The primary role of hemoglobin in blood circulation is to deliver oxygen from lungs to tissues. Hemoglobin molecules comprise four polypeptide chains, e.g. two α and two β chains in hemoglobin A, with a heme group attached to each chain. Each heme group contains one iron atom, which can bind one oxygen molecule. Hemoglobin carrying oxygen molecule, known as oxyhemoglobin, gives out oxygen to tissue cells, and becomes deoxyhemoglobin, which can then load oxygen again. It is critical for the iron atom to remain in reduced state as ferrous (Fe2+) to bind to oxygen. Under oxidative stress, one or more iron atoms are oxidized to ferric (Fe3+) to form a brown color pigment, known as methemoglobin. Methemoglobin is unable to bind oxygen, and elevated level of methemoglobin or methemoglobinemia, can lead to cyanosis and be life-threatening.

A small amount of Fe2+ oxidizes to Fe3+ everyday but at the same time, methemoglobin converts back to hemoglobin through reducing activity of cytochrome b5-reductase. This maintains blood methemoglobin below 1% in normal circumstance. Methemoglobinemia can occur as inherited, mainly due to genetic defects of cytochrome b5-reductase, or as acquired, due to insufficient cytochrome b5-reductase activity under induced oxidative stress. Inherited methemoglobinemia is rare and patient may present mildly cyanotic and asymptomatic. Acquired methemoglobinemia can present acutely, more severe, and is most commonly encountered as a result of exposure to drugs and oxidant chemicals, such as local anesthetics, phenacetin, dapsone, and nitrites. FDA has released multiple warnings on the use of benzocaine-containing products, for its risk of causing serious, life-threatening methemoglobinemia. Benzocaine was found to be the causative agent of local anesthetic-related methemoglobinemia in two-thirds of cases.  

Methemoglobin can be detected and quantified in clinical laboratories with co-oximetry, which is capable of measuring absorbance at multiple wavelengths. The dual-wavelength pulse oximetry only measures oxyhemoglobin and deoxyhemoglobin and is unreliable in the setting of methemoglobinemia. The oxygen saturation calculated from partial pressure of oxygen is also unreliable, because it assumes a normal oxygen dissociation curve.

A case of an 88 year old male who underwent neurosurgery was found to have hypoxemic respiratory failure and subsequently developed acute pulmonary embolism. A blood sample was delivered to the laboratory for blood gas analysis and was soon noticed by a medical student that the blood has a dark brown color. The abnormal observation was discussed with the clinical team and a methemoglobin test was added on the specimen. Result of methemoglobin was as high as 48.6%, while oxygen saturation was falsely normal. Immediate treatment of methylene blue was initiated, and the treatment worked effectively and rapidly. A few hours after treatment, methemoglobin level was reduced to 3.3%. Upon reviewing patient’s history, it was noted that patient was given lidocaine for 3 days post-surgery, which may be the cause of methemoglobinemia. It is important to note that methemoglobinemia is a serious condition and can be fatal. Clinical diagnosis based on symptoms and history, as well as laboratory confirmation, are critical for timely management of methemoglobinemia.


-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.

A Case of Amiodarone Induced Thyrotoxicosis

A 69 year old male presented to the hospital due to worsening back pain and lower extremity weakness.  He had a medical history of follicular thyroid cancer and underwent lobectomy back in 2016. After admission, patient was found to have multiple metastasis of follicular thyroid cancer with lesion in the lumber spine, and his serum thyroglobulin level was elevated at 1,500 ng/mL (1.3 – 31.8 ng/mL) without thyroglobulin antibody detected. Patient did not present hyperthyroidism symptoms. TSH and total T4 were normal, and free T4 was slightly decreased. During hospitalization, patient was prescribed with Amiodarone, to control atrial fibrillation. Amiodarone is an antiarrhythmic drug used for severe ventricular arrhythmias, paroxysmal atrial tachycardia, and atrial fibrillation. It has high content of iodine and a direct toxic effect on thyroid gland, resulting in thyroid dysfunction in 3-5% of patients. In this case, severe hyperthyroidism was observed after amiodarone administration.

Patient’s FT4 level significantly elevated to >5.2 ng/dL (0.9-1.7ng/dL), total T4 was increased to 21.8 ug/dL (4.5-11.7 ug/dL), and TSH was suppressed to below the detection limit. The sudden increase of FT4 suggested Amiodarone induced thyrotoxicosis (AIT). High content of iodine in Amiodarone could raise blood iodine concentration up to 40 folds and enhance thyroid hormone biosynthesis in thyroid cells. This is the main cause of type 1 AIT, which is more common in patients with underlying thyroid diseases, such as Graves’ disease, or autonomous nodular goiter. Type 2 AIT typically happens in patients without underlying thyroid diseases and is caused by a direct toxic effect of amiodarone on thyroid follicular cells. Pre-formed T4 and T3 in thyroid cells are released into the circulation due to destructive thyroiditis in type 2 AIT. Differentiating these two types is important because it has therapeutic implications. However, the distinction may be difficult because patients may have a mixture of both mechanisms. Thyroid function tests are usually not helpful in the differentiation, but ultrasonography and thyroid scan with iodine uptake can help differentiate type 1 from type 2 AIT. 

