Month: October 2019

Microbiology Case Study: An 18 Year Old with Gastrointestinal Bleeding

An 18 year old female with no significant past medical history experienced multiple episodes of gastrointestinal bleeding over the course of a few weeks. The most recent bout included a bloody episode that filled the toilet, for which she provided a picture for the clinician. She denies any other associated symptoms including epigastric pain, nausea, vomiting, fever, or chills. Her travel history is unknown.

Review of her history reveals an unremarkable family and social history. She has never had an incident similar to this in the past and no other family members have ever complained of similar symptoms. Review of systems was unremarkable and within normal limits. Physical exam was unremarkable. A rectal exam was performed and was noted to have brown stool that was guaiac (occult blood) positive. Non bleeding internal hemorrhoids were noted. There were no external hemorrhoids present.

Labs drawn including CBC were within normal ranges with the exception of absolute eosinophils which were at the upper limit of normal range at 0.6 x 103/µL [normal range= 0.0 – 0.6 103/µL].

The patient had an esophagogastroduodenoscopy (EGD) to further investigate the gastrointestinal bleed. The exam was otherwise normal with exception of the ascending colon where they noted a worm on the surface of the mucosa (Image 1-2). The worm was collected and transported to microbiology for examination (Image 3-4).

Image 1. View of a worm seen on the mucosal surface of the ascending colon.
Image 2. Another view of a worm seen on the mucosal surface of the ascending colon.
Image 3. Adult worm viewed under the dissecting microscope.
Image 4. Eggs viewed under the dissecting microscope.

Discussion

Examination of the worm and eggs revealed morphology consistent with Trichuris trichiura, or whipworm.

T. trichiura is most prevalent in warm, moist regions. The worldwide prevalence of infection is estimated to be roughly 800 million, mostly among poorer populations. Infection from T. trichiura is spread via fecal-oral route and caused by ingesting embryonated eggs. This occurs when contaminated dirt is ingested or by consumption of vegetables or fruits that have not been carefully cooked, washed or peeled.

The male and female worms both have the long whip-like structures at the anterior end. T. trichiura worms are 30-50 mm in length and the average life span is 1 year but they can live up to 10 years. The females have a straight and thick head while the males have a curly ended head. The males are typically longer the females. The eggs classically have barreled shaped, brown eggs with thick shells that measure 50-55 µm long by 22-24 µm wide. At each pole is lucent mucoid plug. The can also vary in size as noted in Image 5.

The adult female T. trichiura produces 1,000-7,000 eggs per day. The life cycle begins as unembryonated eggs passed in feces into soil (Figure 1). It takes approximately 21 days in the soil for an unembryonated egg to go through the process of embryonation to become the infective form of the parasite. Once ingested, the embryonated eggs hatch in the human intestine.

Image 5. T. trichiura eggs (CDC DPDx website)
Figure 1. Lifecycle of T. trichiura (CDC, DPDx)

Clinically, symptoms vary depending on the worm biomass present with most infections being asymptomatic. Symptoms include cramping, weight loss, growth restriction in children, bloody stool, and anemia. It can also result in Trichuris dysentery syndrome, which is more common in children. Recurrent rectal prolapse has also been reported. Lab findings include peripheral eosinophilia. T. trichiura is treated with Albendazole for 5-7 days +/- Ivermectin. Our patient was then prescribed albendazole and is being followed in GI clinic.

References

  1. Centers for Disease Control and Prevention. “Laboratory Identification of Parasites of Public Health Concern: Trichuriasis”. https://www.cdc.gov/dpdx/trichuriasis/index.html
  2. Procop, G. W., Church, D. L., Hall, G. S., Janda, W. M., Koneman, E. W., Schreckenberger, P. C., & Woods, G. L. (2017). Koneman’s color atlas and textbook of diagnostic microbiology (Seventh edition.). Philadelphia: Wolters Kluwer Health.

-Sharif Nasr, MD, 4th year anatomic and clinical pathology resident at University of Chicago (NorthShore). Dr. Nasr has an interest in GI pathology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Fighting Fire with Fire

In 1939, the first issue of Marvel Comics introduced the original Human Torch, an android named Jim Hammond who would burst into flames when exposed to oxygen. Fourteen years before that, President Calvin Coolidge proclaimed the first National Fire Prevention Week to commemorate the Chicago fire of 1871 which killed over 300 people 54 years earlier. In that entire span of 68 years, from 1871 to 1939, over 17,000 people died in fires in the United States. Because of fire awareness campaigns over the years, the number of home and work place deaths have greatly decreased, and the risk of fire in your lab goes down when fire safety awareness increases as well.

