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• Abbott's Hepatitis B Test Approved for Blood Screening
• Adoption of new "Critical Values" (9/11/2007)
• Allergy News (6/30/2009)
• Amikacin (4/10/2009)
• CBC with Auto Differential & CBC with Manual Differential
• Change in Reflex Criteria for Urine Culture
• Chemistry and Immunochemistry Instrumentation/New Reference Ranges
• Community-Acquired Methicillin Resistant Staphylococcus Aureus (CA-MRSA)
• Creatinine: Introduction of updated method calibrated to be traceable to IDMS
• Cystatin C Measuring Glomerular Filtration Rate (GFR)
• Cystic Fibrosis
• Diagnosis of Enterohemorrhagic E. coli infection by Detection of Shiga Toxin
• Diagnosing Legionnaire's Disease: Legionella Detection by PCR
• Encourage HIV/Aids Testing
• Epstein-Barr Virus testing update
• ESBLs: The Rise of Antibiotic Resistance (2/24/2009)
• Estimated Glomerular Filtration Rate (GFR)
• Estradiol assay update: minor changes to reference ranges due to change in standardization. (10/2008)
• Factor V Leiden (FVL) Gene Mutation Analysis by Third Wave Invader Assay
• Facts About PTHI
• FDA Clears Lab Test for Bird Flu Infections in Humans
• Glucose Point-of-Care Testing Results
• HPV Testing—New Information
• Immunochemical Fecal Occult Blood Test (FOBT) (4/10/2009)
• Immunological Fecal Occult Blood Test to be Offered
• Intact PTH Assay
• Introduction of HIV-1 Viral Load Testing by PCR at MuirLab (10/2008)
• John Muir Clinical Lab Offers Affirm VPIII
• Laboratory Medicine & Molecular Diagnostics (4/10/2009)
• Latent Tuberculosis (TB)
• MR Contrast Screening Protocols
• Novel Influenza A (H1N1) - Laboratory Perspective (6/30/2009)
• Ordering Occult Blood and PSA Testing
• PLAC® Test
• Reporting of Phenytoin (Dilantin) Concentration
• Stat Gram Stain Specimens
• RSV Testing
• Update to Cardiac Troponin I (cTnI) Reporting
• Urinalysis Department Announces Change in Urine Critical WBC / Culture Reflex Values
• Urinalysis with Microscopic
• Vitamin D - Laboratory Considerations (2/24/2009)

Abbott's Hepatitis B Test Approved for Blood Screening

Abbott Laboratories' PRISM HBsAg assay was approved this month by the Food & Drug Administration to test people who donate blood, blood components, and organs for transplant for the hepatitis B virus. The test also may be used to screen blood from cadavers for organ and tissue donation. A blood test is the only way to determine if a donor has HBV infection. (Source: G2 July 28th 2006)

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Adoption of new "Critical Values": 9/11/2007

Dr. Barry Latner, Medical Director for the Clinical Lab John Muir Health
July 2007

Following the work of a task force of the Department of Medicine headed by Ross Pirkle, M.D., the Medical Executive Committee of the JMMC Walnut Creek Campus approved an updated list of "Critical Values" for laboratory tests. This list is considerably pared down from the previous version. These critical values will be phoned to the ordering physician (outpatient) or responsible licensed caregiver/charge nurse (facility) of clients of MuirLab and the Walnut Creek Campus of JMMC. It is worth noting that some of these critical values differ from those on the Concord Campus of JMMC, which, over time, will be reconciled.

"Critical Values" are abnormal lab values that are generally considered to be potentially life threatening in the correct clinical context. JCAHO has recently amplified the importance of documenting the relay of this critical patient information in a timely manner. As a result, the lab makes every effort to phone these results once they are internally verified. The lab then records such details as person, place and time of the phoning, receipt and read back of these critical values. In addition, similar documentation is made of refusals to accept or receive this information.

If there are any questions or concerns about this update, please do not hesitate to contact me, Dr. Barry Latner at (925) 692-5405 or Barbara Brunell, Regulatory Compliance Manager Laboratory Services, (925) 692-5472, and or your sales representative at (925) 692-5411.

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Allergy News

6/30/2009

Allergy and diseases caused or complicated by allergy, such as asthma, or diseases with symptoms which mimic allergy, are among the most widespread and costly health problems in the world.

John Muir Health, MuirLab will soon provide testing for allergy that is safe, simple, and accurate with groundbreaking technology with the latest generation of automated allergy diagnostic systems.

Detection of immunoglobulin E (IgE) antibodies and determination of specific allergens allow for better outcomes and increased quality of life for allergy patients. To be reliable, the testing system used to detect allergy must be standardized. Phadia allergy test systems, along with all their components, are standardized against World Health Organization reference preparations for reproducible, reliable results. Phadia, a global leader in allergy diagnostics research and development since 1974, offers advanced automated instrumentation to process the ImmunoCAP® technology-based test kits for allergy, and inflammation markers for asthma and anaphylaxis. The ImmunoCAP® instrument provides innovative software and hardware solutions that meet laboratory automation and throughput requirements.

ImmunoCAP® Specific IgE assays are the first to be FDA cleared for the quantitative measurement of specific IgE. The most significant features of ImmunoCAP technology are extensive quality control measures, excellent allergen and reagent source materials, and calibration to WHO reference preparations. In addition, the structural design of ImmunoCAP® allows for automation and thorough elimination of unbound nonspecific material in the reaction complex, resulting in highly reproducible, accurate, and quantitative results.

Childhood Allergy Profile (Food/Environmental) d1 D. pteronyssinus d2 D. farinae e1 Cat dander e5 Dog dander f1 Egg white f13 Peanut f14 Soybean f2 Milk f24 Shrimp f256 Walnut f3 Cod fish f4 Wheat i6 Cockroach m2 Cladosporium herbarum m6 Alternaria alternata Plus Total IgE

Adult Food Profile f1 Egg white f13 Peanut f14 Soybean f2 Milk f207 Clam f24 Shrimp f256 Walnut f3 Cod fish f338 Scallop f4 Wheat f8 Corn f10 Sesame f1 Egg white

Region 14: Central CA Valley d1 D. pteronyssinus d2 D. farinae e1 Cat dander e5 Dog dander g2 Bermuda grass g6 Timothy grass i6 Cockroach m1 Penicillium notatum m2 Cladosporium herbarum m3 Aspergillus fumigatus m6 Alternaria alternata t2 Alder t3 Birch t6 Mountain Cedar t7 Oak t8 Elm t9 Olive t11 Sycamore t70 Mulberry w1 Common ragweed w6 Mugwort w11 Russian Thistle w14 Rough pigweed Plus Total IgE

Region 17: CA, NW; OR, W.; WA d1 D. pteronyssinus d2 D. farinae e1 Cat dander e5 Dog dander g6 Timothy grass i6 Cockroach m1 Penicillium notatum m2 Cladosporium herbarum m3 Aspergillus fumigatus m6 Alternaria alternata t1 Maple t2 Alder t3 Birch t6 Mountain Cedar t7 Oak t8 Elm t10 Walnut t14 Cottonwood t15 White ash w1 Common ragweed w14 Rough pigweed w18 Sheep sorrel w20 Nettle Plus Total IgE

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Amikacin

Amikacin is a semi-synthetic aminoglycoside antibiotic used to treat different types of bacterial infections. This medication can cause serious kidney problems and nerve damage, resulting in permanent hearing loss and balance problems. The Amikacin reagent is a homogeneous, particle-enhanced turbidimetric immunoassay supplied as a liquid, ready to use

Amikacin, in addition to gentamicin and tobramycin, belongs to the aminoglycosides family of antibiotics that are most commonly measured to guide and monitor dosing regimens to evaluate potential toxicity. Features of the Amikacin assasy include:

• Excellent sensitivity of <0.8 µg/mL
• Analytical range is 1.5 to 50 µg/mL
• 35 day on-board reagent stability
• Total Precision of <5% CV
• Acceptable sample types include serum and plasma
• Optimal: 4.0-8.0 µg/mL
  Toxic: 8.1 µg/mL or greater

Previously The Peak and Trough was collected and sent to ARUP for testing with a TAT taking up to 3 Days. Time was a huge factor where the Physician needed the results to regiment the dose and monitor for the toxicity level. In addition, the integrity of the specimen could be jeopardized with the prolonged transport.

MuirLab is adding Amikacin in house. This will improve TAT, The integrity of the specimen, and meeting the needs of the Clients.

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CBC with Auto Differential & CBC with Manual Differential

April 2008

Effective April 1, 2008, "CBC with Auto Differential" and "CBC with Manual Differential" will be combined into a single test, "CBC with Differential". Automated or manual differential results will be reported based upon internal laboratory reflex criteria. If specific morphologic evaluation is required (e.g. for red cell abnormalities, bands, organisms, etc.), ordering "Smear Review" will generate a peripheral smear for review if a manual differential has not already been performed based upon reflex criteria.

These changes bring our laboratory practices in line with those of other local institutions, and will serve to increase result reproducibility, streamline workload, and decrease turnaround time for results.

If you have any questions, please call Dennis Hwang, MD at (925) 947-3351.

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Change in Reflex Criteria for Urine Culture

Nader Shihabi, MD and Bernard Larner, MD
April 2008

We are changing the criteria for reflex urine cultures. In the past, the only criteria for urine cultures was WBC >10 in the urine. We will now also culture urines with positive leukocyte esterase or positive nitrite. These two parameters were added since WBCs may sometimes lyse in transport. Although there are no gold standards as to when to reflex urines for culture, these three criteria are widely used at other laboratories. Please be aware that some organisms do not elicit a typical UTI inflammatory response (such as elevated WBCs). If there is a strong initial suspicion for UTI, or if symptoms persist despite normal screening test results, a urine culture should be ordered rather than a reflex culture.

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Chemistry and Immunochemistry Instrumentation/New Reference Ranges

July 2007

Beginning September 11, 2007 the Clinical Laboratories of John Muir Health will complete the conversion of all of its chemistry and immunochemistry assays (> 70 tests) to a single new platform. This change to Beckman Coulter instrumentation is made because of the end of the expected life (5+ years) of our current excellent analyzers from Dade Behring and Diagnostic Products Corp (DPC), and represents one more step of our transition to total laboratory automation and a new state-of-the-art core laboratory. The Core Lab is currently under construction and is slated for completion and occupancy in the first quarter of 2008.

