This last month, I rotated through our Children’s hospital, which included reviewing hemoglobin electrophoresis tests. I’d learned about them before in residency, but they can be quite more interesting (complicated) than I expected.
Hemoglobin electrophoresis is a blood test to look at different types of hemoglobin to determine if there are any abnormalities. In a children’s hospital it is frequently ordered as a reflex for an abnormal newborn screen or when a child is incidentally found to be anemic. The test is performed in 2 stages. 1st lysed blood samples are run on gel electrophoresis and different types of hemoglobin are separated as they move at different speeds. Several types of hemoglobin will run within the same region, so a secondary method of separation is always employed.
Below, you can see how some bands in the same area of an acidic gel (agarose) are actually very different on the alkaline gel (cellulose acetate) and vice versa.
At our hospital, we use HPLC and measure retention times of the hemolysate to quantify and identify different hemoglobin types present. As a basic primer you should recall that hemoglobin is a tetramer with a pair of alpha globin + a pair of either beta, delta or gamma globin (each separate genes).
Alternative hemoglobins are enriched in populations where malaria is endemic as these variants may provide improved fitness by promoting resistance to the malarial parasite that reproduces inside red blood cells. Thus, many people of African or south east Asian descent may carry these variants.
Our case is that of a 2 year old girl with anemia who had testing sent by her primary care doctor for the following CBC:
This is indicative of microcytic anemia, but unlike some Thalessemias the RBC isn’t very high. More on this later.
Looking at the gel result, there is a large band in the area coinciding with Hgb C. We also see the normal Hgb A2 and a small amount of Hgb F. We know Hgb F can be increased in Hgb SS and thus could also be present if she had Hgb C trait or disease.
Looking at the next HPLC result, we see there is a similar very high level of Hgb C (68%) with corresponding levels of Hgb F and Hgb A2 (note: acetylated Hgb F and Hgb F are added together). Thus, this fits with a homozygous C with some compensatory A1 and F, right?
Remember Hgb C is a β -globin variant and you only have 2 β -globin genes, so if you are homozygous for the C variant on the β-globin gene (HBB), then Hgb A1, which is made of normal β-globin would be impossible to produce. Also you might be bothered by all of these small peaks. However, there are often small peaks that can’t be definitively identified and are likely post-translationally modified hemoglobin. But in the context of an abnormal Hgb A1 that shouldn’t be there, we dug deeper.
One of the most common hemoglobinopathies is Beta Thalassemia (β-Thal), which clinically manifests when less of the beta hemoglobin protein is produced. Heterozygous mutations lead to Beta Thalassemia minor with minimal symptoms, while homozygous mutations lead to β-thal major with symptoms of anemia. Mutations in the β -globin gene, HBB, can lead to complete loss of β-globin (β0 variant) or partial of β-globin (β+ variant).
As this patient has less than 50% of Hgb A present (expected amount), they could also have a β+ variant as well. This would make them compound heterozygous for C and β+.
One of the hallmarks of Thalassemia is an increase in Hgb A2 (normal 2.5-3.5%). Hemoglobin A2 is a normal variant of A that is composed of two alpha and two delta chains (δ2α2). We see in our case that the Hgb A2 is normal at 2.5%. So it seems the patient doesn’t display a typical Thalassemia picture.
One condition that could create this scenario is if there is a variant in the delta chain of A2 that causes it to elute differently. Indeed, there is a delta variant that creates hemoglobin A2 prime (A2’) that moves near the S region of the HPLC. And when we look back at our unknown hemoglobins, Hgb X is marked at 1.03 of the S region and has an abundance of 3.9%. This supports it being the Hgb A2’ and if we add this together with the Hgb A2 we get an elevated 6.6% A2 total, which would be consistent with Beta Thalassemia. Lastly, one would wonder if we could find this third hemoglobin variant A2’ on the alkaline gel. Previous studies have shown the A2’ variant is more negatively charged, so on a basic gel, it should move further from the negative anode than the other hemoglobins. We don’t see anything to the left of the HgbC, but if we flip the gel over and look under the patient label, you can see a faint band that is likely the A2’!
In summary this case arose from 3 separate mutations in a single patient. She was compound heterozygous for a Hgb C and β+ variants in the β-globin gene and she was heterozygous for an A2’ variant on the delta-globin gene. This was certainly a case where paying close attention mattered.