In this case, thyroid scan with technetium showed reduced thyroid uptake in thyroid lobe, and mild uptake within metastatic lesions, suggesting possible thyroiditis due to amiodarone. Type 2 AIT develops as an inflammatory process and anti-inflammatory glucocorticoids are used in the treatment. Amiodarone was discontinued in this patient and his thyroid function tests indicated improvement of thyroid dysfunction. Amiodarone and its metabolites have a long half-life and accumulate in adipose tissues due to its lipophilic property. Therefore, in some cases, amiodarone toxicity effect can last for months, even after drug withdrawal. To be noted, amiodarone can also induce hypothyroidism, especially in patients with underlying Hashimoto’s thyroiditis or positive antithyroid antibodies.


-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.

Overview of Laboratory Tests for Cytomegalovirus


Cytomegalovirus (CMV) is considered the most important pathogen in transplant recipient patients as it can cause significant morbidity and mortality. Anti-CMV treatments have proven to be effective but are not without adverse side effects. Thus, there is a strong need for sensitive and reliable tests to diagnose and monitor active CMV infection. Several testing methodologies are available in today’s clinical laboratories to evaluate a patient’s CMV status: viral culture, serology, histopathology, pp65 antigenemia, and quantitative PCR. In this post, we will review the advantages and limitations of these tests.

Viral culture

Viral culture is performed most commonly by the shell vial assay (also known as rapid culture), in which a cell line (usually human fibroblast cells) is inoculated with patient sample by centrifugation. The virus is then detected by either direct or indirect fluorescent monoclonal antibody, providing results within 1-3 days. The centrifugation step greatly improves turnaround time when compared to traditional tube cell culture technique, which may take 2-3 weeks before a result can be reported as negative.

Culturing CMV has been largely replaced by newer methodologies like quantitative PCR and CMV antigenemia. This is due to relatively weaker sensitivity for diagnosing CMV infection compared to newer tests, as well as slower turnaround time. Viral cultures of urine, oral secretions, and stool are not recommended due to poor specificity; however, for diagnosis of congenital CMV, viral culture of urine or saliva samples is an acceptable alternative if PCR is not available.


CMV serostatus is an important metric to evaluate prior to patients receiving a hematopoietic or solid organ transplant. Serologic testing is done primarily via enzyme immunoassays and indirect immunofluorescence assays. These tests check for presence of anti-CMV immunoglobulin (Ig)M and IgG to provide evidence of recent or past infection. Outside of establishing serostatus (primarily in organ donors and recipients), serologic testing for CMV is not recommended in diagnosing or monitoring active CMV infection.

CMV IgM antibodies can be detected within the first two weeks of symptom development and can be present for another 4-6 months. IgG antibodies can be detected 2-3 weeks after symptoms develop, and remain present lifelong. These antibody measurements are particularly useful in determining risk of CMV acquisition in seronegative patients (negative for IgM and IgG) at time of transplantation. IgG titers can also be measured every 2-4 weeks to assess for CMV reactivation in seropositive patients. Since CMV IgG persistently remains in circulation, testing for it has a higher specificity compared to IgM, and thus is the preferred immunoglobulin to test for in establishing serostatus. Serologic tests can be falsely positive if patients have recently received IVIG or blood products, so testing on pretransfusion samples are preferred if possible.


Under the microscope, cells infected with CMV can express certain viral cytopathic effects. These infected cells classically show cytoplasmic and nuclear inclusions (owl eye nuclei) with cytoplasmic and nuclear enlargement. Additionally, immunohistochemistry (IHC) can stain antibodies specifically for CMV proteins to highlight infected cells, making histologic examination quicker and improving diagnostic sensitivity.

Histopathology can be useful in identifying tissue-invasive disease, such as CMV colitis or pneumonitis. Cases in which PCR testing is negative does not necessarily exclude tissue-invasive disease; thus, the diagnosis of tissue-invasive disease relies on histologic examination (with or without IHC) or possibly viral culture. On the other hand, a negative histologic result does not exclude tissue-invasive disease, possibly due to inadequate sampling, and shows the potential for weak diagnostic sensitivity.

pp65 antigenemia

CMV antigenemia testing uses indirect immunofluorescence to identify pp65 antigen, a CMV-specific matrix protein, in peripheral blood polymorphonuclear leukocytes. Whole blood specimens are lysed and then the leukocytes are cytocentrifuged onto a glass slide. Monoclonal antibodies to pp65 are applied, followed by a secondary FITC-labeled antibody. The slide is then read using a fluorescence microscope for homogenous yellow-green polylobate nuclear staining, indicating presence of CMV antigen-positive leukocytes. Studies have suggested that a higher number of positive cells correlates with an increased risk of developing active disease. The sensitivity of antigenemia testing is higher than that of viral culture and offers a turnaround time within several hours.

This test has been utilized since the 1980s, but has seen less use recently due to the increasing popularity of quantitative PCR. Antigenemia testing is labor intensive, and requires experienced and trained personnel to interpret the results (which can be somewhat subjective). This test also must be performed on whole blood specimens within 6-8 hours of collection due to decreasing sensitivity over time, which limits transportability of specimens. Additionally, It is not recommended to be run on patients with absolute neutrophil counts below 1000/mm3, due to decreased sensitivity. Despite these limitations, CMV antigenemia testing is still considered a viable choice for diagnosing and monitoring CMV infection, especially when viral load testing is not available.

Quantitative PCR

Quantitative real-team polymerase chain reaction (PCR) is the most commonly used method to monitor patients at risk for CMV disease and response to therapy, as well as for diagnosing active CMV infection. The advantages of using a quantitative PCR assay include increased sensitivity over antigenemia testing, quick turnaround time, flexibility of using whole blood or plasma specimens for up to 3-4 days at room temperature, and the availability of an international reference standard published by the World Health Organization (WHO).