With the new instrumentation comes different reagent and detection systems, thus the need to reevaluate reference ranges for each of the assays. While the majority of tests show no significant difference, the enzymes and certain colorimetric tests do differ. Listed below are the results of extensive correlation and reference range studies of the new vs. old platforms for those assays that change significantly.

New = Slope x Old + Intercept

Analyte	Slope	Intercept	Adult Old Ref Range	Adult New Ref Range
Alk phos.	0.8	+4.4	50-136	35-115
ALT	0.9	-14.5	30-65	M: 10-63 F: 10-54
Amylase	1.3	+9.5	25-115	30-140
Creatine Kinase	1.1	+2.5	M: 35-232 F: 21-215	M: 49-397 F: 38-234
CK-MB	1.0 +1.5	0-5	0-6.3
CK-MB %Relative Index	0-4.0	0-3.0
GGTP	0.7	+4.9	M: 15-85 F: 5-55	M: 5-65 F: 5-55
LDH			100-190	100-210
Lipase	0.2	-11.5	114-286	15-51
Iron			35-150	M: 45-182 F: 28-170
TIBC			M: 260-400 F: 260-445	250-450
Uric Acid			M: 3.5-7.2 F: 2.6-6.0	M: 4.8-8.7 F: 2.6-8.0

For each analyte, sex-specific reference ranges have been validated for adults. Considerable collaboration with Children's Hospital Oakland and UC Davis Medical Center, supplemented with literature reviews, has led to assigning age-specific reference range values for pediatrics and adolescents as well. Thus for each assay, lab reports will display the appropriate age and sex-specific reference range for each patient.

As always, if there any questions or concerns, please do not hesitate to contact Barry Latner, M.D. (925) 692-5405, or Connie Houser or Pat Sinz in the Clinical Laboratory at MuirLab (925) 692-5631. Thank you.

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Community-Acquired Methicillin Resistant Staphylococcus Aureus (CA-MRSA)

Presentation:
This pathogen causes skin infections presented as cellulitis, impetigo, folliculitis, furunculosis, carbuncles, abcesses, and infected lacerations. The patient has no history of hospitalization, admission to a nursing home, dialysis, surgery or inserted medical devices in the last year. The diagnosis is made either in the outpatient setting or within the first 48 hours of admission to a medical facility. The patient has no history of MRSA infection.

Treatment:
This begins with incision, drainage and localized care. Diagnosis by culture is useful in recurrent cases or in cases of antibiotic failure. If patient is found to have a MRSA skin infection and antibiotics are indicated, use culture results to select an antibiotic to which the organism is susceptible. If the strain is sensitive to Rifampin, that drug should never be given alone because of rapid development of resistance.

The USA 300 clone is becoming the dominant strain causing 90% of incoming skin and soft tissue infections. The 300 clone gets its name from its identifying markers using pulsed-field molecular epidemiology. Investigators found the strain typically is resistant to beta-lactam drugs and erythromycin but susceptible to clindamycin. Some MRSA isolates that are reported as susceptible to clindamycin and resistant to erythromycin may have inducible resistance to clindamycin. In these cases, additional laboratory testing (the D test) is necessary before treating a serious infection with clindamycin alone.

Transmission prevention:
Skin infections with MRSA are transmitted by close skin-to-skin contact with an infected person or contact with objects or surfaces contaminated with MRSA.

Wash hands regularly. Wear gloves when managing wounds. Then remove gloves and wash hands with soap and water. Carefully dispose of dressings. Clean surfaces of exam room and equipment. Linens should be washed in hot water, detergent and bleach and then dried in a hot dryer. Regular trash disposal is effective.

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Creatinine: Introduction of updated method calibrated to be traceable to IDMS

Barry P. Latner, M.D.
Medical Director, Laboratory Services
April 2008

In 2004 the clinical laboratories of John Muir Health started reporting estimated glomerular filtration rate (eGFR) along with serum/plasma creatinine as recommended by the National Kidney Foundation. The rationale for this was to remind us that approximately 11% of the U.S. adult population has chronic kidney disease, which is defined as either kidney damage (i.e. persistent proteinuria) or decreased kidney function (i.e. decreased GFR) for 3 or more months. It was further noted that adverse outcomes of chronic kidney disease, such as development of kidney failure and cardiovascular disease, could often be prevented or delayed through early detection and treatment.

One of the early recommendations was that "Autoanalyzer manufacturers and clinical laboratories should calibrate serum/plasma creatinine assays using an international standard." An isotope dilution mass spectrometry (IDMS) reference method was chosen, but was not widely available back then. The manufacturer of our assay made reagent systems that are traceable to it available to us in late 2007. Since we have completed our internal validation studies, we will be bringing it on line soon.

In comparison to our old assay, the new IDMS-traceable creatinine runs slightly lower at values <2.00 mg/dL (range 0.04-0.20 mg/dL; average 0.07 mg/dL). This affects the reference range; new, Males: 0.61 - 1.24 mg/dL, Females: 0.44 - 1.10 mg/dL. In addition, the MDRD Study equation to calculate the eGFR also changes slightly. However, in comparison studies, the new IDMS-traceable calculated eGFR does not vary significantly from our old eGFR.

Regarding the laboratory determination of eGFR, the published National Kidney Disease Education Program guidelines also raise additional points:

1. The MDRD Study equation should only be used in individuals age 18 and older. Additionally, the equation has not been validated for use with the elderly (>70 years of age), pregnant women, patients with serious comorbid conditions, or persons with extremes of body size, muscle mass or nutritional status. For most patients, an eGFR is more accurate than a creatinine clearance calculation from serum and urine measurements. Therefore, it is not recommended to perform a measured creatinine clearance procedure for adults except when the patient's basal creatinine production is expected to be significantly abnormal (e.g. obese, severely malnourished, amputees, paraplegics, muscle-wasting diseases, strict vegetarian diets, muscle builders on creatine supplements, etc.).
2. The equation has been most extensively evaluated in people with chronic kidney disease and reduced GFR and is less accurate for persons with normal or mildly impaired kidney function. This is the rationale for the eGFR value of 60 being considered the cutoff for the reference range.
3. It is recommended that creatinine values in mg/dL be reported to two decimal places. This will reduce rounding errors that may contribute to imprecision in the eGFR value.

References:

1. www.nkdep.nih.gov
2. Myers, GL, et al. Recommendations for Improving Serum Creatinine Measurement: A Report from the Laboratory Working Group of the National Kidney Disease Education Program. Clin Chem 2006; 52:5-18.
3. Ceriotti, F, et al. Reference Intervals for Serum Creatinine Concentrations: Assessment of Available Data for Global Application. Clin Chem 2008;54(3):in press.

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Cystatin C Measuring Glomerular Filtration Rate (GFR)

MuirLab introduces the latest addition to our test menu, N Latex Cystatin C, a new diagnostic test for the quantitative determination of Cystatin C in human serum or plasma.

Cystatin C provides a practical alternative to the traditional 24-hour urine creatinine clearance for the assessment of Glomerular Filtration Rate (GFR) with a simple blood collection. GFR is estimate of renal function, which until now, has been a difficult parameter to measure.

N Latex Cystatin C possesses many of the attributes of the ideal GFR marker to help ensure an accurate estimation of GFR. Cystatin C levels are not influenced by age, sex, muscle mass, commonly used prescriptions or over-the-counter drugs, as are traditional tests. There are no lengthy 24-hour urine collections or manual calculations, and the lack of patient compliance becomes a thing of the past!

Cystatin C is test #11893. It is covered by most insurers and results are available the next day.

If your or your staff would like more information or technical data regarding Cystatin C please contact your local MuirLab Representative at: (925) 692-5411

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Cystic fibrosis

Cystic fibrosisis a common autosomal recessive genetic disorder with an incidence of approximately 1:3000 births. Over 1000 mutations in the cystic fibrosis transmembrane regulator (CFTR) gene have been described.

JMMC Clinical Laboratory offers a Cystic Fibrosis Mutation screening test that includes all core mutations recommended by the American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics (ACMG) for population-based CF carrier screening. While some assay platforms may detect rare mutations not included in the standard ACOG/ACMG panel, these mutations are not reported due to lack of consensus by ACOG/ACMG.

A negative test result does not rule out cystic fibrosis disease or carrier status. The risk for mutations other than the ones tested depends greatly on family and clinical history as well as ethnicity. If an individual is asymptomatic and has no family history of cystic fibrosis, the following table is valid for deteminiing their residual risk to carry a CF mutation.

Ethnic Group	Detection Rate	Estimated Carrier Risk Prior to Testing	Estimated Carrier Risk after Negative Test
Ashkenazi Jew	97%	1/25	1 in 830
European Caucasian	90%	1/25	1 in 250
African American	69%	1/65	1 in 207
Hispanic American	57%	1/46	1 in 105

A heterozygous positive result indicates that an individual is at least a carrier for cystic fibrosis. Carrier status assumes that this individual is not clinically affected. If cystic fibrosis is suspected, the individual may have a second mutation not detected by this assay, and testing for additional mutations is recommended. Genetic counseling and additional testing by mutation analysis should be offered to patients, relatives and reproductive partners.

A homozygous positive result indicates that an individual is affected with cystic fibrosis. Genetic counseling and additional testing by mutation analysis for the patient and family members is recommended.

These mutations are detected by linear signal amplification developed by Invader® DNA Technology. The method employs a novel, homogeneous platform that analyzes DNA without prior amplification of the target. The assay uses Cleavase® enzymes to recognize and cleave specific structures formed by the addition of two oligonucleotides to a nucleic acid target. The signals generated in the reaction are detected by a fluorescent microtiter plate reader.

The performance characteristics of this test were validated by John Muir Medical Center Clinical Laboratories. The U.S. Food and Drug Administration (FDA) has not approved this test. However, FDA approval is currently not required for clinical use of this test. The results are not intended to be used as the sole means for clinical diagnosis or patient management decisions. This test is performed pursuant to an agreement with Third Wave Techolgies.

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Diagnosis of Enterohemorrhagic E. coli infection by Detection of Shiga Toxin

July 2007

Enterohemorrhagic E. coli (EHEC), also known as Shiga-toxin-producing E. coli (STEC), is recognized as an important cause of human diarrhea, hemorrhagic colitis, thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). The E. coli serotype O157:H7 has been documented as the most common cause of sporadic and epidemic outbreaks of EHEC disease. However, it has been discovered recently that more than 100 other non-O157:H7 EHEC serotypes are capable of producing the same disease syndromes, since these serotypes may also produce Shiga toxins.

The traditional laboratory diagnosis of EHEC infection has been dependent on the recovery of E.coli O157:H7 followed by immunologic confirmation; however, culture sensitivity is roughly 50 to 80% and does not detect the presence of non-O157 Shiga-toxin-producing E.coli.