- Abdel-Gadir D, Phelan L, and Bain BJ. Haemoglobin A2′ and its significance in beta thalassaemia diagnosis. Int J Lab Hematol. 2009 Jun;31(3):315-9. doi: 10.1111/j.1751-553X.2008.01038.x. Epub 2008 Feb 21.
-Dr. Charles Timmons MD PhD is a pediatric pathologist at Children’s Medical Center in Dallas, TX. His responsibilities include signing out hemoglobin electrophoresis, HPLC and globin sequencing, and has been residency director for 17 years.
-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.
The patient is a 72 year old
woman who presented to her physician’s office with postprandial pain and
unintentional weight loss. A CT scan was performed that showed no obvious
abnormality or cause for the patient’s abdominal pain. The patient subsequently
underwent an EGD and EUS which revealed enlarged gastric folds
without hemorrhage. In addition, there was wall
thickening seen in the body of the stomach within the luminal interface, superficial mucosa, deep mucosa
and submucosa consistent with possible
gastritis versus an infiltrative process. The remainder of the EGD and EUS was
grossly unremarkable. These findings
were concerning for possible linitis plastica. Pathology on the samples taken from the EGD were consistent
with poorly differentiated adenocarcinoma
that was invasive in both the gastric fundus and gastric body. The patient was initially taken to the operating room for
a staging laparoscopy to ensure that there was no metastatic disease before
beginning a preoperative chemotherapy regimen. The staging laparoscopy revealed
a thickened gastric wall from the fundus to the antrum, consistent with linitis
plastica, and no obvious evidence of metastatic disease. The patient then
underwent peritoneal washings which showed no evidence of positive cytology. Based
on these findings, the patient was started on a chemotherapy regimen of
epirubicin, cisplatin and fluorouracil (5-FU), which she tolerated well. The
patient was then taken to the operating room for a total gastrectomy procedure
with Roux-en-Y esophagojejunostomy.
Received fresh for intraoperative consultation is a total gastrectomy specimen with a black stitch designating the proximal side. It was requested by the surgical team to have the proximal esophageal margin frozen to ensure that esophageal tissue was indeed present, as well as to exclude the presence of any carcinoma. The proximal margin was negative for carcinoma with squamous mucosa present. The stomach measures 17.0 cm in length with an internal circumference ranging from 14.7 cm proximally to 9.0 cm distally. There is a 1.0 cm long portion of attached duodenum with an internal circumference of 5.8 cm. The serosal surface of the stomach is glistening, pink-tan and smooth with a scant amount of attached yellow, lobulated adipose tissue and omentum along the length of one entire edge measuring 26.0 x 13.0 x 1.0 cm. The stomach is opened to reveal glistening, tan mucosa with irregular rugal folds which are diffusely nodular, predominantly in the body of the stomach. There is a 6.5 x 5.0 cm are of flattened mucosa in the pyloric region (Image 1). The wall thickness measures 0.5 cm throughout. There are no grossly identifiable masses or nodules. Gross images are taken and the serosal surface is inked entirely in black. The adipose tissue is examined for candidate lymph nodes. Representative sections are submitted as follows:
B1 FS: Frozen section remnants
B2-B6: multiple representative sections from the cardia
B7-B10: multiple representative sections from the body
B11-B12: multiple representative sections from the pylorus
B13: representative perpendicular section through the distal resection margin
B14: seven putative lymph nodes
B15: five putative lymph nodes
B16: three putative lymph nodes
B17: seven putative lymph nodes
B18: six putative lymph nodes
B19: three putative lymph nodes
B20: six putative lymph nodes
Histologically, the specimen consisted of diffuse, poorly differentiated, discohesive cells throughout all the layers of the stomach, penetrating into the serosa, with fibrosis, inflammation and signet ring cells present. In addition, angiolymphatic invasion was present. Based on the gross presentation and histologic appearance, the specimen was signed out as a diffuse gastric adenocarcinoma with a stage of T3.
