clinical laboratory science - ASCLs [PDF]

$-*/*$"-
-"#03"503:
4$*&/$&


)DOO

6OLUME.UMBER

)RFXV&DUGLRYDVFXODU5LVN$VVHVVPHQW 

*/52.!,/&4(%!-%2)#!.3/#)%49&/2#,).)#!,,!"/2!4/293#)%.#%

ASCLS Mission/Vision Statement

The American Society for Clinical Laboratory Science serves as the voice of all clinical laboratory professionals, creating a vision for the advancement of the clinical laboratory practice field, and advocating the value and role of the profession ensuring safe, effective, efficient, equitable, and patient centered healthcare.

ASCLS MEMBER EDITORS Editor-in-Chief Bernadette Rodak MS CLSpH(NCA) Clinical Laboratory Science Program Indiana University Clarian Pathology Laboratory 350 West 11th Street, 6002F Indianapolis IN 46202 317-491-6218, fax 317-491-6163 [email protected] Continuing Education Editor George A Fritsma MS MT(ASCP) The Fritsma Factor http://www.fritsmafactor.com 153 Redwood Drive Trussville AL 35173 205-821-5641, fax 205-975-7302 [email protected] Clinical Practice Editor Libby Spence PhD CLS(NCA) University of Mississippi Medical Center School of Graduate Studies in the Health Sciences 2500 North State Street Jackson, MS 39216 601-984-1204, fax 601-815-9440 [email protected] Research and Reports David L McGlasson MS CLS(NCA) 59th Clinical Research Division/SGRL 2200 Berquist Dr., Bldg. 4430 Lackland AFB TX 78236-9908 210-292-6555, fax 210-292-6053 [email protected] Contributing Editors Eileen Carreiro-Lewandowski/N Dartmouth MA Deborah Josko/Newark NJ Elaine Keohane/Newark NJ Rebecca Laudicina/Chapel Hill NC Connie Mahon/Rockville MD Linda Smith/San Antonio TX Michelle Wright-Kanuth/Galveston TX

Clinical Laboratory Science (ISSN 0894-959X) is published quarterly by the American Society for Clinical Laboratory Science, 6701 Democracy Blvd., Suite 300, Bethesda MD 20817; (301) 657-2768; (301) 657-2909 (fax). Annual Subscription Rates: USA Canada Non-USA Individuals $65 $75 $130 Institutions $80 $80 $130 Questions related to subscriptions should be addressed to: [email protected]. The cost of single copies is $15. Requests to replace missing issues free of charge are honored up to six months after the date of issue. Send requests to ASCLS headquarters. Annual membership dues of ASCLS are $95, $40 of which is allocated to a subscription of CLS. Periodical postage paid at Bethesda, MD and other additional mailing offices.

AMERICAN SCOCIETY FOR CLINICAL LABORATORY SCIENCE 6701 Democracy Blvd, Suite 300 Bethesda, Maryland 20817 (301) 657-2768, (301) 657-2909 (fax) www.ascls.org

REVIEW BOARD Richard Bamberg/Greenville NC Suzanne Campbell/ Liberal KS Eileen Carreiro-Lewandowski/N Dartmouth MA Dianne Cearlock/DeKalb IL Peter Colaninno/Jamaica NY Maria Delost/Youngstown OH Jo Ann Fenn/Salt Lake City UT Maribeth Flaws/Chicago IL Ellis Frohman/St Louis MO Mildred Fuller/Norfolk VA Abraham Furman/Portland OR Richard Gregory/Indianapolis IN Jesse Guiles/Newark NJ Cindy Handley/Liberal KS Lester Hardegree/Bluffton SC Denise Harmening/Baltimore MD Rita Heuetz/St Louis MO Daniel Hoefner/Elon, NC Linda Hogan/Wichita KS Virginia Hughes/Montgomery AL Deborah Josko/Newark NJ Kelly Joyner/Durham NC Linda Kasper/Indianapolis IN Elaine Keohane/Newark NJ Nancy Konopka/Gettysburg PA Robin Krefetz/Cherry Hill NJ Linda Laatsch/Milwaukee WI Hal Larsen/Lubbock TX Donna Larson/Gresham OR Rebecca Laudicina/Chapel Hill NC Louann Lawrence/New Orleans LA Susan LeclairMarcia Lee/Oxford OH Craig Lehmann/Stony Brook NY Elizabeth Kenimer Leibach/Augusta GA Lynn Little/Dallas TX Karen McClure/Houston TX Carol McCoy/Minneapolis MN Sharon Miller/St Charles IL Isaac Montoya/Houston TX Lillian Mundt/North Chicago IL Harriette Nadler/King of Prussia PA Joan Prince/Milwaukee WI Margaret Reinhart/Philadelphia PA Perry Scanlan/Clarksville TN

Advertising for CLS is accepted in accordance with the advertising policy of the ASCLS. Contact the CLS advertising representative at (301) 657-2768. Manuscript Submissions: To encourage consistency in style, refer to guidelines in Scientific Style and Format The Council of Science Editors Manual for Authors, Editors, and Publishers, 2006. Detailed instructions for authors are available on the ASCLS web-site. Contact the CLS Editorial Office for more information. All articles published represent the opinions of the authors and do not reflect the official policy of ASCLS or the authors’ institutions unless specified. Microfilm and microfiche editions of CLS are available from Proquest, 300 N Zeeb Road, Ann Arbor MI 48106.

ASCLS Core Values

Core Values include enhancing quality standards and patient safety; providing professional development opportunities; promoting expanded roles and contributions of clinical laboratory professionals to the healthcare team; increasing the diversity in the profession; and expanding the voice and role of under-represented individuals and groups.

Masih Shokrani/Dekalb IL Stephen Sodeke/Tuskegee AL James Vossler/Syracuse NY Kathy Waller/Columbus OH Lori Woeste/Normal IL Michele Wright-Kanuth/Galveston TX P.A.C.E.® Liaison Diane Kumashiro/Kaneohe HI ASCLS BOARD OF DIRECTORS 2009-2010 Mary Ann McLane, President Marcie Armstrong, President-elect Scott Aikey, Past President Gilma Roncancio-Weemer, Secretary/Treasurer Carol Golyski, Region I Barbara Snyderman, Region II Hassan Aziz, Region III Linda S. Gorman, Region IV Debra Rodahl, Region V Tim Randolph, Region VI Linda Smith, Region VII J.R. Constance, Region VIII Mary Lashinski, Region IX Sheri Gon, Region X Michele Yost, First Year Professional Chair Lisa Martini, Student Forum Chair ASCLS Headquarters Executive Staff Elissa Passiment, Executive Vice President EDITORIAL OFFICE AND PRODUCTION Westminster Publishers 315 Westminster Court Brandon, MS 39047 (601) 214-5028; (202) 315-5843 (fax) [email protected] www.ascls.org/leadership/cls/index.htm Executive Editor David Fowler PhD CLS(NCA) Managing Editor Myra Fowler CLS(NCA) Inclusion in the journal of product names or author opinions does not constitute endorse-ment by either Clinical Laboratory Science or ASCLS. Correspondence related to editorial content should be mailed to: Westminster Publishers, 315 Westminster Court, Brandon MS 39047; (601) 214-5028; (202) 3155843 (fax). Email: [email protected] ©Copyright 2009 American Society for Clinical Laboratory Science Inc. All rights reserved.

ADDRESS CHANGES Postmaster: Send address changes to Clinical Laboratory Science 6701 Democracy Blvd, Suite 300 Bethesda MD 20817

CLINICAL LABORATORY SCIENCE

FALL 2009

194

196 203

208

216

223 226 233

VOLUME 22/NUMBER 4

DIALOGUE AND DISCUSSION The 111th Congress and Health Care Reform Don Lavanty CLINICAL PRACTICE The Roles of the Clinical Laboratory Scientist: Educator, Consultant, Advocate Anne Ranne Clinical Laboratory Tycoon Case Study: Are You Management Material? Amy L Thompson RESEARCH AND REPORTS Immunophenotypic Analysis of Bone Marrow B Lymphocyte Precursors (Hematogones) by Flow Cytometry Jmili N Braham, S Nsaibia, MC Jacob, H Omri, MA Laatiri, S Yacoub, Y Braham, M Aouni, M Kortas Duration of Loxosceles reclusa Venom Detection by ELISA from Swabs David L McGlasson, Jonathon A Green, William V Stoeker, James L Babcock, David A Calcara FOCUS: CARDIOVASCULAR RISK ASSESSMENT Introduction Kevin F Foley Genetic Markers for Coronary Artery Disease Linnea M Baudhuin Heart Failure and B-Type Natriuretic Protein Jonathan Hoyne

240

Hypovitaminosis D: A New Risk Marker for Cardiovascular Disease Joseph P McConnell, Kevin F Foley, Gina M Vargas

247

CONTINUING EDUCATION QUESTIONS

251

2009 ANNUAL INDEX

WASHINGTON BEAT

The 111th Congress and Health Care Reform Don Lavanty President Obama has made health care reform his domestic legislative priority. He has asked the Congress to pass legislation that would extend health insurance coverage to the uninsured and underinsured, and enhance coverage for Medicaid and children’s health care. In his request to Congress, he also asked that the costs necessary to pay for the universal coverage be borne by savings in current health care programs expenditures, and where and if appropriate, by raising revenues. The process began in earnest in the late spring with all five committees of jurisdiction holding hearings. Those Committees were in the Senate: The Committee on Health, Education, Labor and Pensions (HELP) which was chaired by the late Senator Kennedy, and The Senate Finance Committee, chaired by Senator Bacaus. In the U. S. House of Representatives the three committees were: the House Ways and Means Committee, chaired by Mr. Rangel of New York; the Subcommittee in Health, chaired by Mr. Stark of California; the Energy and Commerce Committee, chaired by Mr. Waxman of California and the Committee on Education and Labor, chaired by Mr. Miller, also of California. By the middle of the summer of 2009 and before the August recess, four of the five committees

Washington Beat is intended to provide a timely synopsis of activity in the nation’s capitol of importance to clinical laboratory practitioners. This section is coordinated by Rick Panning, chair of the ASCLS Government Affairs Committee; and Don Lavanty, ASCLS Legislative Counsel. Direct all inquiries to ASCLS, (301) 657-2768 ext. 3022, (301) 657-2909 (fax); or mail to ASCLS, 6701 Democracy Boulevard, Suite 300, Bethesda MD 20817, attn: Washington Beat.

194 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

had acted on legislation to be reported to the full Senate and full House of Representatives, respectfully. In the Senate, the HELP Committee reported a health care reform bill to the full Senate that would require everyone to have health insurance, have a public program established for those who could not afford health insurance and whose employers did not provide coverage. The bill also recommended several changes to the health care work force, with an emphasis on primary care and prevention. The reported bill, however, contained no provisions for savings or revenues to pay for the cost of extending coverage as their committee does not have jurisdiction over the Medicare Program or the tax code (should Tax Code be capitalized? I don’t think so). Jurisdiction for Medicare and taxes rest with the Senate Finance Committee. The Finance Committee has yet to mark up legislation, although at press time they indicated that they would do so by midSeptember. In the House of Representatives, all three Committees have marked up legislation and have reported bills for the full House to consider. The legislation reported by the three committees is very similar and will be merged into one bill by the House leadership for consideration by mid-to-late September. All three House bills require everyone to have health insurance, provide for a public program for those that don’t have coverage and utilize the public program to control costs via competition with private health insurance. On the savings side, both the House Ways and Means Committee and the Energy and Commerce Committee have jurisdiction over Medicare and have proposed reductions in provider payment under Medicare.

WASHINGTON BEAT

Among the proposed reductions are payment controls on imaging procedures, growth limitations on physician payments, except for primary care, and several other procedural changes that would result in Medicare savings, including reductions in Medicare Advantage payments. The clinical laboratory manpower issues are currently addressed, as most committees are proposing increased support for allied health training and education. Although the specifics of support have yet to be finalized, many of the manpower support proposals will be in conjunction with primary and preventive care, which requires laboratory services and personnel. On the reimbursement side, clinical laboratories were scheduled to receive an update tied to the CPI for 2010. Since the economic recession will result in a negative CPI, any increase is unlikely. The three House bills also contain a mechanism that would use a formula to adjust all Medicare payment updates. If this formula or the negative CPI adjustment is applied, laboratories would see further cuts. The Clinical Lab Coalition has been working with the

House committees to cap this formula to the level of the CPI reduction and is hopeful that will prevail. The remaining concern for Laboratories on the reimbursement side comes from draft proposals by the Senate Finance Committee which have suggested that the laboratory co-payment be reinstated to raise revenues to help defer the cost of health care reform. In addition to the likely lower reimbursement due to the CPI update mechanism, the Senate Finance Committee has also proposed that laboratories pay a fee to do business with Medicare and Medicaid. Specifics regarding the amount of the fee and to which laboratories it would apply to were not known at press time. With the Presidential address to the joint session of Congress on health care reform in early September, the action by the three House committees and draft proposals being developed by a non-partisan group of Senators in the Senate Finance Committee, serious legislative action was expected to take final shape by late September or early October. At this stage, it is too early to predict or have a clear view of the final form the legislation will take as it affects clinical laboratories.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 195

CLINICAL PRACTICE

The Roles of the Clinical Laboratory Scientist: Educator, Consultant, Advocate ANNE RANNE ABSTRACT: Advances in clinical laboratory medicine have created an opportunity for clinical laboratory scientists to assume a new role--the role of educator in the integrated healthcare system. A gap created between critical laboratory test results and medical decisions requires the translation of laboratory results into meaningful clinical guidelines. This article suggests three ways the clinical laboratory scientist can fill this gap. INDEX TERMS: computerized provider order entry system, implementation strategies, interpretive reports, system thinker, education Anne Ranne, MS, CLS, is assistant professor, Medical College of Georgia, Lawrenceville Campus, Department of Biomedical and Radiological Technologies, Lawrenceville, GA 30043 Address for Correspondence: Anne Ranne, MS, CLS, Assistant Professor, Medical College of Georgia, Lawrenceville Campus, Department of Biomedical and Radiological Technologies, 1000 University Center Lane, AII72, Lawrenceville, GA 30043, 678-4075536(phone/fax), Email: [email protected] The peer-reviewed Clinical Practice Section seeks to publish case studies, reports, and articles that are immediately useful, are of a practical nature, or contain information that could lead to improvement in the quality of the clinical laboratory’s contribution to patient care, including brief reviews of books, computer programs, audiovisual materials, or other materials of interest to readers. Direct all inquiries to Libby Spence, PhD, CLS(NCA), Clin Lab Sci Clinical Practice Editor, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, [email protected].