One virulence trait of all EHEC strains is their ability to produce two potent cytotoxins called Shiga toxins. Shiga toxin 1 is identical to the toxin produced by Shigella dysenteriae; Shiga toxin 2 is more commonly associated with HUS than Shiga toxin 1. An EHEC may have either or both toxins present. These toxins have a cytotoxic effect on intestinal epithelial cells that probably causes the characteristic bloody diarrhea. Systemic spread of Shiga toxin causes renal endothelial cell toxicity and may be responsible for potentially life-threatening HUS.

EHEC is transmitted primarily by ingestion of contaminated foods such as undercooked meat (especially ground beef), vegetables (spinach, alfalfa sprouts, etc.) and unpasteurized fruit juices or dairy products. Because agricultural products are increasingly processed in mass quantities and are widely distributed, sometimes globally, large numbers of people could potentially be exposed to EHEC. One hamburger patty may contain the meat of several animals from four different countries. One beef carcass ground for hamburger can contaminate 8 tons of finished ground beef.

The Centers for Disease Control and Prevention (CDC) has issued the recommendation that all stool specimens submitted for culture include detection of Shiga toxins. Beginning in August 2007, MuirLab will routinely screen all stool culture specimens for Shiga toxin, in addition to Salmonella, Shigella and Campylobacter spp. The method used will be a rapid immunochromatographic test. Those specimens that test positive for either or both Shiga toxin 1 and 2 will be further processed for isolation of STEC, with confirmation by the public health department. The State of California has also added Shiga toxin to its list of reportable diseases.

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Diagnosing Legionnaire's Disease: Legionella Detection by PCR

January 2008

The genus Legionella includes over 40 different species of fastidious Gram-negative bacilli. While these organisms represent normal environmental flora, many have been shown to cause human disease, most commonly opportunistic pneumonia in immunocompromised patients. The vast majority of such cases are due to L. pneumophila (85%), with the minority most frequently caused by L. micdadei, L. bozemanii, L. dumoffii and L. longbeachae.

For many years the gold standard test for diagnosing Legionella infection has been culture of bronchoalveolar lavage and lung biopsy. A drawback of culture is that results may not be available for several days to longer than a week, since the organism can be quite slow-growing and fastidious. It should also be noted that sputum (expectorated, aspirated or induced) rather than a BAL or lung biopsy is often submitted for Legionella culture and may be less likely to yield positive results.

Rapid testing methods, which include direct histochemical staining of tissue, fluorescent antibody staining of tissue or pulmonary secretions and urinary antigen detection have been useful but frequently lack sensitivity and/or specificity. Urinary antigen detection is specific for L. pneumophila and will not detect the presence of any other Legionella species. Serological diagnosis is highly sensitive, but its utility is generally limited to epidemiological studies, due to the time lag needed to detect seroconversion.

Nucleic acid amplification techniques are attractive tools for detection of Legionella species. Recent studies have shown that Legionella Species by PCR (polymerase chain reaction) offers highly sensitive and specific results for multiple Legionella species with rapid turnaround times of one to two days. Tissue and pleural fluid, as well as respiratory specimens are acceptable for PCR testing for Legionella species. Because of these recent advances in Legionella diagnosis by PCR, MuirLab will no longer offer the culture method.

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Encourage HIV/Aids Testing

January 2008

Despite recent improved access to antiretroviral treatment and care in many regions of the world, the AIDS pandemic claimed between 2.4 and 3.3 million lives in 2005, the highest number since 1981. Half a million or more than 570,000 of these deaths were children less than 15 years old.

The results of a nine country survey released in November 2007 in time for World AIDS Day on December 1st and involving more than 4500 interviews, revealed more than 40% of respondents did not believe that AIDS is always fatal. The countries included the US, UK, Russia, France, China, India, Mexico, Brazil, and South Africa. According to recent World Health Organization statistics, only 28% of the world's HIV/AIDS patients are receiving anti-retroviral drugs. In the United States:

• Approximately 40,000 new cases of HIV diagnosed each year
• Approximately 25% of all HIV cases remain unaware that they are HIV positive.
• Approximately 1/5th of US residents at high risk are tested
• Approximately half of the tests done are at the initiative of the individual and the other half are done at the initiative of a health care provider
• Many high-risk individuals plan to get tested but do not
• Minority females report the highest rates of testing and white males the lowest.

In 2006 the CDC made the following changes in test recommendations:

1. Routine HIV screening for all patients 13-64 years old - regardless of risk level
2. Annual screening for high risk individuals
3. Test all individuals whose blood or body fluids are source of occupational exposures
4. Simplification or elimination of pretest counseling and written consent procedures
5. "Opt-out" testing to make HIV tests part of routine healthcare and remove the stigma of testing
6. Screen all pregnant women early in pregnancy and repeat testing in the 3rd trimester if the women is at high risk or living in areas with high HIV prevalence.

If you have not already done so, perhaps now is the time to consider getting yourself and your partner tested, in order to know your baseline HIV status. For more information or to find an HIV testing site near you

• call 1-800-CDC-INFO 24 hours / day (English and Spanish)
• TTY 1-888-232-6348 (English and Spanish)
• Or visit the National HIV Testing Resources Web site at http://www.hivtest.org

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Epstein-Barr Virus testing update

In addition to its well-known role in infectious mononucleosis (IM), Epstein-Barr virus (EBV) is implicated in several malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Fortunately, increasing demand for viral testing has made sophisticated blood tests for EBV more easily available, and these tests are now being routinely performed at MuirLab.

The traditional Monospot test is still helpful for evaluating patients with acute "mononucleosis syndrome." However, 10-20% of adults (and even more children) with acute IM may have negative Monospot results at the time of presentation, and conversion to a positive Monospot may take months.

Confirmation of EBV infection in these Monospot-negative patients can be obtained with additional testing. Serum antibodies to viral capsid antigen (VCA) are present in most (but not all) IM patients at the time of clinical presentation. As with many other infections, IgM antibodies disappear in a few months while IgG antibodies persist indefinitely. An acute infection can be demonstrated either by the presence of IgM antibody or by a rising level of IgG antibody upon repeat testing.

Another useful antibody is EBNA, directed against Epstein-Barr nuclear antigen. This antibody increases gradually during convalescence from IM, and persists for life. Thus, the appearance of this antibody in a patient who was previously VCA positive but EBNA negative indicates a recent infection.

Antibodies to "early antigens" (EA-R and EA-D), are found transiently in most patients with an acute EBV infection. Persistently elevated antibody titers to EA and EBNA together may suggest reactivation or persistently active EBV infection.

In order to facilitate EBV diagnosis, MuirLab offers a convenient EBV antibody panel, which includes four tests: VCA IgM, VCA IgG, EBNA, and EB Early Antigen D. Since the serologic response to early EBV infection can change over a period of several weeks, repeat testing is recommended if the initial panel results are not clear-cut or do not correlate with the patient's clinical picture.

As an example, consider the testing results for a 15 year old child who recently presented with cervical adenopathy, hepatosplenomegaly, fever, and malaise. After a Monospot test was negative, the patient's pediatrician ordered the EBV panel. All four tests in the panel were negative, and the patient was referred to a specialist. That physician decided to order tests for viral hepatitis and CMV, which were negative. At the same time, the EBV panel was repeated. By then, four weeks had elapsed, and three out of the four EBV tests had converted to positive (only the EBNA was still negative; this would be expected to turn permanently positive at some point during convalescence).

For most patients, the four-test EBV panel (repeated in several weeks if necessary) will provide the necessary diagnostic information. For special situations, PCR testing of actual viral DNA can be performed on serum or plasma, or in-situ hybridization can be performed on tissue biopsies (these tests are sent to reference laboratories).

The commonly ordered EBV panel includes only IgG and IgM antibodies. If EBV-related nasopharyngeal carcinoma is a clinical concern (for example, in patients from demographic groups at high risk), serologic testing should include IgA antibodies to VCA (tested at our reference laboratory), because these are more specific for that malignancy than are the IgG antibodies.

It has been estimated that EBV will infect 90% of the world's people over their lifetimes. The multiple roles of this virus will continue to be the focus of much research. With the four-test EBV panel now available, at least the diagnostic evaluation of MuirLab patients is now more convenient than ever before.

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ESBLs: The Rise of Antibiotic Resistance

Microbes have the incredible ability to quickly respond to antibiotic pressures by developing various mechanisms of resistance. It is with increasing regularity that we read about the "super bugs" such as MRSA, VRE, and multiply resistant M. tuberculosis that are spreading throughout our communities and healthcare facilities. Multiple drug resistance is also becoming more common among enteric Gram negative rods.

Since the introduction in the late 1980s of extended-spectrum beta-lactam antibiotics that include the 3rd and 4th generation cephalosporins, as well as extended-spectrum penicillins such as ticarcillin and piperacillin, there has been a steady increase in resistance to these broad spectrum agents. This resistance is caused by the production of Extended-Spectrum Beta-Lactamase (ESBL) enzymes. While ESBL production is exhibited most frequently by E.coli, Klebsiella spp., and Proteus mirabilis, other Gram negative rods may also produce ESBLs.

MuirLab Microbiology routinely screens for and confirms the presence of ESBL for all clinically significant E.coli, Klebsiella spp., and Proteus mirabilis. For those isolates that are confirmed as having ESBL, the interpretation for all penicillins, cephalosporins and aztreonam are changed to Resistant even though these drugs may appear to be effective with in vitro testing. Cephamycins such as cefoxitin and cefotetan are not affected by ESBLs, although there are resistance mechanisms aside from ESBL that may cause these drugs to be ineffective as well.

ESBLs are of significant concern to infection control practitioners at acute care hospitals and long term care facilities because ESBL resistance is transmitted via plasmids from one organism to another, and thus, from one patient to another. ESBL producing Gram negative rods are generally treated with carbapenems (e.g., imipenem and meropenem), or with fluoroquinolones (e.g., levofloxacin and ciprofloxacin); however, there is every indication that resistance to these agents is also increasing.

This table lists the percentage of isolates that have been confirmed as ESBL producers at MuirLab Microbiology within the past few months.

