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CA 19-9

The CA 19-9 antigen is a tumor-associated antigen synthesized by normal human pancreatic and biliary ductular cells. Ca 19-9 is useful in monitoring pancreatic, hepatobiliary, gastric, heptocellular, and colorectal cancer. Despite the fact that normal pancreatic and bile secretions are rich in this antigen, very little of this marker appears in the blood of normal donors or in patients with benign disorders. On the other hand, most patients with pancreatic cancer have elevated levels of serum CA19-9. Determinations of CA19-9 levels aid in management of patient progress and treatment outcomes in these confirmed cancer patients. However, the CA 19-9 assay value, regardless of level, should not be interpreted as absolute evidence of the presence or absence of malignant disease. Furthermore, results obtained with different assay methods or kits cannot be used interchangeably. Even labs using the same method as John Muir Medical Center (Tosoh Bioscience Nexia AIA Pack, an immunoenzymometric assay) may have different reference ranges since each laboratory establishes its own reference ranges.

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Clinical Usefulness of Serum Tumor Markers

By Dr. Edwin Poore

Laboratory tests that are used to monitor and detect cancer are referred to as "tumor markers." MuirLab performs over 2,500 assays per month for the following tumor markers: prostate-specific antigen (PSA), alphafetoprotein (AFP), CA 125, CA 15.3, CA 27.29, carcinoembryonic antigen (CEA), CA 19 9, and beta human chorionic gonadotropin (ßHCG). In general, these markers are molecules produced by a neoplasm or by the body in response to a tumor. Tumor markers date to the discovery of Bence Jones protein (monoclonal immunoglobulin light chains) in 1846. Thus, monoclonal proteins in serum or urine are also a type of tumor marker, one which we identify today using protein immunoelectrophoresis. Tumor markers are most useful in detecting cancer recurrence, monitoring response to therapy, and predicting prognosis. Most have limited value as screening tests due to low sensitivity and specificity. In the space available here, we will discuss the clinical relevance of two serum markers -namely, CA 15-3 and CA 19 9.

CA 15-3 This serum assay measures a circulating breast carcinoma associated glycoprotein, which is normally found in human milk globule membranes. The CA 15-3 antigen is also known by other names, including the following: CA 27.29, MAM 6, milk mucin antigen, and polymorphic epithelial mucin antigen. In the cells of mammary adenocarcinoma, the CA 15-3 antigen is overexpressed on the entire cell surface and shed into the circulation; however, only a small number of patients with localized disease have elevated CA 15-3. In contrast, CA 15-3 is increased (>39 U/ml) in the sera of over two thirds of breast cancer patients who present with metastatic disease. Its main clinical use is to detect systemic recurrence after therapy for stage II III breast carcinoma. It is also valuable for serial monitoring of response to therapy, because levels of CA 15-3 correlate well with disease progression/regression, respectively.

CA 19 9 This serum marker has been used primarily for detecting and monitoring pancreatic carcinoma. The CA 19 9 antigen is a mucin which is synthesized by normal pancreatic and biliary ductal epithelium. Although pancreatic and bile duct secretions are rich in this antigen, very little appears in the blood of normal persons. However, in patients with pancreatic carcinoma, 70 80 percent have elevated levels of CA 19 9 (=41 U/ml). This antigen lacks specificity for pancreatic carcinoma, in that it may be elevated in 50 60 percent of gastric carcinomas and hepatobiliary carcinomas. If markedly elevated, the test may differentiate pancreatic carcinoma from pancreatitis, especially when used in combination with other tests, such as CEA. However, CA 19 9 may be slightly elevated in non-neoplastic disease, especially inflammatory bowel disease, cirrhosis, and autoimmune disorders. CA 19 9 is primarily used for monitoring disease progression in patients with established diagnoses. CA 19 9 is biochemically related to the Lewis blood group antigens. Patients who lack these antigens (7 percent of the population) will not express CA 19 9.

References

  1. Geraghty, J.G., et al., "CA 15-3 in patients with locoregional and metastatic breast carcinoma," CANCER 1992, 70(12):2831 4.
  2. Rosty, C., et al., "Early detection of pancreatic carcinoma," Hematol. Oncol. Clin. North Am. 2002, 16(1):37 52.

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Diagnosis and Management of the Metabolic Syndrome

The American Heart Association (AHA) and the National Heart, Lung and Blood Institute (NHLBI) have published a synopsis of the full scientific statement on the diagnosis and management of the metabolic syndrome in adults (see Circulation. October 18, 2005). The metabolic syndrome refers to a constellation of metabolic risk factors that appear to directly promote the development of atherosclerotic cardiovascular disease and is strongly associated with risk for the development of type 2 diabetes mellitus. These metabolic risk factors include atherogenic dyslipidemia, elevated blood pressure, elevated plasma glucose, a prothrombotic state and a proinflammatory state.