In the laboratory, fire safety begins with a look at the physical environment. It is important to make sure the department is set up to prevent a fire from starting and to keep one from spreading if a fire ignites. The electrical wiring in the lab plays a large part in fire safety. Frayed cords are the number one cause of laboratory fires, and daisy-chained extension cords or multi-plug adaptors are fire hazards as well. Damaged outlets can also present danger. Because equipment may move often in the environment, it is a good idea to check for safety in the lab electrical set up regularly. In audits I have performed this year alone, I have discovered three damaged electrical cords just waiting to cause a fire. Things change rapidly in the lab physical environment, so looking for these potential safety issues is vital.

The next aspect of the lab physical layout that needs attention is flammable chemical storage. There are complicated regulations about that, and multiple classes of flammable liquids, but you can simplify storage rules to make it easy to understand. In general, there should be no more than one gallon of a flammable liquid out in the lab per every 100 square feet. If there are automatic sprinklers in the department, that amount can go up to two gallons. If safety cans are used, the amount can be doubled again. Any excess volume of flammable liquids should be stored inside of a flammable safety cabinet with self-closing doors. Remember, the point of these storage limits is so that if a fire occurs, there is not a large amount of flammable material in one location. That slows the spread of the fire and allows automatic fire extinguishing systems to be able to perform their job effectively.

Fire-fighting equipment should be available as well, and staff are required to have training to use that equipment if it is available in the department. The best training includes a regular hands-on return demonstration and periodic fire drills. Making sure staff can use fire extinguishers and know how to respond to a fire situation may be the one of the most important safety training policies you can implement. Fire blankets are typically not required per local fire code, but if they are in place, be sure staff is aware of how to use them should the need arise.

The last actions in a departmental fire situation include evacuating and preventing the spread of the fire. To that end, it is important to keep aisles clear and wide for safe travel, and all exit routes and stairwells should be checked to make sure no obstructions exist. Staff should be aware of their primary and secondary evacuation routes, and all exits should be adequately marked. Make sure employees know to close fire and smoke doors during a fire situation.

Even in modern times there are structure fires in the work place, and unfortunately, laboratories are not excluded from that list. The Human Torch could catch fire and not get burned, but we all know that is science fiction, and burns from a fire are no joke. The best practice is to be prepared for a fire-provide training, conduct physical environment rounds, and run drills often. That will protect your staff and make you a true safety super hero.

–Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Tackling the Testosterones: Total, Free, and Bioavailable

When a patient gets their “testosterone test” at the doctor to assess their libido, do they really know what they’re getting? Does your lab test for testosterone, and are you confused about which of these confusingly-named tests are in-house versus send-out? Do you need a refresher on the types of testosterone tests out there and the clinical significance of each?

A Primer on Testosterone

Testosterone, being a fairly hydrophobic member of the steroid-ring family, is the major androgen in males. Apart from its well-known function in promoting the development of primary male reproductive organs and secondary male sex characteristics, it also has important anabolic effects in maintaining muscle mass, bone maturation, regulation of the hypothalamic-pituitary-adrenal axis under stress, and even in promoting platelet aggregation through enhancing platelet thromboxane A2 expression.1 In females, testosterone increases sexual arousal, and is in fact used clinically as treatment for female sexual arousal disorders. So, clearly an important member of the steroid family.

Being hydrophobic, much of the testosterone in the human body is not freely available, but rather bound. Total testosterone signifies the total pool of testosterone available in the human body, and is largely encompassed by the majority of bound testosterone with a small (usually 1.5-2.0%) proportion of free testosterone, which is biologically active. The bound testosterone can further be subdivided into testosterone bound to sex-hormone binding globulin (SHBG), a small glycoprotein that strongly binds various androgens and estrogens, and testosterone bound toalbumin, which is a relatively weak interaction.

Recently, the concept of bioavailable testosterone has been defined,2 based on the understanding that testosterone bound to SHBG (around 2/3rd of the bound proportion) is relatively inaccessible, while testosterone bound to albumin is weakly interacting, and thus potentially bioactive. Therefore, the definition of bioavailable testosteroneincludes both free and albumin-bound testosterone, which comprise the non-SHBG bound proportion.

