Insulin Resistance Caused by Insulin Antibodies

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. 

We recently 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.

Immunologic 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.

  1. 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: 10.1111/j.1464-5491.2008.02621.x.
  2. 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 Oct;146(1):9-14.
  3. 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 Dec 1.

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

Microbiology Case Study: A Male in his Early 20s with Generalized Body Aches

Clinical History

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.   

Laboratory Identification

Results from additional infectious disease testing are in the table below.

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 HIV infection.

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.

Potassium Levels in Transgender Women

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

Figure 1.

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 2).

Figure 2.

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.

Figure 3.

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 co-morbid conditions.

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.


  1. Clark E. Spironolactone Therapy and Gynecomastia. JAMA. 1965;193(2):163-164.
  2. Roberts TK et al.  Interpreting Laboratory Results in Transgender Patients on Hormone Therapy. The American Journal of Medicine. 2014; 127(2): 159-162.
  3. 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 Metab. 2017
  4. 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.

Whether or Not to Report Cytoplasmic Pattern of ANA IFA

Anti-nuclear antibody (ANA) test is commonly used to screen for systemic rheumatic disease. Indirect immunofluorescence assay using HEp-2 cells as substrate, containing approximately 100-150 autoantigens, is still the gold standard for ANA testing (1). Although the test name refers to only anti-nuclear antibody, there are often cytoplasmic staining patterns overserved in this assay. Cytoplasmic patterns result from antibodies against cytoplasmic components, like Jo-1 or Ribosomal P, and have clinical association with various systemic autoimmune disease, like polymyositis, systemic lupus erythematosus or primary biliary cirrhosis.    

There is no standardized recommendation regarding how to report cytoplasmic pattern on ANA IFA, and laboratories independently decides whether to indicate cytoplasmic pattern in their result.  The International Consensus on ANA Patterns (ICAP) workshop discussed this topic in 2015 and proposed two approaches for reporting ANA cytoplasmic patterns (2). Either to regard cytoplasmic pattern as positive or negative, both approaches recommended to include a statement of cytoplasmic staining.

We encountered cases in our laboratory in which reporting cytoplasmic staining had significant clinical values, and our laboratory started to report cytoplasmic staining as an additional comment in the test result a few years ago. Here is one of these cases:

Case: 35 year old woman with a history of hypertension complained about increasing muscle pain, weakness, and swelling. She had difficulties to raise her arms and had multiple falls, and was admitted to hospital three time for rhabdomyolysis. Her initial laboratory assessment were, CK >11,196 U/L, lactic acid 2.5 mmol/L, ALT 152 U/L, AST 416 U/L, and ALKP 42 U/L. Her ANA IFA test didn’t shown any nuclear staining, but there is very strong cytoplasmic staining observed. The clinician was suspecting inflammatory myositis and ordered myositis autoantibody panel to follow up. This panel detects numerous antibodies that are either specific or associated with inflammatory mycosis.

Her myositis autoantibody test result was positive for antibodies against signal recognition particle (SRP). SRP is an abundant, cytosolic, universally conserved ribonucleoprotein that targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes. Antibodies against SRP have been found in 5-8% of adult idiopathic inflammatory myopathies and <1% juvenile myopathies. It is closely associated with necrotizing myositis. Clinically it presents with acute onset, rapidly progressive, severe weakness, with high CK levels and commonly has cardiac and lung involvement. 

Clinically significant antibodies can be present in patients with connective tissue disease that may appear as strong cytoplasmic staining on screening ANA test. It would be helpful to add a comment in these cases to aid the clinician in pursuing further work-up with a strong clinical suspicious of connective tissue disease.


1. Position Statement: Methodology of Testing for Antinuclear. Antibodies American College of Rheumatology. 2009.

2. Damoiseaux J, et al. International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on reporting ANA results. Auto Immun Highlights. 2016 Dec;7(1):1. doi: 10.1007/s13317-016-0075-0. Epub 2016 Jan 30.


-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.

Chemisty Case Study: Heat-Insoluble Cryoglobulin

Case History

A 50 year old female was admitted for acute renal failure on CKD stage IV, present with gross hematuria, anemia (due to blood loss) and hypertension. The patient has a significant history of unresolved cryoglobulinemic vasculitis initially diagnosed in 2016 and has been treated by several rounds of rituximab. Other medical histories include Sjogren’s syndrome, MGUS with monoclonal IgM Kappa, coagulopathy (protein S deficiency, on anticoagulant), hyperviscosity, myalgia, deep vein thrombosis, leg edema with superficial ulcer, pulmonary embolus and membranoproliferative glomerulonephritis (MPGN). Kidney biopsy revealed intraglomerular hyaline thrombi consistent with cryoglobulinemic glomerulopathy, interstitial fibrosis tubular atrophy, arterial sclerosis, suggestive thrombotic microangiopathy. Immunohistochemistry was positive for C3, IgM, Kappa, Lambda and CD68. Bone marrow biopsy shown dyserythropoesis without malignancy. Blood testing shown negative hepatitis panel and undetectable C4.

