive protein is a nonspecific marker of inflammation. Because its level in serum has been found to be a predictor of vascular risk, it is being measured and followed in many research studies and in some clinical settings. The question naturally arises whether clinical assays differ in important ways, and whether the results obtained in one study with one assay are readily applied to the use of a different assay in a different setting. That question is addressed in a manuscript appearing on page 305 of the current issue. Researchers at the Universities of Pisa, Torino and Montpellier compared three commercially-available assays. As test samples, they used normal serum that had been spiked with known quantities of CRP, and they used samples from hemodialysis patients (who commonly have elevated CRP levels and commonly have premature atherosclerosis). A fairly straightforward design was employed. Intra-assay precision was assessed by running the same sample in the same assay, ten times on a single day. Inter-assay (single-sample) precision was assessed by assaying separate aliquots of the same sample on each of several days. Comparability among the assays was examined by preparing and freezing three aliquots from each sample, then performing the assay on freshly thawed samples with each of the three assays under ordinary laboratory conditions. Each of the assays performed within what would ordinarily be considered acceptable limits, but there were differences that could be important in the interpretation of studies. The most precise of the three commercial assays had an intra-assay coefficient of variance of less than 2 (%), and an inter-assay coefficient of variance of less than 4. In contrast, the least precise assay gave coefficients of variance as high as 15% for some samples. The assays agreed reasonably well among themselves and with the known values in the spiked samples, but one of the three had less resolution at the low end of the range. The authors conclude that the performance characteristics of different commercial assays are enough different from each other that they need to be considered carefully in study design. If small differences are to be resolved, an assay with a tight coefficient of variance should be chosen. If low values are to be resolved from “normal” values, an assay should be chosen for its low-end performance rather than assume adequacy from its clinically assessed high-end performance.
J Lab Clin Med 2005;145:285– 8. 0022-2143/$ – see front matter © 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.lab.2005.05.002
285
286
In this Issue
J Lab Clin Med June 2005
Oncogenic mutations and translocations: Can we detect them quickly enough to be more than an afterthought? One of the most exciting developments in the field of hematologic malignancy in the last three decades has been the recognition that cytogenetic abnormalities such as translocations may produce specific identifiable protein abnormalities that are involved in malignant transformation and that may be therapeutic targets. The best-known example is probably that of chronic myeloid leukemia, in which disorder a translocation between chromosomes 9 and 22 leads to the production of a tyrosine kinase that is a key player in the abnormal behavior of the cells; a specific inhibitor of that tyrosine kinase allows the disease to be controlled at low toxicity in a high proportion of patients. Another example is acute promyelocytic leukemia, in which the result of translocation is a dysfunctional retinoid receptor; in that disorder, treatments including large doses of retinoids yield better outcomes than do treatments employing only conventional chemotherapy. Cytogenetic abnormalities are common in many other hematologic malignancies, but without the consistency and specificity we see in CML and APL. If we are to understand where they fit in, and if we are to exploit them therapeutically, we need to have methods for detecting them quickly enough to base treatment decisions on their presence or absence. Flt3 is a class III tyrosine kinase receptor that confers proliferative and antiapoptotic effects on normal and leukemic hematopoietic stem cells. Abnormalities in and adjacent to the Flt3 gene have been reported in a significant fraction of patients with acute myeloid leukemia. Dr Sebastian Scholl and his colleagues from the Friedrich Schiller University, Jena (Germany) describe this month a new approach for rapid screening of Flt3-TKD (tyrosine kinase domain) and Flt3-V592A (activating) mutations using a fluorescence resonance energy transfer assay in 122 patients (see page 295). In most research studies, the genomic area of interest has been amplified by polymerase chain reaction, and restriction enzyme digestion and electrophoresis of the digestate have then been used for final identification of the specific sequence of interest by its size or nucleotide ratios. By use of a fluorescence-based probe for detection of the sequence, the time-consuming later steps can be bypassed if one knows exactly what is being sought. The current authors found that screening for Flt3-TKD mutations with fluorescent probes was equivalent in sensitivity and specificity to conventional screening using standard PCR followed by EcoRV restriction enzyme digestion. This technique was enough quicker to be of potential clinical use in detecting minimal residual disease or in selecting specific therapy. Thus, if a mutation-specific therapy were to be proposed, this type of assay would make it far easier to design and execute a study testing its efficacy.