How is testosterone measured?

Conventionally, total testosterone is measured through either immunoassays (both radioimmunoassays, or more commonly, chemiluminescent immunoassays) or mass spectrometry coupled with gas chromatography (GC/MS) or liquid chromatography (LC-MS/MS). Isotope dilution mass spectrometry (IDMS) is the reference method for testosterone measurement,3 but due to cost and convenience, most labs utilize immunoassays. Sex hormone binding globulin (SHBG) is commonly measured through chemiluminescent immunoassays, and also available for many platforms.4

There are two main approaches to the measurement of free testosterone, which is significantly more challenging. The gold standard for free testosterone measurement is equilibrium dialysis (see inset), a time consuming, expensive, and laborious assay that uses semi-permeable membranes to measure antibody-bound fractions of testosterone. Moreover, results can vary with pH, temperature, and methods of dilution.5 Due to these complications, calculated free testosterone is an attractive alternative used by many laboratories.

What is equilibrium dialysis? Equilibrium dialysis and ultrafiltration are reference methods used to determine true free testosterone calculation. Briefly, a relatively large quantity of serum (500 to 1000 uL) is placed in one chamber of an equilibrium dialysis apparatus, which is comprised of two fluid chambers separated by a semi-permeable membrane. Free-labeled testosterone passes through the membrane, while testosterone bound to SHBG does not. The radioactivity in the free chamber is quantified as a proportion of the total testosterone level, as measured by another assay, such as LC/MS-MS.

What is calculated free testosterone, and how is it calculated?

Recognizing the difficulty of performing equilibrium dialysis on large volumes of testosterone specimens, several researchers have looked into devising good approximations of free testosterone through mathematical expressions modeling the distribution of testosterone among its various compartments. One of the most popular approximations, the Vermeulen equation developed by Dr. Alex Vermeulen,6 models the distribution of testosterone among the SHBG-bound, albumin-bound, and free component through association constants of testosterone among these compartments, and can be modeled by the equation in Figure 1, which depends on the total testosterone, SHBG concentration, and concentration of albumin (although this will be discussed below). The overall concordance of this method with apparent free testosterone obtained through equilibrium dialysis (AFTC), the reference method, is very good, with a correlation coefficient of 0.987 and mean values well within the SEM between the two methods.6

Figure 1. The Vermeulen equation for calculated free testosterone.

In studies of the variation of calculated free testosterone values to the albumin concentration, Vermeulen et al. demonstrated that between “normal” albumin concentrations ranging from 5.8–7.2 × 10−4 mol/L (40 to 50 g/L), the mean calculated free testosterone varied from 340 ± 40.9 pmol/L assuming an albumin concentration of 40 g/L, to 303 ± 35.4 pmol/L assuming a concentration of 50 g/L albumin. Moreover, the concordance of calculated FT results to AFTC concentrations remained very good (correlation coefficient of 0.992) when an intermediate fixed albumin concentration (43 g/L) was used in this calculation, compared to actual albumin levels. Overall, these calculations suggest that for healthy individuals without marked abnormalities in plasma protein composition, such as in nephrotic syndrome or cirrhosis of the liver, or pregnant patients, a fixed albumin concentration could be used without significantly affecting calculated FT results. Of course, in individuals with marked changes in plasma proteins, the actual albumin concentration should be accounted for.

Willem de Ronde et al5 compared five different algorithms for calculating free or bioavailable, which includes the Vermeulen and Sodergard method (which use similar parameters), as well as methods by Emadi-Konjin et al, Morris et al, and Ly et al. In general, there was high concordance between the Vermeulen and Sodergard methods (r=0.98) for measuring free testosterone, and lower, but still reasonable (r=0.88) concordance between Vermeulen and other methods. Fundamentally, the Vermeulen and Sodergard equations were derived from experimentally derived association constants from the law of mass action, as opposed to the other algorithms, which rely on experimentally derived free and bioavailable testosterone measurements that was modeled by regression equations, and thus depends on the accuracy of these measurements. Though the experimental basis underlying the Vermeulen and Sodergard equations is stronger, it is known that supraphysiologic concentrations of other steroid hormones (estradiol or dihydrotestosterone), in competition for binding sites to SHBG, can significantly underestimate free testosterone by any of these methods. Of course, inaccuracies in the measurement of total testosterone or SHBG can significantly affect results, as well as significant perturbations in total serum protein concentrations (as mentioned above).