Several assays from Roche, Abbott, and Qiagen are available and FDA-approved. The targets of these assays vary, with some targeting polymerase and other targeting CMV major immediate early gene. These assays are all calibrated with the WHO international standard, which was developed in 2010 to help standardize viral load values among different labs when results are reported in international units/mL. The goal of this international standard is to decrease the interlaboratory variability of viral load, and determine the appropriate cut-offs for determining clinical CMV disease. There is still improvement to be made in this area, as variability still exists between labs.


There are several tests to determine the CMV status of patients. Some of these tests are suited for particular goals, such as serology for determining serostatus prior to organ transplantation, or histology and IHC to diagnose tissue-specific CMV disease. For diagnosis and monitoring of general CMV disease, the test of choice in most laboratories is quantitative PCR, which offers automated, quick and sensitive results. Antigenemia, while dated and labor intensive, is still an acceptable alternative when PCR is neither available nor cost-effective for smaller labs. Both of these testing methods are preferred over viral culture, which has poorer diagnostic sensitivity and relatively longer turnaround time.

Despite the numerous advantages quantitative PCR has, there is still variability in viral load counts among laboratories. This is due to varying DNA extraction techniques, gene targets used by PCR, and specimen types used. There is still a lot of work to be done in standardizing testing in regards to not just CMV, but also other viral pathogens like Epstein-Barr virus, BK virus, adenovirus and HHV6. Updated standards and increased use of standardized assays will hopefully decrease this variability between labs to improve testing results and in turn, improve patient care.


  3. Kotton CN, Kumar D, Caliendo AM, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2013;96(4):333-60.
  4. Hayden RT, Sun Y, Tang L, et al. Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards. J Clin Microbiol. 2017;55(2):423-430.
  5. Kotton CN, Kumar D, Caliendo AM, et al. The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation. Transplantation. 2018;102(6):900-931.

-David Joseph, MD is a 2nd year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, TX. He is planning on pursuing a fellowship in forensic pathology after completing residency. His interests outside of work include photography, playing bass guitar and video games, making (and eating) homemade ice cream, and biking.

Prozone Effect in the Fecal Immunochemical Test (FIT)

Case History

An 80 year old female had a history of chronic iron deficiency anemia with unknown cause and comorbidities included hypothyroidism, congestive heart failure (CHF), severe aortic stenosis and COPD. The patient presented at the ED with initial presentation with increasing shortness of breast, NYHA class 3-4. She was admitted to the hospital for further treatment for CHF, as well hyperventilation, sleep apnea and COPD. Her serum iron and iron saturation were tested and results were 2 umol/L (reference range for iron: 10-29 umol/L) and 7% (reference range: 14-51%), respectively. Part of her investigations included a qualitative fecal test to screen for gastrointestinal bleeding. The immunochemical fecal occult blood test was performed using a CLIA waived Hema Screen SpecificTM POCT test (Immunostics, Inc, USA) in the hospital lab. Hema Screen Specific test is a qualitative, sandwich dye conjugated immunoassay that uses a combination of monoclonal and polyclonal antibodies to detect the globin component of hemoglobin in the fecal samples. The manufacture recommended using Hema Screen Specific test in routine physical examines, hospital monitoring of bleeding in patients and for screening for colorectal cancer or gastrointestinal bleeding for any source (statement from the product package insert).

 The specimen submitted to the lab was markedly red (Image 1), yet Hema screen test returned a negative result. Since this device is designed to detect occult blood in fecal samples, a prozone effect was suspected, as the stool appeared to contain overt hemorrhage. The specimen was reanalyzed with serial dilutions by a factor of 5, 10, and at 100 × dilution. The FIT result became clearly positive for blood (Image 2). The patient received a colonoscopy, which revealed internal hemorrhoids, severe diverticulosis in the left colon, as well as multiple angiodysplastic lesions. One such lesion was in the ascending colon and was actively bleeding at the time of colonoscopy. The others, which were not bleeding, were distributed in the proximal ascending colon, hepatic flexure, and proximal transverse colon. All angiodysplastic lesions were treated with argon plasma coagulation.

Image 1. Fecal specimen demonstrating overt hemorrhage.
Image 2. Fecal immunochemical test performed on the patient sample submitted. Serial dilutions of fecal specimen were performed. At the dilution factor of 1:100, the result showed positive. Saline was used to dilute the fecal sample.

Moreover, we have tested the device with another bloody fecal sample during the initial evaluation. When an appropriate dilution factor was used, the prozone effect begins to lose its interference as show in Image 3.

Image 3. Fecal immunochemical test (FIT), showing control (C) and test (T) lines, applied to serial dilutions of fecal specimen (B ─ undiluted, C ─ 10 ×, D ─ 100 ×, E ─ 1 000 ×, F ─ 10 000 ×, G ─ 100 000 ×). At 10 × dilution, the prozone effect begins to lose its interference, and at 100 × the test is clearly positive. At dilutions higher than 1 000 ×, the concentration of blood becomes too low to return a positive result. (Image 3 provided by Dr. Andrew Lyon, PhD, DABCC, FCACB, Lab Medicine & Pathology, Saskatchewan Health Authority)


The prozone effect (or Hook effect) has long been appreciated as a source of interference in immunoassays.1 It typically occurs in sandwich assays, of which the FIT test is an example.2 When the concentration of the analyte is excessively high, it oversaturates the capture and detection antibodies in favor of forming single antibody:analyte complexes, rather than sandwiches. This results in a false negative result where the assay is unable to detect the analyte. The solution to the prozone effect is serial dilution to lower the concentration of the analyte.