As of 2018, gastric cancer is the sixth most common cancer with approximately 1.03 million cases, and the third leading cause of cancer deaths worldwide, resulting in 783,000 deaths. Due to a better understanding of epidemiology, pathology, and molecular testing, as well as advances in new forms of treatments, the incidence and mortality in gastric cancer has been declining over the years. Of the gastric cancer types, rates of intestinal type carcinoma have been decreasing, however, the incidence of poorly cohesive gastric carcinoma (PCGC) and signet ring cell carcinoma (SRC) has increased. In order to accurately discuss PCGC, there must first be a discussion about the standardization of gastric cancer subtype definitions. Poorly cohesive, signet ring cell, and diffuse gastric carcinomas have commonly been used interchangeably. In 2010, the World Health Organization defined poorly cohesive gastric carcinoma as being composed of isolated or small groups of tumor cells. If there was a predominance of signet ring cells, then it would be termed a signet ring cell carcinoma. Mariette et al. proposed that a PCGC composed of 90% or more signet ring cells should be classified as SRC. The term “diffuse” corresponds to the same term “poorly cohesive”, and because of this, I will be using the term “poorly cohesive” solely going forward. In addition to this, the term “linitis plastica” would commonly be used interchangeably, but is best used as a term to describe the macroscopic appearance of PCGC or SRC.
Gastric carcinoma is classified as either early or advanced stage to help determine the appropriate type of intervention. Early gastric carcinoma is defined as invasive carcinoma confined to the mucosa and/or submucosa, regardless of lymph node metastases or tumor size. These tumors are generally smaller, measuring less than 5 cm in size, and found most commonly on the lesser curvature of the stomach at the angularis. Histologically, early gastric carcinoma will commonly present as well differentiated, mostly with tubular and papillary architecture. If the biopsies are composed of only mucosa, then distinguishing between well-differentiated carcinoma and carcinoma in situ or high grade dysplasia can be difficult. The presence of stromal desmoplasia in invasive carcinoma can help differentiate it from intramucosal invasion, which can contain single tumor cells within the lamina propria. This is an important distinction to make as intramucosal carcinoma does metastasize. Advanced gastric carcinomas will present grossly as either exophytic, ulcerated, or infiltrative tumors. Histologically, advanced gastric carcinomas will invade the muscularis propria and demonstrate cytologic and architectural heterogeneity, with a combination of patterns.
The 2010 World Health Organization classification determined four major histologic patterns of gastric cancer, which will often present with a combination of elements from the other patterns:
- Tubular: Most common pattern in early gastric carcinoma, with branching, distended or fused tubules containing intraluminal mucus, and nuclear and inflammatory debris
- Papillary: Most common in the proximal stomach with epithelial projections containing an underlying fibrovascular core. Also, it is frequently associated with liver metastases and an increased risk of lymph node involvement.
- Mucinous: Extracellular mucin makes up at least 50% of the tumor volume
- Poorly cohesive (including SRC): Mixture of signet ring and non-signet ring cells. Signet ring cells will have mucin pushing the nucleus to the periphery of the cell.
Helicobacter pylori (H. pylori) is a gram negative infectious bacteria that has been linked to gastric cancer. H. pylori is present in about half of the world’s population and other than gastric cancer, it is also associated with chronic gastritis, peptic ulcer disease, and gastric lymphomas. The bacteria is typically acquired during infancy and will remain for life if left untreated, with reactive oxygen species being generated that are capable of causing DNA damage due to the chronic infection. In addition, H. pylori can induce hypermethylation, resulting in the inactivation of tumor suppressor genes. Although H. pylori infection is considered a strong risk factor for developing gastric cancer, more commonly in intestinal type than diffuse type gastric cancer, only a small portion of those infected with the bacteria actually develop the malignancy. It is believed that approximately 80% of distal gastric cancers are due to a H. pylori infection, whereas there is little association between H. pylori and cardia gastric cancers.
In PCGC, such as this case, it is generally diagnosed in younger patients without a gender bias. Although PCGC can be associated with an H. pylori infection, it is more commonly related to a mutation in the tumor suppressor gene epithelial cadherin, also known as E-cadherin and CDH1. PCGC presents as an infiltrative growth of poorly differentiated, discohesive malignant cells that appear to arise from the middle layer of the mucosa. These cells can infiltrate as individual cells or as small clusters, but usually do not form glands (Image 2). If the gastric wall becomes extensively infiltrated by malignancy, the wall can be thickened and rigid, a macroscopic presentation termed as linitis plastica, which can lead to pyloric obstruction. Within PCGC, numerous signet ring cells can be present, leading to SRC. There is also a hereditary form of poorly cohesive gastric cancer referred to as hereditary diffuse gastric carcinoma, with an autosomal dominant pattern of inheritance. Histologically, it will include hyperchromatic nuclei, occasional mitoses, patchy intramucosal signet ring cells in the lamina propria, and carcinoma in situ associated with pagetoid spread of tumor cells along the preserved basement membrane. Hereditary diffuse gastric carcinoma will present with multifocal tumors under an intact mucosal surface, making diagnosis difficult. In patients with a CDH1 mutation and a family history of gastric carcinoma, a prophylactic gastrectomy is often the recommended treatment option.
