Pathology & Laboratory Medicine Communiqué

JANUARY, 2015 Pathology & Laboratory Medicine Communiqué IN THIS ISSUE COLLECTION TUBE EXPIRATION DATES ARE IMPORTANT OPERATIONS Collection Tubes Expiration Date Container for Urine Culture or CIFP Dr. Maureen Harmon New Director of Surgical Pathology Welcome Dr. Sarah Harm and Dr. Susan Sharp Farewell to Dr. Armando Ciampa TEST UPDATES AFB QNS Sample BK Virus Discontinued CRP Reporting Change Digoxin : Therapeutic Versus Critical Value Free Light Chain, Serum Maternal Fetal Risk Screen Change in Calculation Algorithm New Test Platform For:: Anti-Nuclear Antibody Anti-Neutrophil Antibody Anti-Smooth Muscle Antibody Anti-Mitrochondrial Antibody Anti-Parietal Cell Antibody Autoantibodies to Extractable Nuclear Antigens New Test ENA Panel: RNP Antibody SS-A Antibody SS-B Antibody Sm Antibody New Test: SS-A/SS-B Panel SS-A Antibody SS-B Antibody No one wants to have a patient sample rejected for improper collection. If we receive a sample that has been collected in an expired tube we will reject the sample. Here are some tips to help avoid this problem. Do not overstock tubes. Arrange inventory so that tubes with shorter outdates are used first. Get into the habit of checking tubes expiration date before sample collection. Blood collection tubes should never be used past their expiration date. Date on the outside of a box of tubes Date on each tube Container for Urine Culture or CIFP Collection Please submit urine for culture or culture if urinalysis positive (CIFP) in a 100 ml sterile container. We will not be able to add a urine culture to a urinalysis specimen if it was not collected in a sterile container. Non-sterile containers (food jars, plastic containers) are not acceptable and will be rejected for culture. Sterile urine containers are available from Laboratory Customer Service 847-5121 or 1-800-991-2799. Change in Critical Call Back Values for Vancomycin and Methotrexate Hemagram reporting change: addition of MPV and RDW-SD ATTACHMENT Lynch Syndrome Screening

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 2 Dr. Maureen Harmon New Director of Surgical Pathology We are very pleased to announce that Dr. Maureen Harmon has become the Director of Surgical Pathology. She has been the Pathologist in charge of the gross room overseeing the Pathologists Assistants and processes in the gross room as well as the Pathologist in charge of QA in Surgical Pathology for more than 2 years. She will continue to be the Medical Director of Porter Hospital laboratory. Welcome Dr. Sarah Harm Sarah Harm, MD, MS. has joined Pathology & Laboratory Medicine as assistant director in Blood Bank and Transfusion Medicine. She comes to us from the University of Pittsburgh Medical Center where she was previously a resident, fellow, and then attending pathologist. Welcome Dr. Susan Sharp Dr. Susan Sharp is joining Pathology & Laboratory Medicine and will be working out of Central Vermont Medical Center in Berlin VT. Dr. Sharp will also be the laboratory director of Littleton Hospital in Littleton NH, Cottage Hospital in Woodsville NH, Northeastern Vermont Regional Hospital in St. Johnsbury VT and North Country Hospital in Newport VT. Farewell to Dr. Armando Ciampa Armando Ciampa, MD, has accepted a position at York Hospital in Wells, Maine as part of the Spectrum Group. He will be the Medical Director of the laboratories there. Armando has been a valued colleague in our department for approximately 10 years, first as a surgical pathology fellow and then as an assistant professor of pathology. He has most recently served as the medical director of 2 of our outreach hospital laboratories and one of our surgical pathologists specializing in GI pathology. Armando's professionalism, dedication to the department and commitment to patient care have added so much to our department as has his easy going nature and warm smile. Armando will be starting his new job January 1, 2015 and we are very grateful to him for staying on through the rest of this year. We plan on starting a national search for another GI pathologist very soon.