Health maintenance: Might vaccinations cause problems with screening tests? Vaccination is such an ordinary part of life that we seldom think of its adverse impact in the absence of an unusual circumstance (such as deciding whether to use decades-old crude smallpox vaccine in the event of a bioterrorist attack). Obviously, the intent of vaccination is to stimulate the immune system specifically; headache and low-grade fever remind us that the specificity of the immune response is not absolute. Despite widespread use of the influenza vaccine, relatively little is known about its effect on the inflammatory markers we might measure as part of our coronary risk assessment for a patient. Dr. Michael Tsai and associates from the Minneapolis VA Medical Center addressed this question, and present their findings beginning on page 323. 22 healthy individuals had several markers drawn before receiving an influenza vaccination and 1, 3 and 7 days after the vaccination. There was a significant increase in IL-6 level on the first day
J Lab Clin Med Volume 145, Number 6
In this Issue
after receiving the vaccine. CRP was also elevated compared to baseline on the first and third post-vaccination days. Serum amyloid protein A was elevated only on Day 1. Four other markers were unaffected: interleukin-2, IL-2 soluble receptor, monocyte chemotactic protein 1, tumor necrosis factor ␣. Lipid profiles were also examined; the triglyceride levels were lower than baseline values on Days 1, 3 and 7 after vaccination. The authors conclude that there are transient effects of influenza vaccination upon systemic markers of inflammations and upon lipids—screening these values for risk assessment is best done at a time remote from vaccination.
Green tea—Good for the heart; how about the liver? Green tea is now all the rage, because a number of studies have suggested that it might be good for cardiovascular health. Although some of the early studies did not compare it against black tea (and thus added a bit of irony to the media storm), there are substances of interest that are present in green tea alone and that are now under careful scrutiny. Epigallocatechin-3-gallate (EGCG), one of the polyphenoids in green tea, has been of particular interest because it may have anti-fibrogenic activity. This could of course play a protective role in vascular disease, but it’s attractive to speculate that it could be useful in other disorders characterized by fibrosis, as well. Nobuhiko Higashi and several co-investigators from Kyushu University explored the activity of this compound in the activation of liver stellate cells on page 316. Activated hepatic stellate cells (HSCs) are believed to be central to hepatic fibrosis, and a number of studies have implicated Rho, (a small GTPase) signaling pathways in the activation and proliferation of HSCs. In the current report, the authors investigated the effect of EGCG on Rho signaling pathways in a cell line (TWNT-4) derived from activated human HSC-derived. EGCG inhibited stress fiber formation and changed the distribution of alpha-smooth muscle actin. These inhibitory effects of EGCG were not as prominent in cells transfected with a plasmid to produce overexpression of constituitively active Rho. Activated Rho (GTP-bound state) was strongly inhibited by ECGC. In addition to cell activation, the authors examined cell growth. They found (by BrdU incorporation) that ECGC (100 M) suppressed cell growth by 80%, and caused apoptosis in half of the total cells. EGCG also induced phosphorylation of mitogen-activated protein kinases (Erk1/2, JNK, and p38). The authors conclude that EGCG can influence the morphology and growth of stellate cells via Rho signaling pathways, and they suggest that EGCG and related compounds deserve further scrutiny as agents that might have therapeutic potential in diseases characterized by fibrosis.
Informed consent—25 years after Belmont and almost 40 years after Beecher, and it’s still not easy! In the quarter century since the Belmont Report, respect for persons has had a central position in our thinking about clinical ethics and research ethics. One of the most important manifestations of respect for persons is respect for their autonomy, something we honor by brining them into decisions about health care and research participation. An ideal we pursue is that stated in 1966 by Henry Beecher: that we strive for the truly informed consent of our patients and research subjects whenever we expose them to risk (even though we may not always be able to achieve it). This lofty goal sometimes comes up against some practical limitations. Some people really don’t want a very big voice in decision-making; interestingly, some studies have suggested that most people want to be fully informed, but want the tough decisions made by their caregivers. Sometimes, the dynamics of a care situation effectively preclude informed consent (crushing chest pain or a new
287
288
In this Issue
J Lab Clin Med June 2005
diagnosis of acute leukemia, for example, may distract one from a consent form or discussion). Sometimes, the superficial attractiveness of a novel therapy may lead someone to consider its risks and uncertainties less critically. A very long list could easily be made. In this month’s issue, two essays bearing on the problems of consent are presented. One, by Prof. Carl Schneider of the University of Michigan, is adapted from a presentation at the annual meeting of PRIM&R/ARENA (Public Responsibility in Medicine and Research and the Applied Ethics National Association). The second, by Dr. David Satin, responds to and expands upon Prof. Schneider’s comments. These essays may be found on pages 289 and 292.