Patient location	% of E.coli that produce ESBL	% of Klebsiella spp. that produce ESBL	% of Proteus mirabilis that produce ESBL
Skilled nursing facility	10.1%	13.8%	3.9%
Outpatient	1.5%	3.2%	2.0%
Acute care hospital	2.7%	13.4%	5.8%

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Estradiol assay update: minor changes to reference ranges due to change in standardization. by Barry P. Latner, M.D. Medical Director, Laboratory Services (10/2008)

The manufacturer of our Estradiol assay has updated its standardization to the international reference method of isotope dilution gas chromatography mass spectrometry (ID-GCMS) method from the USP reference material. As a result, there are some changes to the reference ranges for patients as determined by the manufacturer.

	Current Ref Range	New Ref Range
Males	20-75 pg/mL	<48 pg/mL
Post-menopausal Females	20-88 pg/mL	<41 pg/mL
Non-Pregnant Females
Mid follicular phase	24-114 pg/mL	27-122 pg/mL
Mid luteal phase	80-273 pg/mL	49-291 pg/mL
Peri-ovulatory phase	62-534 pg/mL	95-433 pg/mL

Reported values with this assay below 40 pg/mL must be interpreted with caution as the analytical variability (expressed as CV; coefficient of variation) remains >20% at these concentrations. If you would like further information, please call Linda Gaede (925.692.5633), Pat Sinz (925.692.5631), or me (925.674.2508). Thank you.

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Estimated Glomerular Filtration Rate (GFR)

Recently, the National Kidney Foundation published 15 clinical practice guidelines on chronic kidney disease. Their work shows that approximately 11% of the U.S. adult population have chronic kidney disease, which is defined as either kidney damage (i.e., persistent proteinuria) or decreased kidney function (i.e., decreased GFR) for 3 or more months. Adverse outcomes of chronic kidney disease, such as development of kidney failure and cardiovascular disease, can often be prevented or delayed through early detection and treatment.

Guideline 4 relates to the laboratory determination of GFR. Several points are worthy of discussion:

1. "Clinicians should not use serum creatinine concentration as the sole means to assess the level of kidney function." Since serum creatinine concentration is affected by factors other than GFR (e.g. relative state of hydration, creatinine secretion and generation, and extrarenal excretion), there is a relatively wide range of values in normal persons. GFR must decline to about half the normal level before the serum creatinine value rises above the upper limit of the reference range. In the elderly, the age-related decline in GFR is not reflected in the serum creatinine because of the age-related decline in muscle mass that reduces creatinine generation.
2. "Physicians should estimate the level of GFR from predictive equations that take into account the serum creatinine concentration and some or all of the following variables: age, sex, race and body size. The Modification of Diet in Renal Disease (MDRD) study and Cockcroft-Gault equations provide useful estimates of GFR in adults."
   
   To aid in this recommendation, our Clinical Laboratory will calculate and report estimated GFR for every serum/plasma creatinine test performed on adults using the following MDRD study equation:
   
   GFR (mL/min/1.73 m2) = 186 x (Creat.)^-1.154 x (Age)^-0.203 x (0.742 if female) x (1.210 if African American)
   
   The MDRD study equation has many advantages. It is more accurate and precise than other equations, particularly for persons with a GFR less than 90 mL/min/1.73m2. It predicts GFR as measured by an accepted method (urinary clearance of 125I-iothalamate). It was developed on a large database containing persons with various kidney diseases and was tested on a validation database containing more than 500 additional patients. Current studies are underway to validate the equation in a variety of ethnic groups, persons with diabetes and people with normal renal function. It has not been validated in children.
   
   A GFR value less than 60 mL/min/1.73m2 represents loss of half or more of the adult level of normal kidney function. Below this level, the prevalence of complications of chronic kidney disease increases. Thus a GFR value of 60 has been considered the cutoff for the reference range, and values below this will be flagged as abnormal on our reports. In addition, the MDRD equation includes a patient's race (African-American or non-African American), and since this is often not available to the Clinical Lab, values for GFR will be reported for both.
3. 3. "Autoanalyzer manufacturers and clinical laboratories should calibrate serum/ plasma creatinine assays using an international standard." Our assay is a kinetic alkaline picrate (Jaffe) reaction similar to that used in the MDRD study and calibrated to National Institute of Standards and Technology (NIST).
4. 4. "Measurement of creatinine clearance by using timed (e.g., 24-hour) urine collections does not provide more accurate estimates of GFR than do predictive equations." In the MDRD study, the estimated GFR calculation provided a more accurate estimate of GFR than measured creatinine clearance. The guidelines recommend obtaining 24-hour urine collections only for special clinical circumstances; these include extremes of age and body size, diseases of skeletal muscle, paraplegia or quadriplegia, strict vegetarian diet, and rapidly changing kidney function. The guidelines are completely silent on the use of Cystatin C. One small study of 112 patients in Finland (Clin Chem 2003;49:1223-5) concluded that estimated GFR based on Cystatin C determinations were very similar to those from the MDRD study equation. While one study does not a trend make, it is worth considering that Cystatin C is a fairly new test and its place in the testing armamentarium for kidney disease is in evolution.

For more details on all of the guidelines, please see the following references:

1. Ann Intern Med 2003;139:137-47. National Kidney Foundation Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification, and Stratification.
2. Am J Kidney Dis 2002;39:S1-246. K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification, and Stratification. Kidney Disease Outcome Quality Initiative.
3. http://www.nkdep.nih.gov

Barry P. Latner, M.D.

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Factor V Leiden (FVL) Gene Mutation Analysis by Third Wave Invader Assay

by Jeffrey L. Curtis, MD

Normal hemostasis requires a balance between procoagulant and anticoagulant systems which may be perturbed by either inherited or acquired defects. Acquired defects may be environmental or intrinsic. Inherited defects associated with bleeding disorders, such as hemophilia A or B and von Willebrand's disease, have been known and studied for many years. Inherited defects causing thrombosis and hypercoagulability are a more recently recognized phenomenon, and include factor V Leiden, antithrombin III deficiency, protein C and protein S deficiencies and a few others.

Venous thrombosis leads to 50,000 to 100,000 deaths and 500,000 hospitalizations annually in this country, and is a multifactorial problem with both heritable risk factors and environmental conditions implicated. The incidence of symptomatic venous thrombosis is 1:1,000 people per year. Risk factors for venous thrombosis include pregnancy, oral contraceptives, estrogen therapy, malignancy, stroke with paresis of extremities, trauma, surgery, and prolonged immobility. It also may occur in the absence of known risk factors. Genetic causes are implicated in 25% of unselected venous thrombosis cases, and approximately 63% of familial ones.

FVL is the most common genetic risk factor for venous thrombosis, and was discovered in 1993. Factor V is a cofactor in blood coagulation, and is regulated by the protein C system inhibitor complex. The FVL mutation is caused by a single point mutation in the gene for factor V. By destroying a cleavage site in the protein, factor V degradation by activated protein C (ACP), which inactivates factors V and VIII, is inhibited in those with the FVL mutation. This results in a state of hypercoagulability and "activated protein C resistance."

Individuals with FVL have a propensity to develop venous thromboembolic disease (deep venous thrombosis and pulmonary embolism) at a younger age than the normal population. Venous thrombosis is the third most common cardiovascular disease in the United States. The risk of venous thrombosis is increased seven-fold in heterozygotes and 80 fold in homozygotes. Coupling of a heterozygous mutation with oral contraception synergistically increases risk of venous thrombosis thirty-fold. Synergistic risk increases also occur when the mutation is coupled with elevated homocysteine levels or a factor II (prothrombin) 20210G→A mutation. FVL is also associated with arterial thrombosis (particularly in smokers), complications of pregnancy, and increased levels of factor VIII.

Twenty percent of venous thrombosis cases involve FVL, with 3 to 7% of the general Caucasian population and 1.2 % of the African-American population possessing the mutation. Up to 95% of cases of activated protein C resistance and subsequent defective anticoagulation are due to FVL. Populations considered at risk for this mutation include: (1) those with an initial venous thrombotic event at age 50 or younger; (2) relatives of those individuals; (3) young people with histories of recurrent venous thrombotic episodes or episodes in unusual sites (e.g. mesenteric venous thrombosis); (4) pregnant women with venous thrombotic events; (5) women with recurrent pregnancy loss, unexplained severe preeclampsia, placental abruption, intrauterine fetal growth retardation, or stillbirth; and (6) women placed on oral contraceptives or estrogen replacement at menopause who have had venous thrombotic events.

A new microtiter plate based Third Wave Invader assay that does not require PCR, restriction digestion, or gel electrophoresis has recently been added to the laboratory armamentarium. This method of FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods. The new methodology showed complete concordance when 1,369 individuals were FVL genotyped by both the new assay and allele-specific PCR. Mutation analysis is not affected by heparin, oral anticoagulants, pregnancy, oral contraceptives, estrogen replacement therapy, or lupus anticoagulants.

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Facts About PTHI

When ordering Parathyroid Hormone Intact Molecule, TOTAL CALCIUM is needed to produce a result that includes a diagnostic graph which plots parathyroid hormone concentrations against total serum calcium values. This graph is designed to aid in the diagnosis of primary hyperparathyroidism, secondary hyperparathyroidism, hypothyroidism and hypercalcemia of malignancy. It is for this reason that testing laboratories consider the information provided in the PTHI with calcium to be of more value to the physician than the PTHI without the calcium.

If you would prefer to order the PTHI with calcium and have the test without calcium preprinted on the bottom of your MuirLab requisition, please contact your sales representative so that they can make the appropriate changes.

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FDA Clears Lab Test for Bird Flu Infections in Humans

The Food & Drug Administration this month approved a new laboratory test to diagnose the H5 strain of the Avian Influenza virus (a.k.a. bird flu) in people. The test, which was developed by the Centers for Disease Control & Prevention, is called the Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set. It provides results within four hours vs. 2-3 days for previous culture-based methods. If the test detects the H5 strain, further tests must be run to see if the N1 subtype is involved.

The CDC will distribute kits for the new test to all 50 states. The kits will go to labs in the Laboratory Response Network, totaling about 140 labs nationwide. The test has also been shared with the World Health Organization and its collaborating centers worldwide. Results of the test will be used to track cases of illness with the H5 strain. The test is used in patients with symptoms of severe respiratory illness and a risk of exposure, such as direct contact with sick, dead, or infected poultry in a country with outbreaks of the H5N1 virus in poultry.

Since December 2003, more than 160 human cases of bird flu caused by the H5N1 strain have been reported in Thailand, China, Vietnam, Cambodia, Indonesia, Turkey, and Iraq. More than half of those infected have died.

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Glucose Point-of-Care Testing Results

Glucose point-of-care testing results done by RN's are now viewable when physicians/nurses look up lab results in the computer.

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HPV Testing—New Information

By Barry P. Latner, M.D.