Since the publication of the above work, additional calculations for free testosterone accounting for other modes of interaction of SHBG such as allostery and dimerization have been published that may further improve concordance with AFTC;7,8 however, further study is needed to determine if these methods actually result in superior calculated FT measurement for clinical decision making, as well as changes in sensitivity to interference.

Why do accurate free testosterone measurements matter?

Testosterone bound to serum albumin is essentially inactive; therefore, the only testosterone that is biologically relevant is free (and to a lesser extent, bound to SHBG). Current consensus guidelines still support the use of total testosterone for defining hypogonadism in men,9,10 although emerging studies and newer task-force consensus groups11,12 highlight an emerging role for both calculated and free testosterone measurements in addition to total testosterone. The role of direct free testosterone measurement is still hotly debated; a recent analysis of CAP proficiency data indicates considerable heterogeneity among laboratories using the reference methods described above, and suggests considerable cost savings without significant loss of reliability can be achieved by using calculated or FT bioavailable T over direct FT measurement.13 Further standardization of these assays is needed to better understand the tradeoffs here.

References

  1. Ajayi A a. L, Halushka PV. Castration reduces platelet thromboxane A2 receptor density and aggregability. QJM. 2005;98(5):349-356. doi:10.1093/qjmed/hci054
  2. Shea JL, Wong P-Y, Chen Y. Free testosterone: clinical utility and important analytical aspects of measurement. Adv Clin Chem. 2014;63:59-84.
  3. Botelho JC, Shacklady C, Cooper HC, et al. Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Candidate Reference Method for Total Testosterone in Human Serum. Clinical Chemistry. 2013;59(2):372-380. doi:10.1373/clinchem.2012.190934
  4. Dittadi R, Fabricio ASC, Michilin S, Gion M. Evaluation of a sex hormone-binding globulin automated chemiluminescent assay. Scand J Clin Lab Invest. 2013;73(6):480-484. doi:10.3109/00365513.2013.805807
  5. Ronde W de, Schouw YT van der, Pols HAP, et al. Calculation of Bioavailable and Free Testosterone in Men: A Comparison of 5 Published Algorithms. Clinical Chemistry. 2006;52(9):1777-1784. doi:10.1373/clinchem.2005.063354
  6. Vermeulen A, Verdonck L, Kaufman JM. A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum. None. 1999;84(10):3666-3672. doi:10.1210/jcem.84.10.6079
  7. Heinrich-Balard L, Zeinyeh W, Déchaud H, et al. Inverse relationship between hSHBG affinity for testosterone and hSHBG concentration revealed by surface plasmon resonance. Molecular and Cellular Endocrinology. 2015;399:201-207. doi:10.1016/j.mce.2014.10.002
  8. Zakharov MN, Bhasin S, Travison TG, et al. A multi-step, dynamic allosteric model of testosterone’s binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200. doi:10.1016/j.mce.2014.09.001
  9. Margo KL, Winn R. Testosterone Treatments: Why, When, and How? AFP. 2006;73(9):1591-1598.
  10. American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Evaluation and Treatment of Hypogonadism in Adult Male Patients—2002 Update. Endocrine Practice. 2002;8(6):439-456. doi:10.4158/EP.8.6.439
  11. Bhasin S, Cunningham GR, Hayes FJ, et al. Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010;95(6):2536-2559. doi:10.1210/jc.2009-2354
  12. Liu Z, Liu J, Shi X, et al. Comparing calculated free testosterone with total testosterone for screening and diagnosing late-onset hypogonadism in aged males: A cross-sectional study. J Clin Lab Anal. 2017;31(5). doi:10.1002/jcla.22073
  13. Morales A, Collier CP, Clark AF. A critical appraisal of accuracy and cost of laboratory methodologies for the diagnosis of hypogonadism:  the role of free testosterone assays. Can J Urol. 2012;19(3):6314-6318.

-Dr. Jim Hsu is a 2nd year pathology resident currently in training at Houston Methodist Hospital. After completing a M.D./Ph.D at the University of Texas Medical Branch in Galveston, he realized his passions remained in the lab, but wanted to bring that passion into patient care, and soon realized that pathology was the key to achieving both. His love for all things data drew him to pathology informatics, and with the suggestion of his mentor Dr. Wesley Long, to API. In particular, he is interested in the transformative power of data analysis in improving best practices, reducing error, and combating bias. Outside of the lab, he is interested in financial markets, algorithms, neuroscience, reading, and traveling (for the food, of course).