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 3 AFB QNS Sample In August, the Microbiology Laboratory began rejecting specimens for Mycobacteriology (AFB) cultures that do not meet our minimum specimen requirements. Infections with mycobacteria often contain very few organisms, therefore the greater the quantity of sample submitted, the higher the likelihood of growing the organism. Submitting samples that are below the minimum requirements can result in a false negative result. Any specimen volume below the minimum volume listed below will be rejected as Quantity not sufficient. Specimen Minimum volume (ml) Sputum 3 BAL/Bronchial washings 3 Dr. Christina Wojewoda Director of Microbiology Phone : 847-5140 Email: Christina.wojewoda@uvmhealth.org CSF 3 Body fluid (other than CSF) 10 Bone marrow 0.5 Abscess 1 Gastric 10 Urine 10 BK Virus Discontinued SYRINGE DISPOSAL Due to lack of quantitative standards of BK virus in urine, we are unable to meet regulatory requirements for quantitative BK PCR from urine. On 10/17/2014 we discontinued this testing at the University of Vermont Medical Center. Testing is available to be performed at Mayo Medical Laboratories. We still offer quantitative BK PCR on plasma. Fletcher Allen does not accept sharps for disposal from patients. Chittenden Solid Waste District (CSWD) will accept needles that are packaged according to the instructions outlined in their pamphlet GET THE POINT: Be safe with syringes and other sharps. CSWD also has bright orange stickers to attach to a syringe container to warn handlers to be careful. These items are available at any CSWD location. You can also order them so that they are available for patients at your office 872-8111 or visit www.cswd.net C Reactive Protein Reporting Change C-reactive protein (CRP) is a sensitive acute phase reactant that increases in response to inflammatory processes such as trauma, surgery, infections or neoplastic proliferation. It has been used classically to assess and follow such processes. In the mid-1990s a new use for this test was recognized to assess relative risk for cardiovascular disease or ischemic events. This new use was made possible by more sensitive methods for the measurement of CRP and these tests were referred to as high sensitivity C-reactive protein (HSCRP). To add to the confusion of two tests with similar names measuring the same analyte, the units used for the new test were mg/l instead of the traditional mg/dl. (mg/l = 10*mg/dL) The difference in units between the routine CRP and the cardiovascular risk HSCRP has been a continuing source of confusion. The trend in laboratory reporting has been to consolidate reporting on mg/l and as of 2/10/2015; the laboratory will begin reporting results for CRP in mg/l. It is important to recognize that these results for CRP will now be 10X higher than they have been historically. The reference range and critical values for these results will change accordingly and a notice will be added to the results that the units have changed. If you have any questions concerning this change please contact Dr. Greg Sharp (gregory.sharp@uvmhealth.org) in the Laboratory.

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 4 Digoxin: Therapeutic Versus Critical Value A critical alert value is one that represents a value that is so far from the normal expected value as to be life threatening unless an immediate available corrective action is taken. This value does not necessarily represent the limit of the optimal therapeutic or toxic range for a particular analyte. This can be seen for digoxin, where the critical value is 2.0 ng/ml, but the recommended therapeutic range for heart failure is 0.5 to 0.8 ng/ml. 1 and values above this range can cause an increased risk for toxicity. To address this issue the following message is being attached to all digoxin results: Dr. Gregory Sharp Director of Clinical Chemistry Phone: 847 5115 Email: gregory.sharp@uvmhealth.org For heart failure a digoxin level of 0.5 to 0.8 ng/ml is suggested for maximal efficacy and minimal risk of toxicity. If you have any questions concerning this change please contact Dr. Greg Sharp (gregory.sharp@uvmhealth.org) in the Laboratory. 1. Association of serum digoxin concentration and outcomes in patients with heart failure. Rathore SS, Curtis JP, Wang Y, Bristow MR, Krumholz HM JAMA. 2003;289(7):871.0 FREE LIGHT CHAINS, SERUM Test Information: On November 11, 2014, the Chemistry Laboratory changed the instrumentation used for Serum Free Light Chains (Kappa Free Light Chains, Lambda Free Light Chains and Kappa/Lambda Ratio) testing. This assay will be performed on the SPAplus manufactured by The Binding Site. While this assay still uses turbidimetry, the SPAplus further automates the testing on a dedicated instrument. Previously this test was performed by turbidimetry on a nephelometer (Beckman Immage 800) using Binding Site reagent. Serum Free Light Chains testing will be performed Monday through Friday with results available the same