- Adachi Y, Yasuda K, Inomata M, et al. Pathology and prognosis of gastric carcinoma well versus poorly differentiated type. Cancer. 2000;89(7)1218-24.
- Cancer. World Health Organization. Who.int. https://www.who.int/news-room/fact-sheets/detail/cancer. Published September 20, 2018. Accessed September 18, 2019.
- Carcas LP. Gastric cancer review. J Carcinog. 2014;13:14. Published 2014 Dec 19. doi:10.4103/1477-3163.146506
- Hu B, El Hajj N, Sittler S, Lammert N, Barnes R, Meloni-Ehrig A. Gastric cancer: Classification, histology and application of molecular pathology. J Gastrointest Oncol. 2012;3(3):251–261. doi:10.3978/j.issn.2078-6891.2012.021
- Mariette C, Carneiro F, Grabsch HI, et al. Consensus on the pathological definition and classification of poorly cohesive gastric carcinoma. Gastric Cancer. 2019;22(1):1-9 https://doi.org/10.1007/s10120-018-0868-0-
- Pernot S, Voron T, Perkins G, Lagorce-Pages C, Berger A, Taieb J. Signet-ring cell carcinoma of the stomach: Impact on prognosis and specific therapeutic challenge. World J Gastroenterol. 2015;21(40):11428–11438. doi:10.3748/wjg.v21.i40.11428
- Van Cutsem E, Sagaert X, Topal B, et al. Gastric Cancer. Lancet. 2016;388(10060):2654-64. https://doi.org/10.1016/S0140-6736(16)30354-3
- Weisenberg E. Diffuse (poorly cohesive) type carcinoma. Pathology Outlines. http://www.pathologyoutlines.com/topic/stomachdiffuse.html. Revised August 22, 2019. Accessed September 18, 2019
-Cory Nash is a board certified Pathologists’ Assistant, specializing in surgical and gross pathology. He currently works as a Pathologists’ Assistant at the University of Chicago Medical Center. His job involves the macroscopic examination, dissection and tissue submission of surgical specimens, ranging from biopsies to multi-organ resections. Cory has a special interest in head and neck pathology, as well as bone and soft tissue pathology. Cory can be followed on twitter at @iplaywithorgans.
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
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.
- 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
- Shea JL, Wong P-Y, Chen Y. Free testosterone: clinical utility and important analytical aspects of measurement. Adv Clin Chem. 2014;63:59-84.
- 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
- 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
- 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
- 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
- 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
- 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
- Margo KL, Winn R. Testosterone Treatments: Why, When, and How? AFP. 2006;73(9):1591-1598.
- 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
- 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
- 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
- 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).
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.
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.
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%.
A diagnosis of Mantle cell lymphoma, pleomorphic variant was made.
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.
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.
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.
Hello again everyone!
I’m writing to you now back in Manhattan after visiting sunny Phoenix, AZ for this year’s ASCP Annual Meeting. Last month I talked about downtime, pathology emergencies, and introduced you all to our insightful and dynamic colleague, Jalissa Hall. It was great working with her and one of the last things we talked about was getting to go to professional society meetings. We also talked about the upcoming meeting next year in Austin, TX! And that’s exactly what I’d like to talk about with you this time: why going to meetings like ASCP is not only educational, but an excellent way to network with your laboratorian peers from around the country.
I couldn’t go to every single session—there’s just too many—but I did learn so much valuable, practical information at the educational sessions. Here are just a mere few insights from the long list of fantastic speakers I had the chance to visit!