50 Genes? 150 Genes? 500 Genes? Multi-Gene Cancer Panels – How Big Can We/Should We Go?

I first started in my current lab back in 2008. At that time, we did not have a separate section for testing solid tumors in our lab. The small amount of testing we did have were for three different types of sarcomas, and we still used a thermal cycler that didn’t have a heated lid, so we had to put mineral oil over the top of the reactions…

Fast forward eleven years and we now have a “bench” dedicated to solid tumor testing with next generation sequencing as a major part of this testing. We have been running our current solid tumor assay, a hotspot panel of fifty genes, for almost five years now and it has served us well. However, many of our oncologists have been starting to ask for more. We have begun the search for a larger panel to fulfill the needs of our oncologists and our patient population. As a smaller lab, we are somewhat limited in resources and are not quite ready to go completely custom, so we are left with kitted options from major vendors. As we research and evaluate these options, though, certain questions come to light. These panels have more than 150 genes and upwards of 500 genes in order to cover the most relevant genes in a number of different cancers. The areas tested in these genes are important for therapy and/or prognosis, but with the sheer number of bases we are looking at, we are bound to find many variants that do not have a known significance.

So, question one, how do the pathologists deal with trying to interpret the large number of variants of unknown significance (VUS’s)? Currently, with our very limited 50 gene panel, we may get one or two VUS’s, so it doesn’t take much time to assign significance and sign out the report. Our myeloid panel, which is a larger panel of 40 genes, some with full gene coverage, though, can sometimes result in reports with eight to ten VUS’s. These reports take a lot of time to research the potential impact each of these variants will have in the disease. I have seen reports from some of these large gene panels that have upwards of 25 or more VUS’s detected in a single specimen. How are these handled in the pathologists’ workflow? Can time be taken to investigate each of these, or are they just placed in a list in the report?

Question two, how do the oncologists feel when they receive a report with few, if any, variants with known significance, and many variants with unknown significance? Does this help at all, or make it more difficult and frustrating? I’d be interested if anyone has feedback in this area. In our internal tumor boards, when we review testing done at other locations, a great deal of time is spent trying to filter through the results to see how they can help point to the best possible treatment for the patient. If the variants do not point to therapy or clinical trials, those variants are not currently helpful.

Lastly, if and when we bring up a larger panel, do we keep running our smaller 50 gene panel? We believe the answer to this one is easy – yes. The amount of DNA needed for some of these larger panels is more than what we can get sometimes from the smaller biopsies. Also, insurance may not always cover the larger panels. The information we get from the 50 gene panel is still very useful and can point the oncologists to therapy options, as well as clinical trials, so we believe the smaller panel will still have a place in our lab.

rapp_small

-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

Hematopathology Case Study: An 80 Year Old Man with Rapid Onset Cervical Adenopathy

Case History

An 80 year old man presented with rapid onset of cervical adenopathy over a period of few months. The largest lymph node measuring 6 cm was biopsied and sent for histopathological evaluation.

Biopsy Findings

Sections from the lymph node showed effacement of the lymph node architecture by a fairly monotonous population of medium to large sized lymphoid cells arranged in vague nodular pattern. Focally, a starry sky pattern was observed. The cells were 1.5-2 times the size of an RBC, with high N:C ratio, irregular angulated nuclei and small nucleoli. A high mitotic rate of 2-3 mitoses/hpf was seen.

Immunohistochemistry

Immunohistochemical stains showed that the lymphoma cells were positive for CD20, CD5, SOX-11, and negative for Cyclin D1, CD10, CD23, CD30, BCL-1, and BCL-6. Ki67 index was about 70%.

Diagnosis

A diagnosis of Mantle cell lymphoma, pleomorphic variant was made.

Discussion

Mantle cell lymphoma is a peripheral B cell lymphoma, occurring in middle aged or older adults, with a male: female ratio of 7:1. Although Cyclin D1 expression is considered a hallmark of mantle cell lymphoma, yet about 7% cases are known to be Cyclin D1 negative. In these cases, morphological features and SOX-11 positivity helps in establishing a definitive diagnosis.