The FIT test is designed to detect microscopic amounts of blood, hence its function in screening for fecal occult blood. A number of hospital labs use this test in an acute care setting to screening bleeding in patients. However, its capacity is oversaturated in specimens containing overt hemorrhage, as in our patient. In these cases it is nevertheless important to prove that the red color of the specimen is truly due to blood, as bright red stool can be caused by a wide range of dietary factors. Some examples are red food coloring, beets, cranberries, and tomato juice.3 If these possibilities are not ruled out, the patient may become subject to the risks of unnecessary endoscopy. Serial dilution of the specimen is extremely useful in this type of situation.


  1. Dasgupta A, Wahed A. Clinical Chemistry, Immunology and Laboratory Quality Control: A Comprehensive Review for Board Preparation, Certification and Clinical Practice. Amsterdam: Elsevier; 2014. 2.11.
  2. Allison JE, Fraser CG, Halloran SP, Young GP. Population Screening for Colorectal Cancer Means Getting FIT: The Past, Present, and Future of Colorectal Cancer Screening Using the Fecal Immunochemical Test for Hemoglobin (FIT). Gut and Liver. 2014 Mar;8(2):117-30.
  3. Picco MF. Stool color: When to worry [Internet]. Mayo Clinic; 2019 Feb 19 [cited 2019 Feb 23]. Available from:

-Hao Li, MD is a currently a first year anatomical pathological resident at Western University, London ON, Canada. Prior to be a pathology resident, he was a neurosurgery resident at the University of Saskatchewan, Saskatoon SK, Canada. When he was at the University of Saskatchewan, he spent his third year primarily in neuropathology, with also some general anatomical pathology and clinical pathology. Through these experiences, he has come to realize that his passion and calling lay more in pathology than in surgery. He has successfully transferred into pathology, and started a new residency in anatomical pathology in July 2019. Having a background in the clinical neurosciences, he hopes to eventually pursue a fellowship in neuropathology, and possess the skill set to practice both anatomical pathology and neuropathology.

Vitamin D: An Overview


Vitamin D is one of the most commonly ordered laboratory tests in the primary care setting, as well as one of the most widely used forms of dietary supplementation today. While the rationale underlying vitamin D testing and supplementation for deficiency may seem straightforward, in actuality, the metabolism and physiologic functions of vitamin D in the body are quite nuanced and complex, and there remains significant controversy surrounding the appropriate utilization of vitamin D measurement and clinical interpretation of vitamin D test results. In this post, let’s review the basic principles of vitamin D metabolism, its function and mechanisms of regulation in the human body, methods of measurement in the laboratory, and ramifications of vitamin D values on clinical decision-making and management.

Vitamin D Metabolism

Vitamin D is a fat-soluble vitamin and encompasses a group of compounds, all containing a four-ring steroid backbone. The two main forms of vitamin D utilized by humans are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is primarily synthesized in the skin from 7-dehydrocholesterol in the presence of sunlight (UVB rays), while vitamin D2 is synthesized in plants from ergosterol and is used to fortify many foods (milk, bread, cereal, etc.).1

Once synthesized in the skin or ingested from the gastrointestinal tract, both vitamin D2 and vitamin D3 travel in the bloodstream (bound to vitamin D-binding protein) to the liver, where both are converted to 25-hydroxyvitamin D [25(OH)D, calcidiol/calcifediol] by the action of 25-hydroxylase.1,2 While 25(OH)D has only limited biologic activity, it has a very long half-life (2-3 weeks) and is therefore the primary form of vitamin D found in the blood.1 Notably, the half-life of 25(OH)D2 is shorter than that of 25(OH)D3 (possibly due to lower affinity to vitamin D-binding protein) and therefore it is present in significantly lower concentrations than 25(OH)3 in the blood.3

25(OH)D is then further converted to 1,25-dihydroxyvitamin D [1,25(OH)2, calcitriol] via the action of 1-α-hydroxylase primarily in the kidney.1 In contrast to 25(OH)D, 1,25(OH)2D is the biologically active form of vitamin D, but it has a much shorter half-life (5-8 hours) and therefore has much lower circulating levels in the blood.1 25(OH)D may alternatively be converted to 24,25-dihydroxyvitamin D [24,25(OH)2D] by 24-α-hydroxylase, also in the kidney. 24,25(OH)2D is an inactive metabolite and thus serves as an end-product in this degradation-type pathway of 25(OH)D.1,4

Vitamin D Physiology

The overall effect of vitamin D in the body is to increase calcium and phosphate levels in the blood. Via binding of 1,25(OH)2D to nuclear receptors within cells, it acts at three main sites: 1) the intestine, where it increases calcium and phosphate absorption, 2) the bones, where it increases calcium resorption by promoting osteoclast maturation, and 3) the kidney, where it increases calcium reabsorption by enhancing the effects of parathyroid hormone (PTH) on the distal convoluted tubule.1

Conversion of 25(OH)D to 1,25(OH)2D by 1-alpha-hydroxylase is tightly regulated by calcium, phosphate, and PTH concentrations in the body. Decreased calcium or phosphate levels, or increased PTH levels in the blood (most commonly resulting from a fall in calcium) will stimulate 1-α-hydroxylase activity and lead to increased production of 1,25(OH)2D, while increased calcium or phosphate levels or decreased PTH levels will suppress 1-α-hydroxylase activity and thus lead to decreased production of 1,25(OH)2D.1,2