196 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

Clin Lab Sci 2009;22(4):196 CASE HISTORY A 38-year-old married, monogamous female came to the emergency department with aseptic meningitis. She was admitted to the hospital and was seen by a hospitalist who suspected that the patient might have acute HIV and ordered a quantitative HIV by PCR. The test result was positive with a viral load of 32,000 copies/ml, but a note on the report indicated that the batch was “defective” and the test needed to be rerun. The hospitalist never received a report or followed up on the results. The hospital released the patient. Neither the patient nor personal physician knew that an additional HIV test was pending. Six months later the hospitalist found the original test report while cleaning out a desk. The aseptic meningitis was the first manifestation of acute HIV seroconversion. The consequences of this delay caused major emotional distress and the possibility of spreading HIV to her partner due to unprotected sex.1 This case illustrates a disconnect between healthcare professionals and patients. It is also a good example of how the clinical laboratory scientist can be engaged in improving patient care. Even though laboratory tests are now able to predict the progression of diseases and to establish more personalized treatment plans, there is a gap between the clinical laboratory test order and the practitioner’s clinical decision. Clinical laboratory tests are included as part of evidence-based clinical practice guidelines across the 23 main condition/disease categories, but the translation of these test results is often ineffective in supporting patient care.2 Clinical laboratory scientists have the skills to provide accurate and reliable test

CLINICAL PRACTICE

results, and these skills can create a new role that has been missing in the patient continuum. With the current crisis in healthcare, the clinical laboratory scientist has an opportunity to step forward and show how the profession can contribute to the new integrated healthcare system. The time has come for the clinical laboratory scientist to move outside of the laboratory walls and begin translating these critical tests into meaningful information. How many times has the laboratory professional dealt with a similar problem as discussed in the previous case? Clinical laboratory scientists must make sure the correct test is ordered, the correct results reach the correct practitioner(s), and the results have meaning for the practitioner. Day after day, the laboratory professionals provide quality test results, but often they are overshadowed by problems within the pre- and post-analytical phases. The public image of the clinical laboratory scientist is created by the problems that arise when the wrong test is ordered, a wrong sample is collected, or the test results are not clear to the clinician. The clinical laboratory scientists must get involved in all phases of clinical laboratory testing that reflect the quality of their work. This article will discuss three ways the clinical laboratory scientist can take an active role as an educator in the new healthcare system. First, the creation of educational tools can improve communication between the laboratory professionals, nursing units, practitioner, and patients. Secondly, the clinical laboratory scientist as a consultant can establish learning events for other healthcare professionals. Lastly, the laboratory professional participating on multidisciplinary teams can provide an educational role and communicate critical information at the point of care. All three of these approaches used consistently can engage the clinical laboratory scientist, physician, nursing staff, and other healthcare providers in a dialogue that will contribute to improved medical decisions. CREATION OF EDUCATIONAL TOOLS The first step is to create educational tools that are immediately available to answer the healthcare

professional’s questions. The rapid advancement of scientific research has provided specific testing which gives the clinician more knowledge about patient outcomes and treatment plans. The challenge for the clinical laboratory scientist is to create learning tools that ensure they can order the correct test, receive correct results, and that the results give meaning to the patient treatment plan. In discussing the characteristics of a community of practice, Wenger found that adults do not learn in isolation but in a social and cultural environment.3 The socio-cognitive demands of the workplace also shape the daily learning needs. The busy practitioner needs information in a quick and precise manner. The creation of educational tools that will meet this environment should be multidimensional. The tools should create collective and collaborative processes that include all the stakeholders. To understand the clinical laboratory scientist’s role as educator we can use the pre-analytical phase of testing as a good place to begin. In a study by Howanitz including CAP’s Q-Probes and Q-Tracks program data, the error rates of the pre-analytic and post-analytic phases were higher than the analytic phase.4 A questionnaire given to 70 primary healthcare centers and laboratories demonstrated the preanalytical phase was associated with a greater risk of errors affecting patient safety.5 The pre-analytic phase is very complex and involves the clinician, nursing staff, phlebotomists, and laboratory staff. A preanalytical event is the practitioner’s request for a laboratory test. The hospital’s specific mnemonic is not easy to access. Accurate test identification is difficult because it changes and varies between healthcare institutions. To address this issue and other laboratory related problems, Centers for Disease Control and Prevention (CDC) has established a professional workgroup. One goal of the work group is to formulate a single test naming system, so the non-laboratory staff can use the same test mnemonic from one institution to another.6 The universal test mnemonics could be a helpful educational tool to reduce patient errors.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 197

CLINICAL PRACTICE

An educational tool the clinical laboratory scientist could create is an improvement in the computerized provider order entry (CPOE). The clinician would input a specific evidence-based clinical practice guideline, and a template could assist in the selection of the correct test profile. The clinical laboratory scientist would maintain a current listing of tests with research references and additional information guiding the clinician through a decision tree format. In the event a newer test is placed on the laboratory’s test menu, a pop-up window could give additional information on why the older test will be replaced with specific research references, and the date it will be removed. To avoid duplicate test orders, the clinician can see the previous tests ordered on the patient, and a warning that a duplicate test is being ordered within a set timeframe. The system will also ask if the practitioner wants to order a duplicate test referencing the additional healthcare cost. Some healthcare institutions already have some form of electronic test ordering. The information system needs to be refined to make ordering a test easier and reduce incorrect orders that increase healthcare costs and extend hospital stays. To get acceptance of the new system the inclusion of all stakeholders in the creation of the CPOE is vital. In the earlier case study, the practitioner ordered a HIV quantitative PCR instead of a HIV ELISA. The HIV PCR is usually ordered to monitor the viral load of a known HIV positive patient receiving therapy. The HIV ELISA is the initial diagnostic test ordered because it provides faster turn-around-time. Healthcare costs are reduced by selecting this test as the initial step. If the HIV ELISA is positive, the practitioner receives notification and further testing can be requested for confirmation. Educational tools are the way clinical laboratory scientists can eliminate these patient care issues. If research evidence shows that false negative results occur in the HIV ELISA test due to delay in seroconversion, then have a question that asks if the HIV ELISA is being ordered for an initial diagnosis and reference research data on the seroconversion issue. How long will clinical laboratory professionals continue to look at the 198 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

incorrect test order problem as belonging to someone else? The laboratory information system should be reevaluated if information is unclear. The lack of appropriate educational tools plus the absence of collaboration does affect healthcare costs and patient safety.7 The computerized provider order entry provides a learning tool because it occurs when the learner needs the information. Daley demonstrates that learning activities should cause the learner to gain access to their experiences by social activity enhanced by shared inquiry.8 When using an interactive computerized provider order entry system not only is a test ordered correctly, but also new knowledge is received. Daley’s research also demonstrates that reflection and metacognition are aspects of constructing new knowledge and meaning. The test ordering activity will reinforce the new knowledge and give meaning to the experience each time it is repeated. The role of the laboratory professional is to disseminate, synthesize, and identify the information into an accessible and practical format (educational tools) that encourages the practitioner to use it. An additional educational strategy is to create a quick and easy on-line training program to demonstrate the ease of test ordering. A follow-up reminder via text messages or emails could be helpful in encouraging the practitioner to begin using the electronic order system. If the clinician continues to write orders then another educational opportunity occurs. How can the clinical laboratory help the nursing staff order the correct test? The continuous telephone request for interpretation of a clinician’s order that is unclear has been a longterm issue. The test order may require further information that the nursing staff may not have, thus delaying patient care. The nursing unit has to receive information in an efficient manner that will positively influence test ordering. A pre-analytical event is the introduction of new tests that requires complex changes across hospital services in patient preparation and specimen collection.

CLINICAL PRACTICE

In the past, clinical laboratories provided an actual laboratory manual for the nursing unit and quickly found it was not an efficient learning tool. The manual is placed on a shelf where it remains and quickly becomes outdated. Another educational tool, the electronic, interactive laboratory manual, provides the staff member with immediate information that is more inclusive and current. The manual can include directions on collection of the test, patient preparation, and specimen handling. If the order is still unclear, the next educational tool is a call to a laboratory professional to clear up the questions. The nursing unit can log into an electronic call center to get answers. In most laboratories, the person representing the laboratory and answering questions has limited knowledge on the laboratory ordering protocols. Even though this is a cost cutting measure for the laboratory, it can eventually create additional healthcare costs for the patient. A clinical laboratory scientist answering these questions can provide correct information as well as establish a collaborative relationship with the nursing staff. A call log will also indicate repeat questions over time and provide additional educational interventions. A quality indicator of a certain number of calls in two days from the same nursing unit concerning the same ordering question would create a short electronic educational note to the nursing unit, thus reinforcing the information provided by the call center. In the questionnaire sent out by Söderbert, Brulin, Grankvist, and Wallin 60% of the healthcare respondents did not consult the online laboratory manual prior to ordering a test.5 Another educational tool would be outreach visits to the nursing units that have consistent problems. The outreach visits will need to be a collaborative process with the nursing department and include all staff members. Dissemination of new information to the appropriate nursing managers does not mean the educational goal is complete. The previous examples represent the need for continuous education at different levels and using a variety of learning tools such as electronic reminders, site visits, and advanced CPOE.

EDUCATIONAL CONSULTANT PROVIDING LEARNING EVENTS The clinical laboratory scientist can be an advocate for the patient by providing consultation to other healthcare professionals as well as educational support. All healthcare professionals should be dedicated to lifelong professional learning and because laboratory medicine is rapidly changing patient care, it is the responsibility of the clinical laboratory scientist to provide learning opportunities. Grol and Grimshaw have done extensive research on effective implementation strategies to improve patient care.9 They note that complex changes in practice are not easy especially if it requires collaboration between services and change in organization of care. The clinical laboratory scientist knows how vital laboratory medicine is to patient safety, but the important issue is to understand how new knowledge becomes meaningful to other healthcare professionals. The goal should be to develop learning strategies that translate evidence into behavior changes in the clinical settings. To be an educational consultant the laboratory professional must provide continuous learning events that support patient care. The “us versus them” environment is not conducive to resolving patient safety issues. In the post-analytical stage, the creation of an interpretive report provides additional educational consultation. According to Dupree and Kemp, the narrative interpretation translates data into knowledge and educates the physician at the point of practice.10 Advanced technology, the IPOD or PDA, can provide the ability to receive patient information when an informed decision is required. Simply giving a numerical result is not providing the best patient care. The current problem is the absence of a software program that can accurately transfer the interpretative report to the electronic medical record (EMR). A PDF format with no reformatting may provide a solution.11 Visual charts and graphs can provide a quick summary of additional research information. The practitioner can also receive links to websites that may answer specific patient questions. Dupree and Kemp suggest including laboratory VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 199

CLINICAL PRACTICE

results from previous patient visits using electronic medical records. In the RAND Health Institute Study, 30% to 45% of patients in the U.S. and the Netherlands are not receiving care supported by scientific evidence. The report also shows that 20% to 25% of patient care is not needed or potentially harmful.2 The clinical laboratory’s contextualized patient report could provide at the point of practice more scientific research information assisting in a clinical decision.12

sessions are best for the initial training, but a clear and attractive message adapted to the target audience is essential with this training. There is enough experiential data to support the need for the clinical laboratory scientist to involve a collaborative multidisciplinary group to identify the training objectives and goals.15 In most institution “throwing training” at a problem is suppose to eliminate the problem, but unclear training objectives often compound the problem.

How can important new laboratory information be introduced to the clinician or nurse? In a metaanalysis on the effectiveness of continuing professional education the didactic interventions (lecture format) was the least effective.13 To maximize educational activities we must analyze where the professional learns. The transplantation of performance skills or new information into actual work environments plus reinforcement with discussion is more likely to become a part of the participant’s behavior. Active learning events are more effective than passive strategies. Active learning events include simulations, small group discussions, and individual audits.9

Grol and Grimshaw demonstrate from their research on introducing evidence and clinical guidelines into routine daily practice that barriers to change can come from three levels; individual, team and organization.9 In the practice environment, barriers such as lack of time or financial reimbursement keep new knowledge from reaching the staff. The social barriers such as opinion leaders not agreeing with the new approach has a major affect on successful implementation. Finally, the professional context will hinder implementation due to information overload and a lack of interest in change. Identification of the barriers is important in creating a change in professional behavior. Program planning by the multi-disciplinary group should develop educational strategies to address these barriers. Clinical laboratory scientists, nursing staff, and nursing leaders can develop educational events that allow for a continual and open discussion of specific barriers that are interfering with quality POCT.

An excellent example of using active learning events can occur when a new point-of-care test (POCT) is selected for use on the nursing units. The POC coordinator is responsible for educating the practitioner on the importance of quality control procedures and consistent use of the testing instrument or kit. In a historical review of POCT instruments, the first attempts placed many nonlaboratory trained individuals into the patient testing arena, and the struggles to provide accurate test results fell on the POCT coordinator following CLIA and CAP guidelines.14 With the introduction of conductivity and electronic requirements, testing is blocked if the quality control is not completed prior to testing. The quality of POCT has improved with these advancements, but the education of the nursing staff continues to be a challenge due to high volume of retraining, new POCT, and the influx of new employees. The group interactive educational 200 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

The results of a recent survey of family practice practitioners support the consultant role for the clinical laboratory scientist.16 In a response to the statement, “My clinical performance would benefit if there was a mechanism for simple and effective consultation on the selection of laboratory tests, particularly the more complex assays” 92% of the practitioner totally agreed. “A lab medicine consult service, involving the provision of advice on test selection and result interpretation needs to be available 24 hours a day, 7 days a week.” This statement received 52% of respondents who totally agreed and 32% strongly agreed. The clinical

CLINICAL PRACTICE

laboratory scientist’s opportunity to provide a consultative role with learning events would certainly be a positive factor for the clinician. MULTI-DISCIPLINARY TEAMWORK The previous discussion has presented the need for an integrative approach to delivering valuable laboratory medicine services. This discussion would not be complete without a look at the role of the clinical laboratory scientist on multi-disciplinary teams. Some healthcare institutions have begun to include clinical laboratory scientists on the patient rounding teams. The advantage of having a laboratory professional on the team is the quick resolution of problems that have traditionally taken hours or days to resolve or they never get resolved. In this article, we have examined examples of educational opportunities that involve laboratory services. To have a knowledgeable clinical laboratory scientist immediately available to answer questions or provide solutions to laboratory related problems would establish a learning moment for the other team members and enhance a collaborative relationship. It has been said that the laboratory professional is invisible to the public and other healthcare professionals. First, the laboratory professionals are identified as a place (“the laboratory”) and secondly, “the laboratory” is the cause for patient errors because the healthcare team members do not see the clinical laboratory scientist adding to valuable diagnostic information. The clinical laboratory scientist can become an active member of the healthcare institution’s infection control committee, quality improvement team, and hospital safety committee because the CLS’s scope of practice will expand the other team members’ knowledge on nosocomial infection, quality improvement, and safety. In the ASCLS Scope of Practice (2001), the CLS is responsible for “developing a Quality Management System, correlating and interpreting test data, and promoting awareness and understanding of the use of the clinical laboratory”.17 In a recent ASCLS TODAY article, Mary Ann McLane challenged clinical laboratory

scientists to submit real-life scenarios to the AHRQ Web Morbidity and Mortality that demonstrate how important they are in patient safety.18 As a member of multi-disciplinary teams, the CLS can provide unique skills that can translate the clinical laboratory knowledge essential to patient care. CONCLUSION Clinical laboratory medicine is a significant part of evidence-based clinical practice guidelines. The turnaround-time indicators, quality management programs, and other quality measurements are already integral functions of clinical laboratory science. The gap between critical laboratory results and medical care can be reduced by active educational strategies that allow the clinical laboratory scientist to provide vital information to the practitioner and other healthcare professionals. This is the time for the clinical laboratory scientist to take on new roles. New advanced laboratory testing will identify disease progression and individualized patient treatment that is not currently available. Will these advanced tests be utilized properly for the best patient care? Congress appropriated $50 million to the Agency for Healthcare Research and Quality in December 2000 to investigate tools for reducing medical errors.19 The clinical laboratory scientist can help reduce medical errors by providing support in the form of improved computerized provider order entry, consultation to establish learning events, and multi-disciplinary teamwork. Today with the U.S. healthcare system under critical scrutiny, it is an appropriate time for the clinical laboratory scientist to step forward to promote the valuable resources available through laboratory medicine. Of course, educational opportunities can only occur if the organization values the contributions of the individual, is open to innovative change, and has decentralized decision-making.20 A learning organization does not look for the individual who failed but how to improve the system. The clinical laboratory scientist must be a system thinker and look at the pattern of behavior. To achieve long-term change requires reviewing consistent errors to understand VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 201

CLINICAL PRACTICE

whether a pattern exists and seeking educational solutions that are part of an integrated total system of patient care.21 “The greatest personal revolution is the discovery that human beings, by changing the inner attitudes of their minds, can change the outer aspects of their lives.” Someone who wants to change will find a way; one who does not will find an excuse.22 Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this article. Email responses to [email protected]. In the subject line, please type “CLIN LAB SCI 22(4) RE RANNE”. Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be edited for length and clarity. We look forward to hearing from you. REFERENCES

1. Wachter, R. Lost in the black hole. AHRQ Web Morbidity and Mortality, 2003. Available from http://www.webmm. ahrq.gov/ case.aspx?caseID=31 2. The value of laboratory medicine. Chapter 1. Laboratory medicine: A national status report. 2008. Available from https://www.futurelabmedicine.org/reports/Laboratory_Med icine_National_Status_%20Report_08-09_Update--PatientCentered_Care.pdf 3. Wenger E, Snyder W. Communities of practice: The organizational frontier. Harv Bus Rev 2000 JanuaryFebruary; 78(1): 139-45. 4. Howanitz P. Errors in laboratory medicine: Practical lessons to improve patient safety. Arch Pathol Lab Med 2005 Oct;129(10): 1252-61. 5. Söderberg J, Brulin C, Grankvist K, Wallin O. Preanalytical errors in primary healthcare: A questionnaire study of information search procedures, test request management, and test tube labeling. Clin Chem Lab Med. 2009; 47(2): 195-201. 6. Centers for Disease Control and Prevention. Institute for Laboratory Medicine Integration Workgroup. Available from http://wwwn.cdc.gov/cliac/pdf/CLIAC0209.pdf 7. Kitson A. The need for systems change: Reflections on knowledge translation and organizational change. J Adv Nurs 2009; 65(1): 217-28. 8. Daley B. Learning and professional practice: A study of four professions. Adult Educ Q 2001; 52(1): 39-54.