While it is often referred to as a discrete entity, the metabolic syndrome likely has more than one cause. It shows considerable variation in the components between different individuals and this variation is even greater among different ethnic groups. In the United States, the most important underlying risk factors are abdominal obesity and insulin resistance. Other associated conditions include physical inactivity, aging, hormonal imbalance, and genetic (ethnic) predisposition.

The newly released scientific statement reaffirms the utility and validity of the criteria developed by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) in 2001 with minor modifications and clarifications. These modifications include the following:

  1. Reducing the threshold for counting increased fasting glucose from ≥110 mg/dL to ≥100 mg/dL, in accordance with the American Diabetes Association's revised definition of impaired fasting glucose.
  2. Allowing for adjustment in waist circumference to lower thresholds in individuals or ethnic groups who are prone to insulin resistance (especially Asians).
  3. Counting as abnormal - triglycerides, HDL-C and blood pressure - when a person is taking drug treatment for these factors.
  4. The definition of elevated blood pressure is a level that exceeds the threshold for either systolic or diastolic pressure.

From the laboratory testing viewpoint, the reference values for the various lipid and glucose tests have been reviewed and adjusted to conformance as needed. A new line item, non-HDL-Cholesterol, has been added. This calculation will allow for easier use, as the non-HDL-C is reaffirmed to be the secondary target of treatment if triglycerides are ≥200 mg/dL and after the primary LDL-C goal is achieved. The non-HDL-C goal is 30 mg/dL higher than that specified for LDL-C.

Thanks are due to those who brought this timely subject to our attention. As always, please do not hesitate to call with questions or concerns.

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Diagnosing Legionnaire's Disease: Legionella Detection by PCR

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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The Difference in Differentials

Making The Most Of Automated White Cell Differentials

The white blood cell (WBC) differential count is one of the traditional mainstays of clinical laboratory testing. Over the years, many different methodologies have been used to generate these data, ranging from the classic, technologist-performed manual differential, to the powerful automated analyzers in widespread use today. Currently, two Coulter Gen-S instruments are in use at MuirLab, performing an average of 700 complete blood counts and automated differentials per day. An additional 135 manual differentials are also generated daily.

The Coulter Gen-S instrument uses a modified flow cytometric assay, called VCS (an acronym for Volume, Conductivity, and Scatter), to determine the characteristics of the WBCs in the sample and generate the differential count. In this method, the blood sample is mixed with two agents to lyse the red cells while maintaining the WBCs in a near-native state. Using hydrodynamic focusing, the resulting leukocyte suspension is channeled into a quartz crystal flow cell, through which cells pass one at a time. Volume (V) is determined by measuring the electrical impedance produced by the displacement of the diluent fluid by the cell, thereby not only measuring the size of the cell but also providing a correction factor for the other two parameters. Conductivity (C) is assessed by passing a radiofrequency (RF) alternating current through the cell membrane, providing information about the cytoplasmic composition and nuclear volume. Finally, scatter (S) is measured with the use of an elliptical helium-neon laser beam and a detector covering a range of 10-70 degrees, thus generating information about cellular granularity, nuclear complexity, and cell surface characteristics.

In a single sample, these three parameters are measured for each of over 8,000 leukocytes.

Results from the analyzed cells are plotted on a three-dimensional axis, in which cell populations with similar characteristics will form distinct clusters. Assisted by various hardware and software tools that correct for variables such as ambient temperature and morphologically-overlapping populations such as variant lymphocytes and monocytes, the cell populations are separated and classified. By assessing the three measured parameters, all of the normal leukocyte populations may be identified; for instance, large cells (V) with numerous small cytoplasmic granules (C) and complex nuclei (S) are classified as neutrophils. The differential analytic algorithm proceeds in a binary fashion, identifying eosinophils first, then monocytes, then neutrophils, then finally separating lymphocytes and basophils.

In the presence of this technology, the manual differential count assumes a distinct but limited role. Samples in which various parameters exceed or fall below certain critical numeric values, as well as cases in which the automated analyzer returns a "suspect" message (in the presence of difficult-to-classify cells such as blasts and variant lymphocytes), are automatically flagged for a "reflex" manual differential. A manual differential may also be useful if the presence of a particular cell type is in question. However, the limitations of the manual differential are well-known and include small sample size (100 manually-counted white cells versus over 8,000 analyzed leukocytes), interobserver variation in cell classification, and slow turnaround time. In a recent internal review of fifty consecutive manual differential orders, it was found that roughly half were essentially equivalent to the results generated by the automated analyzer; furthermore, the analyzer identified all of the remaining samples as requiring reflex manual differentials.

