Chemistry Case Study: Protein Bands in All Lanes of the Immunofixation Electrophoresis

Waldenstrom Macroglobulinemia (WM) is defined as lymphoplasmacytic lymphoma (LPL) with IgM paraprotein and bone marrow involvement. The IgM paraprotein is an important serum marker for WM diagnosis, symptom prediction, disease burden assessment, treatment decision and drug response evaluation. Serum protein electrophoresis (SPEP) in conjunction with immunofixation electrophoresis (IFE) are the routine laboratory tests for IgM paraprotein detection, quantitation and characterization. A monoclonal protein typically presents as a sharp band on SPEP and selective lanes of IFE, allowing characterization of the immunoglobulin heavy chain isotypes and light chain classes. In rare situations, a monoclonal band is seen on all immunofixation lanes, suggesting cryoglobulin and/or soluble immune complex. We encountered a recent case of WM with a strong demarcated band on all immunofixation lanes.

The patient is a 76-year-old Chinese man diagnosed as WM/LPL by bone marrow biopsy. Peripheral blood showed pancytopenia with rouleaux formation. The serum IgM was up to 6900 mg/dL. Serum viscosity was increased up to 3.1 cP (normal range 1.5-1.9 cP). Serum rheumatoid factor was negative (<10 IU/ml). Serum protein electrophoresis (SPEP) on Sebia Hydrasys 2 showed a wide smearing pattern (Fig 1A). Serum protein immunofixation electrophoresis (IFE) showed a monoclonal band on all lanes with equal intensity, preventing isotype identification (Fig 1B). This pattern is generally believed to be due to cryoglobulin and/or polymerization of monoclonal proteins, similar to rheumatoid factor activity. Urine electrophoresis was consistent with an overflow pattern and urine immunofixation showed monoclonal free lambda light chain.

Previously it was demonstrated that cryogolublin dissolution was achieved by pre-treatment of serum samples with Fluidil. The IgM polymer can be disrupted by adding reducing agents such as beta-mercaptoethanol (bME) to disrupt the disulfide bonds (1-2). In our case, despite pretreatment with Fluidil and bME, no isotype resolution was achieved on serum IFE, prompting us to develop a novel method through the addition of sodium dodecyl sulfate (SDS) to the pretreatment process. Different combinations of reaction conditions were tested, including SDS concentration ranging from 0.01 to 1%, three different temperatures (37, 56 and 95 °C), three different concentrations of bME (1%, 2% and 4%) and three different serum volume (25 µL, 50 µL and 75 µL). Optimal isotype resolution was achieved using 0.1% SDS/0.25%bME/Fluidil incubated at 56°C for 30 mins (Fig 1C).




  1. Attaelmannan M, Levinson SS. Understanding and identifying monoclonal gammopathies. Clin Chem. 2000 Aug; 46(8 Pt 2):1230-8.
  2. Yusra Othman. Protein Bands in All Lanes of the Immunofixation Electrophoresis Pattern of Serum From a 50-Year-Old Saudi Woman. Lab Med (2006) 37 (3): 152-154.



-Huifei Liu, MD, PhD. Former PGY4 resident in the Department of Pathology and Genomic Medicine, Houston Methodist Hospital. She currently serves as the associate medical director at Hematologics, Inc., Seattle, WA.  


-Xin Yi, PhD, DABCC, FACB, is a board-certified clinical chemist, currently serving as the Co-director of Clinical Chemistry at Houston Methodist Hospital in Houston, TX and an Assistant Professor of Clinical Pathology and Laboratory Medicine at Weill Cornell Medical College.

Microbiology Case Study: A 14 Year Old Female with Neck Swelling

Case History

A previously healthy 14-year-old female presents to the emergency department with three days of progressive facial and neck swelling. The swelling started on the left side. Two days ago she visited her primary care physician where she had negative monospot and mumps IgM testing.  She is fully vaccinated, but was exposed to a mumps outbreak at school.


Our patient was diagnosed with mumps by positive RT-PCR from a buccal swab. The mumps virus is a member of the Paramyxoviridae family which includes notable human pathogens parainfluenza, Hendra, and Nipah viruses. Members of this family are enveloped, helical viruses with single-stranded, non-segmented RNA genomes with negative polarity. Mumps is an obligate human pathogen that replicates in the epithelial cells of the upper respiratory tract and subsequently moves to regional lymph nodes. It is spread from person to person via direct contact with respiratory secretions or contact with contaminated fomites. Mumps is a highly contagious disease with as high as 85% of naïve individuals becoming infected after contact with a mumps infected individual. It spreads most efficiently in areas where there is close contact among individuals for prolonged periods of time such as college campuses and close-knit religious communities.