202 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

9. Grol R, Grimshaw J. From best evidence to best practice: Effective implementation of change in patients’ care. Lancet 2003; 362(9391): 1225-30. 10. Dupree W, Kemp, K. “A one-stop shopping center” which transforms data into information and knowledge. Lab Med 2005 Feb; 36(2): 78-80. 11. Wagner K. Lost in translation: When clinical systems reformat lab reports. CAP Today 2009; 23: 18-9. 12. Plebani M. Decreasing postanalytical errors with interpretive report. Clin Lab News; 2008 October. 13. Davis D, O’Brien M, Freemantle N, Wolf F, et al. A. Impact of formal continuing medical education: Do conferences, workshops, rounds, and other traditional continuing education activities change physician behavior or health care outcomes? JAMA 1999; 282(9): 867-74. 14. Paxton A. Point of care sagas: A tale of three cities. CAP Today 2009 January. 15. Holdcraft S. Bridging the gap between laboratory and nursing: Helpful hints for effective communication. Lab Med; 2005 36(11): 685-6. 16. Innovative laboratory services survey. Alabama Academy of Family Physicians Mid-Winter Conference. ILM Integration Workgroup minutes 2008. Available from http://wwwn. cdc.gov/cliac/pdf/CLIAC0209.pdf. 17. Scope of practice. American Society of Clinical Laboratory Science 2001. Available from http://www.ascls.org/position/ scope_of_practice.asp.. 18. McLane M. Making a difference in patients’ lives. ASCLS Today 2008 September; 22(8): 11. 19. Agency for Healthcare Research and Quality 2001 Budget Allocations. Available from http://www.ahrq.gov/about/ budbrf01.htm. 20. Rycroft-Malone J, Kitson A, Harvey G, McCormack B, et al. Ingredients for change: Revisiting a conceptual framework. Qual Saf Health Care 2002; 11(2): 174-80. 21. Bierema L. Systems thinking: A new lens for old problems. J Contin Ed Health Prof 2003; 23(S1): S27-33. 22. Bach R. Illusions: The Adventure of a Reluctant Messiah. 2001 Dell Publishing, New York, New York.

CLINICAL PRACTICE

Clinical Laboratory Tycoon Case Study: Are you management material? AMY L. THOMPSON OBJECTIVE: Many clinical laboratory scientists find themselves in a management position during their career, but have limited training to assume this role. The Clinical Laboratory Tycoon case study was designed to submerge students into the laboratory business by having the participant act as a laboratory manager with the capacity to make all the decisions about his or her own lab. DESIGN: The student participant completed a set of modules that each related to varying aspects of managing a laboratory including selecting what tests to offer, hiring employees, marketing the services, setting goals, budgeting, and evaluating overall success. SETTING: This Clinical Laboratory Tycoon case study was used to instruct clinical laboratory science students in a university based clinical laboratory science program as part of their seminar in teaching, research, and management course. The size of these classes range from ten to twenty students and results reported in this paper are collected from a class size of sixteen students. The study could also be adapted for use in a hospital based program or as continuing education for laboratory employees. MAIN OUTCOME MEASURE: At the conclusion The peer-reviewed Clinical Practice Section seeks to publish case studies, reports, and articles that are immediately useful, are of a practical nature, or contain information that could lead to improvement in the quality of the clinical laboratory’s contribution to patient care, including brief reviews of books, computer programs, audiovisual materials, or other materials of interest to readers. Direct all inquiries to Libby Spence, PhD, CLS(NCA), Clin Lab Sci Clinical Practice Editor, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, [email protected].

of the study, the student participants reported what they had learned during their time as managers and how their perspective changed. CONCLUSION: Student participants reported that they gained an understanding and appreciation for the responsibilities of the laboratory manager. For some, this study helped to define career goals. INDEX TERMS: Management; Laboratory Education; Teaching Tools Clin Lab Sci 2009;22(4):203 Address for Correspondence: Amy L. Thompson, PhD, MT(ASCP), 43 Quarry Road Montgomery, NY 12549, Email [email protected] INTRODUCTION Often clinical laboratory scientists find themselves being promoted to supervisory and managerial positions with limited exposure to what these positions entail. Students and laboratory employees set goals to eventually move into a managerial assignment and may not realize all that is involved with this career choice. A manager typically leaves the bench and takes on the business side of laboratory medicine. As a faculty member, it is often difficult to engage students in management topics and the instructor may need to challenge him or herself as a scientist to teach this important part of clinical laboratory science. The Clinical Laboratory Tycoon case study introduces student participants to managerial topics, while encouraging active learning. Students designed and managed their own small laboratories in an attempt to become a Clinical Laboratory Tycoon. This modular study requires the student to learn VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 203

CLINICAL PRACTICE

about a topic through reading assignments, lectures, and discussion and then apply their new knowledge to a related scenario, thus reinforcing hierarchical learning. Topics included organizational laboratory structure, leadership styles, ethics, human resources issues, education and training, finances and marketing, regulations and compliance, laboratory information systems and career planning. MATERIALS AND METHODS The Clinical Laboratory Tycoon case study contains modular assignments that address various topics relating to laboratory management. Students complete a reading assignment using a laboratory management textbook and related articles and participate in a lecture/discussion prior to engaging in modular activities. Lecture sessions conducted similar to continuing education seminars given to laboratory professionals and discussions that incorporate brainstorming further give the student the feel of working as a manager in a clinical lab. Each module is introduced with directions and leading questions. Condensed modular assignments are included below and can be manipulated as needed. MODULAR ASSIGNMENTS: Introduction to Clinical Laboratory Tycoon Case Study You have been hired to create a small, but functional clinical laboratory to service the university student community in conjunction with health services (lab size ~10 ft X 10 ft). The lab will be open 40 hours per week (M-F 8:00 AM-4:00PM) and you must have it staffed for this timeframe. You have been authorized to hire 2 full time employees or 1 full time employee and 2 part time employees. You have no equipment except for an old Clinical Laboratory Science Program microscope and your budget, although not disclosed to you, is limited. To set up this lab, you must choose instrumentation, hire employees, create policy and procedures, and maintain the lab’s daily activities. You are under serious time constraints and must have this lab up and running by the end of the semester.

204 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

Throughout the semester, you will have assignments to aid you in creating the ultimate clinical lab so that you might become a clinical lab tycoon. For each assignment, completely address the questions using your textbook and other resources. All assignments should be typed and created to reflect your professionalism as a laboratory manager. Module 1: Laboratory Organization A carefully designed and organized lab is essential to clinical function. How will your lab be spatially organized? Consider that two or even three employees may be present in this small space and each must be able to carry out their necessary work functions. A model or models should be used to address this question. Module 2: Leadership Style Complete a leadership self assessment prior to completing this assignment*. As the manager (and leader) of the new university clinical lab, discuss your leadership style. What did you learn about your leadership style from the leadership self assessment? What will you do to develop your leadership qualities that are weak? What leadership challenges do you expect to overcome? How will you plan, organize, implement, and control your lab? How much time will you spend on each task and how you involve your employees in each process? * There are numerous leadership assessments available. A useful website with an assessment is found in the reference section and Denise Harmening has several assessments available in her book1, 2.

Module 3: Laboratory Workflow As the manager of the university laboratory, you are charged with the task of creating a workflow chart to demonstrate how specimens will be received, tested, and results reported. Will you collect specimens yourself (i.e. Will you draw blood?) or will health services do this? How will results be reported? Demonstrate your problem solving skills when completing this task. Keep in mind that you are limited to the software that is available (Microsoft Office, D2L, and E-mail). Module 4: Ethical Principles Create a code of ethics for your lab. This should be a list of 5 to 6 ethical principles that you expect your employees to follow.

CLINICAL PRACTICE

Module 5: Job Advertisement Design a one page advertisement for the positions that you have available in your clinical lab. It should include all criteria relating to the position including essential job functions. Your advertisement should reflect whether the position requires a Bachelors or Associates degree and whether the position is parttime or full time. Module 6: Interview Questions Prepare interview questions to ask prospective applicants. When preparing the questions, remember what is acceptable and unacceptable or even illegal to ask. Module 7: Applicant Review You have been authorized to hire 2 full time employees or 1 full time employee and 2 part time employees. After placing a job advertisement in the newspaper and in several journals, you receive numerous applications and interview five applicants (see table 1). You may ask the applicant more questions if needed. (A discussion board is provided for this purpose. You must identify the applicant and your question. The applicant’s answer will be posted.) Finally, who will you hire and why? Module 8: Educational Objectives You have hired your employees and now must train them. Write 5 educational (training) objectives that they must master to begin work. Apply appropriate taxonomy levels depending on employee degree level.** ** A NAACLS approved resource on writing instructional object-tives is available.3

Module 9: Employee Evaluation Your employees have been working and you are now tasked with doing a 90 day review with each of them. Design your review. Include a description of how you will deliver this review. Describe how you will discuss the review with your employees and how you will receive feedback from them. Module 10: Lab Testing Cost Analysis You have been running the university lab for a few months and you are considering dropping the HIV screening test since it is so labor intensive. Analyze the cost/benefit of this test and determine a cost per test for the HIV procedure. Is it worth the effort to

continue screening your population of patients for HIV? The patient pays $15 per HIV screening test performed. Keep in mind that this test requires venipuncture and that these charges are not calculated into the charge per test generated above. Also, note that while you can quit offering the test, you cannot raise the price of the testing since you signed a contract with Health Services saying that you would not raise testing prices for 3 years. Module 11: Considering Financial Reports and Creating a Budget Using the provided financial reports, create a generic lab budget for the upcoming fiscal year. Identify ways that you might cut back to increase profit. This should be a basic budget with salary based on the employees that you have hired ($20 per hour for Bachelors and $17 per hour for Associates). Also include projected non-salary items on your budget. This budget should contain basic items based on what information you have been provided. Module 12: Healthcare Reimbursement Your lab does not take any healthcare plans. Anyone using the lab’s services is required to file their own paperwork for healthcare reimbursement. As lab manager you are trying to decide what cost/benefit there would be if you were to file healthcare reimbursement for your patients. Discuss the pros and cons of this decision and how it might impact your overall budget and staffing needs. Module 13: Marketing of Services Your lab is not making as much profit as you would like. Develop a marketing plan/strategy for your lab. Describe this plan/strategy and develop a flyer that you will distribute to “sell” your product. Where will you distribute this flyer and how does it reflect your marketing plan/strategy? Module 14: Regulatory Agencies There are numerous regulations that affect labs and laboratory testing. Which regulatory agencies affect the university lab? How will you as lab manager address the requirements of each of these agencies? Module 15: Laboratory Information Systems Several local labs donated old lab equipment to get VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 205

CLINICAL PRACTICE

TABLE 1. University Lab Applicants Applicant Name

Education

Part/Full Time

Experience

Interview Notes

Susie Sample

Bachelors

Full time

Generalist in Hospital for 5 years setting, seems to be know it all

Ready for change from hospital

Joe Phlebotomy

Associates Recent graduate

Part time

Phlebotomist for 6 months while in school; no MLT experience

Excited about job; seems very naïve

Lucy Lyme

Bachelors

Part time-full time for a short duration, if needed

Retired as MT with 20 years at local hospital lab

Does not seem very flexible; nervous about new setting

Christopher Chemistry

Associates

Full time

Worked at local clinic for 3 years

Ready for change; dislikes college setting, thinks will enjoy small lab

Melanie Micro

Bachelors

Full time or part Time

Worked part time at small, local lab involved personal life

Familiar with university setting,

your lab up and running, but you must still come up with a computer system. Your lab has a very limited budget for computer based hardware and software. You do have 2 computers with printers that have internet access. Address how you will record, maintain, and transmit patient results to Health Services. You can utilize any system that is currently in place including Blackboard (Desire 2 Learn), Excel, and Microsoft Word. Keep in mind security and confidentiality when completing this module. Module 16: Trends How will you respond to the current ever-changing trends in laboratory science and medicine? Since you are a small lab, do you expect these to impact you? Module 17: Laboratory Annual Report Over the past year you have aspired to become a Clinical Laboratory Tycoon. It is now your job, as laboratory manager, to evaluate the success of your lab. How do you feel as manager and what have you learned? Do 206 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

you feel that the lab was successful under your leadership and what would you have done differently? Have your future career goals changed based on what you learned in this study? Can we count on you for another year as Clinical Laboratory Tycoon lab manager? RESULTS Student participants in the Clinical Laboratory Tycoon case study completed topical modules that explored various aspects of laboratory management. These tycoons reported that the study opened their eyes to all of the duties and responsibilities involved in being a laboratory manager. Several students decided that becoming a manager is not for them and they instead would prefer to stay at the bench. Other students felt that after some time working in the main laboratory, they would enjoy taking on supervisory responsibilities and even eventually assuming the lead

CLINICAL PRACTICE

role as manager. Still other participants found that they enjoyed one aspect of management such as education or test cost analysis and wish to explore these avenues either as clinical laboratory scientists or by furthering their education. The majority of applicants agreed that they would prefer to gain experience on the bench prior to taking on extensive managerial related duties. CONCLUSIONS Some clinical laboratory scientists aspire to become laboratory managers, while others gain this position by default. Besides personnel actions, many clinical laboratory scientists and especially students are not exposed to the job duties and responsibilities of laboratory managers. The Clinical Laboratory Tycoon case study is designed to show participants a different side of laboratory medicine, the side that managers face on a daily basis. By completing this study, participants hopefully will gain a new respect for this

challenging position and will be able to make an informed decision about whether this role is for them. Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this article. Email responses to [email protected]. In the subject line, please type “CLIN LAB SCI 22(4) RE THOMPSON”. Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be edited for length and clarity. We look forward to hearing from you. REFERENCES

1. Harmening, D. Laboratory Management Principles and Processes. 2nd ed. St., Petersburg, Florida: D.H. Publishing & Consulting, Inc.; 2007. 2. Leadership Self Assessment. Available from http://www. nwlink.com/~donclark/leader/survlead.html. Accessed 2009 March 20. 3. NAACLS Writing Instructional Objective by Kathy V. Waller. Available from http://www.naacls.org/docs/an nouncement/writing-objectives.pdf. Accessed 2009 March 20.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 207

RESEARCH AND REPORTS

Immunophenotypic Analysis of Bone Marrow B Lymphocyte Precursors (Hématogones) by Flow Cytometry N BRAHAM JMILI, S NSAIBIA, MC JACOB, H OMRI, MA LAATIRI, S YACOUB, Y BRAHAM, M AOUNI, M KORTAS ABSTRACT: The aims of this flow cytometry study were to quantify B lymphoid precursors known as hématogones across age and clinical conditions and to study the immunophenotypic profile of these benign immature B cells. A total of 406 consecutive marrow specimens were analyzed for hématogones using 4color flow cytometry during a 19 month period (60% males and 40% females). The age range was 3 months to 89 years. Hématogones were present in 80% of the specimens. Morphologic analysis of the smears from each patient showed small numbers of hématogones (5% of bone marrow cells) in several clinical conditions (Table 3). Table 3. Clinical conditions with increased hématogones for the 406 specimens. Disease Neoplastic disease:

Number of cases 70/406 cases

Known myeloid leukaemia Other neoplasias (Nonhaematopoietic neoplasms) Cytopenia:

50 20

257/406 cases

Idiopathic thrombocytopenia purpura Megaloblastic anaemia Others Infectious disease:

86 74 97 79/406 cases

DISCUSSION Hématogones were identified by 4-color flow cytometry using optimal antibody combinations in many bone marrow samples 3,6,7. Bone marrow hématogones were separately assessed as hematogone 1 populations of early stage and hématogones 2 of mid-stage precursor B cells, respectively. In some (about 30%) of the hématogones, a third type of hématogones could be assessed in the bone marrow samples (Figure 4)6. Our study showed that intermediate hématogones predominated. Increased information about benign B lymphocyte precursors, especially the existence of a third type hématogones could provide a basis for better discrimination of B leukaemia cells even in very small amounts. In a 212 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

multidisciplinary study, Rimsza, has demonstrated that hematogone-rich lymphoid proliferations exhibit a spectrum of B- lymphoid differentiation of antigen expression with predominance of intermediate and mature B lineage cells8. Flow cytometry revealed in this study that intermediately differentiated cells (CD10+,CD19+) predominated and followed in frequency by CD20+ 8. Hématogones may morphologically resemble the neoplastic lymphoblasts of precursor B ALL, and their immunophenotype also has features in common with neoplastic lymphoblasts. Thus, distinction of hématogones and neoplastic lymphoblasts of B cells present in bone marrow may cause diagnostic problems due to their morphologic and immunophenotypic similarities with neoplastic lymphoblasts of acute lymphoblastic leukaemia 5,6,9,10. In the medical literature that we reviewed, the neoplastic lymphoblasts in precursor B ALL deviated from the normal B-lineage maturation spectrum and exhibited maturation arrest and over-, under-, and asynchronous expression of antigens observed on normal B-cell precursors. They often aberrantly expressed myeloid-associated antigens 5. Hematogone populations always exhibit a continuous and complete maturation spectrum of antigen expression typical of the normal evolution of Blineage precursors and they lack aberrant or asynchronous antigen expression5. (Table 4) Hematogones are precursors which were defined by CD19 positivity and CD45 bright. The expression of antigen immaturity includes HLA DR and CD34, and the co-expression of the more mature markers CD19, CD20, CD22. These cells are blended and confused with those of mature B lymphocytes (CD10 negative) on CD45/SSC and could be better recognized on CD10 gating6. Leukemic cells can be distinguished from normal haematopoietic cells on the basis of morphology, of chromosomal or molecular abnormalities and immunophenotype. With flow cytometry using opti-