The flow cytometry-based VCS technology used in the Coulter Gen-S instrument enables MuirLab to offer rapid, accurate WBC differentials, while remaining sensitive enough to identify samples that require further evaluation by a technologist. The automated differential method is thus appropriate for the vast majority of clinical situations in which a leukocyte differential is required; orders for manual differentials are usually unnecessary.

Environmental Care and Sampling (10/2008)

Prior to 1970, hospitals regularly and routinely cultured air and environmental surfaces for microbial contamination. By 1970, both the CDC and the American Hospital Association were discouraging this practice. The 2003 CDC Guidelines for Environmental Control in Healthcare Facilities and the CDC Guidelines for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings 2007, do not recommend routine, random microbial sampling of the environment in healthcare facilities for several reasons, the foremost being that infection transmission was deemed less from the environment and more from hand contamination.

The CDC Guidelines for Environmental Control in Healthcare Facilities (published in 2003) state that environmental microbial sampling is indicated in 4 situations: 1) during the “…investigation of an outbreak of disease or infections when environmental reservoirs or fomites are implicated epidemiologically in disease transmission…”, 2) when warranted in a research study, 3) “…to monitor a potentially hazardous environmental condition to confirm the presence of a hazardous chemical or geological agent and validate the abatement of the hazard…”, and, 4) “…to evaluate the effects of a change in infection control practice or to ensure that equipment or systems perform according to specifications and expected outcomes.” The Guidelines add that “…sampling on an extended basis in the absence of an adverse outcome, is usually unjustified.”

Environmental surfaces can be a source of hand contamination and thus disease transmission. To reduce environmental hand contamination facilities should:

  1. Facilitate healthcare staff adherence to Standard Precautions and good Hand Hygiene as these remain the best method of reducing microbial transmission during routine healthcare activities. Routine hand washing should ensure a 20-second cleaning and scrubbing, include all surfaces of the hands and wrists, and utilize water and liquid soap or appropriate alcohol hand sanitizers. Patients should also be instructed in good hand hygiene and encouraged to wash their hands with soap and water or an alcohol hand sanitizer before leaving their rooms.
  2. Identify high-touch surfaces in patient rooms and common areas and ensure their cleansing and disinfection at least daily and when visibly soiled. Checklists and direct observation help housekeeping staff to not miss these frequently touched areas or items. Housekeeping staff should change or disinfect mop-heads and cleaning implements immediately after cleaning an isolation room or the room of a patient infected or colonized with difficult to eradicate organisms, such as Clostridium difficile, norovirus, or Acinetobacter baumannii.
  3. Ensure regular cleansing and disinfection of personal care and medical equipment, when visibly soiled, and prior to use on another patient. Patients infected or colonized with MDROs (Multiple Drug Resistant Organisms) or with difficult to eradicate organisms should have equipment dedicated to their use.
  4. Carefully consider roommate selection, especially when patients are colonized with an MDRO or a potential environmental contaminant. Patients colonized with an MDRO should have the opportunity to bathe or shower frequently and access to a fresh change of clothes at least daily.
  5. Ensure that manufacturer’s instructions are carefully followed when equipment, such as whirlpools and bathing tubs, hydrotherapy tanks and pools, ice machines, water dispensers, etc. are cleaned and disinfected.

Homocysteine: A Predictor of atherosclerosis and thromboembolism

Recent clinical studies show that moderately elevated total homocysteine (tHcy) levels are an independent predictor for atherosclerosis and thromboembolism. tHcy is a graded risk factor; consequently the risk for vascular disease increases progressively with tHcy concentration. A meta analysis of 27 studies found that the risk associated with a 5umol/L increase in tHcy is equivalent to that found with a 20mg/dL increase in cholesterol. This corresponds to a relative risk of 1.5.

MuirLab measures tHcy by Florescence Polarization Immunoassay. The test is performed daily (except weekends). Our normal range is 4-12 umol/L.

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Mars & Venus

Tips for Testing Testosterone
By: Marian Scott, MD

We all know that certain lab tests have different normal ranges for men, women, and children. Now it's time to take that concept a bit further, and consider a test situation where the appropriate test methodology itself differs.

Immunoassay has long been the standard methodology for quantifying serum testosterone. This method is still available and is very appropriate for testing healthy adult male patients. However, it is much less accurate at lower serum levels of testosterone.

Fortunately, a newer method is now also available, for better evaluation of the lower levels of testosterone found in children and adult female patients.