Through comprehensive epidemiologic and molecular biologic studies over the last 10-15 years, it has become clear that infection with specific types of Human Papilloma Virus (HPV) is required for the development of cervical caner and its high-grade precursors (high grade SIL; CIN 2, 3). This has led to the development of sensitive molecular methods to identify HPV DNA in clinical specimens, which are available for clinical use. Such methods include Southern Blot, Hybrid Capture, PCR, and In-Situ Hybridization. The laboratories at John Muir Health are pleased to announce that we have evaluated and adopted one of these methods, In-Situ Hybridization, into our practice. Beginning early July, requests for HPV testing will be performed by this method and the results integrated into our GYN cytology (Pap smear) report.

This testing is accomplished from the same liquid based medium that is currently being used for the Pap test, so no additional specimen collection is needed. It does require, however, that the specimen collection be diligent and proper. No problem! The test performed will be for "high risk" HPV types utilizing a probe mixture designed to identify HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 68. Because "low risk" HPV types are uncommonly found in association with high grade SIL (CIN 2, 3) and cancer, testing for these types is not considered useful and will not be performed. [The use of "low risk" HPV testing has a role in the selected instance of when a pathologist needs to determine whether a lesion that does not have the classic histological features of a condyloma acuminata is an HPV associated lesion or another type of lesion.]

The main advantage of the In-Situ Hybridization test is its ability to correlate DNA probe results with morphology. It is sensitive to 10-50 copies of target DNA in cell nucleus and does not suffer from false negatives due to insufficient cellularity in the sample. A study from authors at Columbia University Medical Center, Cleveland Clinic, and Associated Regional and University Pathologists (Diagnostic Cytopathology 2003; 29:149-55) demonstrated excellent negative predictive value (99%) and significantly better sensitivity specificity and positive predictive value than the Hybrid Capture II method.

In 2001, consensus guidelines were published on the use of HPV DNA tests in the management of women with cervical abnormalities and CIN. The American Society for Colposcopy and Cervical Pathology (ASCCP) is slated to meet again this Fall to update these consensus guidelines.

If there are any questions, please do not hesitate to contact your local client service representative or contact me directly at (925) 692-5405.

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Immunochemical Fecal Occult Blood Test (FOBT)

by Barry P. Latner, M.D., Medical Director, Laboratory Services

Fecal occult blood testing (FOBT) is used as a method to screen for colorectal cancer and to test for gastrointestinal bleeding in general. Traditionally, it has been performed using guaiac-based methods that detect the pseudoperoxidase activity of the hematin portion of hemoglobin. The guaiac-based methods are limited by a low sensitivity to anything but a clinically significant amount of gastrointestinal bleeding and the major drawback of a high incidence of false positive results especially when patients have not followed a restricted diet prior to testing. Immunochemical tests for FOBT have been studied as an alternative to guaiac-based tests. They are specific for human hemoglobin, avoid false positive results from peroxidase-containing foods in the diet, and are sensitive to relatively low concentrations of blood. One such immunologic FOBT, the FDA-approved Polymedco OC-Auto Micro 80, has been available through MuirLab since December 2005 and continues to meet clinical expectations.

The automated FOBT detects the presence of human hemoglobin by immunoassay using a photometric reading of the presence of an antibody-hemoglobin complex. There are many advantages of this over guaiac-based methods. Firstly, the photometric reading eliminates the subjectivity of interpreting a guaiac card test ("Is that blue?"). Secondly, as it is specific for human hemoglobin, there are essentially no false positives due to dietary peroxidase-containing foods. This eliminates the need for dietary restrictions for several days prior to testing, which potentially avoids a costly GI bleed workup. Finally, the immunologic FOBT is significantly more sensitive to the presence of hemoglobin than are guaiac-based methods, so it requires only ONE sample, rather than the three consecutive specimens recommended for the guaiac-based tests.

Regarding the sensitivity comment, it is known that in a normal state, a small amount of blood (i.e. 2-3 mL) is lost into the intestine each day. In a study by McRae and St. John, it was concluded that any amount over 300 µg hemoglobin/g stool was associated with gastrointestinal pathology. Thus it would be clinically useful to know the actual analytical performance of the various FOBTs that are commercially available, notwithstanding the claims in their package inserts. A well designed study was recently published by Tannous et al from the Massachusetts General Hospital/Harvard Medical School comparing a well known guaiac-based method to four different immunochemical methods including the one performed here at MuirLab. Using known concentrations of blood to spike a known weight of a normal occult blood negative fecal sample, they produced samples ranging from 1500 to 8 µg Hgb/g stool and tested each FOBT method in duplicate. The immunochemical method in use here at MuirLab showed an analytical sensitivity of 300 µg/g, whereas the well known guaiac method had an analytical sensitivity of 1500 µg/g. The other three immunochemical methods showed analytical sensitivities of 1500, 75 and <8 µg/g. This study reaffirms that the immunochemical method performed on site has the appropriate cutpoint to provide the necessary high sensitivity and low incidence of false positive results to optimize screening programs for colorectal cancer.

References:
McRae F, St. John D. Relationship between patterns of bleeding and Hemoccult sensitivity in patients with colorectal cancers or adenomas. Gastroenterol 1988;94:A5.

Tannous B, et al. Comparison of conventional guaiac to four immunochemical methods for fecal occult blood testing: Implications for clinical practice in hospital and outpatient settings. Clinica Chimica Acta 2009;400:120-2.

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Immunological Fecal Occult Blood Test to be Offered

The clinical laboratory at John Muir Medical Center, Walnut Creek Campus will be introducing a new immunochemical screening test for colorectal cancer this fall. Our new test, FOBT (fecal occult blood test) is an automated immunoassay performed on Polymedco's newly FDA approved OC-Auto Micro 80. Developed in Japan 15 years ago, this test is based on the immunological reaction between human hemoglobin and its specific antibody. It will offer many advantages over the currently used guaiac occult blood cards. In the guaiac test, false positives are often seen due to interfering substances from ingested items such as meat, some raw vegetables, vitamin C and aspirin. Our new FOBT test has no dietary restrictions and is specific for human hemoglobin.

Other advantages are: ease of collection, fewer required samples, and safety for the handling clinical scientist performing the test. The guaiac test requires multiple specimens and the collection procedure is messy and offensive to some people. FOBT, because of enhanced sensitivity requires only one sample from a single stool specimen. The sample is collected using a small brush. Once collected the brush is inserted into a closed vial which remains closed throughout the testing process. The collection devices are supplied with mailers so that specimens can be collected at home and mailed to us just as the occult blood cards were.

A study performed at Flinders Medical Center in Japan showed that the brush sampling technique achieved better compliance by patients due to ease of sampling and lack of dietary restrictions. Medicare reimbursement for this test is about $22, a rate nearly 5 times that of the occult blood card test.

The new FOBT will be available in November of this year. We will offer a gradual roll-out, starting with our inpatient population and then moving on to outpatients and nursing facilities. Any inquires can be directed to Dorie Ruhe, Supervisor Transfusion Services, Hematology, Urinalysis (947-3335 ext 5816) or Karen Lewis, Technical Specialist Hematology & Urinalysis (947-3335 ext 2297).

References:
Colorectal Cancer Screening: Immunological FOBT used for mass screening in Japan, Nagase Medical Update, Vol 1, Oct 2001
New Analyzer Standardizes FOBT for Greater Compliance, Better Outcomes, and Improved Bottom Lines, Clinical Lab Products, April 2005
A Randomized Trial of the Impact of New Faecal Haemoglobin Test Technologies on Population Participation in Screening for Colorectal Cancer, Journal of Medical Screening, Volume 10 Number 3, 2003

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Introduction of HIV-1 Viral Load Testing by PCR at MuirLab (10/2008)

In our continuing commitment to provide innovative laboratory services for patients and physicians, John Muir Health and MuirLab are pleased to offer the next evolution in HIV-1 viral load testing, the COBAS® AmliPrep/Taqman HIV-1 test by Roche Diagnostics. This FDA-approved real time PCR is the first in the U.S. to offer full automation sample preparation, amplification and quantitation and delivers full Group M subtype coverage, including the rare non-B subtypes.

The dynamic range of this test delivers standard of care sensitivity and quantitation between 48 copies and 10,000,000 copies. In addition, there is no need to re-baseline or recalibrate patients because a high correlation between this test and other HIV-1 viral load methodologies (including branched-chain DNA assays) has been demonstrated. These studies are available upon request.

If there are any questions, please do hesitate to contact Nick Byrne, M.D. (925-947-5390), Barry Latner, M.D. (925-692-5405) or Cindy Liedstrand, Supervisor Molecular Testing, (925-692-5681).

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Intact PTH Assay

MuirLab is pleased to announce that we will be offering the intact PTH assay as an in-house test. PTH is one of the primary hormones for calcium metabolism and maintenance of optimal calcium ion concentrations, and works directly on bone and kidney. Therefore, measurement of PTH is the most important test for the differential diagnosis of hypercalcemia.

Special Specimen Requirements
The test requires 1-2 ml of serum only, drawn in an SST tube. Because of the nocturnal rise in intact PTH levels, samples should preferably be collected in the morning, after 7 AM, but specimens drawn throughout the day will be accepted. Once the specimen is drawn and is allowed to clot for 30 - 60 minutes (or at most 2 hours) at room temperature, it will be necessary to centrifuge and immediately place the specimen on ice. The specimen will then be handled as a Critical Transport and should be transported on wet ice to INPATIENT processing at John Muir Medical Center (Walnut Creek) within 5 hours of the collection time. The specimen will need to be brought directly to Special Chemistry on ice or in a cold block.

Feedback Mechanism for Calcium Regulation
In healthy individuals, PTH is secreted according to a negative feedback mechanism. A lowered circulating calcium level, triggers a pronounced increase in PTH secretion. Higher than normal calcium levels inhibit PTH secretion. PTH is synthesized and secreted by the parathyroid glands located near the thyroid glands. PTH raises serum ionized calcium levels by increasing the rate of calcium ion flow from bone to the extracellular fluid, and increases both the renal tubular readsorption of ionized calcium and the renal excretion of phosphate. Long-term regulation of total body calcium by PTH occurs in combination with its stimulation of vitamin D metabolism, resulting in enhanced intestinal adsorption of ionized calcium.