From a clinical perspective on vitamin D physiology, there are numerous causes of abnormal vitamin D levels in the body. Here are some common causes of low vitamin D levels:

  • Inadequate intake of vitamin D (whether from diet, inadequate sunlight, or malabsorption)
  • Decreased PTH (hypoparathyroidism, hyperphosphatemia, hypercalcemia of malignancy)
  • End-organ resistance to PTH (pseudohypoparathyroidism)
  • Decreased 1-α-hydroxylase activity (renal failure, vitamin D-dependent rickets type 1)5

Conversely, causes of high vitamin D levels are listed below:

  • Excessive intake of vitamin D (usually from supplements)
  • Increased PTH (primary hyperparathyroidism)
  • Increased extrarenal 1-α-hydroxylase activity (seen in granulomatous diseases such as sarcoidosis, as well as some lymphomas)
  • End-organ resistance to vitamin D (vitamin D-dependent rickets type 2)5

Vitamin D Measurement in the Laboratory

25(OH)D is the most commonly measured vitamin D metabolite in laboratory assays, since (as mentioned above) it has a longer half-life and a larger concentration in the blood compared to 1,25(OH)2D. In addition, its concentration does not fluctuate as significantly as that of 1,25(OH)2D, since its production from 25-hydroxylase in the liver is not so tightly regulated as 1-α-hydroxylase activity in the kidney.1,6 Nevertheless, 1,25(OH)2D measurement is indicated in a few specific clinical circumstances, including workups for idiopathic hypercalcemia and bone/mineral disorders, and for evaluation of vitamin D status in the setting of renal failure (where 1-α-hydroxylase activity is decreased).1,7

While the gold standard for vitamin D measurement is liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), most laboratories utilize immunoassays (including radioimmunoassays, chemoluminescent immunoassays, and enzyme-linked immunoassays) for vitamin D quantitation.1,7 One significant difference between these two methods is that while LC-MS/MS can differentiate vitamin D3 and vitamin D2 metabolites, immunoassays cannot.6 In addition, the antibodies used in many 25(OH)D immunoassays often have lower cross-reactivities with 25(OH)D2 and therefore may underestimate this form when giving the total 25(OH)D value.6 These antibodies also have varying cross-reactivities with other vitamin D metabolites and so may result in an overestimation of the total 25(OH)D due to positive interference from these metabolites.6

Another advantage of the LC-MS/MS method is that it can detect C3 epimers of 25(OH)D, while immunoassays cannot.8 The physiologic significance of these epimers has not yet been clearly delineated, but recent evidence has shown that while these epimers do not affect calcium concentrations, they do contribute to suppression of PTH levels.8 In addition, while these epimers comprise a low proportion (about 2-3%) of the overall 25(OH)D concentration in adults, they have been found in significantly higher proportions (up to 60%) in infant and pediatric populations.8,9 Thus, the detection of these epimers (and their quantitation, which is possible through high-performance LC-MS/MS) may be more important in these patient populations.

Interpretation of Vitamin D Results

The optimal serum levels of 25(OH)D are not universally established. First of all, levels vary with factors affecting sunlight exposure including latitude, skin pigmentation, and sunscreen use.1 Levels also demonstrate significant seasonal variation, with winter measurements up to 40-50% lower than summer measurements.1 Recommended minimum 25(OH)D levels for optimal bone health differ among various national organizations and generally range from 20 ng/mL to 30 ng/mL; as mentioned above, these thresholds are controversial and there is no established consensus.10-12

Vitamin D deficiency is very common, with the majority of patients exhibiting no clinical symptoms and normal calcium and phosphate concentrations. However, a significant proportion of these asymptomatic patients will have increased PTH levels and concomitant increased risk of osteopenia/osteoporosis and fractures; therefore, all patients with vitamin D deficiency should be treated with repletion.13 If deficiency is severe and persistent, bone demineralization with rickets (in children) and osteomalacia (in adults and children) can develop. In contrast, vitamin D toxicity is very rare and is usually associated with over-supplementation; patients develop hypercalcemia with related symptoms including confusion, muscle weakness, nausea and vomiting, and polydipsia and polyuria.14

Recent studies have linked vitamin D deficiency (usually with residency at higher latitudes) to a wide variety of clinical disorders ranging from autoimmune diseases (multiple sclerosis, rheumatoid arthritis, type I diabetes), to cancers (including colon, breast, and prostate), to psychiatric illnesses (schizophrenia, depression), and cardiovascular disease (including hypertension and congestive heart failure).15 Whether these links possess a causal basis or are merely associative needs to be further investigated. Nevertheless, what is certain is that understanding the functions of vitamin D in the body and methodologies of vitamin D measurement in the laboratory is crucial in appreciating its clinical significance and various, ever-expanding applications in disease pathophysiology and management.