RESEARCH AND REPORTS

mal antibodies in combination, the distinction can nearly always be made. However, we have to emphaTable 4. Maturational sequence of bone marrow B cell precursors (hématogones). Stage 1 hématogones correspond to the least mature (top horizontal row). Stage 2 includes middle rows and stage 3 the bottom hematogone row. Mature marrow B lymphocytes are shown for comparison(6). Hématogones Stage 1 TdT CD34 Stage 2

Stage 3

Mature B Cells

CD10

CD19

CD22 CD38 (dim) (bright)

CD10

CD19

CD10

CD19

CD22 CD38 (dim) (bright) CD22 CD38 CD20 Slg* (dim) (bright) (dim)

CD10

CD19

CD22 CD38 CD20 Slg* (dim) (bright)

CD19

CD22

CD38

CD20 SIg* (bright to négative)

* The appearance of surface immunoglobulin is variable among individual cells occurring from shortly before to after acquisition of high level of CD20 expression.

size the difficulties in distinguishing these cells from residual marrow blasts after chemotherapy.9,10,11 Hématogones were identified by 4-color flow cytometry using optimal antibody combinations in most bone marrow specimens. They were more commonly found in higher numbers in children and there was a general decline in hématogones with increasing age12,13,14. They are often increased (> 5%) in regenerating marrow and in some clinical conditions 3,4,6. In our study there was a significant decline in hématogones with increasing age, but a broad range was found at all ages, although, some adult’s bone marrow contained relatively high numbers (Table 2 and 3).

In a study by Kallkury, flow cytometric analysis revealed 1% to 20% precursor B cells based on expression of 1 or more pan B cell antigens in addition to CD10, CD34 and terminal deoxynucleotidyl transferase (TDT) 11. In Caldwell’s study hématogones were most commonly observed in young children, comprising up to 21% of marrow cells in normal infants 15. It has been reported that the number of hématogones in bone marrow is variable; the hématogones are present in higher numbers in children and they are often increased in regenerating marrow and in some clinical conditions particularly in patients with cytopenias and neoplastic diseases6,16,17. It has also been reported that there is a decline in the mean percentage of hématogones with increasing marrow involvement by neoplastic cells 17. The reason for the decline is uncertain but may relate to encroachment on the hematogone compartment by the neoplastic infiltrate: alteration of factors that regulate B lymphocytogenesis may also play a role. A study has shown that even though total hématogones may be decreased there is an increased proportion of stage 3 hématogones in marrow involved by lymphoma or leukaemia compared to un-infiltrated marrows 18. Furthermore, hématogones are the predominant lymphoid population in the bone marrow of preterm infants (for10 to 60%; mean = 34%) of all cells. Flow cytometry revealed a level of 3.8 % of immature cells in a < 1 week- old neonate and 25.7% in a 19 week old infant 9. They are reported to occur in large numbers in some healthy infants and young children and in a variety of diseases in both children and adults 12,13,14,19. Hématogones may be particularly prominent in the regeneration phase following chemotherapy or bone marrow transplantation and in patients with autoimmune and congenital cytopenias, neoplasms, and acquired immunodeficiency syndromes. In some instances they constitute 5 % to more than 50 % of cells 6,20,21,22. Immune mediated thrombocytopenia is a VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 213

RESEARCH AND REPORTS

clinical condition characterized by increased platelet destruction due to the sensitization of platelets by antibodies. A statistically significant increase in the percentage of hématogones was demonstrated in their bone marrow. An increased percentage of hématogones was demonstrated; with a mean of 18+/15.2%, CD19+ with a mean of 27+/- 16.3% and CD 34+ with a mean of 18+/- 15.2%. This could be the sequence of an immunological response to the cause which determinates the disease, or the regeneration of the stem cell compartment following transient damage 23,24. The presence of benign immature B cells has been noted to interfere with the flow cytometric analysis of cases of suspected acute lymphoblastic leukaemia because their immunophenotype (positive for CD19, CD10, CD34 and terminal deoxynucleotidyl transferase) is similar to that of pre B cells lymphoblasts and they simulate acute lymphoblastic leukaemia or lymphoma 20,21,25. The percentage of marrow hématogones may fluctuate with disease status or persistent elevations may occur. Persistent elevations have been observed for 2 years following cessation of chemotherapy for ALL by one group of investigators and another group found elevations for more than a year following marrow transplantation 26,27,28. The presence of hématogones in clinical samples should be recognized so as not to adversely influence prognostic studies 22, 25. Flow cytometry is reported to distinguish between these cell populations in nearly all instances Identification of normal hématogones B contribute to better clarification of the detection of small numbers of blasts B of acute lymphoblastic leukaemia 29,30,31,32. In conclusion, these findings suggest that it is important to continue this study by flow cytometric analysis of the lymphoblasts of ALL with the same 4 combinations of antibodies in order to clarify the optimal combination which clearly distinguishes B leukemic cell from hématogones. Such comple214 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

mentary investigations are necessary for the recognition of early relapsed ALL and disease progression. Thus these differences in hématogones and lymphoblasts of ALL would be very important and could be utilized for analysis of minimal residual disease after chemotherapy treatment of B ALL. Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this article. Email responses to [email protected]. In the subject line, please type “CLIN LAB SCI 22(4) RE BRAHAM”. Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be edited for length and clarity. We look forward to hearing from you. REFERENCES

1. Vogel P, Bassen FA. Sternal marrow of children in normal and pathologic states. American Journal of Disease of childhood. 1939, 57, 245-68. 2. Vogel P, Erf LA. Haematological observations on bone marrow obtained by sternal puncture. American Journal of Clinical Pathology. 1937, 7, 436-47. 3. Longacre TA, Foucar K, Crago S, Chen IM, et al. Hematogones: a multiparametric analysis of bone marrow precursor cells. Blood. 1989, 73, 543-52. 4. Vandersteenhoven AM, Williams JE, Borowitz MJ. Marrow B-cell precursors are increased in lymphomas or systemic diseases associated with B cell dysfunction. American Journal of Clinical Pathology. 1993, 100, 60-6. 5. Babusikova O, Zeleznikova T, Mlcakova A, Kusenda J, Stevulova L. The knowledge on the 3rd type hematogones could contribute to more precise detection of small numbers of precursor B-acute lymphoblastic leukaemia. Neoplasma, 2005, 52(6), 502-9. 6. McKenna RW, Asplund SL, Kroft SH. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) and neoplastic lymphoblasts by 4- color flow cytometry. Leukaemia Lymphoma. 2004, 45(2), 277-85. 7. Riley RS, Massey D, Jackson-Cook C, Idowu M, Romagnoli G. Immunophenotypic analysis of acute lymphocytic leukaemia. Hematol Oncol Clin North Am. 2002, 16(2), 245-99. 8. Rimsza LM, Larson RS, Winter SS, Foucar K, et al. Benign hematogone-rich lymphoid proliferations can be distinguished from B lineage acute lymphoblastic leukaemia by integration of morphology, immunophenotype, adhesion molecule expression and architectural features. American Journal of Clinical Pathology. 2000, 114(1), 66-75. 9. Rimsza LM, Douglas VK, Tighe P, Saxonhouse MA, et al. Benign B-cell precursors (hematogones) are the predominant

RESEARCH AND REPORTS

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

lymphoid population in the bone marrow of preterm infants. Biol Neonate. 2004, 86(4),247-53. Chen W, Karandikar NJ, McKenna RW, Kroft SH. Stability of leukaemia- associated immunophenotypes in precursor Blymphoblastic leukaemia: a single institution experience. Am J Clin Pathol. 2007, 127(1), 39-46 Kallkury BV, Hartmann DP, Cossman, Gootenberg JE, Baag A. Posttherapy surveillance of B cell precursor acute lymphoblastic leukaemia.Value of polymerase chain reaction and limitation of flow cytometry. American Journal of Clinical Pathology. 1999, 111(6), 759-66. Lucio P, Parreira A, Van den Beemed MW, Van Lochem EG, et al. Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal disease in precursor-B-ALL. Leukemia. 1999, 13, 419-27. Dworzak MN, Fritsch G, Fleischer C, Printz D, et al. Multiparameter phenotype mapping of normal and postchemotherapy B lymphopoiesis in pediatric bone marrow. Leukemia. 1997, 11, 1266-73. Loken MR, Slah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. Normal B lymphocyte development. Blood, 1987, 70, 1316-24. Caldwell CW, Poje E, Helikson MA. B cell precursors in normal paediatric bone marrow. American Journal of Clinical Pathology. 1991, 95, 816-23. Jelic TM, Raj AB, Kurczynski EM, Tolamat N, Chang HH. Expression of CD5 on hematogones in a 7-year old girl with Shwachman-Diamond Syndrome. Pediatr Dev Pathol. 2001, 4(5), 505-11. McKenna RW, Waschington LT, Aquino DB, Picker LJ, Kroft SH. Immunophenotypic analysis of hematogones (Blymphocyte precursors) in 662 consecutive bone marrow specimens by 4- color flow cytometry. Blood. 2001, 98(8), 2498-507. Wright B, McKenna RW, Asplund SL, Kroft SH. Maturating B cell precursors in bone marrow: a detailed subset analysis of 141 cases by 4- color flow cytometry. Modern Pathology. 2002, 15: 270A. Klupp N, Simonitsch I, Mannhalter C, Amann G. Emergence of unusual bone marrow precursor B-cell population in fatal Shwachman-Diamond Syndrome. Arch Pathol Lab Med. 2000, 124(9), 1379-81. Vargas SO, Hasegawa SL, Dorfman DM. Hematogones as an internal control in flow cytometric analysis of suspected acute lymphoblastic leukemaia. Pediatr Dev Pathol. 2001, 4(5), 505-11. Davis RE, Longacre, Cornbleet PJ. Hematogones in the bone marrow of adults. Immunophenotypic features, clinical settings, and differential diagnosis. American Journal of Clinical Pathology. 1994, 102(2), 201-11. Davis BH, Scwartz M. ZAP-70 expression is low in normal precursor B cells or hematogones. Cytometry B clin cyto2006, 70B(4), 314-8.

23. Guiziry DE, El GW, Farhat N, Hassab H. Phenotypic analysis of bone marrow lymphocytes from children with acute thrombocytopenic purpura. Egypt J Immunol. 2005, 12(1), 9-14. 24. Fisgin T, Yarali N,Duru F, Kara A. CMV induced immune thrombocytopenia and excessive hematogones mimicking an acute B precursor lymphoblastic leukaemia. Leuk Res. 2003, 27(2), 193-6. 25. Hurford MT, Altman AJ, DiGiuseppe JA, Scherburne BJ, Rezuke WN. Unique pattern of nuclear TdT immunofluorescence distinguishes normal precursor B cells (Hematogones) from lymphoblasts of precursor Blymphoblastic leukaemia. Am J Clin Pathol. 2008, 129(5), 700-5. 26. Sandhaus LM, Chen TL, Ettinger LJ, Hirst Allen A, et al. Significance of CD10 positive cells in non malignant bone marrow of children. American Journal of Pediatric Hematology Oncology. 1993, 15, 65-70 27. Leitenberg D, Rappeport JM, Smith BR. B cell precursor bone marrow reconstitution after bone marrow transplantation. American Journal of Clinical Pathology. 1999, 102, 231-6. 28. Babusikova O, Zeleznikova T, Kirschnerova G, Kankuri E. Hematogones in acute leukaemia during and after therapy. Leuk Lymphoma, 2008, 49(10), 1935-44. 29. San Miguel JF, Vidriales MB, Lopez Berges C, Diaz Medivilla J, et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukaemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood. 2001, 98, 2456-62. 30. Coustan Smith E, Gajjar A, Hijiya N, Razzouk Bl, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukaemia after relapse. Leukemia. 2004, 18, 499-504. 31. Coustan Smith E, Sancho J, Behm FG, Hancock ML, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukaemia. Blood. 2002, 100, 52-8. 32. Krampera M, Perbellini O, Vincenzi C, Zampieri F, et al. Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia. Haematologica. 2006, 91(8), 110912.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 215

RESEARCH AND REPORTS

Duration of Loxosceles reclusa Venom Detection by ELISA from Swabs DAVID L McGLASSON, JONATHON A GREEN, WILLIAM V STOECKER, JAMES L BABCOCK, DAVID A CALCARA BACKGROUND: Diagnosis of Loxosceles reclusa envenomations is currently based upon clinical presentation. An enzyme-linked immunosorbent assay (ELISA) can detect surface Loxosceles venom at the envenomation site, allowing diagnostic confirmation. The length of time that venom on the skin is recoverable non-invasively is unknown.

the ELISA venom test appears capable of detecting venom on most patients presenting with Loxosceles envenomations. This detection system will allow the physician more accurate determination of whether the lesion is from a brown recluse spider or some other agent that can cause this type of necrotic ulcer. Clin Lab Sci 2009;22(4):216

MATERIALS AND METHODS: To investigate duration of recoverable venom antigen, whole venom and fractionated sphingomyelinase D venom aliquots were injected subcutaneously in New Zealand White rabbits. Cotton and Dacron swabs were compared for venom recovery over a 21-day period using a surface swab technique. RESULTS: Significant amounts of Loxosceles reclusa antigen were found on the surface of rabbit skin after experimental injection of whole venom and sphingomyelinase D. The duration of recoverable antigen using this experimental model appears to be at least two weeks and as long as 21 days in some cases. CONCLUSIONS: Because the duration of the recoverable antigen is seen to be at least two weeks,

The peer-reviewed Research and Reports Section seeks to publish reports of original research related to the clinical laboratory or one or more subspecialties, as well as information on important clinical laboratory-related topics such as technological, clinical, and experimental advances and innovations. Literature reviews are also included. Direct all inquiries to David L McGlasson MS, MLS, 59th Clinical Research Division/SGRL, 2200Berquist Dr., Bldg. 4430, Lackland AFB TX 78236-9908,david.mcglasson@lackland. af.mil 216 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

David L. McGlasson, MS, MLS, is clinical research scientist, Wilford Hall Medical Center, Lackland AFB, TX Jonathan A. Green PhD is an associate professor, Department of Animal Sciences, University of MissouriColumbia, Columbia, MO William V. Stoecker, MS, MD is president and CEO, Stoecker & Associates, Rolla, MO James L. Babcock, MD, PhD is staff pathologist, Ozark Pathology Associates, Mountain Home, AR David A. Calcara, BS is a medical student, Department of Animal Sciences, University of MissouriColumbia, Columbia, MO Address for Correspondence: David L. McGlasson, MS, MLS, 59th Clinical Research Division, CPSG/SGVUL, 2200 Bergquist Drive, Bldg. 4430, Lackland AFB, TX 78236-9908, Tel: 210-292-6555 Fax: 210-292-2897, [email protected] ACKNOWLEDGEMENT: The assistance of Tina Parks in protein preparations and ELISA determinations is gratefully acknowledged. The assistance of Dr. Hugh H Harroff, DVM, ACLAM, Ms. Jackie E Sutton, RLATG and Mr. Daniel T Sellers, RALAT in the