I participated in an interactive session on the ASCP/CAP/ASH guidelines for lymphoma workup…
Drs. Kroft, Sever, and Cheung discussed updates from the WHO 2016 guidelines as well as relating any changes in concurrent literature to appropriate diagnostic accuracy with evidence-based guidelines. If it sounds familiar, it’s because I talked about these guidelines a few months ago! In my month clerkship at The Mayo Clinic in Rochester, MN I presented a therapy-related AML case in the setting of Li-Fraumeni disorder. In my discussion I stressed the utility and importance of having organized and algorithmic guidelines to diagnose patients accurately, effectively, and timely. This time, instead of just talking about the guidelines, I got to listen to some of the folks who actually put them together—and, according to them, it’s no easy task!
I learned about culturally appropriate leadership training…
This year’s ASCP president, Dr. Melissa Upton moderated this fantastic panel and opened with an old proverb: “If you want to go fast, go alone. If you want to go far, go together.” This was definitely a theme for each of the mini-sessions’ discussions. ASCP’s own Lotte Mulder discussed her research on culturally applicable leadership training using her Leadership Institute Initiative. She talked about countries that are culturally different and developmentally different up and down the spectrum can all benefit from leadership development and opportunity. Next came Dr. Edda Vuhahula, an accomplished physician, educator, and advocate in Tanzania. She related her experiences of women in leadership roles, and challenges on the horizon as more women rise to these positions every day. Dr. Malak Abed AlThagafi talked about her “hats:” as an entrepreneur, a medical director, and a researcher in her whirlwind story of empowerment and accomplishment. Finally, medical laboratory scientist and former Philippine Army colonel, Filipinas Papas gave her personal perspectives on sexism, education, bias, and opportunity.
Celebrated my colleagues and my contributions to the 6th Choosing Wisely list of recommendations…
The Choosing Wisely initiative, partnering with the American Board of Internal Medicine and many other specialty organizations, is one of my favorite programs at ASCP. To date, our lab medicine organization has the highest number of effective test utilization recommendations. ASCP seeks active contributions to our expanding lists of recommendations to eliminate wasteful, unnecessary testing and to improve patient outcomes. This talk was also a great opportunity to honor the ASCP 2019 Choosing Wisely Champions: Dr. Gary W. Procop from the Cleveland Clinic, Dr. Lucy Nam from the Inova Lab best practice team, and Dr. Alyssa Ziman from UCLA Health. Want to read the most updated list of recommendations ASCP made to the Choosing Wisely initiative?
I watched some cutting-edge exchanges about cellular therapy…
One really effective take-home message from this seminar was that, if we’re going to rely on cellular therapy in the future—especially as it relates to “individualized medicine”—then who do you think should be in charge? Who’s got the most experience and knowledge when it comes to cell storage, transfusion protocol, patient outcomes, and high reliability? Short answer: it’s us. Long answer: go back and check out a piece I wrote about high-stakes responsibility in and out of the lab!
Popped into fascinating hematologic cases at our neighboring SHEAHP2019 meeting…
Listen, I like hematopathology, I’ll be the first to tell you that. There were so many people giving presentations in this near standing-room-only meeting, that I recognized from papers, abstracts, and journals that I’ve read in the past year alone! There were so many interesting sessions at this meeting, I wish I could have seen more…
Learned how to create an impactful dialogue with patients directly…
What do you do as a pathologist when a patient wants to speak to you? Yes, you. Not a typo! This was the last talk I went to and it was a great way to close out this awesome conference.
In their own ways these patient advocates demonstrated that if you want to represent our lab profession as one of accuracy, answers, and hope, we’ve got the skills and resources to do it! Dr. Sanford sees so many patients in her transfusion services and discusses their care plans regularly. Mr. Reed is an ASCP patient champion who, after being diagnosed with ESRD, became a learned lab ally. Dr. Sitorius is a family medicine physician at a pathology conference, talking about empathy and connection! Ms. Mitchell has done fantastic work with her pathology colleagues after beating cancer and fighting for patient education every day! These folks have taken our field of laboratory medicine to its outer edges, touching patients’ lives directly—and I left energized to take it further in the future.
And of course, I learned so much about the utilization of social media as a practical tool for education, advocacy, and outreach…
I can’t list every single session, lecture, keynote, presentation, or panel in this article. This was just a glimpse of what meetings like this have to offer. You will learn, obviously, but you’ll also gain access to new perspectives and meet people who reinvigorate your passion for your profession in ways you didn’t even consider. One of the most fulfilling experiences of this meeting was being on the ASCP Social Media Team! Posting to Instagram, Facebook, and Twitter with the hashtags #ASCP2019, #ASCPSoMeTeam, or the scavenger hunt #ASCPiSpy was a great way to bolster our enthusiastic network. This was my third ASCP Annual Meeting, and I met so many wonderful people I can’t wait for the next one! Here’s a few of my favorite snaps from the meeting:
Social media has become so valuable in our field. Not just for networking, but sharing cases, impressions, publications, and more! It’s so easy to rally behind a hashtag and support a cause in so many instances—why not in our profession? Get involved, be an active voice for your own practice as well as your colleagues.