  1. McPherson RA, Pincus MR. Henry’s Clinical Diagnosis and Management by Laboratory Methods. Elsevier Health Sciences; 2017.
  2. Brown AJ. Regulation of vitamin D action. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association-European Renal Association. 1999 Jan 1;14(1):11-6.
  3. Armas LA, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. The Journal of Clinical Endocrinology & Metabolism. 2004 Nov 1;89(11):5387-91.
  4. Cashman KD, Hayes A, Galvin K, Merkel J, Jones G, Kaufmann M, Hoofnagle AN, Carter GD, Durazo-Arvizu RA, Sempos CT. Significance of serum 24, 25-dihydroxyvitamin D in the assessment of vitamin D status: a double-edged sword?. Clinical chemistry. 2015 Apr 1;61(4):636-45.
  5. Clarke W. Contemporary practice in clinical chemistry. Amer Assn for Clinical Chemistry; 2016.
  6. Zerwekh JE. Blood biomarkers of vitamin D status. The American journal of clinical nutrition. 2008 Apr 1;87(4):1087-91.
  7. Hollis BW. Assessment and interpretation of circulating 25-hydroxyvitamin D and 1, 25-dihydroxyvitamin D in the clinical environment. Endocrinology and Metabolism Clinics. 2010 Jun 1;39(2):271-86.
  8. Lutsey PL, Eckfeldt JH, Ogagarue ER, Folsom AR, Michos ED, Gross M. The 25-hydroxyvitamin D3 C-3 epimer: distribution, correlates, and reclassification of 25-hydroxyvitamin D status in the population-based Atherosclerosis Risk in Communities Study (ARIC). Clinica chimica acta. 2015 Mar 10;442:75-81.
  9. Singh RJ, Taylor RL, Reddy GS, Grebe SK. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. The Journal of Clinical Endocrinology & Metabolism. 2006 Aug 1;91(8):3055-61.
  10. Del Valle HB, Yaktine AL, Taylor CL, Ross AC, editors. Dietary reference intakes for calcium and vitamin D. National Academies Press; 2011 Apr 30.
  11. Vieth R. What is the optimal vitamin D status for health?. Progress in biophysics and molecular biology. 2006 Sep 1;92(1):26-32.
  12. American Geriatrics Society Workgroup on Vitamin D Supplementation for Older Adults. Recommendations abstracted from the American geriatrics society consensus statement on vitamin D for prevention of falls and their consequences. Journal of the American Geriatrics Society. 2014 Jan;62(1):147-52.
  13. Valcour A, Blocki F, Hawkins DM, Rao SD. Effects of age and serum 25-OH-vitamin D on serum parathyroid hormone levels. The Journal of Clinical Endocrinology & Metabolism. 2012 Nov 1;97(11):3989-95.
  14. Ozkan B, Hatun S, Bereket A. Vitamin D intoxication. Turk J Pediatr. 2012 Mar 1;54(2):93-8.
  15. Holick MF. Vitamin D deficiency. New England Journal of Medicine. 2007 Jul 19;357(3):266-81.

-Michelle Lin, MD, is a second-year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, Texas.

Hemoglobin Electorphoresis in Children

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.

InkedBlog 1B_LI

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.


  1. 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.

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.


  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).

Laboratory Test of Anti-Neutrophil Cytoplasmic Antibody in Sinonasal Inflammatory Disease

Case History

A 44 year old male with history of cocaine use presented with 1 year history of headache and progressive frontal lobe syndrome, including symptoms like apathy, personality changes, lack of ability to plan, poor working memory for verbal information or spatial information, Broca aphasia, disinhibition, emotional lability, etc. CT scan found extensive destruction of osteocartilaginous structures of the nasal cavity and MRI showed extensive edema of the frontal lobe. Biopsy showed chronic inflammation but negative for granulomatous inflammation. Patient’s CSF laboratory analysis was normal but ANCA was tested positive, in a P-ANCA pattern without MPO detectable. Patient was diagnosed as CIMDL. After stopping cocaine use, patient was doing better but still has mild frontal lobe syndrome.


Anti-neutrophil cytoplasmic antibody (ANCA) are a group of autoantibodies that directed toward antigens expressed mainly in neutrophil granulocytes, such as proteinase 3 (RP3) and myeloperoxidase (MPO). The presence of ANCA is mainly associated with a distinct form of small vessel vasculitis, known as ANCA-associated vasculitis, but is also detected in other disease, like autoimmune hepatitis, primary sclerosing cholangitis, ulcerative colitis, and other chronic inflammatory disease. The gold standard laboratory method to screen ANCA is indirect immunofluorescence assay (IFA or IIF), which qualitatively capture antibodies in serum/or plasma bound to fixed human neutrophil granulocytes.

Two form of ANCA-associated vasculitis, granulomatous with polyangiitis (GPA) and eosinophilic granulomatous with polyangiitis (EGPA), are systemic diseases that commonly associated with necrotizing granulomatous vasculitis. GPA has a primary involvement of the upper and lower respiratory tract and kidney. Autoantibodies to PR3 are found in 90% of active GPA cases, which generates a cytoplasmic-ANCA (C-ANCA) pattern on ANCA IFA test. EGPA is a rare form of systemic necrotizing vasculitis characterized by asthma and eosinophilia. A perinuclear-ANCA (P-ANCA) IFA pattern directing towards MPO antibody are often seen in EGPA cases.

Both GPA and EGPA may also present with sinonasal involvement, causing non-infectious inflammatory lesions of the sinonasal tract. Sinonasal inflammatory disease can also result from bacterial and fungal infections, or other non-infectious process, such as sarcoidosis, polychondritis, or obstruction. ANCA is detected in the majority of GPA and EGPA case, therefore it provides useful information in differential diagnosis of sinonasal inflammatory disease. Both GPA and EGPA are autoimmune diseases, corticosteroids and immunosuppressive agents are effective treatment.