day. If you have any questions concerning this change, please contact Dr. Gregory Sharp in the laboratory (gregory.sharp@uvmhealth.org) Method: Evaluating the concentration of a soluble antigen by turbidimetry involves the addition of the test sample to solution containing the appropriate antibody in a cuvette. A beam of light is passed through the cuvette and, as the antigenantibody reaction proceeds, the light passing through the cuvette is scattered increasingly as insoluble immune complexes are formed. Light scatter is monitored by measuring the decrease in intensity of the incident beam of light. The antibody in the cuvette is in excess so the amount of immune complex formed is proportional to the antigen concentration. A series of calibrators of know concentrations are assayed to produce a calibration curve of measured light scatter versus antigen concentration. Samples of unknown concentration can then be assayed and the results read from the calibration curve. Clinical Significance: Immunoglobulin molecules consist of two identical heavy chains which define the immunoglobulin class and two identical light chains (kappa or lambda). Each light chain is covalently linked to a heavy chain and the two heavy chains are linked covalently at the hinge region. In healthy individuals, the majority of light chain in serum exists in this form, bound to heavy chain. However, low levels of free light chain (flc) are found in serum of normal individuals due to the over-production and secretion of free light chain by the plasma cells. Elevated serum levels of monoclonal free light chain are associated with monoclonal gammopathies such as multiple myeloma, Waldenstrom s macroglobulinemia, monoclonal gammopathy of undetermined significance, primary amyloidosis and light chain deposition disease. This automated, turbidimetric assay is reported to be more sensitive than immunofixation for detection of monoclonal free light chains. The specificity of this assay for detection of monoclonal light chains relies on the ratio of free kappa and lambda light chains. Once an abnormal free light chain kappa/lambda ratio has been demonstrated and a diagnosis made, the

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 5 (Con nued from page 4) quantitation of the monoclonal light chain is useful for the monitoring of disease activity. Limitations: Moderate lipemia may interfere with the ability to perform testing. Diagnosis cannot be made and treatment must not be given on the basis of free light chain measurement alone. Clinical history and other laboratory findings must be taken into account. Monoclonal free light chains can be highly variable: the amino acid composition of the light chain produced during an individual B cell disease state will influence the level at which a sample may show antigen excess with the Freelite assay. This assay has not been validated for the pediatric population. Ordering Information Test Name: Free Lt Chains, Serum Test Code: SERFLC Sample Requirements: Submit 0.5 ml serum. Stable for 7 days refrigerated. Markedly lipemic or hemolyzed samples will not be accepted. Days Performed: Monday through Friday (run starts at 10am) Analytic Time: Same Day Reference Range: Kappa free light chain: 0.33 1.94 mg/dl Lambda free light chain: 0.57 2.63 mg/dl Kappa/Lambda FLC ratio: 0.26-1.65 CPT Code: 83883 X2 References: Freelite human kappa free kit for use on the SPAplus product insert, INC256 June 2014. Freelite human lambda free kit for use on the SPAplus product insert, INC257 June 2014. Maternal Fetal Risk Screen (QUAD Marker): Change in Calculation Algorithm The Quad Marker screen uses a combination of four biochemical markers and maternal demographics in a mathematical model to identify pregnancies that are at higher risk for Trisomy 21 (Down syndrome), Trisomy 18 and open spina bifida (OSB). New guidelines recommend, that the SURUSS 2003 2 model be used, which provides a slightly increased detection rate, with a slightly lower false positive rate. The Chemistry laboratory changed to the new algorithm on October 6, 2014. A second change that took place on the same day is in regard to samples that are collected at 14 weeks of gestation. Current guidelines from New York State recommend that these samples be analyzed for trisomy risk since that can be calculated for 14 weeks gestation, but that they continue to be rejected for calculation of the risk for OSB. The reports reflect this process. If you have any questions concerning these changes please contact Dr. Greg Sharp (gregory.sharp@uvmhealth.org) in the laboratory. References: 1. Wald NJ,Hackshaw AK, George LM. Assay precision of serum alpha fetoprotein in antenatal screening for neural tube defects and Down s syndrome. J Med Screen 2000; 7:74-77. 2. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty L, Mackinson AM. First and second trimester antenatal screening for Down s syndrome: the results of the Serum, Urine, and Ultrasound Screening Study (SURUSS). J Med Screen 2003; 10:56-104.