PTH Fragments
Intact PTH contains 84 amino acids and has a molecular weight of 9425. PTH undergoes proteolysis to a lesser extent in the parathyroid glands but mostly peripherally- especially in the liver but also in the kidneys and bone- to yield N-terminal fragments and longer lived C-terminal and midregion fragments. The N-terminal fragment contains the region that confers bioactivity. C-terminal and N-terminal fragments are initially generated in equivalent amounts, but the N-terminal fragments disappear rapidly. The C-terminal fragment has a half-life of several hours. In renal failure, C-terminal fragment clearance is impaired, so that high levels are found. C-terminal assays, as well as midregion assays, are especially unreliable in chronic renal failure, where increased PTH is typically just a reflection of impaired renal clearance. In normal renal function, intact PTH is the greatest part of circulating PTH-like bioactivity.

Clinical Significance
In hypercalcemia due to primary hyperparathyroidism production, the majority of patients have elevated PTH levels. By contrast, in hypercalcemia due to malignancy or other causes, the PTH levels are typically low or within normal ranges. A finding of increased PTH in patients with hypercalcemia and malignancy suggests coexisting hyperparathyroidism and malignancy, since ectopic PTH production appears to be extremely rare. PTH levels are also usually high in secondary hyperparathyroidism- usually associated with renal failure- as a result of constant stimulation of the parathyroid gland by low calcium levels. Hypocalcemia together with a low PTH level, on the other hand is to be expected in hypoparathyroidism, either postsurgical or idiopathic.

Types of Assays
Immunoassays for various specific PTH fragments have been developed, by relying on antisera specific for a discrete region such as the C-terminal, the N-terminal, or midmolecule. The antisera employed in these assays recognize not only the specific region in the intact molecule, but similar fragments as well. Recent assays for intact PTH have the necessary sensitivity for detecting circulating intact PTH (amino acid sequence 1-84) in normals and for discriminating between normals and those with primary hyperparathyroidism. These intact assays also appear to discriminate better between primary hyperparathyroidism and hypercalcemia of malignancy compared with previous assays, and do so without significant overlap between the groups.

The intact PTH assay at MuirLab is a solid-phase, two-site chemiluminescent enzyme-labeled immunometric assay.

References:

1. IMMULITE 2000 Intact PTH Package Insert (PIL2KPP-14, 2005-04-05)

2. Burtis C, Ashwood E, Bruns D. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics 2006: 913-919

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John Muir Clinical Lab Offers Affirm VPIII

The clinical laboratory at John Muir Medical Center will be offering the Affirm™ VPIII test for Bacterial Vaginosis/Vaginitis in October of 2006. The Affirm VPIII test is a molecular probe-based system for the rapid diagnosis of the most common causative agents of bacterial vaginosis (BV) and vaginitis; Gardnerella vaginalis; Candida species; Trichomonas vaginalis.

The Affirm VPIII Microbial Identification Test is based on the principles of nucleic acid hybridization. The test uses two distinct single-stranded nucleic acid probes for each organism, a capture probe and a color development probe, that are complementary to unique genetic sequences of the target organisms. Collection: Collect a vaginal specimen using the swab in the ATTS (Ambient Temperature Transport System) set. Label tube with patient name and DOB. In extensive clinical trials, the overall accuracy of Affirm VPIII compared to standard clinical laboratory methods was: 95.8% for Trichomonas (compared to culture and wet mount); 90.2% for Candida (compared to clinically significant culture levels); 97.5% for Gardnerella (compared to Gram stain scored for BV). Use of the Affirm VPIII alleviates the need for examination of the wet mount within 1 hour and reduces the turnaround time for routine genital culture for Gardnerella and Candida species from three days to one.

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Laboratory Medicine & Molecular Diagnostics

R. Scott Liff, VP / COO MuirLab

I recently had the opportunity to attend and present in a Molecular Diagnostics conference held in Ft. Lauderdale Florida. Although I typically shy away from these "technical" types of gatherings, I was quite surprised to find the majority of the presentations and discussions moving away from the scientific developments, which have dominated this area for the past couple of years, to the real-world health care uses for these new discoveries.

Since the '60s and '70s, widely considered the "golden age" of molecular biology, development around the understanding of DNA and its ultimate translation into the proteins - the building blocks of cells - has been a tremendously steep curve. Early manual methods developed to extract, magnify, and measure these proteins have given way to more automated platforms capable of measuring multiple assays on multiple samples at one time. It has been widely publicized that although originally costing in the millions, the current cost to fully map an individual human genome is now approaching $1,000.

Scientifically speaking, this is all great news! But what does it mean for the health care community, and to each of us as individual human beings? Because, science and technology-advances aside, the ultimate applicable translation of these findings into better health care is what we are striving to achieve. It is this translation that is now beginning to take shape and find its way into advances in health care.

The uses for molecular-level diagnostics are many; ranging from more specific diagnosis of illness, to predetermination of specific diseases, to determination of the most effective therapeutic agent to use with both a specific disease and specific individual patient. It is this last use that is commonly referred to as "personalized medicine." The ability to now more accurately pinpoint the disease, as well as determine which medication will give the individual patient the best prognosis, has huge positive ramifications for both health care in general, as well as for individual patients.

Historically speaking, drugs have been given to each patient on a trial and error basis. Since the drug given has been shown to work on many patients with a same disease, it should therefore work in every individual case. However, we now know that no two individual patients react the same to any specific medication. In fact, the same medication found to help many patients may actually harm others. This harmful reaction is what we now call an Adverse Drug Reaction (ADR).

Recent studies have called out ADRs as the fourth leading cause of death in the U.S., with more than 106,000 patient deaths per year resulting from drug reactions. More striking, it is believed that up to 28% of inpatients suffer ADRs - including nearly 2.2 million serious but non-fatal occurrences each year. The cost of these unfortunate instances adds more than $150 billion to our nation's health care costs.

Clearly, we are at an incredibly important crossroad in laboratory medicine with the developing knowledge and application of personalized medicine. Its global application in health care to minimize ADRs and control costs are only overshadowed by its individual use to better manage each patient's health care needs. While this exciting area continues to unfold, we at MuirLab, under our Medical Director, Dr. Barry Latner, and his colleagues in the Contra Costa Pathology group, will continue to review applications of these new scientific discoveries to assist our physicians in providing the best possible health care to our patients.

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Latent Tuberculosis (TB)

Latent TB infection (LTBI) generally results in the conversion of the TB skin test (TST) from negative to positive within two to ten weeks after exposure. Persons with a positive TST are infected, but if they have no symptoms and a negative chest x-ray, they cannot transmit TB to others. They are, however, at risk of progressing from latent to active disease unless they receive anti-TB therapy. Health care workers who convert during employment should: 1) complete a symptom-screen questionnaire, 2) have a CXR — within 7 days is recommended, and 3) be seen by a health care provider or local public health department for treatment. A person with one unexplained symptom on the questionnaire should be excluded from work until active TB has been ruled out. An annual chest x-ray for asymptomatic TST positive employees is not necessary nor recommended.

To identify employees who may have LTBI and to establish a baseline for annual or exposure testing, all new employees should have a two-step TST administered and interpreted prior to beginning work. A TST is not required if there is written documentation of 1) a positive TST on a 1st or 2nd test; a negative 2-step within 90 days prior to employment and 3) history of active TB disease. While several TST screening tests are available, it is important for a facility to stay with one brand or method of TST since variations in reactions could render comparisons with previous reactions inaccurate.

The TST is based on the Mycobacterium tuberculosis ability to produce a delayed-type hypersensitivity skin reaction to certain components of the organism. Components of the bacterium are the core elements of the classic tuberculin PPD. A PPD skin reaction is initiated when specialized immune cells, called T-cells, which have been sensitized by prior infection, are recruited to the skin site. There they release chemical messengers called lymphokines, which induce induration (a hard, raised area with clearly defined margins at and around the injection site) through local vasodilation, edema, fibrin deposition, and other inflammatory cells.

The FDA has approved the use of QuantiFERON-TB Gold Test (QFT) for detecting active TB disease or LTBI. The 2005 Department of Health Services and California TB Controllers Association Joint Guidelines for the Prevention and Control of TB in California Long-Term Health Care Facilities require approval by the facility's patient care committee for use of QFT on new admissions and a grant of program flexibility from Licensing and Certification for use on employees, since the TST is required by CCR Title 22 §72535.

Symptoms of active TB include but are not limited to: Coughing up blood, hoarseness lasting three weeks or more, persistent cough lasting three weeks or more, unexplained, excessive fatigue, unexplained, persistent fever lasting three weeks or more, unexplained, excessive sweating at night, unexplained weight loss.

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MR Contrast Screening Protocols

To date, there have been approximately 226 patients diagnosed with Nephrogenic Systemic Fibrosis/Nephrogenic Fibrosing Dermopathy (NSF/NFD), possibly associated with intravenous Gadolinium MR Contrast administration. In the past 20 years, millions of Gadolinium containing contrast injections have been administered. The FDA believes that there is some risk of this complication (NSF/NFD) related to these contrast agents. All such patients have had moderate to end stage renal dysfunction.

Based on this alert, and after consultation with nephrology, a new screening policy will be instituted at John Muir Magnetic Imaging Center, Contra Costa Imaging Center and John Muir Medical Center, Brentwood Campus for all patients referred for contrast enhanced MRI.

All patients referred for examinations with Gadolinium based contrast media will be screened for the following indications:

• Over the age of 60
• Under the age of 60 with:
  • Known or suspected renal insufficiency
  • Diabetes mellitus
• Scheduled for MRA arteriography including but not limited to:
  • MRA aortofemoral runoff
  • MRA for asymmetric renal arterial stenosis

All Outpatients meeting any of the above criteria will be required to have a Creatinine/eGFR Lab work within 30 days prior to a contrast enhanced MRI. Inpatients will require the same lab work within 2 days. It is the ordering physician's responsibility to order the necessary lab work prior to the MRI exam.

Please contact us if you have any questions about our new screening policy.

Contra Costa Imaging Center
Phone (925) 687-5600

John Muir Magnetic Imaging Center
Phone (925) 295-1545

John Muir Medical Center, Brentwood Campus Medical Imaging
Phone (925) 308-8123

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Novel Influenza A (H1N1) - Laboratory Perspective
Barry Latner, M.D., Medical Director Laboratory Services, John Muir Health

6/30/2009

At the time of this writing, the World Health Organization (WHO) has changed the phase of pandemic alert for the novel Influenza A (H1N1) virus from Phase 5 to Phase 6. This indicates a global pandemic is underway in which there is little or no immunity in the population. While the upgrade designation is an indication to world leaders to implement strategies that reduce the spread, such response has already occurred in our area. It does NOT mean that the virus is more virulent.