Sinonasal inflammation can also been seen in a subset of patients with cocaine abuse, who normally present with midline destructive lesions, known as cocaine-induced midline destruction lesions (CIMDL). Long-term cocaine use has been associated with ischemia of mucosal tissue, cartilage and bone, and cocaine abuser using intranasal inhalation route can have midline deformity and septal perforation. Interestingly, ANCA are also found in a large portion of CIMDL, and in contrast to GPA or EGPA, ANCA in CIMDL are primarily directed against neutrophil elastase, generate a P-ANCA or atypical P-ANCA pattern, without detection of MPO. Therefore, ANCA serology testing could help the differentiation between CIMDL and GPA although these two can overlap clinically and histopathologically. Also, CIMDL does not respond well to immunosuppressive therapy and only consistent removal of stimuli (cocaine) can halt the disease process.


  1. Montone KT. Differential Diagnosis of Necrotizing Sinonasal Lesions. Arch Pathol Lab Med. 2015 Dec;139(12):1508-14. doi: 10.5858/arpa.2015-0165-RA.
  2. Trimarchi M, Bussi M, Sinico RA, Meroni P, Specks U. Cocaine-induced midline destructive lesions – an autoimmune disease? Autoimmun Rev. 2013 Feb;12(4):496-500. doi: 10.1016/j.autrev.2012.08.009. Epub 2012 Aug 24.
  3. Madani G, Beale TJ. Sinonasal inflammatory disease. Semin Ultrasound CT MR. 2009 Feb;30(1):17-24.
  4. Timothy R. Helliwell Non-infectious Inflammatory Lesions of the Sinonasal Tract. Head Neck Pathol. 2016 Mar; 10(1): 32–39.

-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.

Hormone Blockers = Blood Letting for Female Athlete with high T?

Caster Semenya celebrates as she wins gold in the women’s 800 meters in the Commonwealth Games on April 13, 2018, on Australia’s Gold Coast (1). Jason O’Brien/Getty Images

I will continue this month along the thread of last month’s post, which addressed the controversy surrounding South African female mid-distance runner Caster Semenya. Caster has won many international mid-distance races (400-800m), but she has been suspected of naturally producing higher levels of testosterone.

Since last month, I’ve learned the reason for the higher testosterone is uncertain: it could be due to natural production (hyperandrogenism) or rumors of her being intersex1. Regardless, what I will discuss here is how the proposed actions of the International Olympic Committee would be expected to affect Semenya’s performance. Specifically, how would lowering testosterone levels affect her athletic performance?

Last month, we saw that muscle mass might be expected to decrease, but this may not affect athletic performance significantly.

Another important effect of testosterone is on red blood cell levels including hemoglobin, which by carrying oxygen to muscle is a central part of calculating VO2max. VO2max is maximal oxygen consumption. This is strongly linked to performance in cardiovascular athletic events.

Mid-distance running requires a large cardiovascular capacity. Maybe not the same level of Tour-de-France long distance bikers in the Alps, but still substantial. As a runner that feels pretty proud at having run a sub-3 minute 800m, I can say Caster’s feat of running it in less than 2 minutes is incomprehensible. From the burning feeling in my lungs and thudding, maximum heart rate at the end of the half-mile, I can attest that this event requires substantial cardiovascular efficiency.

Maximal oxygen consumption (VO2max) by exercising skeletal muscle is principally limited most by cardiac output and oxygen-carrying hemoglobin levels. This has been shown quite convincingly in a series of experiments in the 1950’s-70’s2,3 that probably wouldn’t be approved by the IRBs of today charged to protect research subject rights.

First, transfusing blood increased hemoglobin concentration and similarly the VO2max and exercise endurance of participants.  (This practice was exploited most notably later on in the Tour de France).  In other studies3, blood was removed from participants before assessing their exercise tolerance (10% loss of hemoglobin à 13% reduction in VO2max). Another study removed 400mL, 800mL and 1,200mL over several days, which decreased hemoglobin by 10%, 15%, and 18% respectively. There was a concomitant decrease in endurance time (-13%, -21%, -30%) and VO2max as well (-6%, -10%, -16%)3.  A summary of blood transfusion and hemodilution studies is shown in Figure 1 from Otto JM et al4.

Figure 1. Reproduced from Otto JM et al (4)

In transgender women (gender incongruent with sex assigned male at birth), hormone therapy to increase estrogen levels (oral estradiol) and block testosterone (anti-androgen: spironolactone) reduces hemoglobin by 9% on average (from 15.2 g/dL to 13.9 g/dL)5. I would expect a smaller decrease for Semenya as she will likely not get a full dose hormone regimen used for transgender transition and because her testosterone levels wouldn’t be as high as biologic males’.  However, she would still be expected to have lower hemoglobin- similar to donating a half or whole unit of blood. If hemoglobin decreased even just 5%, that could affect her performance substantially when the difference between competitors boils down to seconds in mid-distance races.

Arguably, forced blood donation could produce the same effects as testosterone-lowering therapy. But it would be far too dramatic to suggest something like bloodletting by the International Olympic Committee.

In the end, I don’t feel qualified to say what should be done in this case. All I can say is that I don’t think lowering Caster Semanya’s testosterone levels will have the intended effect of decreasing muscle mass. On the other hand, it would decrease hemoglobin levels tempering her performance. But who should determine the point where her hormone levels should be? There is such a strong biologic connection between hormone levels and physiology that manipulating them for athletic fairness could be akin to playing puppeteer.