VOLUME 145
JUNE 2005
NUMBER 6
THIS MONTH IN J Lab Clin Med Issue Highlights for June 2005
CRP— consistency in measurement C-reactive protein is a nonspecific marker of inflammation. Because its level in serum has been found to be a predictor of vascular risk, it is being measured and followed in many research studies and in some clinical settings. The question naturally arises whether clinical assays differ in important ways, and whether the results obtained in one study with one assay are readily applied to the use of a different assay in a different setting. That question is addressed in a manuscript appearing on page 305 of the current issue. Researchers at the Universities of Pisa, Torino and Montpellier compared three commercially-available assays. As test samples, they used normal serum that had been spiked with known quantities of CRP, and they used samples from hemodialysis patients (who commonly have elevated CRP levels and commonly have premature atherosclerosis). A fairly straightforward design was employed. Intra-assay precision was assessed by running the same sample in the same assay, ten times on a single day. Inter-assay (single-sample) precision was assessed by assaying separate aliquots of the same sample on each of several days. Comparability among the assays was examined by preparing and freezing three aliquots from each sample, then performing the assay on freshly thawed samples with each of the three assays under ordinary laboratory conditions. Each of the assays performed within what would ordinarily be considered acceptable limits, but there were differences that could be important in the interpretation of studies. The most precise of the three commercial assays had an intra-assay coefficient of variance of less than 2 (%), and an inter-assay coefficient of variance of less than 4. In contrast, the least precise assay gave coefficients of variance as high as 15% for some samples. The assays agreed reasonably well among themselves and with the known values in the spiked samples, but one of the three had less resolution at the low end of the range. The authors conclude that the performance characteristics of different commercial assays are enough different from each other that they need to be considered carefully in study design. If small differences are to be resolved, an assay with a tight coefficient of variance should be chosen. If low values are to be resolved from “normal” values, an assay should be chosen for its low-end performance rather than assume adequacy from its clinically assessed high-end performance.
J Lab Clin Med 2005;145:285– 8. 0022-2143/$ – see front matter © 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.lab.2005.05.002
285
286
In this Issue
J Lab Clin Med June 2005
Oncogenic mutations and translocations: Can we detect them quickly enough to be more than an afterthought? One of the most exciting developments in the field of hematologic malignancy in the last three decades has been the recognition that cytogenetic abnormalities such as translocations may produce specific identifiable protein abnormalities that are involved in malignant transformation and that may be therapeutic targets. The best-known example is probably that of chronic myeloid leukemia, in which disorder a translocation between chromosomes 9 and 22 leads to the production of a tyrosine kinase that is a key player in the abnormal behavior of the cells; a specific inhibitor of that tyrosine kinase allows the disease to be controlled at low toxicity in a high proportion of patients. Another example is acute promyelocytic leukemia, in which the result of translocation is a dysfunctional retinoid receptor; in that disorder, treatments including large doses of retinoids yield better outcomes than do treatments employing only conventional chemotherapy. Cytogenetic abnormalities are common in many other hematologic malignancies, but without the consistency and specificity we see in CML and APL. If we are to understand where they fit in, and if we are to exploit them therapeutically, we need to have methods for detecting them quickly enough to base treatment decisions on their presence or absence. Flt3 is a class III tyrosine kinase receptor that confers proliferative and antiapoptotic effects on normal and leukemic hematopoietic stem cells. Abnormalities in and adjacent to the Flt3 gene have been reported in a significant fraction of patients with acute myeloid leukemia. Dr Sebastian Scholl and his colleagues from the Friedrich Schiller University, Jena (Germany) describe this month a new approach for rapid screening of Flt3-TKD (tyrosine kinase domain) and Flt3-V592A (activating) mutations using a fluorescence resonance energy transfer assay in 122 patients (see page 295). In most research studies, the genomic area of interest has been amplified by polymerase chain reaction, and restriction enzyme digestion and electrophoresis of the digestate have then been used for final identification of the specific sequence of interest by its size or nucleotide ratios. By use of a fluorescence-based probe for detection of the sequence, the time-consuming later steps can be bypassed if one knows exactly what is being sought. The current authors found that screening for Flt3-TKD mutations with fluorescent probes was equivalent in sensitivity and specificity to conventional screening using standard PCR followed by EcoRV restriction enzyme digestion. This technique was enough quicker to be of potential clinical use in detecting minimal residual disease or in selecting specific therapy. Thus, if a mutation-specific therapy were to be proposed, this type of assay would make it far easier to design and execute a study testing its efficacy.