The CDC, California Department of Public Health (CDPH) and our local county health services departments have confirmed the widespread incidence of this infection in our community. In the early stages of the outbreak, local county health services and CDPH asked us to test patients with influenza-like illness (ILI) via rapid antigen methods for Influenza A & B, and to forward specimens that tested positive for Influenza A for further speciation testing with real-time PCR. At that time, we were trying to do quick surveillance and identify if and when the novel H1N1 virus would enter the community. Of the 22 rapid test Influenza A positive specimens that the laboratories of John Muir Health forwarded, 21 were confirmed positive for the novel H1N1 Influenza virus (i.e. 95% specificity). As of June 3, 2009 the CDPH reported 1014 total cases of the novel H1N1 Influenza in California (796 confirmed, 218 probable - pending confirmation).

Now that the virus is definitely here, there is no longer a need to test all people with ILI for the novel H1N1 virus, and the practice of referring for confirmation all rapid screen positive Influenza A specimens has been discontinued. However, county health services departments and CDPH are now seeking to determine the severity of disease and how many hospitalizations it is causing. Thus, testing for the novel H1N1 Influenza virus should continue only on people with severe ILI who are hospitalized regardless of rapid test results. For these cases in which further testing for novel H1N1 Influenza virus is required, please notify the John Muir Health laboratory where the specimen was submitted. MuirLab: Microbiology (925) 692-5670; John Muir Med. Center, Walnut Creek Campus: (925) 947-5285 (or ext. 35285); John Muir Med. Center, Concord Campus: (925) 674-2184 (or ext. 22184).

Use of Rapid Influenza Diagnostic Tests: The reliability of rapid influenza diagnostic tests depends largely on the conditions under which they are used. For detection of seasonal influenza virus infection, the sensitivity of the rapid diagnostic test in use at the John Muir Health laboratories ranges from 70% to around 80% and specificity approaches 95%. While it is reasonable to assume that these rapid diagnostic tests can detect novel H1N1 Influenza A in respiratory specimens as nucleoprotein antigens are highly conserved across Influenza A viruses, data are not available on a large scale to determine the sensitivity of the tests for this novel virus. However, our own experience of 95% specificity (see above) is encouraging.

Interpretation of a positive rapid test result: A patient testing positive for Influenza B likely has been infected with seasonal Influenza B virus or is a false-positive result. Such a patient is unlikely to have novel H1N1 virus infection. There are several possibilities when a patient tests positive for Influenza A by rapid antigen test.

• Might have novel H1N1 virus infection, or
• Might have seasonal Influenza A virus infection, or
• Might have false positive test result.

Interpretation of a negative rapid test result: Novel H1N1 Influenza virus infection cannot be excluded. Further testing and treatment should be based upon clinical suspicion, severity of illness, and risk for complications. If there is no epidemiologic link to confirmed cases of novel H1N1 infection and the patient has mild illness, further testing and treatment are not recommended.

For further questions or concerns regarding the laboratory aspect of influenza testing, please contact MuirLab Microbiology Supervisor, Janet Long, MS, MT (ASCP) SM at (925) 692-5671, MuirLab Microbiology Medical Director, Nicholas Byrne, MD at (925) 947-5356, or John Muir Health Medical Director of Laboratory Services, Barry Latner, MD at (925) 692-5405. Thank you.

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Ordering Occult Blood and PSA Testing

Very shortly we will be distributing new requisitions to our clients. You will notice a change in your order options for the Occult Blood and PSA test. The new Occult Blood test (EFOBT) will give you the option of ordering either an Occult Blood Screen (EFOBT) or an Occult Blood Diagnostic (EFOBT) test on your patient. The appropriate test should be ordered on all patients. Medicare does not pay for Occult Blood screening and we will ask the patient to sign an ABN for this test. You will also have the option of ordering a PSA Screen or a PSA Diagnostic test on your patient. The same concept applies to the PSA test. The appropriate test should be ordered on all patients. Medicare will only pay for PSA Screening once every twelve months on a male who is 50 years or older. Medicare patients will be asked to sign an ABN for all screening PSA tests.

Please remember, we need appropriate ICD-9 codes to match the test you are ordering. NCD books have been distributed to our clients outlining which ICD-9 codes Medicare will accept when ordering diagnostic testing for the Occult Blood and PSA Diagnostic tests. If you have not received your NCD book, please contact your sales/service representative. Note: Please remind your patients that the new Occult Blood (EFOBT) test must be received by the laboratory within 5 days of collection.

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PLAC® Test

Predicting Risk for Coronary Heart Disease and Ischemic Stroke Associated with Atherosclerosis
July 2007

About PLAC®: The PLAC test is a blood test that measures the level of Lp-PLA₂, an enzyme highly specific for vascular inflammation and implicated in the formation of rupture-prone plaque. The PLAC test is cleared by the FDA to aid in assessing risk of both coronary heart disease and ischemic stroke associated with atherosclerosis.

Clinical Significance: Lipoprotein-associated Phospholipase A2 (Lp-PLA₂) is produced within the plaque itself. Because elevations in Lp-PLA₂ are independent from traditional cardiovascular risk factors, including obesity, the results of the PLAC test provides valuable additive information to help determine appropriate patient care.

Results from the Atherosclerosis Risk in Communities (ARIC) study indicated that individuals with elevated levels of Lp-PLA₂ had a twofold increased risk of suffering an ischemic stroke over a period of 6-8 years compared to individuals with low levels of Lp-PLA₂. Patients with elevated levels of Lp-PLA₂ and high blood pressure were six-fold more at risk for ischemic stroke.

Appropriate Use of the Test: The test helps identify patients who have "hidden" cardiovascular risk due to the formation of rupture-prone plaque. Suitable patients may include those with two or more risk factors, such as metabolic syndrome or hypertension, even if their overall lipid profiles appear normal.

The PLAC test may be used as a management tool in patients with borderline to high risk for coronary heart disease or ischemic stroke events. It is not recommended for screening low risk patients.

Methodology: The diaDexus PLAC test is a high complexity, enzyme linked immunosorbent assay using two specific monoclonal antibodies for the quantitative determination of Lp-PLA₂ in human serum. This test is now performed at MuirLab / John Muir Health Lab Service.

Specimen: Collection: Submit one 5 mL gold-top SST. Transport: Routine courier pickup. Storage: Store at 2-8°C for up to 7 days.

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RSV Testing

In an attempt to better serve your needs, erase confusion, and eliminate testing errors with regards to (pediatrics) RSV testing, please take note of the following:

The STAT Respiratory Syncytial Virus (RSV) test is the RSV Direct Antigen Screen #15845. If we receive a NP swab aspirate, we perform the RSV Antigen Screen.

Respiratory Syncytial Virus DFAs are not performed STAT. These tests are run at 1:00 PM daily. NP slides must be made and submitted for DFA testing.

Please be specific when ordering. The title "RSV test" is too ambiguous, and we run the risk of not performing the actual test desired. Please specify the test required, RSV Direct Antigen Screen, or RSV DFA.

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Reporting of Phenytoin (Dilantin) Concentration

by Barry Latner, MD
July 2007

In an attempt to remind clinicians of the importance of the degree of protein binding in the action of phenytoin and in the interpretation of serum/plasma phenytoin concentrations, the Clinical Laboratories of John Muir Health will soon report two additional "corrected" phenytoin concentration calculations when serum/plasma albumin falls below 4.0 mg/dL. The rationale for the "corrected" values relates to changes in the bound vs. unbound fractions as explained below.

As a review, phenytoin (diphenylhydantoin) acts by modulating the synaptic sodium channel by prolonging inactivation, which reduces the ability of the neuron to respond at high frequency. The physiologic effect of this action is reduction in central synaptic transmission, aiding in control of abnormal neuronal excitability. The optimal therapeutic concentration for seizure control is 10-20 µg/mL. Total phenytoin concentrations >20 µg/mL do not usually enhance seizure control and are often associated with nystagmus and ataxia. Concentrations >35 µg/mL have been shown to actually precipitate seizure activity. Development of gingival hyperplasia, a side effect of phenytoin, does not appear related to serum/plasma concentration.

Once absorbed, phenytoin is tightly bound to protein (90-95%) and has a half-life around 20 hours. As with all drugs, the pharmacological effect is directly related to the amount present in the free (unbound) state. Only free phenytoin is available to cross biological membranes and interact at biologically important binding sites. The degree of protein binding can be reduced by hypoalbuminemia, uremia, and by the presence of other drugs that compete for protein-binding sites (e.g. salicylate, valproic acid, sulfisoxazole and sulfonylureas). In these conditions, an increased effect is observed (due to increased free form) at the same total drug concentration as in serum/plasma from normal patients. Virtually all phenytoin assays commercially available measure total phenytoin concentration. Unfortunately, the measurement of free phenytoin is challenging and not readily available in house, thus not available in a timely fashion.

The Clinical Laboratories will utilize two equations that have been shown to be useful in predicting free phenytoin concentration in patients with hypoalbuminemia (defined as <4.0 mg/dL) and normal function and those with renal failure (Creatinine Clearance <10mL/min).

For normal renal function:		For renal failure:
Corrected phenytoin =	Measured total phenytoin (0.2 x Albumin) + 0.1	Corrected phenytoin = Measured total phenytoin (0.1 x Albumin) + 0.1

To help the Clinical Laboratory assist you in this patient safety initiative, we ask that an Albumin be ordered whenever Phenytoin concentration is desired.

The foregoing has been endorsed by the Pharmacy and Therapeutics Committees of John Muir Health. If there are any questions or concerns, please do not hesitate to contact me. Thank you.

References:

Evans WE, et al. Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring. Vancouver: Applied Therapeutics. 1992.

Winters ME. Basic Clinical Pharmakokinetics. Vancouver: Applied Therapeutics. 1994.

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Stat Gram Stain Specimens

April 2008

On March 31, 2008, the Microbiology Department will be moving to the new MuirLab Core Lab in Concord. In order to expedite STAT gram stains on specimens that will need to be read in the hospital, we are requesting that two swabs of the specimen be submitted to the Laboratory. One swab will be used to make the gram stain and the second one will be used to inoculate the media for culture. This is required in order to insure that the culture specimen is not contaminated in the process of making the slide for the gram stain.

If there are questions about this process, please contact Janet Long, Microbiology Supervisor at (925) 692-5671 or Susan Sheldon at (925) 674-2180. Thank you.