We observed unusual cryoprecipitate test results from this patient: gelatinous appearance precipitate which accounts for more than 40% of volume was observed in both plasma and serum and cannot be cleared at 37C° after several hours of incubation. Further testing shown incubation at 56°C for 30min cleared up the serum but not the plasma. After checking the test history, we found that there was a similar situation for the patient’s cryoprecipitate test a few months back earlier in 2018, and was reported negative for cryoglobulins due to the heat-insoluble nature of the precipitate. Patient was transfused for anemia. No plasmapheresis was done. Due to the patient’s incomplete response to rituximab, Cytoxan was also added to help improve the symptoms.


  • Definition: Cryoprecipitates (or cryoproteins) are blood proteins that form precipitates or gels at temperatures lower than 37°C and typically re-dissolve after warming up to 37°C. There are two types:
  • Cryoglobulin (CG): precipitate from both serum and plasma; either immunoglobulins or a mixture of immunoglobulins and complement components
  • Cryofibrinogen (CF): precipitate from plasma only; typically composed of a mixture of fibrinogen, fibrin, fibronectin, and fibrin split products
  • Lab Testing done in our hospital:
    1. Blood are collected in two pre-warmed tubes (one serum, one EDTA plasma) and kept in warm water (37°C) until the serum tube clots.
    2. The plasma and serum are extracted at room temperature, and then stored in refrigerator for 72 hours.
  • If cryoprotein is present, a precipitate or gel will be seen. An aliquot of the serum is rewarmed at 37°C to verify the cryo-nature.
  1. The precipitate as a percentage of the original serum volume is measured in an ESR tube to determine the cryocrit.
  2. Immunofixation is ordered per pathologist to identify the immunoglobulin compositions of the cryoglobulin.


  • Classification

Strictly speaking, cryoglobulinemia refers to the presence of cryoglobulin (CG) in a patient’s serum, which could be either asymptomatic or present with apparent clinic syndromes (i.e. cryoglobulinemic vasculitis). Cryoglobulinemia can be classified into three types (see table below [1]), with mixed cryoglobulinemia (type II and type III) representing 80% of the cases.


  • Clinical Manifestations

Type I cryoglobulinemia is frequently asymptomatic, while mixed cryoglobulinemia manifests clinically by a classical triad of purpura, weakness and arthralgias, as well as some other conditions including MPGN, chronic hepatitis, peripheral neuropathy, lymphoma, Raynaud’s, Sjogren’s syndrome, etc.

The presence of heat-insoluble cryoglobulins is rare, and its pathogenesis is poorly understood. On the other side, it may indicate sever clinical consequence as seen in our case and some others as mentioned above.


  1. Mixed Cryoglobulinemia, Ferri, C; Orphanet Journal of Rare Diseases 2008, 3:25

 Further reading

  1. Essential type II cryoglobulinemia with cryoglobulin-occlusive MPGN and MGUS (Clin Chim Acta. 2009 Aug;406(1-2):170-3):79 y.o. female admitted due to edema and renal failure, cryoglobulin re-dissolved at 56°C, composed of monoclonal IgG-Kappa and polyclonal IgM.
  1. HCV associated thrombotic microangiopathy and cryoglobulin-occlusive MPGN (Am J Med Sci. 2013 Oct;346(4):345-8):57 y.o. female, cryoglobulin re-dissolved at 47°C, composed of monoclonal IgM-Kappa and polyclonal IgG. Symptoms only partially resolved upon treatment of plasmapheresis, corticosteroids and antiviral therapy of peginterferon plus ribavirin.
  1. Essential type I cryoglobulinemia with massive cryoglobulin-occlusive glomerulonephritis (Am J Kidney Dis. 1995 Oct;26(4):654-7):54 y.o. male progressed to ESRD prior to the detection of cryoglobulin. Cryoglobulin with white gelatinous appearance re-dissolved at 54°C, composed of monoclonal IgG-Kappa.
  1. Primary Sjogren’s syndrome with type II cryoglobulinemia and mesangiocapillary glomerulonephritis (Nephrol Dial Transplant. 2000 Jun;15(6):917-8):82 y.o. patient with IgM-MGUS, negative BM, deposition of IgG, IgM and C3 on kidney biopsy, decreased complement levels, negative HCVAb, HBsAb, HBsAg. cryoglobulin re-dissolved at 47°C, composed of monoclonal IgM-Kappa and polyclonal IgG-Kappa.



-Rongrong Huang, PhD is a first year clinical chemistry fellow at Houston Methodist Hospital. Her interests include general clinical chemistry, genetic biochemistry and applications of mass spectrometry in clinical laboratories.