If you want to learn more about the sessions you may have missed, download the ASCP2019 app from the Apple App Store or Google App Store!
Thanks for reading! See you on social media, because when we communicate and collaborate, we are #StrongerTogether! I’m on twitter at @CKanakis, until next time!
–Constantine E. Kanakis MSc, MLS (ASCP)CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student actively involved in public health and laboratory medicine, conducting clinicals at Bronx-Care Hospital Center in New York City.
The patient is a 41 year old male with a history of smoking who presents with a tender, slowly growing mass on the angle of the left mandible for the past 1 to 1.5 years. The patient also complains of otalgia, but no dysphagia or weight loss. A computed tomography (CT) scan was performed, which demonstrated a 3.2 x 2.6 cm enhancing mass in the superficial lobe of the left parotid gland with no significantly enlarged lymph nodes and a patent Stensen’s duct (Image 1). A fine needle aspirate (FNA) was performed that showed acinic and ductal cells, but was not diagnostic. The decision was made to take the patient to surgery in order to perform a parotidectomy.
Received in the surgical pathology laboratory for intraoperative consultation was a 0.3 x 0.2 x 0.2 cm biopsy of the left superficial lobe parotid gland mass. The tissue was frozen, stained and read out as an “acinic cell neoplasm”. Following the frozen diagnosis, the main specimen was received for routine processing, weighing 19.0 gm and measuring 4.0 x 4.0 x 3.0 cm. The specimen was unoriented and entirely inked black. It was serially sectioned revealing a 2.5 x 2.5 x 2.4 cm tan, friable, well-circumscribed mass and surrounding tan-brown, beefy-appearing parotid tissue (Image 2). A second tan-brown nodule measuring 1.5 x 1.0 x 0.8 cm abuts the larger tan mass. Representative sections of the larger mass are submitted in cassettes 1-8, and the smaller nodule is entirely submitted in cassettes 9 and 10.
Microscopy demonstrates a low-grade, very well differentiated tumor consistent with an acinic cell carcinoma with complete inked surfaces (i.e. the mass has not been transected and excision appears complete). There is a small focus of capsular “disruption”/parenchymal hemorrhage, which most likely corresponds to the area sampled for intraoperative consultation. In addition, there are two separate benign periparotid lymph nodes.
Acinic cell carcinoma (ACC) is a rare tumor of the parotid gland, representing 2 to 4% of all primary parotid gland neoplasms. It is the second most common childhood salivary gland malignancy behind mucoepidermoid carcinoma, but has been found throughout the age range. There is a gender predilection, as ACC is found in females more than males in a 3:2 ratio. One of the first cases of ACC dates back to 1892, in which the tumor was diagnosed as being a “blue dot tumor”, thought to be called this due to the intracytoplasmic zymogen granules.
Clinically, ACC presents as a slowing growing mass in the salivary glands, most commonly in the parotid gland. Other symptoms are not commonly found until late in the diagnosis, and include pain, facial nerve palsy, and nodal disease. There have also been cases of ACC that arise in the minor salivary glands. Unlike minor salivary gland carcinomas that arise in the palate, ACC of the minor salivary glands will mostly be found in the buccal mucosa and upper lip.
Grossly, ACC presents as a round, well-circumscribed to variably encapsulated mass with a rubbery, gray-tan, solid to cystic cut surface, commonly with areas of hemorrhage and necrosis. Histologically, the mass will be composed of acinar type cells with basophilic granular cytoplasm, clear cells with glycogen or mucin, intercalated ducts, non-specific glandular cells and a few mitotic figures. ACC is defined by the World Health Organization as a malignant epithelial neoplasm of the salivary glands in which at least some of the neoplastic cells demonstrate serous acinar cell differentiation, which is characterized by zymogen secretory granules, and can also include salivary ductal cells (Image 3). It is common for sections taken of ACC to show microscopic invasion of the capsule with nests of tumor cells outside the capsule. There are four histologic patterns that were described by Abrams et al in 1965 that are still applicable today: solid, microcystic, papillary cystic and follicular. Immunohistochemical stains, if needed, will be positive for keratin, alpha-1-antichymotrypsin and alpha amylase. It can be difficult to distinguish ACC from normal acini or benign salivary gland tumors (leading to a false negative result) on cytology due to the absence of any hallmark malignancy features such as necrosis and pleomorphism, but centrally placed large nuclei, distinct nucleoli, binucleated cells, and ill-defined cell borders can help make this distinction. The same caution applies to aspirates because if the tumor is cystic, it may be interpreted as being hypocellular and deemed to be a benign salivary cyst.