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 New Test Platform For: Anti-Nuclear Antibody Anti-Neutrophil Antibody Anti-Smooth Muscle Antibody Anti-Mitrochondrial Antibody Anti-Parietal Cell Antibody 6 On January 15, 2015, the Immunology Laboratory will begin using INOVA Diagnostics (San Diego, CA) NOVA Lite Assays on the QUANTalyser analyzer for the above tests. This instrumentation utilizes indirect immunofluorescence to detect the antibody. One of the advantages of this new methodology includes the use of an affinity purified anti-human IgG specific conjugate versus the polyclonal specific conjugate currently in use. Virtually all clinically significant autoantibodies exhibit IgG subclass specificity even in the presence of IgM and IgA specific ANA. In contrast, ANA found in healthy blood donors are generally of the IgM and IgA subclass only. This makes the assays more disease specific. In addition, the fixation process used in preparing the slides does not destroy certain nuclear antigens while the fixation process employed by some other manufacturers has been shown to do so. These reagent parameters allow the NOVA Lite assays to detect clinically relevant autoantibodies which can remain undetected by some other commercial kits. In addition, the IgG conjugate specificity eliminates physiologic false positive results due to normally occurring low titer IgM autoantibodies, often found in older, but otherwise healthy persons. In addition to changing methodology, we will begin confirming positive ANCA s on formalin fixed slides to determine the ANCA specificity. With implementation of these changes as well as bringing testing for Extractable Nuclear Antibodies (SS-A, SS-B, Sm, and RNP) in-house, the following reflex testing will be added. All positive ANA s with a titer of greater than or equal to 1:160 (with the exception of the centromere pattern) will reflex the ENA panel as well as a dsdna unless the reflex testing is declined by the ordering provider. If you have any questions concerning this change please contact Dr. Greg Sharp (greg.sharp@uvmhealth.org) in the laboratory. References ANA: NOVA Lite HEp-2 ANA Kits/Substrate Slides Product Insert, INOVA Diagnostics, Inc., 9900 Old Grove Road, San Diego, CA 92131, December 2012, Rev. 21. ANCA: 1.) NOVA Lite ANCA, INOVA Diagnostics, Inc., 9900 Old Grove Road, San Diego, CA 92131, June 2013, Rev. 18. 2.) NOVA Lite ANCA Kits, Substrate Slides, INOVA Diagnostics, Inc., 9900 Old Grove Road, San Diego, CA 92131, December 2012, Rev.1. MIT, SMO, PRT: NOVA Lite ANA Plus (Mouse Kidney & Stomach), INOVA Diagnostics, Inc., 9900 Old Grove Road, San Diego, CA 92131, July 2010, Rev. 15. Test Name Test Code CPT Reference Range Anti-Nuclear Antibody ANA 86038 <40 dils Anti-Neutrophil Antibody ANCA 86255 Negative Anti-Smooth Muscle Antibody SMO 86255 < 20 dils Anti-Mitrochondrial Antibody MIT 86255 <20 dils Anti-Parietal Cell Antibody PRT 86255 <20 dils Lab Division Sample Requirements Days Performed Analytical time Instrument NY Certified Immunology Collect 4 ml SST, submit 1 ml serum, minimum volume 0.4 ml Monday - Friday 4 days Inova Quantalyser Yes

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 AUTOANTIBODIES TO EXTRACABLE NUCLEAR ANTIGENS 7 New Test ENA Panel: Test Code: ENAPNL includes RNP Antibody, SS-A Antibody, SS-B Antibody, Sm Antibody New Test: SS-A/SS-B Panel: Test Code: SSABPL INCLUDES SmSS-A Antibody, SS-B Antibody, On January15, 2015, the Immunology Laboratory will begin to offer the INOVA Diagnostics (San Diego, CA) QUANTA Lite SS-A, SS-B, Sm, and RNP assays. These assays will be offered individually, the SS-A and SS-B will be offered as a panel and all four will be offered as part of an Extractable Nuclear Antibody Panel. While the ANA test is an excellent screening test for SLE (a negative result virtually rules out SLE) it is by no means specific. Autoantibodies to SS-A