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-globingene 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
-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
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 bioavailabletestosterone 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
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
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
Ajayi A a. L, Halushka PV. Castration reduces platelet thromboxane A2
receptor density and aggregability. QJM. 2005;98(5):349-356.
Shea JL, Wong P-Y,
Chen Y. Free testosterone: clinical utility and important analytical aspects of
measurement. Adv Clin Chem. 2014;63:59-84.
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.
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.
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.
Verdonck L, Kaufman JM. A Critical Evaluation of Simple Methods for the
Estimation of Free Testosterone in Serum. None. 1999;84(10):3666-3672.
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.
Margo KL, Winn R.
Testosterone Treatments: Why, When, and How? AFP. 2006;73(9):1591-1598.
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
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).
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).
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
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.
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.
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.
Madani G, Beale TJ. Sinonasal inflammatory disease. Semin Ultrasound CT MR. 2009 Feb;30(1):17-24.
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.
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
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
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
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.
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.
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.
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.
Insulin antibodies are seen in two conditions: 1, in insulin-naïve type-1
diabetic patients, insulin antibodies are developed together with some other
autoantibodies against pancreatic islet cells; 2, in patients being treated
with insulin, antibodies can be developed against exogenous insulins, in both
type-1 and type-2 diabetes. These antibodies against exogenous insulins are
found in >95% of patients treated with porcine and bovine insulins (1).
Although the prevalence has decreased after the introduction of human insulin
and insulin analogues, it is still not uncommon to detect these antibodies in
insulin treated patients (2). However, these antibodies are rarely of clinical
significance and laboratory test for insulin antibodies in insulin-treated
patients has limited clinical value, except in rare cases where these
antibodies are found to have immunologic role, causing insulin resistance. In
some of these cases, postprandial hyperglycemia and nighttime hypoglycemia are
both described due to reversible binding of insulin from antibodies (3), and
patients were reported to respond to immunosuppressive therapies, and plasmapheresis
in severe cases.
worked up a case for possible immunologic insulin resistance caused by insulin
antibodies. In this case, patient is a 45 years old female with uncontrolled
type-1 diabetes. She was found to have all four antibodies positive, including
zinc transporter 8, islet
antigens glutamate decarboxylase 65 (GADA), IA-2A, and insulin antibodies. Patient
has been on multiple dose insulin injection (MDI) therapy, including insulin
determir, aspart and lispro. She was reported to be compliant with medications
and low carb diet. However, patient has poor glycemic control and presents with
recurrent diabetic ketoacidosis. She was given high doses of insulins, but
still presented with recurrent DKA and occasional hypoglycemia. Her HbA1c was
consistently at >10% with daily glucose measured up to 500 mg/dL.
insulin antibody and insulin receptor antibody were considered after ruling out
more common causes of her uncontrolled diabetes. These two tests were then performed
at a reference laboratory and patient was found to have positive insulin
antibodies to analog insulin (determir and lispro) and negative insulin
receptor antibodies. Significant insulin resistance by insulin antibodies was
not found and the antibodies level did not suggest immunosuppressive therapy. Still,
given her poor controlled diabetes, patient’s insulin was switched to human
insulin and she was also recommend for pancreas transplant.
Greenfield JR, Tuthill A, Soos
MA, Semple RK, Halsall DJ, Chaudhry A, O’Rahilly S. Severe insulin
resistance due to anti-insulin antibodies: response to plasma exchange and
immunosuppressive therapy. Diabet Med. 2009 Jan;26(1):79-82. doi:
Hall TR, Thomas JW, Padoa CJ, Torn
C, Landin-Olsson M, Ortqvist E, Hampe CS. Longitudinal epitope analysis of
insulin-binding antibodies in type 1 diabetes. Clin Exp Immunol. 2006
Hao JB, Imam S, Dar
P, Alfonso-Jaume M, Elnagar N, Jaume JC. Extreme Insulin
Resistance From Insulin Antibodies (Not Insulin Receptor Antibodies)
Successfully Treated With Combination Immunosuppressive Therapy. Diabetes
Care. 2017 Feb;40(2):e19-e20. doi: 10.2337/dc16-1975. Epub 2016
-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.
An African American male in his early 20s presented to the emergency
department (ED) with complaints of a sore throat, headache, generalized body
aches, and fatigue for the past week. He also noted intermittent fever and
chills as well as some nausea with a decrease in his appetite. He had been seen
multiple times in the ED recently for similar symptoms. His past medical
history was non-contributory and he noted no significant travel or exposure
history with the exception of attending a local party 10 days ago. His
temperature was 100.5°F and vitals were otherwise normal. His physical exam was
normal with the exception of dry mucous membranes indicating mild dehydration. Initial
laboratory testing showed a leukopenia (white blood cell count of 1.5 TH/cm2)
with 39% lymphocytes and rapid antigen testing for group A Streptococcus, influenza, and infectious mononucleosis were
negative. The patient was admitted for further work up due to the prolonged
nature of his symptoms.
Results from additional infectious disease testing are in the table
This pattern of results is most consistent an acute HIV infection.
Human immunodeficiency virus (HIV) is an enveloped, single stranded RNA
virus which belongs to the family Retroviridae.