Imaging by ultrasound, CT and magnetic resonance imaging (MRI) can prove to be worrisome as similar with cytology, the scans can demonstrate a mass with benign features, and thus a more favorable diagnosis. On ultrasound, ACC will appear lobular, well-defined, hypoechoic and poorly vascularized. Ultrasound can be useful help to determine the size and location of the mass, as well to help with ultrasound guided fine needle biopsies. On CT, the mass will appear non-specific with limited heterogenous enhancement but can be used to demonstrate the relationship of the mass to the facial nerve, and to identify any distant metastases. On MRI, ACC can have a nonspecific intensity pattern similar to benign salivary gland neoplasms, but low T1 and T2 signals can help suggest vascularity, fibrosis and calcification within the mass. In addition, MRI can help in assessing the parotid gland, stylomastoid foramen, and any possible facial nerve invasion or perineural invasion.
Risk factors for the development of ACC include radiation exposure and familial predisposition. Risk factors for the development of salivary gland tumors, but not necessarily ACC, include radiation exposure, the use of iodine 131 in the treatment of thyroid disease (isotope is concentrated in the salivary glands), and working with materials in certain industries, such as those that use asbestos and rubber manufacturing, metal in the plumbing industries, and woodworking in automobile industries.
Complete surgical excision is considered the primary treatment option, with postoperative radiotherapy in cases of incomplete removal, recurrence, undifferentiated ACC, positive margins, and cervical lymph node metastasis. Removal of the facial nerve may be necessary in T3 and T4 cases, as well as a possible neck dissection. As of now, ACC has been considered chemo-resistant, and treatment with chemotherapy is not suggested. Around 35% of tumors will recur, and that percentage rises to 80-90% if the tumor is incompletely excised. ACC has a 5 year survival rate of 90%, a 10 year survival rate of 88%, and there have even been of cases of recurrence occurring up to 30 years after the initial procedure. If metastasis was to occur, although rare, the spread tends to be more hematogenous than lymphatic, with the most common sites being the lungs and bones.
- Al-Zaher N, Obeid A, Al-Salam S, Al-Kayyali BS. Acinic cell carcinoma of the salivary glands: a literature review. Hematol Oncol Stem Cell Ther. 2009;2(1):259-64.
- Bury D, Dafalla M, Ahmed S, Hellquist H. High grade transformation of salivary gland acinic cell carcinoma with emphasis on histological diagnosis and clinical implication. Pathol Res Pract. 2016;212(11):1059-1063. DOI: 10.1016/j.prp.2016.08.005.
- Rosero DS, Alvarez R, Gambó P, et al. Acinic Cell Carcinoma of the Parotid Gland with Four Morphological Features. Iran J Pathol. 2016;11(2):181–185.
- Vander Poorten V, Triantafyllou A, Thompson LD, et al. Salivary acinic cell carcinoma: reappraisal and update. Eur Arch Otorhinolaryngol. 2016;273(11):3511-3531. DOI: 10.1007/s00405-015-3855-7
- Zahra Aly F. Acinic Cell Carcinoma. Pathology Outlines. http://www.pathologyoutlines.com/topic/salivaryglandsaciniccell.html. Revised April 30, 2019. Accessed August 23, 2019.
-Cory Nash is a board certified Pathologists’ Assistant, specializing in surgical and gross pathology. He currently works as a Pathologists’ Assistant at the University of Chicago Medical Center. His job involves the macroscopic examination, dissection and tissue submission of surgical specimens, ranging from biopsies to multi-organ resections. Cory has a special interest in head and neck pathology, as well as bone and soft tissue pathology. Cory can be followed on twitter at @iplaywithorgans.