antigen are found in 40-60% of patients with Sjogren s Syndrome and in 25-35% of patients with ANA positive Systemic Lupus Erythematosis (SLE). Autoantibodies to SS-B antigen are found in 5-15% of SLE patients and 25% of random Sjogren s Syndrome patients. SS-B antibodies are found in approximately 60% of Sjogren s patients with sicca complex. As with SS-A, these antibodies are often found in patients with clinical features of SLE, but are ANA negative. SS-B antibodies are usually found to occur simultaneously with SS-A, while SS-A can occur alone. Patients producing both SS-B and SS-A as opposed to SS-A alone generally have a milder disease with lower incidence of nephritis and antibodies to dsdna. Sm antibodies are highly specific for SLE and, as with antibodies to dsdna, are considered a marker antibody. Sm antibodies have been included in the Arthritis and Rheumatism subcommittee criteria (REF?) for the classification of SLE and antibody titers were found to be a reliable measure of disease activity. Antibodies to RNP are found in up to 45% of patients with SLE and also in some other connective tissue diseases. RNP antibodies are associated with a relatively benign disease course with lower incidence of renal and CNS disease. RNP antigen contains epitopes or antibody sites that are unique to RNP but it also contains epitopes that are immunologically identical to free Sm. Since the RNP antigen coated onto the solid phase of this test has Sm antigenic epitopes, the patient Sm antibody response must be considered when interpreting the RNP results. A positive RNP result indicates the presence of antibodies reactive with the RNP/Sm complex but cannot distinguish between anti-sm and anti-rnp activity. When the Sm antibody is measured in conjunction with the RNP antibody activity, the RNP antibody activity can be inferred. If a sample is negative in the Sm ELISA, but positive in the RNP ELISA, then the reactivity is largely or exclusively due to RNP antibodies. If the reactivity in the Sm ELISA is equivalent to the reactivity of the RNP, then the activity indicates the sample is a mixture of Sm and RNP antibodies. The RNP activity can be estimated by subtracting the Sm activity over the cut-off (i.e. over 20 Units) from the total RNP ELISA value. Reference values for SS-A, SS-B, Sm, and RNP will change from those used by Mayo Medical Laboratories and are listed in the table below. Interpretation Negative <20 Weak Positive 20-39 Units Specimen Information Test Name SQ Code CPT RNP Antibody RNPA 86235 SS-A Antibody SSAA 86235 SS-B Antibody SSBA 86235 Sm Antibody SMAA 86235 Moderate Positive 40-80 Strong Positive >80 Lab Dividion Method Sample Requirements Days Performed Analytical Time Instrument NY Certified Immunology ELISA Collect 4 ml SST, process sample and send 0.5 ml serum refrigerated, minimum volume is 0.4 ml. SST must be spun within 8 hours, refrigerated serum must be received within 48 hours, for longer storage freeze. Tuesday and Thursday Same day Inova Quantalyzer Yes

PATHOLOGY & LABORATORY MEDICINE COMMUNIQUÉ JANUARY 2015 8 Pathology & Laboratory Medicine Communique is produced quarterly. COMMUNIQUE EDITORS Lynn Bryan, Laboratory Manager Monica Sullivan, Laboratory Manager Colleen Williams, Lab Communication Strategist PATHOLOGY & LABORATORY MEDICINE 111 Colchester Avenue Mail Stop 233MP1 Burlington, VT 05401 PHONE (802) 847-5121 Change in Critical Call Back Values for Vancomycin and Methotrexate With clinical input from Pharmacy, Infectious Disease and Oncology, the Laboratory has decided to alter select critical call back values. The current critical call value for vancomycin trough levels will shift from 20 to 25 ug/ml. The laboratory will eliminate critical call values for random vancomycin levels. The current methotrexate critical call value will shift from 1000 to 100 umol/l. Routine methotrexate calls will be eliminated. The