August 2011

Editor:
Fredrick L. Kiechle, MD, PhD

Q. Is there an algorithm for protein electrophoresis interpretation? In our laboratory in Brazil, we observe gamma globulins elevated in percentages and inside of range for absolute interpretation. For example, gamma globulins: 20.8 percent, reference range: 11.1 to 18.8, and 1.48 g/dL, reference range: 0.67 to 1.50 g/dL. Total protein: 7.1 g/dL. Our instrument is the Sebia Electrophoresis Capillarys 2.

A. A significant elevation or decrease of an individual protein fraction may affect the percentages of other protein fractions without a deviation from an absolute concentration that falls within the reference interval for that particular fraction. Therefore, a laboratory interpretation should consider the absolute concentration rather than a percentage value when determining whether the fraction occurs in reference range. Thus, an absolute gamma globulin concentration of 1.48 g/dL falling within a reference range of 0.67 to 1.50 g/dL of this particular laboratory does not represent an abnormal concentration of gamma globulins.

Despite a normal concentration of gamma globulins, a laboratory must determine whether a monoclonal gammopathy is present. Monoclonal gammopathies produce a dense band that usually falls within the gamma region, but the band may migrate more toward the anode into the beta region or even into the alpha-2 region.

When monoclonal gammopathies occur, a laboratory then evaluates the identity of the monoclonal fraction using a technique such as immunofixation electrophoresis, the concentration of the monoclonal fraction by a method such as nephelometry, the type of light chain restriction (kappa or lambda), and the presence or absence of free light chains. When a monoclonal gammopathy is identified and reported after electrophoresis testing, clinicians often order urine electrophoresis to determine whether the monoclonal gamma globulin or free light chains appear in the urine.

Astion, et al.,¹ describe an algorithm for interpreting electrophoresis assays of various body fluids. Their article, which addresses serum and urine monoclonal gammopathies, includes a schematic diagram to explain the presence of monoclonal gammopathies. However, not all cases of increased concentrations of immunoglobulins are associated with monoclonal disorders. Polyclonal gammopathies, characterized by an increased absolute concentration of gamma globulins without a monoclonal pattern by electrophoresis, may explain the increased concentration of immunoglobulins.² On this page is an algorithm, based on the two references, for evaluating the increased serum concentrations of immunoglobulins.

The differentiation of the various causes of monoclonal gammopathy requires a quantitative assessment of the amount of monoclonal immunoglobulin, identification of the type of immunoglobulin—for example, IgM in Waldenstrom macroglobulinemia—and correlation with clinical findings and other laboratory results. The CAP Diagnostic Immunology Resource Committee Diagnostic Immunology Topic Center provides excellent references that describe the criteria for diagnosis, staging, risk stratification, and response assessment in patients with multiple myeloma. Readers can find the center by visiting www.cap.org, navigating to the Committees and Leadership tab, scrolling down to and selecting the Diagnostic Immunology Resource Committee, and then selecting, under Related Links, the Diagnostic Immunology Topic Center.

References

Stanley J. Geyer, MD
Pathologist, Geyer Pathology Services
Pittsburgh

Chair, CAP Diagnostic Immunology
Resource Committee

Q. I never see very high (toxic) drug levels in blood, other body fluids, or tissue samples in coroner autopsy cases, even in the clear-cut circumstances of drug overdose. Is this because drug metabolism continues after death? If so, which cells or tissues are responsible for such low levels? Are there other reasons for these levels?

A. There are several possible explanations.

First, in many overdoses in which a person has slipped into a coma because of the overdose, he or she continues to metabolize drugs and ethanol up until the point of death. Technically, drugs don’t metabolize after death because that would require cellular energy. That being said, some drugs are not necessarily stable after death.

Second, peripheral blood samples are the best specimens to use. The drug concentrations in postmortem peripheral blood, if sampled within 24 hours of death, should approximate antemortem levels. As decomposition begins, some drug levels may drop.

Third, in many overdoses, there may not be a clear-cut lethal level of one drug. Most of the fatalities I have encountered in forensic practice are “combined drug toxicity” involving the synergistic effects of two or more drugs at slightly elevated to toxic levels.