Differential Diagnosis

In the assessment of morphological features of lymphoma, the cell size is an important starting point. In this case, the lymphoma cells ranged from medium to large sized. The following differential diagnoses were considered:

  • Burkitt lymphoma

This case showed a “starry sky” pattern focally. A medium sized population of cells, high mitotic rate and a high Ki67 index (70%) favoured a Burkitt lymphoma. However, although commonly seen in Burkitt lymphoma, a “starry sky” pattern is not specific for this type of lymphoma. Also, the lack of typical “squaring off” of nuclei, basophilic cytoplasmic rim were against the diagnosis of Burkitt lymphoma. The nuclei in this case showed 0-1 small nucleoli, unlike the typical basophilic 2-3 prominent nucleoli of Burkitt lymphoma. Moreover, Ki67 index, even though high was not enough for Burkitt lymphoma where it approaches 100%. The cells were negative for CD10 and Bcl-6, which are almost always found in a Burkitt lymphoma. Hence, a diagnosis of Burkitt lymphoma was ruled out.

  • Diffuse Large B cell Lymphoma

The presence of interspersed large cells with nucleoli, irregular nuclei, high mitotic rate, and a high Ki67 index with a history of very rapid enlargement of lymph node suggested a diagnosis of Diffuse Large B cell lymphoma. However, the scant cytoplasm, lack of bizarre cells, and absence of CD10, BCl-2, BCl-6 were against a diagnosis of DLBCL.

  • Lymphoblastic lymphoma

A diagnosis of lymphoblastic lymphoma was favoured by the irregularly angulated nuclei, and presence of nucleoli. However, the cells of lymphoblastic lymphoma have a more delicate nuclear chromatin, higher mitotic rate as against the relatively condensed chromatin and the low to high variable mitotic rate of Mantle cell lymphoma. Also, lymphoblastic lymphomas are more commonly of the T cell subtype and occur commonly in younger individuals. In this case, B cell markers were positive (CD 20), and the patient was 80 year old, disfavouring a lymphoblastic lymphoma. The blastoid variant of mantle cell lymphoma is practically indistinguishable from lymphoblastic lymphoma, except that it is Tdt negative.

Cyclin D1 negativity in Mantle cell lymphoma

In the cases of Cyclin D1 negative mantle cell lymphomas, morphology plays a critical role in coming to a diagnosis of mantle cell lymphomas. In this case, points that favoured the diagnosis of mantle cell lymphoma were clinical features such as older age (80 years), and male gender, and morphological features such as a vaguely nodular pattern of growth, irregular nuclei, and 0-1 small nucleoli. Due to the presence of variably sized cells with distinct nucleoli, a pleomorphic variant was considered. Even though Cyclin D1 was found to be negative, the cells were positive for SOX-11.

SOX-11 is a transcription factor that is not normally expressed in B cells, but is sensitive and fairly specific for mantle cell lymphomas. It is important to note that SOX-11 is also positive in 25% Burkitt lymphoma, 100% lymphoblastic lymphoma, and 66% T-prolymphocytic leukemia. Herein lies the importance of recognising morphological features, as all of these lymphomas that may express SOX-11 were ruled on the basis of morphology. A more specific antibody, MRQ-58 may be used for greater specificity. The presence of SOX-11 is considered a specific biomarker for Cyclin-D1 negative mantle cell lymphomas. In these cases, there is upregulation of Cyclin D2 or D3 that may substitute for Cyclin D1 upregulation. But, immunohistochemical detection of Cyclin D2 or D3 is not helpful for establishing a diagnosis, as other lymphomas are commonly positive for these markers. Hence, it is important to perform SOX-11 immunohistochemistry to diagnose the Cyclin D1 negative variant of mantle cell lymphoma.

SOX-11 can be used not just for the diagnosis, but also for determining prognosis of mantle cell lymphoma. Indolent MCL usually lack SOX-11 expression. The pattern of SOX-11 staining has also been used a marker of prognosis. Cytoplasmic expression of MCl, seen in only a few cases was associated with a shorter survival as compared to the more common nuclear staining of SOX-11.