HIV is most commonly sexually transmitted via body fluids such as blood, semen,
and vaginal secretions directly contacting mucosa membranes. HIV can also be
transmitted due to needle stick injuries, blood transfusions, and
transplacentally from infected mother to fetus or by breast feeding. Acute HIV
illness presents as a mononucleosis-like syndrome with fever, pharyngitis,
arthralgias, malaise, and weight loss. During this acute illness, the HIV RNA
viral load is extremely high. After a period of clinical latency, which on
average is approximately 10 years, there is a deterioration of the immune
system, the CD4 count drops, and the patient is at risk for opportunistic
infections and neoplastic diseases.
Based on the 2014 CDC/APHL guidelines, the initial screening test for
HIV is an antigen-antibody combination assay. These immunoassay based tests
detect the p24 antigen and antibodies to HIV-1 and HIV-2 (see image below). By
testing for the p24 antigen in addition to HIV antibodies the time to a
positive patient result is decreased (window period) as p24 is one of the first
viral proteins to appear, even before antibodies are present.
If the antigen-antibody test is repeatedly positive, the second step in
the testing algorithm is an antibody differentiation assay. This test has taken
the place of the Western blot and Western blot is no longer recommended in the
diagnosis of HIV. If the antibody differentiation test is positive, the
diagnosis of HIV-1 or HIV-2 is confirmed. As this step only detects the
presence of antibodies, the differentiation test will be negative in an acute
If there is a discrepancy between the first two steps in the testing
algorithm or an indeterminate result is obtained, the final step involves
nucleic acid amplification testing (NAAT) to detect viral RNA. Viral RNA is the
first HIV-1 specific marker to appear following infection. In the case of an
acute or untreated long term infection, the viral load can approach levels up
to 100 million copies.
When additional history was obtained from our patient, he said he was
sexually active with a new male partner in the past few weeks and did not use
protection. He stated he had been treated with Chlamydia in the past. Further testing for CD4 count, other
opportunist & sexually transmitted infections, and HIV genotype testing was
performed and outpatient HIV care was arranged for the patient.
-Lisa Stempak, MD, is an Assistant Professor of Pathology at the
University of Mississippi Medical Center in Jackson, MS. She is
certified by the American Board of Pathology in Anatomic and Clinical
Pathology as well as Medical Microbiology. She is the Director of
Clinical Pathology as well as the Microbiology and Serology
Laboratories. Her interests include infectious disease histology,
process and quality improvement, and resident education.
For transgender women, taking pills of estradiol is
insufficient to counteract the endogenous levels of testosterone produced by
their bodies. To counteract the undesired testosterone, anti-androgens are
employed. These include cyproterone acetate (approved only in Europe) or
spironolactone. Spironolactone is a potassium sparing diuretic that could have
unintended consequences like gynecomastia.1 This effect comes from
off-target binding of spironolactone to the androgen receptor. Like the
intended spironolactone target (mineralocorticoid receptor), the androgen receptor
localizes to the nucleus when activated and acts as a transcription factor.
Taking daily high doses of spironolactone
(100mg- 300mg daily) has been shown to be safe,1 but can increase
Potassium levels. In a cohort of 55 transgender women, potassium was actually
not higher (Figure 1).2 This was the first time a study had
rigorously measured electrolytes like potassium in transgender patients.
Current guidelines recommended checking electrolyte levels in transgender women
taking spironolactone.3 Full electrolytes were included for 126 TW
in our study and what we found was not what we were expecting.4
We found no increased potassium levels in TW who had taken
hormone therapy for at least 6 months (p>0.05). However, we did see a
decrease in sodium which is consistent with the diuretic effect (p<0.0001, Figure
We wondered if variability in spironolactone dosing could explain why no significant potassium change was found. Luckily, we had a large number of patients who were taking various doses of spironolactone for comparison. One-way ANOVA with Tukey post-hoc tests revealed no difference in potassium levels (p>0.05)- even between the lowest (0mg daily) and highest dose (200-300 mg daily) (Figure 3). While the sodium level trended to decrease with higher spironolactone, it was not statistically significant.
One reason that potassium levels did not increase is a
difference in study populations. The original population studied for
spironolactone involved patients with heart failure and hypertension whereas
our study’s population was mostly in their 20’s and 30’s with very few
Although sodium levels are decreased, they did not fall
below the lower limit of normal (135 mmol/L). Low sodium would put transgender
women at risk of dizziness and syncope (passing out) from low blood pressure.
Thus, the takeaway is: sodium should be clinically monitored as it can decrease
in transgender women.
Clark E. Spironolactone Therapy and
Gynecomastia. JAMA. 1965;193(2):163-164.
TK et al. Interpreting Laboratory
Results in Transgender Patients on Hormone Therapy. The American Journal of Medicine. 2014; 127(2): 159-162.
Hembree WC, Cohen-Kettenis PT,
Gooren L, Hannema SE, Meyer WJ, Murad MH, et al. Endocrine Treatment of
Gender-Dysphoric/Gender-Incongruent Persons: An Endocrine Society* Clinical
Practice Guideline. J Clin Endocrinol
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.