-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.

Error Codes in Blood Gas Analysis

We recently received a venous blood sample for blood gas analysis from the operation room. We analyzed the specimen according to manufacturer’s instructions on the ABL800 FLEX blood gas instrument (Radiometer, Copenhagen, Denmark). Multiple error codes were present for the results of ctHb, sO2FO2Hb, FCOHb, FHHb, and FMetHb. Text messages accompanying the report read, “Detection of SHb” and “OXI spectrum mismatch.” The sample was re-tested on the ABL800 but the same error codes were flagged.

A closer look at the patient’s chart revealed that patient is heterozygous for hemoglobin M-Saskatoon variant,  which causes the replacement of histidine by tyrosine in position 63 on the beta chain of hemoglobin (beta codon 63, CAT>TAT/His63Tyr). This renders the NADH methemoglobin reductase system incapable of reducing oxidized iron. A group of mutations in the globin chain gene can result in such dysfunction of ferric iron reduction and are referred to as methemoglobin forming hemoglobin variants (Hgb M).

HbM variants usually have a different absorbance spectrum from the physiologic methemoglobin. Modern day CO-oximeters use more than 100 wavelengths and can detect most unknown substances. We speculated that Hgb M in the patient is the reason the ABL800 reported error codes. The clinical team collected another venous blood sample and  it was tested on the GEM5000 blood gas instrument (Instrumentation Laboratory, Bedford, MA, USA). This specimen also reported with error codes.

Non-invasive pulse-oximetry devices use two wavelengths (660 nm and 940 nm) to calculate hemoglobin oxygen saturation based on oxyhemoglobin and deoxygenated hemoglobin, and thus are unable to report interferences from dyshemoglobins. In a nut shell, Hgb M variants can possibly interfere with CO-oximetry measurements. Caution is needed to interpret the results. Pulse oximetry usage should be avoided for these patients.


  1. Schiemsky T, Penders J, Kieffer D. Failing blood gas measurement due to methemoglobin forming hemoglobin variants: acase report and review of the literature. Acta Clin Belg. 2016 Jun;71(3):167-70.
  2. Stucke AG, Riess ML, Connolly LA. Hemoglobin M (Milwaukee) Affects Arterial Oxygen Saturation and Makes Pulse Oximetry Unreliable. Anesthesiology 4 2006, Vol.104, 887-888.



-Jayson Pagaduan, PhD, is a senior year clinical chemistry fellow Texas Children’s Hospital in Houston, TX.


-Jing Cao, PhD, DABCC, FACB, is a board-certified clinical chemist, serving as the Associate director of Clinical Chemistry at Texas Children’s Hospital in Houston, TX and an Assistant Professor of Pathology and Immunology at Baylor College of Medicine.

Is Your Clinician Concerned About Biotin Interference?

Many clinical laboratorians received questions in the past few months from clinicians about biotin interference on laboratory tests. Although biotin interference is not something new to most clinical chemists, it became more of a concern for clinicians since FDA released a safety communication warning to the public and healthcare professionals that “Biotin May Interfere with Lab Tests” in Nov 2017.

Why does biotin interfere with some laboratory tests?

Immunoassays employed in clinical laboratories often use biotin-streptavidin linkage to separate bound antibody-antigen complex from unbound components.  For example, in a sandwich immunoassay setting, analytes bind to signal antibodies and biotinylated capture antibodies, which are immobilized on streptavidin-coated solid phase via biotin-streptavidin binding. In the excess of exogenous biotin, it interferes the binding of biotinylated antibodies and streptavidin, causing erroneous results.

Owing to assay design, tests that utilize the biotin–streptavidin linkage have different tolerance on biotin interference. There has been recent publications that summarized the tolerance level of biotin on commonly used immunoassays from different manufacturer platforms (1, 2). The recommended daily intake (RDI, 30 µg/day) of biotin do not typically interferes with laboratory testing. However, many over-the-counter dietary supplements may contain biotin much higher than the RDI, and the level used for treatment of multiple sclerosis or some other diseases can be even higher. These levels of biotin can cause either falsely high or falsely low test results.

As high-dose biotin use has been increased among general population for nutraceutical purposes, it requires clinicians’ awareness of biotin interference and communication with laboratories to identify incorrect laboratory results. It may require patients to discontinue dietary supplements containing high dose biotin for a period of time before blood drawn to minimize potential biotin interference with testing.


  1. Li D, Radulescu A, Shrestha R, Root M, Karger A, Killeen A, et al. Association of Biotin Ingestion With Performance of Hormone and Nonhormone Assays in Healthy Adults. Jama. 2017;318:1150–1160.
  2. Colon P, Greene D. Biotin Interference in Clinical Immunoassays. J Appl Laboratory Medicine Aacc Publ. 2018;2:941–951.



-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.