This newer method, called liquid chromatography-tandem mass spectrometry (LC-MSMS), offers significantly improved sensitivity and accuracy and is very strongly recommended for testing women, pre-pubertal children, and certain adult male patients such as those undergoing hormonal treatment for prostate cancer.

In adult males, serum testosterone shows a diurnal variation (highest in early morning, lowest in evening) and can be affected acutely by exercise.

Testosterone circulating in the bloodstream is largely protein-bound (40% loosely to albumin and 60% more tightly to sex hormone binding globulin (SHBG)). Thus, the measurement of total testosterone alone may not provide the most clinically relevant number. Instead, either free testosterone (less than four percent of the total) or "bioavailable" testosterone (free plus albumin-bound) is thought to be more relevant in many clinical correlations.

An increase in free testosterone could arise from either an increase in total testosterone or a decrease in SHBG (or both). Thus, a normal total value does NOT rule out a clinically significant increase in free testosterone. In women, increased free testosterone has been associated not only with tumors but also with polycystic ovaries, acne, male pattern balding, and hirsutism.

Similarly, a normal total value may be accompanied by increased SHBG and therefore does NOT rule out decreased levels of free or bioavailable testosterone. Commonly, increased SHBG is found in women treated with oral estrogens (in contraceptives or hormone replacement).

In sum, the testing choices for testosterone have become a bit more complex, but your patience in understanding them will reward you with far more accurate measurements than were earlier available.

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Pertussis Update

The year 2006 marks the 100-year anniversary of the discovery that the bacterium Bordetella pertussis is the causative agent of whooping cough. In the early- to mid-1900's, pertussis was one of the most common childhood diseases and a major cause of childhood mortality in the United States.

After the introduction of pertussis vaccine in the 1940s, pertussis cases decreased from the average incidence of 150/100,000 to only 1/100,000 of the US population in 1980. However, since the 1980s, the number of cases has been steadily climbing. The pertussis vaccine is far from perfect and cases have been appearing in fully immunized children and adults.

Cases among adults are particularly problematic as the presentation is atypical, with the patient having little more than a chronic cough. Infected adolescents and adults may introduce pertussis into households where susceptible preschool-age children could be exposed.

Laboratory Diagnosis

The diagnosis of pertussis is usually based on a characteristic history and physical examination. However, laboratory tests may be useful with young infants, atypical cases and cases modified by vaccine or previous antibiotic therapy. B. pertussis specifically binds to ciliated epithelial cells. Since the nasopharynx is lined with ciliated epithelial cells, culture of this site has a higher yield than culture from any other specimen source. However, culture is slow; it may take as long as 10 days to isolate this organism. Therefore, it is of limited value in an outbreak setting where the organism can be rapidly spread from person to person via inhalation.

Direct fluorescent-antibody (DFA) for B. pertussis yields a quicker response than culture, but it has a sensitivity of only 50 to 65%, and false-positive results occur. Ordering DFA for pertussis is not recommended.

Polymerase Chain Reaction (PCR) has become the method of choice for diagnosing pertussis. This is especially important in settings where specimens may be delayed in transport, because such transport compromises the sensitivity of culture but not the sensitivity of PCR. PCR is rapid (usually less than 48 hours turn-around-time), is far more sensitive than culture, and has a high negative predictive value. False positives may occasionally occur because of cross-reactivity with organisms similar to B. pertussis. In some situations, it is recommended that culture for B. pertussis be performed in addition to PCR so that the organism is available for susceptibility testing and molecular epidemiology studies.

The optimal specimen for pertussis is a nasopharyngeal swab collected during the first two weeks of disease. A separate NP swab should be collected for PCR and culture.

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Vancomycin resistance found in a Ca-Mrsa Strain

A USA300 strain of CA-MRSA has developed intermediate resistance to vancomycin. This strain is the predominant US strain currently found in 40 states. It is hardy and easily transmitted. The mechanism of resistance is a thickening of the cell wall which occurred after six weeks of treatment with vancomycin. The mechanism of resistance in all US cases of VRSA has been genetic transfer from VRE to MRSA. The USA300 clone has the necessary plasmids to be receptive to a genetic transfer of vancomycin resistance from VRE according to the published complete genome sequence of this strain. It was discovered that this strain carries two of the three plasmids that are resistance markers. One of the plasmids carries clindamycin resistance and also caries mupiricin resistance. There are reports that CA-MRSA USA300 strains are displacing nosocomial MRSA strains in some hospitals. This will give the pathogen more exposure to VRE. Some CA-MRSA strains carry four different classes of resistance making treatment options increasingly more difficult. In general CA-MRSA may cause more severe infections than the typical hospital acquired strains. This makes the situation very serious. Some physicians are requesting a consistent antibiotic management strategy for CA-MRSA.

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