RESEARCH AND REPORTS

injection of venoms and swab collection was instrumental in the accomplishment of this protocol. INTRODUCTION Loxosceles reclusa (common name: Brown Recluse Spider) and related arachnids are indigenous American spiders that possess a venom capable of causing painful necrotic ulcers with surrounding inflammation and possibly severe systemic effects1,2,3,4. The diagnosis of a brown recluse spider bite is made clinically, based on the appearance of the lesion1,2,3,4. Definitive diagnosis is usually not possible because few patients bring the offending spider to the clinician for identification. Even then, misidentification of the spider is a distinct possibility5. The appearance of significant envenomation with cutaneous necrosis is the usual basis for diagnosis but is not specific for Loxosceles species envenomation1,3,6. Indeed, a variety of treatable illnesses can also give rise to cutaneous necrotic ulcers, including staphylococcal, and streptococcal infections such as impetigo and cellulitis, herpes simplex infection, factitial injury, squamous cell carcinoma, toxicodendron dermatitis (poison ivy), diabetic ulcer, fungal infection, localized vasculitis, erythema nodosum, Stevens-Johnson syndrome, toxic epidermal necrolysis, and other arthropod bites including bites from ticks, triatomid bugs, hymenoptera, blister beetles, crickets, and grasshoppers5, sporotrichosis7, and even Lyme disease8. A test for Loxosceles envenomation is desirable for cases with significant systemic findings, for often misdiagnosed cases in nonendemic areas, and for cases of nonhealing ulcers and other wounds. All of these are often falsely attributed to loxoscelism5,7,9,10. A polyclonal ELISA, derived from New Zealand White rabbits, was reported by Gomez et al11. In that study, 17 North American arthropod venoms elicited no cross-reactivity when assayed at relevant venom amounts12. The present study was designed to test the hypothesis that venom could be detected by using a swab assay and to determine how long, up to three weeks, venom could be detected after subcutaneous venom injection. The present study included controls

for both injection (a saline-only injection was performed) and for the type of swab (both Dacron and cotton swabs were used). MATERIALS AND METHODS Venom necrotic fraction purification Purification of the fraction of the venom that causes necrosis in rabbits proceeded as previously described13. Polyacrylamide gel electrophoresis14 was conducted using seven percent 5mm diameter acrylamide gels at pH 9.5. All electrophoresis reagents were purchased from Bio-Rad Laboratories, Richmond, CA. Stacking gels were 1.0 cm long and separating gels 4.1 cm long. Electrophoresis with ice water cooling was initiated at 2 mA/gel and continued at 4 mA/gel after samples entered the separating gel. Protein collection was accomplished by using dialysis tubing, with samples marked by bromphenol blue tracking dye. Proteins were concentrated using CF25 Centriflo ultra-filtration membrane cones. Purification of an enriched venom necrotic fraction was accomplished by sequential fractionation over diethylaminoethyl cellulose (DE 52), carboxymethyl cellulose (CM 52) (Whatman, Clifton NJ) and BioGel P-100 gel resin (Bio-Rad, Richmond CA). All chromatography steps were performed at 4°C. All dialysis tubing (Fisher, St. Louis MO) was boiled for 0.5 hr in a 0.3 M ethylenediamine tetraacetate (EDTA) solution followed by washing with deionized distilled water to avoid a major loss of lethal activity as measured in mice13. The protein purified by the method of Babcock et al. is a single subunit protein of 33 kD. This purified protein is the major fraction in the venom, comprising about 40% by weight15. The fraction has the lethal effect on mice of whole venom, is dermonecrotic to rabbits13 and has sphingomyelinase D activity16. Accordingly, we have labeled this fraction the sphingomyelinase D fraction. Rabbit Inoculation Animal testing and euthanization following the procedure was approved by the animal care committee (IACUC) of Lackland Air Force Base. All of the test animals were shaved in the area of the midVOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 217

RESEARCH AND REPORTS

dorsal spine prior to injection. Three New Zealand White rabbits (Oryctolagus cunniculus) were injected with a 4.0 or 5.0 µg aliguot of whole venom of Loxosceles reclusa (SpiderPharm, Yarnell, AZ) in 0.2 mL saline, in the mid-dorsal back area subcutaneously. In addition, three rabbits were injected with 5.0 µg of sphingomyelinase D fraction extract, purified from whole venom as noted above. Three control rabbits were injected with 0.2 mL normal saline. Swab specimens were collected daily for 21 days. Each type of individual swab (Dacron and cotton) was dipped in normal saline and the inoculation site was then swabbed for 30 seconds. Biopsies were obtained at 24 and 72 hours at 1 cm from the injection site. Biopsies and swabs were frozen in liquid nitrogen and kept frozen at (-70°C) and transported under dry ice to the laboratory for ELISA testing. The swab was thawed and the absorbent end was removed from the swab stick and was placed in a 1.5 mL microcentrifuge tube. The swab was centrifuged at 10,000g for 10 minutes to recover the saline from the absorbent material. The presence of venom proteins in the solution was detected with an ELISA designed to detect Loxosceles venom. The assay was originally described by Gomez et al11; the assay employed for these experiments was modified slightly from the original format. Polyclonal antibodies for recognition of whole venom were raised in New Zealand white rabbits with unfractionated Loxoceles reclusa venom. Antibodies were purified from serum by means of protein A column liquid chromatography17. The concentration of blocking agents as noted in Gomez et al,11 were increased and nonfat milk solids were added to the blocking buffer. The detection agent was changed from horseradish peroxidase to alkaline phosphatase after standard curves showed slightly greater sensitivity with the alkaline phosphatase in the current assay design. Product generation was monitored at 405 nm on a model ELx808, BIO-TEK, Inc. microplate reader. Sensitivity and specificity were determined by the following methods: 218 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

Sensitivity = Specificity =

True Positives × 100% True Positives + False Negatives

True Negatives × 100% True Negatives + False Positives

RESULTS The lesions in the rabbits caused by the experimental whole venom and sphingomyelinase injections were characterized by more hemorrhage and less cutaneous necrosis than is seen in humans. A typical 24-hour post inoculation lesion in rabbits is shown in Figure 1.

Figure 1. 24 hours post envenomation with brown recluse spider venom in rabbit model.

The assay results showed that the cotton swabs allowed for more detectable venom recovery than were obtained from the Dacron swabs (Figures 2 and 3). The average signal detected from the animals treated with venom was significantly above the background signal detected from the saline treatment for up to 21 days after the initial treatment (Figure 2, 3, and 4). The sphingomyelinase D injections showed greater separation of amounts of recoverable antigen, when compared to saline injections, than did the whole venom injections (Figures 3 and 4). The average amounts of venom detected by ELISA are shown in figures 2-5. The averages were determined from the amount detected in each of six rabbits for figures 2 and 3 and for each of three

RESEARCH AND REPORTS

Figure 2. Venom recovery using Dacron swabs.

Figure 3. Venom recovery using cotton swabs. VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 219

RESEARCH AND REPORTS

Figure 4. Comparisons of recoverable antigen with sphingomyelinase D vs saline injections.

Figure 5. Comparisons of recoverable antigen with venom vs saline injections.

220 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

RESEARCH AND REPORTS

rabbits for figures 4 and 5. ELISA performed on control biopsy extracts exhibited no detectable venom immuno-reactivity. The sensitivities of the ELISA for the whole venom with the cotton swabs up to and including 7, 10, 14, and 21 days were 67%, 65%, 62%, and 60% respectively. For the sphingomyelinase D, the sensitivity of the ELISA test was 95%, 90%, 83%, and 77%. The overall specificity remained high throughout the tests, at 95%, 96%, 93%, and 92% on days 7, 10, 14, and 21, respectively. The values were above three times the standard deviation of the background signal. The small number of test animals did not allow establishment of a confidence interval between venom amounts and background. DISCUSSION Patients with suspected spider bites bring in the culpable spider in only a minority of cases. In one series, 19 of 274 (7%) of patients diagnosed with brown recluse spider bites between 1987 and 1993 brought in the spider18. The spider may be found after a significant delay, leading to uncertainty that the arachnid presented is the offending agent. Therefore, the diagnosis of most spider bites is generally dependent upon bite morphology. Many bites lack the moderately specific ‘red, white and blue’ sign3 and the atrophic, bluish patch1. Additional confusion is created by the diagnosis of “spider bite,” often for nonspecific necrotic wounds, in areas where L. reclusa and similar species have never been verified5. A sensitive and specific Loxosceles species venom assay is clinically needed. With so many alternative diagnoses, diagnostic error in spider bites remains high. These alternative diagnoses in our clinic have included staphylococcal or streptococcal infection, herpes simplex, herpes zoster, pyoderma gangrenosum, granulomatous rosacea, and squamous cell carcinoma. A sensitive and specific assay would provide the “more strict diagnostic criteria” called for by Vetter and Bush 10. Misdiagnosing loxoscelism

“may lead to unnecessary, expensive, or even harmful therapy”10. Additionally, it may lead to delays in appropriate care that may lead to adverse, and possibly fatal, consequences. This, in turn, can lead to increased medical-legal risk if there is a treatment for the actual diagnosis5. It is our experience that patients with suspected spider bites report the bite most commonly 2-8 days after the appearance of the possible bite. Therefore, to correctly evaluate those who delay reporting the bite, it is essential to have a clinical assay that is able to detect the venom after at least one week. This study demonstrates that the Loxosceles venom antigens that are detectable by ELISA persist for up to two weeks. The sensitivity for the whole venom was between 60 and 70% over the test interval. If we exclude the results for a single rabbit, the sensitivity of both the whole venom and the sphingomyelinase D tests would have been above 85%. The remaining rabbits had no significant difference in the venom sensitivity. In summary, our research has established an ELISA for the detection of brown recluse venom present in swab samples. The assay is effective in identifying venom up to two weeks after exposure. Further refinement of the polyclonal ELISA may make the assay even more sensitive. Studies are currently underway to determine if it is possible to obtain an increase in the sensitivity and specificity of the assay if venom-affinity purified antibodies are employed in the ELISA. CONFLICTS OF INTEREST Dr. Stoecker owns a controlling interest in SpiderTech, which develops tests for spider bites and spider traps. Drs. Stoecker, Green, and McGlasson have filed a provisional patent for a diagnostic test for Loxosceles envenomations. This publication was made possible by Grant Number SBIR 1R 43AR 055683-01 of the National Institutes of Health (NIH). Partial funding was obtained from the United States Air Force Surgeon VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 221

RESEARCH AND REPORTS

General Office. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH or the United States Air Force. Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this article. Email responses to [email protected]. In the subject line, please type “CLIN LAB SCI 22(4) RE MCGLASSON”. Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be edited for length and clarity. We look forward to hearing from you. REFERENCES

1. Anderson PC. Spider bites in the United States. Dermatol. Clin. 1997:15:307-11. 2. Atkins JA, Wingo CW, Sodeman WA, Flynn JE. Necrotic arachnidism. Am J Trop Med Hyg 1958:7:165-84. 3. Sams HH, Dunnick CA, Smith ML, King LE Jr. Necrotic arachnidism. J Am Acad Dermatol 2001:44:561-73. 4. Wasserman GS, Anderson PC. Loxoscelism and necrotic arachnidism. J Toxicol Clin Toxicol 1983-1984:21:451-72. 5. Swanson DL, Vetter RS. Bites of brown recluse spiders and suspected necrotic arachnidism. N Engl J Med. 2005:352: 700-7. 6. Vetter RS. Envenomation by a spider, Agelenopsis aperta (family: Agelenidae) previously considered harmless. Ann Emerg Med. 1998;32:739-41. 7. Moaven LD, Altman SA, Newnham AR. Sporotrichosis mimicking necrotising arachnidism. 1999:Med J Aust. 171: 865-68. 8. Rosenstein ED, Kramer N. Lyme disease misdiagnosed as a

222 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

brown recluse bite [letter]. Ann Intern Med. 1987:107:782. 9. Rees R, Campbell D, Rieger E, King LE . The diagnosis and treatment of brown recluse spider bites. Ann Emerg Med. 1987:16: 945-9. 10. Vetter RS, Bush SP. The diagnosis of brown recluse spider bite is overused for dermonecrotic wounds of uncertain etiology. Ann Emerg Med. 2001:39: 554-6 11. Gomez HF, Krywko DM, Stoecker WV. A new assay for the detection of Loxosceles species (brown recluse) spider venom. Ann Emerg Med. 2002:39: 469-74. 12. Gomez HF, Miller MJ, Waggener MW, Lankford HA, Warren JS. Antigenic cross-reactivity of venoms from medically important North American Loxosceles spider species. Toxicon. 2001:39: 817-24. 13. Babcock JL, Civello DJ, Geren CR. Purification and characterization of a toxin from brown recluse spider (Loxosceles reclusa) venom gland extracts. Toxicon 1981: 19:677-89. 14. Weber K. and Osborn M. The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 1969:244: 4406-12. 15. Kurpiewski G, Forrester L., Barrett J and Campbell B. Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa. Biochemica et Biophysica Acta, 678 1981: 467-76. 16. Rekow MA, Civello DJ, Geren CR. Enzymatic and hemolytic properties of Brown Recluse spider (Loxoceles reclusa) toxin and extracts of venom apparatus, cephalothorax and abdomen. Toxicon 21:441-4. 17. Harlow E, Lane D. Antibodies: A laboratory manual. 1988; Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1988:726 pp. 18. Sams HH, Hearth SB, Long LL, Wilson DC, et al. Nineteen documented cases of Loxosceles reclusa envenomation. J Am Acad Dermatol. 2001a: 44: 603-8.

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

INTRODUCTION KEVIN F FOLEY INDEX TERMS: Cardiovascular risk, proBNP, CRP, CAD. Clin Lab Sci 2009;22(4);223 Kevin F. Foley PhD, MT is a clinical chemist, Kaiser Permanente NW Laboratories, Portland, OR. Address for Correspondence: Kevin F. Foley PhD, MT, Kaiser Permanente NW Laboratories, 13705 NE Airport Way Portland, OR, 97230 503 258-6902, [email protected]. Kevin F. Foley PhD, MT is the Focus: Cardiovascular Risk Assessment guest editor. There is no area of laboratory medicine that requires more interpretive knowledge than cardiovascular risk assessment. The clinical laboratory has moved far from the days when triglycerides, total cholesterol, HDL and a calculated LDL were the only laboratory tests that could be used to assess the cardiovascular risk for patients. In this series we will look at four different topics in cardiovascular laboratory medicine. One can think of cardiovascular laboratory medicine

The Focus section seeks to publish relevant and timely continuing education for clinical laboratory practitioners. Section editors, topics, and authors are selected in advance to cover current areas of interest in each discipline. Readers can obtain continuing education credit (CE) through P.A.C.E.® by completing the continuing education registration form, recording answers to the examination, and mailing a photocopy of it with the appropriate fee to the address designated on the form. Suggestions for future Focus topics and authors, and manuscripts appropriate for CE credit are encouraged. Direct all inquiries to the Clin Lab Sci Editorial Office, Westminster Publishers, 315 Westminster Court, Brandon MS 39047. (601) 214-5028, (202) 315-5843 (fax). [email protected].

as having two arms; tests used to assess cardiovascular risk, and tests used to assess myocardial infarct. Of course these two arms overlap significantly in that myocardial infarct is a major cardiovascular event for which we want to gauge risk. Measuring markers such as CK-MB or troponins allows us to confirm or rule out myocardial infarction. In contrast, risk markers are a tool we can use to assess a person’s risk for cardiac morbidity or mortality. When considering markers for myocardial infarct, most laboratorians know that CK-MB and troponin can provide valuable diagnostic and sometimes even prognostic information. But is it time to phase-out CK-MB testing given the performance and demonstrated superiority of troponin testing? In this series we review the use of troponin and CK-MB in contemporary laboratory practice. This journal also discusses the value and supporting data for genetic assessment of patients at risk for coronary artery disease. Clinical laboratorians are no doubt aware that a genetic component exists when predicting cardiovascular risk. The linkages, specific genes and utility of genetic testing with regard to cardiovascular disease is particularly timely for those in laboratory medicine given the increasing role that molecular diagnosis has on the clinical laboratory and the increasing overlap between genetic and conventional clinical chemistry testing. Given the multiple variables that can influence or contribute to cardiovascular disease, gene association studies aimed at identifying genetic risk factors can be difficult to design and interpret. The following article on genetic markers of coronary artery disease (CAD) attempts to review some recent findings in light of these challenges. A biomarker that can be used predict the risk of a future cardiovascular event would be a powerful tool for any physician given the global prevalence of heart VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 223