  1. North, Anna. ““I am a woman and I am fast”: what Caster Semenya’s story says about gender and race in sports” Vox. May 3, 2019
  2. BALKE B, GRILLO GP, KONECCI EB, LUFT UC. Work capacity after blood donation. J Appl Physiol. 1954 Nov; 7(3):231-8.
  3. Ekblom B, Goldbarg AN, Gullbring B. Response to exercise after blood loss and reinfusion. J Appl Physiol. 1972 Aug; 33(2):175-80.
  4. Otto JM, Montgomery HE, Richards T. Haemoglobin concentration and mass as determinants of exercise performance and of surgical outcome. Extrem Physiol Med. 2013; 2: 33.
  5. SoRelle JA, Jiao R, Gao E et al. Impact of Hormone Therapy on Laboratory Values in Transgender Patients. Clin Chem. 2019; 65(1): 170-179.

-Jeff SoRelle, MD is a Molecular Genetic Pathology fellow 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 advancing quality in molecular diagnostics.

Sex Hormones in Competitive Athletics

Image 1. Photo from NBC News.

Given my previous work in lab value changes in transgender individuals on hormone therapy, I was recommended to consider discussing the case of Olympic mid-distance runner, Caster Semenya. Although she is not transgender, this professional runner from South Africa has won her last 30 races and been scrutinized for her muscular build as having potentially higher levels of testosterone, a condition called hyperandrogenism. The International Olympic Committee’s (IOC) regulations require testosterone levels to be below a certain threshold for female athletes. 

While no competitor can achieve great victories without hard work and practice, there are certainly examples of outliers whose genetics give them an advantage. However, I don’t think we would endorse shortening Michael Phelps’ arms or lobotomizing chess master Bobby Fisher to decrease their inborn advantages for a level playing field.

But this gets into an area of ethics that I’m not an expert on, so instead I will stick to my area of science and examine what evidence may exist to support the IOC’s policy. Then I will extrapolate the results from our study of transgender individuals to see if hormone regulation may impact contributions to athleticism. The most strongly shifted lab values in hormone therapy for transgender individuals are red blood cells (including oxygen-carrying hemoglobin) and creatinine (byproduct of muscle used to monitor kidney function, but also reflects total muscle mass).

Once looking more closely at this topic, I realized there is a lot to say about the contributions of 1) muscle mass and 2) red blood cells to athleticism. So, I will discuss muscle mass this month and wait until next month to discuss hemoglobin levels (including athletic performance by blood removal/ doping).

Mid-distance running, which is Caster Semenya’s sport, is a mix of anaerobic and aerobic activity. This means having more muscle would be advantageous. This is supported by a study that was commissioned by the IAAF (International Association of Athletics Federation), which shows a 1.8-2.6% increased competitive advantage in short distance track events (400m, 800m and, 400m hurdles)1. However, this study had several limitations. First, the sample size was quite low with only 22 female athletes. Next, they use a p-value of 0.05 for significance without correction for multiple hypothesis testing (21 hypotheses tested representing each event), which increases the likelihood of a false positive result by chance.

What makes me curious is whether following the International Olympic Committee’s recommendations of lowering testosterone levels would even have a meaningful impact and improve competitiveness?

From my research, I know that adding testosterone to individuals assigned female at birth to transition to transgender males (TM ) does substantially increase creatinine (p<0.005, Figure 1)2 to male levels (baseline TW). This is likely not due to changes in kidney function (although this has not yet been proven), but rather due to increased muscle mass.

Figure 1.

However, the inverse is not quite true for transgender women who take combinations of estrogen for feminization and spironolactone to block the effects of testosterone. In these patients, we see a slight decrease in the creatinine (TW). While this decrease is statistically significant, the range is not clinically different from male creatinine levels. This concurs with the observations that musculature in transgender women does not change substantially upon taking hormone altering medication.

A more rigorous examination of muscle mass, performed by MRI measurement, determined that after 1 year of hormone therapy testosterone increased muscle mass in transgender men to biological male levels3, similar to our observations of creatinine. Further, they saw a significant reduction in muscle mass from baseline of transgender women on hormone therapy for 12 months, but it was still much higher than the muscle mass of biologic females4.

Therefore, were Casten Semenya to take testosterone blocking medication, I suspect there would be little impact on her overall muscle mass. Which is one of, if not the explicit purpose of taking testosterone lowering medicine. The strength of my conclusions is limited by the fact that we don’t know Casten Semenya’s testosterone levels, and furthermore a hyperadrogenic female is not the same as a male-to-female transgender woman.

As mentioned above, I will continue this discussion next month with an exploration of how testosterone lowering therapy could affect red blood cell levels, which would affect athletic performance differently.


  1. Bermon S and Garnier P. Serum androgen levels and their relation to performance in track and field: mass spectrometry results from 2127 observations in male and female elite athletes. British Journal of Sports Medicine. 2017; 51(17): 1309-1314.
  2. SoRelle JA, Jiao R, Gao E et al. Impact of Hormone Therapy on Laboratory Values in Transgender Patients. Clin Chem. 2019; 65(1): 170-179.
  3. Gooren LJ, Bunck MC. Transsexuals and competitive sports. Eur J Endocrinol. 2004; 151(4): 425-9.
  4. Jones BA, Arcelus J, Bouman WP, Haycraft E. Sport and Transgender People: A Systematic Review of the Literature Relating to Sport Participation and Competitive Sport Policies. Sports Med. 2017;47(4):701-716.

-Jeff SoRelle, MD is a Molecular Genetic Pathology fellow 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 advancing quality in molecular diagnostics.