Reference

Stephen J. Cina, MD
Associate Medical Director
University of Miami Tissue Bank
Miami

Chair, CAP Forensic Pathology Committee

Q. If you perform a stool culture on material collected after a bowel prep for colonoscopy, will this affect the recovery rate of organisms—Salmonella-Shigella, Campylobacter jejuni, Yersinia enterocolitica, for example? Will it affect results of examination for ova and parasites?

A. The literature is mixed on the effect that bowel preparations have on the rate of isolation of stool pathogens. Phillips, et al.,¹ found that purgative magnesium sulfate caused loss of the nor-mal flora in the ileal and cecal crypts, while the flora was relatively unchanged in the colonic crypts. In particular, spiral or curved bacteria (likely Campylobacter species) appeared to be eliminated after bowel preps.

Beck, et al.,² used quantitative cultures to look at the effect of bowel preps on mucosal bacteria. Though the preparations resulted in removal of the solid, intraluminal wastes, there was no significant change in the mucosa-associated colony counts (aerobic and anaerobic cultures) before and after GoLytely, a laxative commonly used for bowel preparation. Antibiotic-containing preparations did demonstrate slight, though not statistically significant, decreases in bacterial counts. GoLytely had the added benefit of decreasing the production of combustible gases by microorganisms, which is a good thing if electrocautery is to be used.

Nichols, et al.,³ demonstrated that mechanical cleansing (laxatives and enemas) removed most of the feces from the gut lumen. This was accompanied by a decrease in the number of coliform bacteria in four of six patients treated in this manner but did not cause significant changes in the counts of other aerobes or the intestinal anaerobes compared with untreated individuals. Antibiotic-containing regimens led to significant changes in the bacterial counts but did not completely sterilize the gut. Bartlett, et al.,⁴ likewise found significant decreases in intestinal colony counts in patients who had been treated with antibiotics and mechanical cleansing compared with those who had mechanical cleansing alone.

Zhang, et al.,⁵ studied Campylobacter species in children with Crohn’s disease. Based on PCR assessments of bowel contents, they concluded that some Campylobacter species were found in higher concentrations in patients with Crohn’s disease compared with those without Crohn’s disease. Culture results from specimens collected after bowel preparations did not produce high rates of Campylobacter isolation, however. The authors hypothesized that the decrease in organism isolation may have been due to the osmotic effect of the bowel preparation, though no data were presented in this study to substantiate this hypothesis.

References

Julie A. Ribes, MD, PhD
Department of Pathology
and Laboratory Medicine
University of Kentucky
College of Medicine
Lexington

Member, CAP Microbiology
Resource Committee

Q. When will DNA laboratories be regulated? What would the requirements be for such labs?

A. If DNA laboratories refers to laboratories that perform molecular diagnostic testing on patient DNA, then the answer is that they are regulated under CLIA ’88. The Centers for Medicare and Medicaid Services or a deemed accreditor such as the CAP oversees the implementation and performance of CLIA-stipulated laboratory standards.

Molecular diagnostic laboratories generally perform high-complexity testing and must undergo biennial inspections that assess compliance with CLIA requirements, including personnel qualifications, testing standards, and record keeping. The laboratories are also required to perform proficiency testing.

For laboratories that perform clinical diagnostic testing using laboratory-developed tests (LDTs), formerly known as homebrew tests, additional federal regulations will be forthcoming in the near future. The Food and Drug Administration intends to exercise its authority over LDTs, which the agency considers to be a class of in vitro diagnostics that are manufactured—including being developed, validated, and offered—within a single laboratory. In July 2010, the FDA sponsored a public hearing for stakeholders to comment on its intent to enforce regulations  pertaining to LDTs. The public commentary can be found at www.regulations.gov/#!documentDetail;D=FDA-2010-N-0274-0111.

The FDA has drafted three guidance documents outlining the new regulations to be imposed. However, the documents have not been released publicly and the date of release is not known. The CAP created a risk-based proposal for the oversight of LDTs and has met with the FDA commissioner, director of the FDA’s Center for Devices and Radiological Health, and members of the FDA’s Office of In Vitro Diagnostic Device Evaluation and Safety about the proposal. Conversations are ongoing.

Gail H. Vance, MD
Professor of Medical
and Molecular Genetics
Director of the IU Genetic Testing Laboratories
Departments of Medical and Molecular Genetics and Laboratory Medicine and Pathology
Indiana University School of Medicine
Indianapolis

Member, CAP Board of Governors