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

disease and atherosclerosis. There are dozens of cardiovascular risk markers which can, to various degrees, indicate cardiovascular pathology. In 2009 the National Academy of Clinical Biochemistry released practice guidelines for cardiovascular risk markers1. This expert panel evaluated clinical data associated with the risk markers listed in Table 1. Although there are many more putative risk markers than appear on this list, the list reflects those risk markers that have been most studied. As of now, only high sensitivity C-reactive protein (hsCRP) has been endorsed by the NACB as meeting all the risk marker requirements needed for routine clinical practice1. In this series we discuss another of the markers on this list: B-natriuretic peptide (BNP). BNP measurement has been available since 1997 and is found on most clinical chemistry laboratory testing menus. The value of BNP as a marker for heart failure is a current topic of debate and the clinical utility of this marker is discussed in this series2 Vitamin D testing volumes are increasing dramatically in clinical laboratories due to our increasing knowledge of the ubiquitous role that this vitamin/hormone appears to have in overall health. Studies showing that vitamin-D levels correlate with the prevention of osteoporosis, and measure risk for developing cancer, autoimmune disease and type 1 diabetes are plentiful3. In addition to these diseases, studies are now revealing that cardiovascular diseases may be associated, to varying degrees, with vitamin-D deficiency. Our series summarizes some of these recent findings in the context of cardiovascular risk. Although well-validated, sensitive and specific cardiovascular risk markers are valuable, risk can also be assessed without using novel cardiovascular risk biomarkers. The Framingham risk score is a proven tool used to gauge cardiovascular risk. Although the Framingham Heart Study has validated various clinical factors for several cardiovascular disease outcomes, general cardiovascular risk can be calculated based on a patient’s age, gender, total cholesterol, HDL, systolic blood pressure and a 224 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

person’s diabetic and smoking status. This calculated score will predict the likelihood of an adverse cardiac event in the subsequent ten years. Given this easy to use and well-validated risk formula, an obvious question to ask is ‘why do we need additional cardiovascular risk markers?’ The reason is multifold. It is possible that different populations of patients will have different patterns of risk markers which may correlate to different pathologies. For example, Lp(a) is an independent marker for cardiovascular disease yet in African American populations Lp(a) values tend to be as much as three times higher than those found in Caucasians.4 Thus one marker does not always work to the same degree in all populations. Further, risk prediction is a statistic, not a certain prognosis. Therefore with each new risk marker there is the potential that we can increase the predictive power of our risk estimates. Perhaps a biomarker exists which can strongly predict a cardiovascular event in the next year or month rather than in the next ten years. It is also likely that multiple risk markers, when used together can provide additional information to predict risk and guide therapy. For example, a recent study by Dai et al. showed that for major adverse cardiac events, elevated CRP had an odds ratio of 2.4 whereas elevated NT-proBNP carried an odds ratio of 5.25. When both risk markers were used, an odds ratio of 7.04 was found5. So despite the fact that traditional risk factors such as LDL, blood pressure and total cholesterol are cheap and readily assessed, we can likely increase our detection of cardiac pathologies and risk when we employ additional cardiovascular risk markers. Their application is most justified for those patients with intermediate risk: 10– 20% chance of an event in the next 10 years as measured by the Framingham risk score. Finally, many patients present with a major adverse cardiovascular event with no previous history of cardiovascular disease and lipid levels within the normal ranges recommended by the National Cholesterol Education Program. A risk marker that could be used to screen these patients so that effective

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

intervention could be initiated early on, would be invaluable. The landmark JUPITER study (The Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin) provides an example of how a novel cardiovascular risk marker can function.6 In this trial, individuals with low LDL cholesterol but elevated C-reactive-protein (CRP) levels were given rosuvastatin (Crestor), a cholesterollowering statin. The study found that this treatment significantly reduced the chances of nonfatal myocardial infarct, nonfatal stroke, hospitalization for unstable angina, revascularization, and confirmed death from cardiovascular causes. The treatment group had a risk reduction of 44% compared with placebo-treated individuals. This decrease was large enough to justify halting the study so that all patients could benefit from the treatment. This trial demonstrates that even in patients with normal LDL levels, cardiovascular risk may be present and can be reduced with conventional statin treatment. Studies such as this validate the use of cardiovascular risk markers, in this case, hsCRP; and show that such markers can drive changes in the standard of care to achieve better outcomes. Because of data such as these from the JUPITER study, the NACB practice guidelines support the use of hsCRP as a proven and validated biomarker for risk assessment in primary prevention. Although hsCRP is the only novel risk marker currently endorsed by the NACB, other biomarkers may provide significant predictive power, warranting future studies. An overview of the leading novel cardiovascular risk markers (Table 1) is not within the scope of this series. However we have endeavored to touch on several important areas in cardiovascular lab medicine: myocardial infarct markers, genetic testing, BNP (a risk marker and marker for heart failure) and the potential influence of Vitamin-D.

Table 1. Cardiovascular Risk Markers • • • • • • • • •

Apolipoprotein A1 Apolipoprotein B high sensitivity C-reactive protein (hsCRP) Fibrinogen White blood cell count Homocysteine B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) Lipoprotein (a) Lipoprotein subclasses and particle concentration

REFERENCES

1. Myers GL, Christenson RH, Cushman M, et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice guidelines: emerging biomarkers for primary prevention of cardiovascular disease. Clin Chem 2009;55:378–84. 2. Rollins G. The BNP debate. Clinical Laboratory News 2009;35:1. 3. Holick MF. Vitamin D status: measurement, interpretation, and clinical application. Ann Epidemiol. 2009;19:73–8. 4. Howard BV, Le NA, Belcher JD, et al. Concentrations of Lp(a) in black and white young adults: relations to risk factors for cardiovascular disease. Ann Epidemiol 1994;4:341–50. 5. Dai DF, Hwang JJ, Lin JL, et al. Joint effects of N-terminal pro-B-type-natriuretic peptide and C-reactive protein vs angiographic severity in predicting major adverse cardiovascular events and clinical restenosis after coronary angioplasty in patients with stable coronary artery disease. Circ J 2008;72:1316–23. 6. Ridker PM, Danielson E, Fonseca FA, et al. Rosuvastatin to prevent vascular events in men and women with elevated Creactive protein. N Engl J Med 2008;359:2195–207.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 225

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

Genetic Markers for Coronary Artery Disease LINNEA M BAUDHUIN INDEX TERMS: association, atherosclerosis, coronary artery disease, genetics, genome wide association, genomics, linkage, myocardial infarction

Linnea M. Baudhuin, PhD, DABMG is assistant professor of laboratory medicine, Mayo Clinic, Jacksonville, FL.

LEARNING OBJECTIVES Upon completion of this activity, participants should be able to: 1. Compare linkage studies with association studies and recognize aspects regarding the utility and limitations of these studies in identifying markers associated with complex disease. 2. Describe genome wide association studies and their advantages and disadvantages for identifying genetic markers. 3. Discuss various aspects regarding the 9p21 risk allele, its association to CAD, and its potential as a clinical marker for CAD. 4. Describe microRNAs and their potential utility as CAD markers. 5. Discuss aspects of genetic markers that would lend to their ability to enter the clinical realm as risk markers for complex disease.

Address for correspondence: Linnea M. Baudhuin, PhD, DABMG, Assistant Professor of Laboratory Medicine, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, (507) 284-2511, (507) 266-4176 (fax), baudhuin.linnea@ mayo.edu . The hereditary component of coronary artery disease (CAD) is widely recognized. However, identifying clinically useful genetic markers for a complex disease like CAD has been challenging. Linkage-based and association-based genetic studies have pointed to some interesting findings, but many of these studies have lacked reproducibility or statistical significance. Recently, genome-wide association (GWA) and microRNA discoveries have uncovered some potentially promising new markers for CAD. The current status of genetic markers for CAD and their utility in the clinical arena are summarized here.

Clin Lab Sci 2009;22(4);226

The Focus section seeks to publish relevant and timely continuing education for clinical laboratory practitioners. Section editors, topics, and authors are selected in advance to cover current areas of interest in each discipline. Readers can obtain continuing education credit (CE) through P.A.C.E.® by completing the continuing education registration form, recording answers to the examination, and mailing a photocopy of it with the appropriate fee to the address designated on the form. Suggestions for future Focus topics and authors, and manuscripts appropriate for CE credit are encouraged. Direct all inquiries to the Clin Lab Sci Editorial Office, Westminster Publishers, 315 Westminster Court, Brandon MS 39047. (601) 214-5028, (202) 315-5843 (fax). [email protected].

226 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

Coronary artery disease (CAD) is a multifactorial disease that can be influenced by a multitude of environmental and heritable risk factors. While there are many traditional and novel analyte risk markers associated with CAD, a large gap for CAD risk prediction remains. Epidemiological evidence points to an approximate 50% genetic susceptibility to CAD. Many different genetic associations with CAD have been identified through family and populationbased analyses, and genetic risk markers may be important for better defining individuals at risk for CAD and CV events. Some potentially promising and interesting markers produced from such studies are highlighted here.

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

Linkage Analysis and CAD Linkage analyses are family-based studies investigating genetic variants that segregate with disease in affected vs. non-affected family members. Multiple linkage studies have been performed in CAD cases, investtigating the occurrence of myocardial infarction (MI) or subclinical atherosclerosis.1 While several loci of interest have been identified through linkage studies, the majority of them have not been replicated or directly implicated in CAD. One gene that was identified through linkage studies is the MEF2A gene, encoding the transcription factor myocyte enhancer factor 2A. A seven amino acid sequence deletion from exon 12 of MEF2A was identified as the causative mutation in a large autosomal dominant CAD-MI family.2 Subsequent studies have variably confirmed or refuted the involvement of MEF2A in CAD and/or MI.3-6 The lack of consistency in findings could point to variable penetrance of the alleles, modifier gene effects, or other genetic variants within the linkage interval. In a linkage study of Icelandic families, the ALOX5AP gene [encoding 5-lipoxygenase activation protein (FLAP)] was identified for its association with MI (OR = 1.8) and stroke (OR = 1.9).7 FLAP is involved in the biosynthesis of leukotrienes, which promote chemotaxis and increase vascular permeability. Further fine mapping identified an association with leukotriene A4 hydrolase (LTA4H) in the Icelandic cohort.8 It has been, however, challenging to determine which specific ALOX5AP SNPs or haplotypes are associated with MI in various ethnic groups and the association between these leukotrieneassociated genes and MI has not been consistently replicated.9-12 Nonetheless, several studies have highlighted a potentially key role for leukotrienes in atherosclerosis. Importantly, a small molecular FLAPinhibitor had the ability to reduce both leukotriene production and C-reactive protein (a CAD biomarker) levels in a placebo-controlled, randomized trial.13

Candidate Gene Association Analysis and CAD

Candidate gene association analyses are populationbased studies investigating variants in candidate genes (identified via linkage studies or based on biological knowledge) that segregate with disease in cases and controls. Many candidate genes have been investtigated for association with CAD, including genes coding for apolipoproteins (e.g. APOE), matrix metalloproteinases, paraoxonase, cytokines, and other proteins involved in coagulation, blood pressure or lipoprotein regulation (e.g. PCSK9), and/or atherosclerotic processes.

Similar to linkage analyses, candidate gene association analyses have been hampered by the general inability to replicate findings in follow-up studies. A genetic analysis of 103 candidate genes in a cohort of 1400 individuals from a founder population demonstrated a lack of association with CAD status.14 Similar observations were made in a study of 70 candidate genes in 811 acute coronary syndrome patients.9 The lack of reproducibility speaks to both the complex nature of CAD and the design flaws of candidate gene studies, which are often single-gene, underpowered studies that may be, in part, afflicted with false positive associations. Because of the limitations of these types of studies, as well technological and genomic haplotype mapping advances, the field has been moving towards large-scale genome-wide (GWA) association studies.

Genome-wide Association (GWA) Studies and CAD GWA studies are unbiased large-scale populationbased studies evaluating the association of hundreds of thousands of markers, generally single nucleotide polymorphisms (SNPs), across the genome with a particular phenotype. Perhaps one of the more interesting loci that has been identified in multiple GWA CAD studies, and confirmed in numerous follow-up case-control analyses is the locus at chromosome 9, band p21.3.15-22 Markers at the 9p21 locus have been shown to give a 15–20% increased risk for CAD in the 50% of Caucasian individuals VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 227

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

heterozygous for the allele, and a 30–40% increased risk of CAD in the 25% of individuals homozygous for the allele.16 A meta-analysis of case-control studies showed that the odds ratio per copy of the 9p21 risk allele was 1.29 (95% CI 1.22-1.37, p=0.0079).21 In addition to CAD, the 9p21.3 locus has been associated with stroke, abdominal aortic aneurysms, and intracranial aneurysms, suggesting potential involvement for the 9p21 risk allele with plaque stability and/or vessel wall integrity processes.21, 23-28 While the 9p21 association with CAD has been replicated on multiple occasions, the biological relevance of 9p21 is unclear at this time. The 9p21.3 risk-allele locus spans 50–60 kb and is in linkage disequilibrium (LD) with the 3’ end of CDKN2B, encoding the cyclin-dependent kinase inhibitor tumor suppressor p15INK4B, with weaker LD extending through CDKN2B to CDKN2A, which encodes another tumor suppressor p16INK4B. The cyclin dependent kinases are involved in cell cycle regulation and transforming growth factor-β (TGF-β) cell cycle arrest.29 TGF-β has been shown to have impaired signaling and reduced expression in atherosclerotic lesions, overexpression in abdominal aortic aneurysms, and variable expression in different stages of plaque development.30-32 A gene encoding a large antisense non-coding RNA (ANRIL) spans almost the entire 9p21-CAD association region.33 A speculated mechanism for the 9p21 risk allele involves antisense regulation of CDKN2B (and/or CDKN2A), potentially affecting signaling of TGF-β and/or additional cytokine(s) involved in cell cycle arrest/proliferation.23, 25, 34 Recently, a SNP in the 9p21 risk allele was shown to be associated with significantly reduced expression of CDKN2B, CDKN2A, and ANRIL.35 Other loci potentially involved in CAD that have been identified through multiple, independent GWA studies and confirmed in follow-up association analyses include 1p13, 1q41, and 10q11.15, 17, 19, 20 The 1p13 risk allele is in a 97-kb region of LD containing the CELSR2, PSRC1, and SORT1 genes, and has been 228 VOL22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

found to be strongly associated with low-density lipoprotein (LDL) and total cholesterol concentrations.36-42 Of particular interest in this gene cluster is SORT1, which encodes sortilin, a pro-neurotrophin receptor involved in adipocyte and muscle glucose metabolism. Sortilin is downregulated in obesity and has been implicated in insulin resistance.43 Examples of other CAD-associated loci identified through GWA studies include 19p13 (LDLR), 1p32 (PCKS9), and 12q23-24 (DRIM, SH2B3, HNF1A-C12orf43).22 Additionally, a genome-wide haplotype analysis utilizing a sliding-windows approach identified a haplotype of four SNPs in the SLC22A3-LPAL2-LPA gene cluster associated with CAD.44 LPA, encodes apolipoprotein (a), the protein component of lipoprotein (a) [Lp(a)] which is associated with an increased risk for CAD and MI. In addition to the loci described here, other loci associated with atherosclerosis-related phenotypes have been identified via GWA studies and are summarized in Table 1.

MicroRNA

MicroRNAs (miRNAs) are endogenous, small (approximately 22 nucleotides), non-coding RNAs that modulate gene expression and have been shown to play roles in cardiovascular disease pathogenesis, including cardiac hypertrophy, heart failure, and myocardial infarction.45, 46 Recently, it was shown that members of the miR-29 family are downregulated in acute MI in mice and humans.46 It was further shown that miR-29 downregulation leads to enhanced fibrotic response, indicating a role for miR-29 family members in cardiac fibrosis, an important aspect of post-MI remodeling. Since miRNAs are expressed in a tissue-specific manner, miRNA-based diagnostics in the clinical setting may be the most immediately available and applicable tests in diseases where tissue is readily available and regularly examined, such as in cancer. However, it is now being recognized that serum-based miRNA biomarker diagnostic tests may have great value in cancer and other disease states.47 In the

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

Table 1. Genome-Wide Association Studies of Atherosclerosis-related Phenotypes (p5% prevalence, these types of studies are not very useful for identifying less common variants (50 years were accepted to yield appropriate sensitivity and specificity for establishing acute HF (BNP 100 pg/mL, sensitivity = 90%, specificity = 76%; NT-proBNP 900 pg/mL, sensitivity = 90%, specificity = 85%). Both groups warn that since BNP levels are higher in women and people over 60, clinicians should evaluate BNP levels in relation to patient demographics. Additionally, they recommended investigating the possibility of HF in patients with elevated BNP and suitable symptoms. BNP levels correlate with the staging of HF. As Table 1 demonstrates, BNP levels increase as patients’ progress in NYHA classification level. Although reference intervals differ across institutions, values of 200–400 pg/mL generally correspond to moderate HF while values > 400 pg/mL indicate severe HF. 236 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

However, there is wide variation of BNP levels for patients within a NYHA functional class and overlap between them. Specific reference intervals are recommended and are available which consider both gender and age. Beta-natriuretic Peptide; Current Research Studies in 2008–2009 have investigated the utility of BNP or NT-proBNP for HF prognosis27-29, to screen elderly populations for early signs of HF30, to guide HF therapy31 or to evaluate conditions other than HF32, 33. Both the ACC/AHA and NACB guidance committees published guidance in 2009 on the proper role of BNP and NT-proBNP in these situations3,34. While both committees endorse continued research and note some promising studies, they recommend that BNPs not be used for assessment of cardiovascular disease or HF risk. Although BNPs have a role in HF assessment, they have not yet been validated as cardiovascular risk markers in the same sense as hsCRP. Since 1998, BNP and NT-proBNP have become more important in the assessment of potential HF pa-

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

Table 1. Classification and staging systems A. NYHA HF Classification by Functional Capacity Class I: Patients with cardiac disease but without l imitation of physical activity.