Conclusion

In this age, lymphoma diagnosis relies heavily on the use of immunohistochemical markers. However, this case highlights the importance of morphological features in diagnosing lymphomas with unusual immunohistochemical marker profile. Although, this case was negative for Cyclin D1, considered a hallmark of Mantle cell lymphoma, yet, the combination of morphological features with SOX-11 staining helped in clinching the diagnosis. To avoid a misdiagnosis, it would be prudent to perform SOX-11 staining in all lymphoma cases morphologically resembling MCL, but lacking Cyclin-D1.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Laboratory Medicine, Medical Education and Applied Health Sciences at Loyola University Chicago Stritch School of Medicine and Parkinson School for Health Sciences and Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018 and placed #5 in the #PathPower List 2019. Follow him on twitter @kmirza.

Microbiology Case Study: A 70 Year Old Male with Multiple Myeloma

Case History

The patient is a 70 year old male who was diagnosed with Kappa free light chain multiple myeloma. He was initially seen after he had a fall in the woods and underwent imaging which showed multiple lytic lesions and blood work showing monoclonal proteins and thrombocytopenia. He was found to have a lesion on his right scapula for which he received radiation. Bone marrow biopsy was performed which showed 60% plasma cells. To date he has completed radiation therapy, 5 cycles of chemotherapy, and is in the process of collecting stem cells for autologous stem cell transplant. Routine fungal culture of the stem cell collection grew a single tan white dry appearing colony on potato flake agar. A Gram stain of the organism revealed gram positive cocci mixed with filamentous structures.

Laboratory Identification

Image 1. Single tan white dry colony on potato flake agar.
Image 2. Modified acid fast stain (left) and Gram stain (right).
Image 3. Filamentous branching on Gram stain.

Based on the colony morphology and Gram stain results the organism was suspected to be in the Streptomyces genus. Identification with MALDI-TOF was attempted and did not yield a result as this bacteria is not in the data base.

Discussion

Streptomyces is a genus of gram positive aerobic saprophytic bacteria that grows in various environments, and has a filamentous form similar to fungi (1). The morphologic differentiation of Streptomyces involves identification of complex multicellular architecture with germinating spores that form hyphae, and multinuclear aerial mycelium, which forms septa at regular intervals, creating a chain of uninucleated spores (2,3). They are able to metabolize many different compounds including sugars, alcohols, amino acids, and aromatic compounds by producing extracellular hydrolytic enzymes (helping with degradation of organic matter). Their metabolic diversity is due to their extremely large genome which has hundreds of transcription factors that control gene expression, allowing them to respond to specific needs (3).

Streptomyces is also considered to be one of the most medically important bacteria because of its ability to produce bioactive secondary metabolites. These metabolites are used in the creation of antifungals, antivirals, antitumoral, anti-hypertensives, and many antibiotics and immunosuppressives. They are responsible for 2/3 of all the worlds naturally occurring antibiotics (1).

Streptomyces is usually considered a laboratory contaminant though they can cause infections in immunocompromised patients and are chiefly responsible for granulomatous lesions in skin also known as actinomycotic mycetomas (1,2). Invasive pulmonary disease has been seen in HIV patients, splenectomized patients with sarcoid, and rarely in immunocompetent hosts (1). More rare presentations include brain abscesses can be seen in patients with cerebral trauma, peritoneal infections have been shown to occur in patients undergoing multiple pericenteses, and bacteremia in patients with indwelling catheters (1). Infection with Streptomyces is not common so susceptibility data is limited. Available data shows that organisms were consistently susceptible to amikacin; frequently susceptible to imipenem, clarithromycin or erythromycin, minocycline, and trimethoprim-sulfamethoxazole; and infrequently susceptible to ciprofloxacin and ampicillin (4).

Our patient had not received the stem cell unit that this grew from, so another aliquot was requested. The second aliquot did not grow any organisms, so the Streptomyces was considered a contaminant.

References

  1. Procop, Gary W., et al. Konemans Color Atlas and Textbook of Diagnostic Microbiology. 7th ed., Wolters Kluwer Health, 2017.
  2. Tille, Patricia M. Bailey & Scotts Diagnostic Microbiology. 13th ed., Elsevier, 2014.
  3. Chater KF. Recent advances in understanding Streptomyces. F1000Res. 2016;5:2795. Published 2016 Nov 30. doi:10.12688/f1000research.9534.1
  4. Mona Kapadia, Kenneth V.I. Rolston, Xiang Y. Han, Invasive Streptomyces Infections: Six Cases and Literature Review, American Journal of Clinical Pathology, Volume 127, Issue 4, April 2007, Pages 619–624, https://doi.org/10.1309/QJEBXP0BCGR54L15

-Casey Rankins, DO, is a 3rd year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.