Health maintenance: Might vaccinations cause problems with screening tests? Vaccination is such an ordinary part of life that we seldom think of its adverse impact in the absence of an unusual circumstance (such as deciding whether to use decades-old crude smallpox vaccine in the event of a bioterrorist attack). Obviously, the intent of vaccination is to stimulate the immune system specifically; headache and low-grade fever remind us that the specificity of the immune response is not absolute. Despite widespread use of the influenza vaccine, relatively little is known about its effect on the inflammatory markers we might measure as part of our coronary risk assessment for a patient. Dr. Michael Tsai and associates from the Minneapolis VA Medical Center addressed this question, and present their findings beginning on page 323. 22 healthy individuals had several markers drawn before receiving an influenza vaccination and 1, 3 and 7 days after the vaccination. There was a significant increase in IL-6 level on the first day
J Lab Clin Med Volume 145, Number 6
In this Issue
after receiving the vaccine. CRP was also elevated compared to baseline on the first and third post-vaccination days. Serum amyloid protein A was elevated only on Day 1. Four other markers were unaffected: interleukin-2, IL-2 soluble receptor, monocyte chemotactic protein 1, tumor necrosis factor ␣. Lipid profiles were also examined; the triglyceride levels were lower than baseline values on Days 1, 3 and 7 after vaccination. The authors conclude that there are transient effects of influenza vaccination upon systemic markers of inflammations and upon lipids—screening these values for risk assessment is best done at a time remote from vaccination.
Green tea—Good for the heart; how about the liver? Green tea is now all the rage, because a number of studies have suggested that it might be good for cardiovascular health. Although some of the early studies did not compare it against black tea (and thus added a bit of irony to the media storm), there are substances of interest that are present in green tea alone and that are now under careful scrutiny. Epigallocatechin-3-gallate (EGCG), one of the polyphenoids in green tea, has been of particular interest because it may have anti-fibrogenic activity. This could of course play a protective role in vascular disease, but it’s attractive to speculate that it could be useful in other disorders characterized by fibrosis, as well. Nobuhiko Higashi and several co-investigators from Kyushu University explored the activity of this compound in the activation of liver stellate cells on page 316. Activated hepatic stellate cells (HSCs) are believed to be central to hepatic fibrosis, and a number of studies have implicated Rho, (a small GTPase) signaling pathways in the activation and proliferation of HSCs. In the current report, the authors investigated the effect of EGCG on Rho signaling pathways in a cell line (TWNT-4) derived from activated human HSC-derived. EGCG inhibited stress fiber formation and changed the distribution of alpha-smooth muscle actin. These inhibitory effects of EGCG were not as prominent in cells transfected with a plasmid to produce overexpression of constituitively active Rho. Activated Rho (GTP-bound state) was strongly inhibited by ECGC. In addition to cell activation, the authors examined cell growth. They found (by BrdU incorporation) that ECGC (100 M) suppressed cell growth by 80%, and caused apoptosis in half of the total cells. EGCG also induced phosphorylation of mitogen-activated protein kinases (Erk1/2, JNK, and p38). The authors conclude that EGCG can influence the morphology and growth of stellate cells via Rho signaling pathways, and they suggest that EGCG and related compounds deserve further scrutiny as agents that might have therapeutic potential in diseases characterized by fibrosis.
Informed consent—25 years after Belmont and almost 40 years after Beecher, and it’s still not easy! In the quarter century since the Belmont Report, respect for persons has had a central position in our thinking about clinical ethics and research ethics. One of the most important manifestations of respect for persons is respect for their autonomy, something we honor by brining them into decisions about health care and research participation. An ideal we pursue is that stated in 1966 by Henry Beecher: that we strive for the truly informed consent of our patients and research subjects whenever we expose them to risk (even though we may not always be able to achieve it). This lofty goal sometimes comes up against some practical limitations. Some people really don’t want a very big voice in decision-making; interestingly, some studies have suggested that most people want to be fully informed, but want the tough decisions made by their caregivers. Sometimes, the dynamics of a care situation effectively preclude informed consent (crushing chest pain or a new
287
288
In this Issue
J Lab Clin Med June 2005
diagnosis of acute leukemia, for example, may distract one from a consent form or discussion). Sometimes, the superficial attractiveness of a novel therapy may lead someone to consider its risks and uncertainties less critically. A very long list could easily be made. In this month’s issue, two essays bearing on the problems of consent are presented. One, by Prof. Carl Schneider of the University of Michigan, is adapted from a presentation at the annual meeting of PRIM&R/ARENA (Public Responsibility in Medicine and Research and the Applied Ethics National Association). The second, by Dr. David Satin, responds to and expands upon Prof. Schneider’s comments. These essays may be found on pages 289 and 292.