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Update to Cardiac Troponin I (cTnI) Reporting

Barry P. Latner, M.D.
Medical Director, Laboratory Services, John Muir Health
January 2008

In September (2007) the Clinical Laboratories of John Muir Health introduced a new assay for cTnI and implemented reporting practice guidelines published in the laboratory literature and endorsed by all major cardiology organizations (including ACC and AHA). A significant guideline for cTnI was the decision-limit for myocardial injury/myocardial-cell necrosis being set at the 99th percentile of a population of normal, healthy individuals without a known history of heart disease (>0.06 ng/mL with our new cTnI assay). Although these guidelines have been discussed in the literature for a few years, and updated in April 2007, their introduction locally has induced a "firestorm" of controversy.

Much of the controversy surrounds the high sensitivity of the decision-limit and the desire to have a cTnI value that is reasonably predictive of acute myocardial infarction (AMI) due to thrombotic coronary artery disease, regardless of the pretest probability of having it. To this end, it is determined that with our new assay, a cTnI value >0.50 ng/mL has a 96% clinical sensitivity and 94% clinical specificity for AMI. This information will be included in our laboratory reports.

Contemporary cTnI assays are quite sensitive and can detect very small episodes of myocardial necrosis (<1 g). In patients with a high pretest probability or clinical suspicion of acute coronary syndrome (ACS), the diagnostic and prognostic value of cTnI is well known. Even small increases above normal (i.e. >99th percentile decision-limit) in these patients have clinical significance. The available data from over 25 studies indicate an ~4 fold higher risk of death and recurrent MI among patients presenting with suspected non-ST elevation acute coronary syndrome (NSTEACS; which encompasses both unstable angina and non-ST elevation myocardial infarction [NSTEMI]) and an increased cTnI compared to patients with a cTnI that is within normal limits.

However, cTnI testing is also being used as a screening tool in patients with a low pretest probability of thrombotic coronary artery disease. Thus given the high sensitivity of cTnI for detecting even minimal myocardial-cell necrosis, cTnI may become "positive" even in the absence of thrombotic acute coronary syndromes, hence the "firestorm". The causes of cTnI elevations that are unrelated to coronary thrombosis is the subject of an excellent review article from cardiologists from the Beth Israel Deaconess Medical Center and Harvard Medical School (Ann Intern Med. 2005;142:786-91).

Finally, the optimal timing of sample acquisition is relevant for the diagnosis of AMI. It derives from cTnI kinetics (begins to rise within 3-4 hours, peaks at 12-24 hours, and can remain increased for up to 4-7 days) and patient-related factors (timing and duration of symptoms relative to presentation, and overall probability of ACS). Given the good analytical performance of contemporary cTnI assays, testing on presentation and up to 6 9 hours after presentation is expected to deliver optimal sensitivity in most patients. A significant rise in cTnI over this time period is indicative of AMI and unexpected in causes that are unrelated to coronary thrombosis. Repeat testing at 12-24 hours should be considered in patients for whom these initial cTnI values are negative and there is an intermediate or high clinical index of suspicion, or in whom plausibly ischemic symptoms have recurred.

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Urinalysis Department Announces Change in Urine Critical WBC / Culture Reflex Values

With the automation of our urine microscopic exams we have changed our critical value levels for white blood cells. Please note the following changes:

Previous Critical Values		New Critical Values
Adult males:	>10 WBCs/HPF	Adult males:	>15 WBCs/HPF
Adult females:	TNTC WBCs/HPF	Adult females:	>49 WBCs/HPF (Previously >50)
Children under 10:	>10 WBCs/HPF	Children under 10:	> 9 WBCs/HPF (Previously >10)

In addition, our Conditional Urinalysis test will now reflex to a urine culture based entirely on WBC count. Please note the following changes:

Previous Reflex Values		New Reflex Values
Males:	>3 WBCs/HPF, and/or: 3+ bacteria, 3+ yeast	Males:	>9 WBCs/HPF (Previously >10)
Females:	>10 WBCs/HPF, and/or: 3+ bacteria, 3+ yeast	Females:	>9 WBCs/HPF (Previously >10)
		Children under 10:	> 9 WBCs/HPF

New Expert Rule: Conditional UA #15015: Reflex culture if WBC is 10-15 or higher. Add "Culture to follow" or "Culture not indicated" to test comment.

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Urinalysis with Microscopic

by IRIS iQ200 Automated Analyzer

John Muir Medical Center Laboratory Urinalysis Department announces the conversion of manual urine microscopic analysis to an automated flow cell system that counts & characterizes the separate elements in the specimen.

Particle microscopy significantly aids in the diagnosis of renal & urinary tract disease. This is a non-invasive method used as a routine diagnostic screen, a monitor of disease progression & of therapeutic efficacy. The urine microscopic reflexes from our Urine Screen when any of the following chemistries are positive: glucose, blood, nitrite, leukocyte esterase or protein. It is also performed as part of our Conditional Urine test.

The IRIS iQ200 uses proprietary flow imaging and auto-particle recognition technology to classify and quantify 13 urine particles: RBCs, WBCs, WBC clumps, hyaline casts, pathological casts, squamous epithelial cells, non-squamous epithelial cells, bacteria, yeast, crystals, mucus, sperm and artifacts. These particles are digitally imaged with a DDC camera and subject to visual review by the resulting Clinical Laboratory Scientist.

Performed: 7 days/24 hrs. Reported: Same day. Specimen required: One orange/yellow-top conical Vacutainer tube with preservative. Transport at room temperature.

A complete MuirLab Marquee covering this material is available by calling (925) 692-5411.

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Vitamin D - Laboratory Considerations

By: Barry P. Latner, M.D., Medical Director, Laboratory Services

Vitamin D has received increasing attention over the past few years. In fact, testing for Vitamin D deficiency is one of the fastest growing tests across the country, if not the fastest, with many clinical labs reporting doubling and tripling of requests year over year. Once many foods were fortified with Vitamin D and the incidence and prevalence of rickets in children plummeted, most clinicians thought the major health problems resulting from Vitamin D deficiency had been resolved. However, in recent years we have learned that Vitamin D deficiency is not uncommon. In adults it can precipitate or exacerbate osteopenia and osteoporosis, cause osteomalacia and muscle weakness, and increase the risk of fracture. The discovery that most tissues in the body have a Vitamin D receptor and a large variety of cells possess the enzymes necessary to convert the primary circulating form (25-hydroxyvitamin D) to the active form (1,25-dihydroxyvitamin D), has provided new insights into its function. Attention has turned to the role Vitamin D can play in reducing the risk of many illnesses, including common cancers, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and cardiovascular disease.

Vitamin D is a prohormone, rather than a true vitamin, as one of its forms (Vitamin D3, cholecalciferol) is synthesized in the skin after solar UVB exposure. Aside from sunlight, the best nutritional sources of Vitamin D3 are oily fish, primarily salmon and mackerel. The other form of Vitamin D, Vitamin D2 (ergocalciferol), generally is a minor contributor to total Vitamin D and is derived only from the nutritional sources of some vegetables, yeast, and fungi. Some vegetarian diets, thus, are an exception to this usually small contribution. Both forms of Vitamin D are converted in the liver to 25-hydroxyvitamin D, which is the major circulating form. The accepted standard for clinical assessment of Vitamin D status is the measurement of total circulating 25-hydroxyvitamin D concentration.

Much of the ground breaking work on Vitamin D deficiency was based on RIA assays in which the total 25-hydroxyvitamin D was determined by the sum of 25-hydroxyvitamin D3 and D2. Our assay at John Muir Health and MuirLab is a newer version of this concept utilizing chemiluminescent immunoassay (CLIA) technology rather than radioactive isotopes, and achieves excellent accuracy and precision exhibited by within-assay variability <5% and between-assay variability <10% for total circulating 25-hydroxyvitamin D. Other newer assays, such as liquid chromatography mass spectroscopy (LC-MS), can measure the 2 forms separately. However, data from the College of American Pathologists and the UK-based DEQAS (external quality assessment surveys) have shown the LC-MS assays to have wide variability in the range of 20% between labs, thus supporting our decision to stick with our current assay. Much of the variability problem stems from the fact that there is no agreed upon reference standards, and the preparation of reagents for in-house LC-MS assays is conducted by individual labs per their own practices.

Another complexity follows as a result of the lack of agreed upon standardization, and that is the controversy on the optimum reference intervals for 25-hydroxyvitamin D to classify patients with moderate to severe deficiency. These controversies related to Vitamin D measurement are not unlike other issues in laboratory medicine that have been dealt with in the past. Such examples include the standardization of cholesterol measurement 20 years ago, the use of INRs related to prothrombin time and the anticoagulant effects of Warfarin, HbA1c traceability to the DCCT assay, and more recently creatinine to achieve standardization in eGFR calculations. Until such time as standardization is achieved, we will provide the following ranges for the classification of total 25-hydroxyvitamin D status as suggested in the literature:

Vitamin D status Total 25-hydroxyvitamin D

Deficiency < 10 ng/mL

Insufficiency 10-30 ng/mL

Optimum 30-100 ng/mL

Toxicity > 100 ng/mL

Finally, it is worth stressing the importance of the total 25-hydroxyvitamin D concentration rather than the individual D3 and D2 forms for clinical decision making. An interesting survey performed at the University of Wisconsin and the Medical University of South Carolina showed that reporting total 25(OH)D, 25(OH)D3 and 25(OH)D2 can confuse ordering clinicians, thereby leading to incorrect clinical decisions. For one of their scenarios of a hypothetical 82-year-old person with a hip fracture, and lab results of 25(OH)D3 <5 ng/mL and 25(OH)D2 = 40 ng/mL, 23% of the clinicians interpreted these results to indicate vitamin D or D3 deficiency requiring vitamin D treatment. Another 12% of clinicians interpreted the scenario of the same patient with 25(OH)D3 = 32 ng/mL and 25(OH)D2 < 5 ng/mL as being deficient in vitamin D or D2. In both scenarios, the total 25(OH)D was in the optimum range, thus neither warranted vitamin D treatment.

Should there be any questions or concerns about our total 25(OH) D assay, please do not hesitate to contact me (925.674.2508) or the supervisor of immunology at MuirLab, Cindy Liedstrand (925.692.5681). Thank you.

References:
Holick, MF. Vitamin D Deficiency. N Engl J Med 2007;357:266-81.
Binkley, N et al. Laboratory Reporting of 25-Hydroxyvitamin D Results: Potential for Clinical Misinterpretation. Clin Chem 2006;52(11):2124-5.
Singh, RJ. Are Clinical Laboratories Prepared for Accurate Testing of 25-Hydroxyvitamin D? Clin Chem 2008;54(1):221-3.

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