Prior to vaccination for mumps in the 1960s, greater than 150,000 cases of mumps occurred each year in the US. The incubation period for infection is 16-18 days, with the majority of infected persons being asymptomatic or having mild respiratory symptoms. Orchitis causing sterility in post-pubescent males is the main concern of mumps infection but other rare but serious complications include mastitis and oophoritis in females, meningoencephalitis, pancreatitis, and deafness.

Due to sporadic outbreaks of measles since the introduction of the vaccine, the vaccine schedule has been revised from one dose of the MMR (measles, mumps, and rubella) vaccine at age 12-15 months to include another MMR booster at age 4-6 years. We are currently in the middle of yet another outbreak with nearly 6,000 cases of mumps reported to the CDC in 2016 and a high rate of infections reported thus far in 2017 (Figures 1 and 2).

Figure 1. Number of cases identified by the CDC in 2017 by state. (Figure courtesy of the CDC Mumps website at Content source: National Center for Immunization and Respiratory Diseases [NCIRD], Division of Viral Diseases)
Figure 2. Number of cases of mumps per year identified by the CDC.
(Figure courtesy of the CDC Mumps website at Content source: National Center for Immunization and Respiratory Diseases [NCIRD], Division of Viral Diseases)


Diagnostic Testing for Mumps

Serological testing for IgM and RT-PCR from a buccal swabs are the mainstay of mumps diagnosis. IgM becomes positive in the first 3-4 days after symptom onset and will remain positive for 8-12 weeks. IgG becomes positive 7-10 days following symptom onset and will remain at high levels for many years and detectable for life. In a vaccinated individuals, IgM testing has less utility as it may be non-reactive or weakly positive following a secondary immune response.

RT-PCR from a buccal swab specimen is the most sensitive test for diagnosis of mumps. It should be performed as soon as a patient is symptomatic, as testing by this method is the most sensitive in the first few days following symptom onset and becomes less sensitive as time goes on.

Urine specimens can be used to isolate mumps in viral culture. Urine is not positive for mumps until greater than 4 days post symptom onset and is less sensitive than PCR performed on the bucal swab. For these reasons, viral isolation from urine is no longer a commonly used test for diagnosis of mumps, although viral culture is still considered the gold standard for mumps conformation.


The patient and her family were counseled on the infectious nature of mumps. She was instructed to remain in isolation at home for 6 days after resolution of swelling.



  1. Manual of Clinical Microbiology, 11th edition
  2. CDC Mumps Website (


Erin McElvania TeKippe, PhD, D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.

Serum Protein Electrophoresis in Children

Although most of the testing performed and the methodologies utilized in a clinical laboratory which serves a pediatric institution are very similar to those found in adult laboratories, a few differences stand out. Differences include devising ways to deal with small test volumes and different test menus than those found in laboratories that serve adult patients. One such test menu differences is the lack of serum protein electrophoresis (SPEP) testing in pediatric labs.

SPEP’s are essentially performed for the main purpose of helping to diagnose and then monitor the treatment of multiple myeloma. SPEPs provide this help by detecting, identifying by reflex immunofixation electrophoresis (IFE), and quantifying monoclonal gammopathies. Children don’t get multiple myeloma. After 20 years of signing out SPEP results at the county hospital next door, the youngest person with a diagnosis of multiple myeloma that I’ve seen was 24 years old. Thus in general SPEP’s are not ordered on children, nor performed in pediatric labs.

Recently however, I learned that although children don’t get multiple myeloma, they do in fact get monoclonal gammopathies. An SPEP ordered on a 7 month old patient in my institution came back with a very clear biclonal gammopathy, identified by IFE as an IgG kappa and an IgA kappa. This child has no bone marrow indication of abnormality, although she does have a deficiency of B-cells along with plasma cell infiltrates in the liver and duodenum.

A little searching determined that apparently, monoclonal spikes on SPEP’s in children are not at all unusual. A study published in 2014 (1) looked at 695 children who had SPEPs performed, and 11% of those children had a monoclonal gammopathy, although none of them had multiple myeloma. The most common associated diagnosis was ataxia-telangiectasia (22%), with a wide range of other diagnoses being found in these children, including some immunodeficiencies, autoimmune diseases, various hematological disorders and a few solid organ malignancies.

Thus it appears that monoclonal gammopathies are present in children and have an entirely different meaning than they do in adults. In addition, currently monoclonal gammopathies in children have no clear diagnostic utility. Perhaps that is the real reason we don’t routinely perform them in the pediatric population.

  • Karafin MS, Humphrey RL, Detrick B. Evaluation of monoclonal and oligoclonal gammopathies in a pediatric population in a major urban center. AJCP 141:482-487. 2014

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.