effective date for these changes will be January 15, 2015. If you have any questions concerning these changes, please contact Dr. Jill Warrington (jill.warrington@uvmhealth.org) or Dr. Greg Sharp (gregory.sharp@uvmhealth.org) in the Chemistry Laboratory. (800) 991-2799 FAX (802) 847-5905 HEMAGRAM REPORTING CHANGE: ADDITION OF MPV AND RDW-SD On January 11, 2015 the mean platelet volume (MPV and the red cell distribu on width standard devia on (RDW SD) will be included in the hemagram report. The MPV is a measure of the mean platelet volume in femtoliters (fl). This parameter has poten al value in assessing bone marrow platelet produc on. Younger, recently released platelets are larger in size which is reflected by an increase in the MPV. In contrast, platelets of greater age are smaller in size. This can poten ally be exploited in dis nguishing between thrombocytopenia due to peripheral destruc on, such as in immune thrombocytopenia, in which the marrow a empts to compensate by releasing many young platelets that have higher MPVs, from thrombocytopenia due to decreased thrombopoiesis in the bone marrow. Recent studies have also shown that the MPV may poten ally have a predic ve role for venous thrombosis, cardiovascular disease, malignancies metasta c to bone marrow, etc. Reference Range: 7.5 11.2 fl Dr. John Lunde Medical Director Hematology Phone: 802 847 5135 Email: John.lunde@uvmhealth.org The RDW CV is a calcula on based on the width of the RBC distribu on curve and the RBC Mean Corpuscular Volume (MCV). The RDW SD is a direct measurement of the width of the red cell distribu on curve, reported in femtoliters (fl). In so doing the RDW SD avoids the ar factual errors that may occur in the RDW CV at extremes of the MCV, both high and low. Since the RDW SD is a direct measurement, it is not influenced by the MCV and more accurately reflects the red cell size variance. Reference Range: 36.5 45.9 fl Male 37.6 50.3 fl Female Please contact Russ Brown (russell.brown@uvmhealth.org) or John Lunde, M.D. (john.lunde@uvmhealth.org) should you have any questions.

PATHOLOGY AND LABORATORY MEDICINE JANUARY 2015 Lynch Syndrome Screening Effective October 1, 2014, University of Vermont Medical Center GI Pathology began performing Universal Screening for Lynch Syndrome on biopsy specimens found to be positive for colorectal cancer. This screening was formerly performed on resection specimens. This change allows for clinical decision making to be made prior to surgical intervention. It is important to note that the initial screening test, immunohistochemical (IHC) staining with antibodies against four mismatch repair proteins, done at the University of Vermont Medical Center is NOT considered a molecular test. However, any follow up molecular testing (e.g. MLH1-Promoter Methylation) requires preauthorization. The most common scenario in which this is encountered is in colon cancers that show the following IHC results: Loss of MLH1/PMS2 and retention of MSH2 and MSH6 proteins. In these cases the following comment will always be present in the surgical pathology report: The majority of colon cancers that have loss of MLH1/PMS2 protein are associated with somatic changes rather than an inherited mutation (Lynch syndrome). However, if additional testing to rule out Lynch syndrome is warranted in this individual, additional molecular testing, specifically MLH1 Promoter Methylation can be ordered upon obtaining preauthorization. MLH1 Promoter Methylation allows clinicians to definitively delineate Lynch Syndrome-associated cancer from microsatellite unstable (aka MSI-high) tumors that are sporadic (non-familial). However, this test will only be performed following preauthorization obtained from the treating clinician. If you have any questions concerning this change please contact Dr. Rebecca Wilcox, 802-847-9477, Rebecca.wilcox@uvmhealth.org. UVMHealth.org/MedCenter