Median BNP Concentration40 83.1 pg/mL

Class II: Patients with cardiac disease resulting in slight limitation of physical activity. Comfortable at rest. Symptomatic with ordinary physical activity.

235 pg/mL

Class III: Patients with cardiac disease resulting in marked limitation of physical activity. Comfortable at rest. Less than ordinary activity causes symptoms.

459 pg/mL

Class IV: Patients with cardiac disease resulting in symptoms even at rest.

1119 pg/mL

B. ACC/AHA staging system Objective assessment based on evidence of cardiovascular disease A: No evidence of CV disease B: Evidence of minimal CV disease C: Evidence of moderate CV disease D: Evidence of severe CV disease

tients. It has also become increasingly apparent that our methods for quantifying these peptides need to be more fully characterized. The biochemistry of BNP translation, secretion and proteolytic processing was reviewed in 200835. The proteases corin and furin were shown to cleave proBNP into BNP and NTproBNP. Furin is a membrane-bound protease. Corin is expressed in the Golgi and shuttles between the Golgi and the plasma membrane. Thus, NT-proBNP could be processed during maturation by the Golgi or during secretion into the circulation. Recent reports have shown that intact, unprocessed proBNP circulates36, as do proteolytically clipped and glycolsylated forms of NT-proBNP37,38. There are also reports of proteolytically clipped molecules derived from BNP circulating in blood39. Due to these post-translational modifications and proteolytic cleavage events, laboratory tests differ in their detection of the circulating forms of BNP and NT-proBNP. This difference introduces uncontrolled error between tests. Additionally, it leads to a decrease

in the correlation between what we measure and what we’d like to measure. Finally, it raises the question of which species of BNP would be best to measure. There is much work to be done to ascertain and define the complete role for BNPs in the evaluation and treatment of HF. Areas of research include identifying and characterizing the different species of BNPs and their interaction with various commercial tests that are in use in the laboratory. Additionally, research into the use of BNPs in evaluating other cardiac conditions as well as in the prognosis and potential to guide therapeutic decisions will continue. Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this Focus section. Email responses to westminsterpublishers @comcast.net. In the subject line, please type “CLIN LAB SCI 22(4) FO CARDIOVASCULAR RISK”. Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 237

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

edited for length and clarity. We look forward to hearing from you. REFERENCES

1. Abraham WT, Ayesha H, Poole-Wilson P. Chapter 26. Diagnosis and Management of Heart Failure. In: Fuster V, O'Rourke R, RA W, Poole-Wison P, eds. Hurst's The Heart. 12th Edition ed. [Internet]. New York: McGrawHill; 2009 May [cited 2009 Jun 1]. Available from: http:// www.accessmedicine.com/content.aspx?aID=3065126. 2. Francis GS, Sonnenblick EH, Tang WHW, Poole-Wilson P. Chapter 24: Pathophysiology of Heart Failure. In: Fuster V, O'Rourke RAW, Richard A., Poole-Wilson, Philip, eds. Hurst's The Heart, 12th Edition. 12 ed. [Internet]. New York: McGraw-Hill; 2009 Apr [cited 2009 Jun 1]. Available from: http://www.accessmedicine.com/ content.aspx?Aid =3060544 3. Hunt SA, Abraham WT, Chin MH, Feldman AM, et al, American College of Cardiology F, American Heart A. 2009 Focused update incorporated into the ACC/AHA 2005 Guidelines for the Diagnosis and Management of Heart Failure in Adults A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines Developed in Collaboration With the International Society for Heart and Lung Transplantation. J Am Coll Cardiol. 2009;53(15):e1-e90. 4. Lloyd-Jones D, Adams R, Carnethon M, De Simone G, et al, American Heart Association Statistics Committee and Stroke Statistics S. Heart disease and stroke statistics--2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.[erratum appears in Circulation. 2009 Jan 27;119(3):e182]. Circulation. 2009;119(3):480-86. 5. Association. TCCotNYH. Diseases of the heart and blood vessels: nomenclature and criteria for diagnosis. Boston: Little, Brown; 1964. 6. Association TCCotNYH. Diseases of the Heart and Great Vessels: Nomenclature and criteria for diagnosis. 9th ed. Boston: Little, Brown & Co; 1994. 7. Hunt SA, Abraham WT, Chin MH, Feldman AM, et al, American College of C, American Heart Association Task Force on Practice G, American College of Chest P, International Society for Heart and Lung T, Heart Rhythm S. ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation. 2005;112(12):e154-235.

238 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

8. Dar O, Cowie MR. Chapter 25: The Epidemiology and Diagnosis of Heart Failure. In: Furster V, O'Rourke R, Walsh R, Poole-Wilson P, eds. Hurst's The Heart. 12th ed. [Internet]. New York: McGraw-Hill; 2009 May [cited 2009 Jun 1]. Available from: http://www.accessmedicine.com/ content.aspx?aID=3046486 9. Tang WHW, Francis GS, Morrow DA, Newby LK, et al, National Academy of Clinical Biochemistry Laboratory M. National Academy of Clinical Biochemistry Laboratory Medicine practice guidelines: Clinical utilization of cardiac biomarker testing in heart failure. Circulation. 2007;116(5):e99-109. 10. Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature. 1988;332(6159):78-81. 11. Protter AA, Wallace AM, Ferraris VA, Weishaar RE. Relaxant effect of human brain natriuretic peptide on human artery and vein tissue. Am J Hypertens. 1996;9(5):432-36. 12. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci. 1981;28(1):89-94. 13. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Reprinted from Life Sci. 28:89-94, 1981. J Am Soc Nephrol. 2001;12(2):403-409; discussion 403-8. 14. Kisch B. A Sginificant Electron Microscopic Difference between the Atria and the Ventricles of the Mammalian Heart. Experimental Medicine & Surgery. 1963;21:193221. 15. de Bold AJ, Raymond JJ, Bencosme SA. Atrial specific granules of the rat heart: light microscopic staining and histochemical reactions. J Histochem Cytochem. 1978; 26(12):1094-102. 16. de Bold AJ. Heart atria granularity effects of changes in water-electrolyte balance. Proc Soc Exp Biol Med. 1979;161(4):508-11. 17. Sudoh T, Minamino N, Kangawa K, Matsuo H. Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain. Biochem Biophys Res Commun. 1988;155(2):726-32. 18. Minamino N, Aburaya M, Ueda S, Kangawa K, Matsuo H. The presence of brain natriuretic peptide of 12,000 daltons in porcine heart. Biochem Biophys Res Commun. 1988;155(2):740-6. 19. Saito Y, Nakao K, Itoh H, Yamada T, et al. Brain natriuretic peptide is a novel cardiac hormone. Biochemical and Biophysical Research Communications. 1989;158(2):360-8. 20. Goetze JP. Biochemistry of pro-B-type natriuretic peptidederived peptides: the endocrine heart revisited.[see comment]. Clin Chem. 2004;50(9):1503-10.

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

21. Burnett JC, Jr., Kao PC, Hu DC, Heser DW, et al. Atrial natriuretic peptide elevation in congestive heart failure in the human. Science. 1986;231(4742):1145-7. 22. Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, Shirakami G, Jougasaki M, Obata K, Yasue H, et al. Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest. 1991;87(4):1402-12. 23. Cioffi G, Tarantini L, Stefenelli C, Azzetti G, et al. Changes in plasma N-terminal proBNP levels and ventricular filling pressures during intensive unloading therapy in elderly with decompensated congestive heart failure and preserved left ventricular systolic function. J Card Fail. 2006;12(8):60815. 24. Barclay JL, Kruszewski K, Croal BL, Cuthbertson BH, et al. Relation of left atrial volume to B-type natriuretic peptide levels in patients with stable chronic heart failure. Am J Cardiol. 2006;98(1):98-101. 25. Maisel A. B-type natriuretic peptide levels: diagnostic and therapeutic potential. Cardiovasc Toxicol. 2001;1(2):15964. 26. Troughton RW, Prior DL, Pereira JJ, Martin M, et al. Plasma B-type natriuretic peptide levels in systolic heart failure: importance of left ventricular diastolic function and right ventricular systolic function. J Am Coll Cardiol. 2004;43(3):416-22. 27. Pascual-Figal DA, Penafiel P, de la Morena G, Redondo B, et al. Relation of B-type natriuretic peptide levels before and after exercise and functional capacity in patients with idiopathic dilated cardiomyopathy. Am J Cardiol. 2007;99(9):1279-83.. 28. Waldo SW, Beede J, Isakson S, Villard-Saussine S, Fareh J, Clopton P, et al. Pro-B-type natriuretic peptide levels in acute decompensated heart failure. J Am Coll Cardiol. 2008;51(19):1874-82. 29. Miller WL, Hartman KA, Grill DE, Burnett JC, Jr., Jaffe AS. Only large reductions in concentrations of natriuretic peptides (BNP and NT-proBNP) are associated with improved outcome in ambulatory patients with chronic heart failure. Clin Chem. 2009;55(1):78-84. 30. Eggers KM, Lindahl B, Venge P, Lind L. B-type natriuretic peptides and their relation to cardiovascular structure and function in a population-based sample of subjects aged 70 years. Am J Cardiol. 2009;103(7):1032-8. 31. Godkar D, Bachu K, Dave B, Niranjan S, Khanna A. B-type natriuretic peptide (BNP) and proBNP: role of emerging markers to guide therapy and determine prognosis in cardiovascular disorders. Am J Ther. 2008;15(2):150-6. 32. Hejmdal A, Boesgaard S, Lindholm MG, Goetze JP. B-type natriuretic peptide and its molecular precursor in myocardial infarction complicated by cardiogenic shock. J Card Fail. 2007;13(3):184-8. 33. Garrido IP, Pascual-Figal DA, Nicolas F, Gonzalez-Carrillo MJ, et al. Usefulness of serial monitoring of B-type

34.

35. 36.

37.

38.

39.

40.

natriuretic peptide for the detection of acute rejection after heart transplantation. Am J Cardiol. 2009;103(8): 1149-53. Members NLC, Myers GL, Christenson RHM, Cushman M, Ballantyne CM, et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice guidelines: emerging biomarkers for primary prevention of cardiovascular disease. Clin Chem. 2009;55(2):378-84. Mair J. Biochemistry of B-type natriuretic peptide--where are we now? Clin Chem Lab Med. 2008;46(11):1507-14. Hammerer-Lercher A, Halfinger B, Sarg B, Mair J, et al. Analysis of Circulating Forms of proBNP and NT-proBNP in Patients with Severe Heart Failure. Clinical Chemistry. 2008;54(5):858-65. Ala-Kopsala M, Ruskoaho H, Leppaluoto J, Seres L, Skoumal R, Toth M, Horkay F, Vuolteenaho O. Single assay for amino-terminal fragments of cardiac A- and B-type natriuretic peptides. Clin Chem. 2005;51(4):708-18. Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, et al Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin Chem. 2008;54(5):866-73. Brandt I, Lambeir A-M, Ketelslegers J-M, Vanderheyden M, et al Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin Chem. 2006;52(1):82-7. Wieczorek SJ, Wu AHB, Christenson R, Krishnaswamy P, et al. A rapid B-type natriuretic peptide assay accurately diagnoses left ventricular dysfunction and heart failure: a multicenter evaluation. Am Heart J. 2002;144(5):834-9.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 239

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

HYPOVITAMINOSIS D: A NEW RISK MARKER FOR CARDIOVASCULAR DISEASE JOSEPH P MCCONNELL, KEVIN F FOLEY, GINA M VARGAS INDEX TERMS: vitamin D, cholecalciferol, hypovitaminosis, 25-hydroxyvitamin D.

Laboratory Medicine, The Mayo Clinic and Foundation, Rochester, MN.

LEARNING OBJECTIVES 1. Differentiate and define the various forms of vitamin-D, including Vitamin D2, D3 25(OH)D and 1,25(OH2)D. 2. Describe current opinion concerning vitamin-D dosing recommendations as well as issues around establishing a normal reference range. 3. Identify several diseases and pathologies for which hypovitaminosis D has been implicated. 4. Explain the correlations between serum 25(OH)D levels and the cardiovascular risk factors of hypertension and metabolic syndrome. 5. Describe the overall relationship between hypovitaminosis D and cardiovascular disease and the need for future studies to demonstrate causality.

Kevin F. Foley PhD, MT is a clinical chemist, Kaiser Permanente NW Laboratories, Portland, OR.

Clin Lab Sci 2009;22(4);240 Joseph P. McConnell PhD, is director, Cardiovascular

The Focus section seeks to publish relevant and timely continuing education for clinical laboratory practitioners. Section editors, topics, and authors are selected in advance to cover current areas of interest in each discipline. Readers can obtain continuing education credit (CE) through P.A.C.E.® by completing the continuing education registration form, recording answers to the examination, and mailing a photocopy of it with the appropriate fee to the address designated on the form. Suggestions for future Focus topics and authors, and manuscripts appropriate for CE credit are encouraged. Direct all inquiries to the Clin Lab Sci Editorial Office, Westminster Publishers, 315 Westminster Court, Brandon MS 39047. (601) 214-5028, (202) 315-5843 (fax). [email protected]. 240 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

Gina M. Vargas PhD, is director, Importadora y Laboratorio Clinico ATM SRL: Cochabamba, Bolivia Address for Correspondence: Joseph P. McConnell PhD, Mayo Clinic; Cardiovascular Laboratory Medicine, Rochester, Minnesota, (507) 284-0524, Fax: (507) 266-2888, Email: [email protected] INTRODUCTION Vitamin D has a well-established role in calcium and phosphorus metabolism and bone mineralization. Vitamin D deficiency causes rickets in children, and in adults can lead to osteomalacia, resulting in muscle and bone weakness. Data are emerging that link hypovitaminosis D, as assessed by measurement of 25-hydroxyvitamin D [25(OH)D], with cardiovascular pathology. Vitamin D deficiency has been associated with hypertension, some inflammatory markers, and metabolic syndrome. More recently, low serum 25(OH)D has been associated with increased incidence of cardiovascular events and all-cause mortality. In this review, we discuss the role of vitamin D in health, and describe recent evidence linking hypovitaminosis D to cardiovascular disease. We describe controversies surrounding recommended daily intake and optimal serum levels, as well as discuss the need for further research relating vitamin D deficiency with cardiovascular disease. Vitamin D deficiency Vitamin D, which has also been referred to as the “sunshine vitamin” is a lipid-soluble vitamin obtained from both exogenous and endogenous sources. Some

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

Vitamin D2 and D3 are biologically inactive and are activated by two hydroxylation reactions, the first occurring in the liver to form 25(OH)D and the second occurring in the kidney to form the biologically active 1,25-dihydroxyvitamin D [1,25 (OH)2D] (see Figure 1). Since only the hydroxylated forms are active and because these active molecules are produced within the body and have effects in multiple tissues and cell types, it is more accurate to consider 25(OH)D and 1,25(OH2)D as hormones rather than vitamins.

foods, such as eggs, fatty fish, and liver naturally contain vitamin D, but other dietary sources of vitamin D are from fortified foods, like milk and cereals, or from nutritional supplements1. Most of the body’s vitamin D is produced endogenously following exposure of skin to sunlight, thus geography, season, skin tone, and sun exposure are primary predictors of vitamin D nutritional status2. Vitamin D obtained from sun is in the form of vitamin D3 (cholecalciferol), while vitamin D2 (ergocalciferol) or D3 may be obtained from dietary sources. Vitamin D2 differs from vitamin D3 in that it is derived from dietary ergosterol; it contains a double bond between carbon 22 and 23 and it has an additional methanol group at position 24, while vitamin D3 is produced by the action of sunlight on 7-dehydrocholesterol. Both

The main function of vitamin D is to promote calcium absorption in the gut and to maintain adequate blood levels of calcium and phosphorus. If the body is deficient in vitamin D, normal bone

HO

HO

Vitamin D3 (Cholecalciferol)

Vitamin D2 (Ergocalciferol)

OH HO

OH

OH

HO

HO HO

HO

HO

25-Hydroxy Vitamin D2 (Ercalcidiol)

1-alpha, 25-Dihydroxy Vitamin D2 (Ercalcitriol)

HO

25-Hydroxy Vitamin D3 (Calcidiol)

1-alpha, 25-Dihydroxy Vitamin D3 (Calcitriol)

Figure 1. Inactive and active forms of Vitamin D2 and D3.

VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 241

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

mineralization is compromised and bones may become thin and brittle. Severe deficiency can lead to rickets, with associate skeletal deformities, in children and osteomalacia in adults, which results in both weak bones and muscles. Despite the clear association between vitamin D deficiency and bone disease, much controversy still exists regarding appropriate vitamin D doses needed to avoid disease. Currently, the US Institute of Medicine-National Academy of Sciences recommends a daily intake of 200 IU (5 mg) for individuals < 50 years of age, 400 IU (10 mg) for those 50 to 60 years, and 600 IU (15 mg) for those over 70 years of age.3 However, many experts are recommending that higher daily intakes would be beneficial to most adults. A meta-analysis of 13 randomized placebo controlled studies for hip fracture and non-vertebral fracture risk demonstrated that vitamin D supplementation between 700–800 IU/day reduced the risk of fractures while a dose of 400 IU/day was not sufficient for fracture prevention.4 The current upper limit of vitamin D intake is 2000 IU/day due to toxicities that may occur at higher doses. Toxicities are rare but are associated with symptoms caused by marked hypercalcemia. Many have also suggested that the 2000 IU/day recommendation is too conservative. A review of recent clinical trials by Hathcock, et.al.5, applying risk assessment methodology used by the Food and Nutrition Board, demonstrated an absence of toxicity in trials conducted on healthy individuals using a vitamin D dose of ≥ 10,000 IU/day. The authors suggested this value could be used as the upper limit. However concerns about possible toxicity lead most patients and clinicians to use more conservative dosing regimens. Vitamin D intoxication has been clearly documented in experimental studies in animals and in human case reports.6 Hypervitaminosis D is associated with increased absorption of calcium and phosphorus, which can lead to hypercalcemia, hypercalciuria, vascular calcification, renal, and even renal failure. It is important to note that most case reports of vitamin D toxicity have resulted calcification from vitamin D intake far above 10,000 IU/day (5). 242 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

Most of the attention concerning vitamin D has been generated over the assessment of vitamin D deficiency. Vitamin D nutritional status is best determined by measurement of 25(OH)D.7 At the 13th workshop consensus for vitamin D nutritional guidelines in 2007, studies were summarized indicating that adults should maintain a blood 25(OH)D concentration of approximately 75 nmol/L (30 ng/mL).8 There was general consensus that serum vitamin D concentration should meet, or hopefully exceed a minimum desirable concentration of 50 nmol/L (20 ng/mL). Using these levels, it has been estimated that as many as half of the elderly persons in North America are not getting adequate vitamin D to facilitate healthy bone density.8 In addition to its primary action on bone metabolism, it is now clear that vitamin D has many non-skeletal actions, and that vitamin D deficiency may be associated with chronic diseases including cancer, autoimmune disease, cardiovascular disease and even psychological illnesses such as schizophrenia and depression.9 Here we describe some of the more recent findings associating vitamin D deficiency with various aspects of cardiovascular disease. Vitamin D deficiency has been associated with hypertension, inflammatory markers, diabetes, and the metabolic syndrome, and has recently been associated with increased cardiovascular events and all cause mortality. Vitamin D and Hypertension It is well documented that hypertension is a risk factor for cardiovascular disease. For most individuals with hypertension, the cause is unknown and its origin is likely multifaceted. It has been hypothesized that vitamin D deficiency is associated with hypertension, possibly through activation of the renin-angiotensin system. This is due to the finding that 1,25(OH)2D is a negative endocrine regulator of the renin-angiotensin system.10 A study describing data from the third National Health and Nutrition Examination Survey (NHANES, 1988-1992) including 16,135 participants > 19 years of age, found that systolic blood pressure (SBP) is inversely

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

associated with serum vitamin D concentrations in nonhypertensive white persons in the United States.11 Although adjusting for age resulted in loss of significance for the association, concentrations of 25(OH)D > 80 nmol/L decreased the age-related increase in SBP by 20% compared with individuals having 25(OH)D < 50 nmol/L. The authors suggest that future studies should be undertaken to determine the effects of vitamin D supplementation on hypertension.11 Vitamin D, Diabetes, and the Metabolic Syndrome The association between diabetes and cardiovascular disease is well-established and the presence of diabetes is considered as a coronary heart disease risk equivalent in the National Cholesterol Education Program Adult Treatment Panel III guidelines.12 A body of research now indicates that vitamin D may play a role in insulin resistance13 and diabetes.14 Although many studies have been published, a few will be highlighted here. Hyppönen and colleagues collected data on the dose and frequency of vitamin D supplementation during the first year of life in 10,366 children born in 1966.14 By the end of December 1997, 81 of the 10,366 children were diagnosed with diabetes during the study. Regular supplementation with vitamin D during the first year of life was associated with a decreased frequency of type I diabetes (rate ratio = 0.12, 95% CI = 0.030.51) even after adjustment for neonatal anthropometric and social characteristics. In 15,088 participants (7186 male and 7902 female) from the NHANES III who had serum 25(OH)D measures, the adjusted prevalence of diabetes mellitus was significantly higher in the first than in the fourth quartile of serum 25(OH)D levels (OR = 1.98, P 15 ng/mL. In a nested case control analysis of The Health Professionals Follow-up Study 18,225 men (age 4075 years) were followed for 10 years.28 Nonfatal myocardial infarction or fatal coronary heart disease occurred in 454 men. Men with 25(OH)D concentrations < 15 ng/mL were at increased risk for myocardial infarction when compared to 900 controls without events who were matched for age, date of blood collection, and smoking status and had 25(OH)D values > 30 ng/ml (relative risk 2.09, 95% CI 1.24-3.54). This difference was significant even after adjustment for family history, lifestyle, laboratory, and other risk factors. Most recently, Vitamin D deficiency has been linked with both cardiovascular mortality and all cause mortality. In a prospective cohort study, 3256 consecutive male and female patients scheduled for coronary angiography were followed for a median of 7.7 years with 737 deaths including 463 from cardiovascular causes.29 Compared to patients with 25(OH)D concentrations in the highest quartile (median 28.4 ng/mL or 71 nmol/L), patients in the lower two quartiles (median 7.6 and 13.3 ng/mL or 19.0 and 33.3 nmol/L) had higher hazard ratios for all cause mortality (HR = 2.08, 95% CI = 1.60-2.70 and HR = 1.53, 95% CI = 244 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

1.17-2.01, respectively) and for cardiovascular mortality (HR = 2.22, 95% CI = 1.57-3.13 and HR = 1.82, 95% CI = 1.29-2.58, respectively). Although these studies demonstrate significant associations between 25(OH)D concentrations and cardiovascular events, they do not confirm a direct causal relationship. Consideration should be given to whether vitamin D deficiency is a cause or an effect of cardiovascular disease. Groups at risk for developing adverse cardiovascular events include older adults and individuals with obesity and a sedentary lifestyle who have poor dietary habits. These same individuals are likely at risk for vitamin D deficiency because their sedentary life style includes limited sun exposure and poor dietary habits that may result in decreased intake of vitamin D. Determining a causal effect between vitamin D deficiency and cardiovascular events would require large prospective trials designed to demonstrate that correcting vitamin D deficiency reduces adverse cardiovascular events. Monitoring Vitamin D status Because the prevalence of vitamin D deficiency in the general population is been well-publicized, laboratory requests for the measurement of vitamin D have increased dramatically. The most widely used indicator of vitamin D status is 25(OH)D concentration in plasma or serum. However, several studies have reported discrepancies between the assays used.30,31 Methods used include low and high throughput RIAs, automated chemiluminescent immunoassays, HPLC, and liquid chromatography tandem mass spectrometry (LC-MS/MS).7 Although the reasons for the noted discrepancies in methods are not fully understood, possible causes include the ability of the assays to respond equally to 25(OH)D2 and 25(OH)D3 as well as the lack of a standard reference material (SRM). It is also important to note that some labs and assays report total vitamin-D whereas others discriminate between 1,25(OH2)D and 25(OH)D. In response to the apparent lack of standardization, the National Institute of Standards and Technology (NIST) and the National Institutes of Health’s Office of Dietary Supplements have been

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

working together to develop a reference material consisting of four pools of fresh-frozen serum, each pool having different concentrations of 25(OH)D2, 25(OH)D3 and one pool containing the recently discovered metabolite, 3-epi-25(OH) D3.32 This new SRM should be very helpful as it can be utilized by investigators to validate new methods, as well as serve as a reproducible point of comparison for existing methods, facilitating method standardization. CONCLUSIONS Vitamin D deficiency has been associated with hypertension, inflammatory markers, diabetes, metabolic syndrome, and has recently been associated with increased cardiovascular events and all cause mortality. This is an exciting finding, as it leads to the possibility that correction of vitamin D deficiency could improve cardiovascular outcomes. However, although lower concentrations of vitamin D have been associated with adverse outcomes, we do not yet have prospective controlled studies demonstrating that supplementation of vitamin D in patients with apparent deficiency is associated with improved outcomes. Although observational studies suggest this may be the case, caution should be taken not to draw causal inferences from observational studies, even if the sample size is large and the duration of follow-up long. We need only to be reminded about the results of studies looking at folate/B vitamin supplementation and hormone replacement therapy to realize the importance of carrying out such studies. Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this Focus section. Email responses to westminsterpublishers @comcast.net. In the subject line, please type “CLIN LAB SCI 22(4) FO CARDIOVASCULAR RISK”. Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be edited for length and clarity. We look forward to hearing from you. REFERENCES 1. Endres DB and Rude RK, in Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th edition. Burtis CA, Ashwood ER, Bruns DE, eds. 2008; 1920–6.

2. Bischoff-Ferrari H, Boucher BJ, et.al. The urgent need to recommend an intake of vitamin D that is effective. Am J Clin Nutr 2007;85:649–50. 3. Food and Nutrition Board, Institute of Medicine. Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Washington, DC: National Academy Press, 1997. 4. Bischoff-Ferrari HA, Willett WC, Wong JB, et al. Fracture prevention with vitamin D supplementation: a meta-analysis of randomized controlled trials. JAMA 2005;293:2257–64. 5. Hathcock JN, Shao A, Vieth R, Heaney R. Risk assessment for vitamin D. Am J Clin Nutr 2007;85:6–18. 6. Zittermann A, Koerfer R. Protective and toxic effects of vitamin D on vascular calcification: Clinical implications. Mol Aspects Med. 2008 Dec;29(6):423-32. 7. Singh RJ. Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D? Clin Chem 2008;54:22-3. 8. Norman AW, Bouillon R, Whiting SJ, et al. 13th Workshop consensus for vitamin D nutritional guidelines. J Steroid Biochem Mol Biol 2007;103:204–5. 9. Holick MF. Vitamin D deficiency. N Engl J Med 2007;357:266–81. 10. Li YC, Kong J, Wei M, et al. 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 2002;110:229–38. 11. Judd SE, Nanes MS, Ziegler TR, et al. Optimal vitamin D status attenuates the age-associated increase in systolic blood pressure in white Americans: results from the third National Health and Nutrition Examination Survey. Am J Clin Nutr 2008;87:136–41. 12. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285:2486–97. 13. Chiu KC, Chu A, Go VL, Saad MF. Prevalence of cardiovascular risk factors and the serum levels of 25hydroxyvitamin D in the United States: data from the Third National Health and Nutrition Examination Survey. Arch Intern Med 2007;167:1159–65. 14. Hyppönen E, Läärä E, Reunanen A, et al. Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study. Lancet. 2001;358(9292):1500–3. 15. Martins D, Wolf M, Pan D, et al. Prevalence of cardiovascular risk factors and the serum levels of 25hydroxyvitamin D in the United States: data from the Third National Health and Nutrition Examination Survey. Arch Intern Med 2007;167:1159–65. 16. Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis. J Clin Endocrinol Metab 2007;92:2017–29. 17. Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74. VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE 245

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

18. Bhalla AK, Amento EP, Clemens TL, et al. Specific highaffinity receptors for 1,25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes following activation. J Clin Endocrinol Metab 1983;57:1308–10. 19. Cantorna MT, Yu S, Bruce D. The paradoxical effects of vitamin D on type 1 mediated immunity. Mol Aspects Med. 2008 Dec;29(6):369-75. 20. Cantorna MT, Zhu Y, Froicu M, Wittke A. Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system. Am J Clin Nutr 2004;80(6 Suppl):1717–20S. 21. Dobnig H, Pilz S, Scharnagl H, et al. Independent association of low serum 25-hydroxyvitamin D and 1,25dihydroxyvitamin D levels with all-cause and cardiovascular mortality. Arch Intern Med 2008;168:1340–9. 22. Targher G, Bertolini L, Padovani R, et al. Serum 25hydroxyvitamin D3 concentrations and carotid artery intima-media thickness among type 2 diabetic patients Clin Endocrinol (Oxf) 2006;65:593–7. 23. Peterson CA, Heffernan ME. Serum tumor necrosis factoralpha concentrations are negatively correlated with serum 25(OH)D concentrations in healthy women. J Inflamm (Lond) 2008;5:10. 24. Bonakdaran S, Varasteh A. Correlation between serum 25 hydroxy vitamin D3 and laboratory risk markers of cardiovascular diseases in type 2 diabetic patients. Saudi Med J 2009;30:509–14.

246 VOL 22, NO 4 FALL 2009 CLINICAL LABORATORY SCIENCE

25. Ewers B, Gasbjerg A, Zerahn B, Marckmann P. Impact of vitamin D status and obesity on C-reactive protein in kidney-transplant patients. J Ren Nutr 2008;18:294–300. 26. Michos ED, Streeten EA, Ryan KA, et al. Serum 25hydroxyvitamin D levels are not associated with subclinical vascular disease or C-reactive protein in the old order Amish. Calcif Tissue Int 2009;84(3):195–202. 27. Wang TJ, Pencina MJ, Booth SL, et al. Vitamin D deficiency and risk of cardiovascular disease. Circulation 2008;117:503–11. 28. Giovannucci E, Liu Y, Hollis BW, Rimm EB. 25hydroxyvitamin D and risk of myocardial infarction in men: a prospective study. Arch Intern Med 2008;168:1174–80. 29. Dobnig H, Pilz S, Scharnagl H, et al. Independent association of low serum 25-hydroxyvitamin d and 1,25dihydroxyvitamin d levels with all-cause and cardiovascular mortality. Arch Intern Med 2008;168:1340–9. 30. Carter GD, Carter R, Jones J, Berry J. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Clin Chem 2004;50:2195–7. 31. Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab 2004;89:3152–7. 32. Phinney KW. Development of a standard reference material for vitamin D in serum. Am J Clin Nutr 2008;88:511S– 512S.

FOCUS: CARDIOVASCULAR RISK ASSESSMENT

Continuing Education Questions FALL 2009 ®

To receive 2.0 contact hours of basic level P.A.C.E. credit for the Focus: Cardiac Risk Assessment questions, insert your answers in the appropriate spots on the answer sheet that follows; then complete and mail the form as directed.

Questions 1. The percent genetic contribution to CAD is approximately: a. 80% b. 70% c. 50% d. 30% 2. Family-based genetic studies are known as: a. Linkage studies b. Candidate gene association studies c. Genome-wide association studies d. B + C 3. A 21-bp deletion in this gene was identified as being involved in a large autosomal dominant family with CAD and MI: a. ALOX5AP b. APOE c. MEF2A d. LPA 4. Results from candidate gene association and linkage analyses are hampered by the general inability to replicate findings in follow-up studies. This could be due to: a. The studies are underpowered b. The markers identified are false positives c. The markers identified are specific only to the group studied d. All of the above

5. A small molecular FLAP-inhibitor was shown to reduce levels of this CAD biomarker in a placebocontrolled randomized trial: a. CRP b. LDL cholesterol c. Lp(a) d. Homocysteine 6. Which of the following is not true regarding genome-wide association studies? a. GWA studies were instrumental in identifying the 9p21 CAD risk allele. b. GWA studies are useful for identifying markers with