By Diva D. De León-Crutchlow, MD, MSCE

Case history: An 18-month-old female presented to her local emergency room with hypoglycemia. She had not eaten for at least 16 hours, and parents were concerned because she was sleepy. On arrival to the emergency room her plasma glucose was 38 mg/dL. She had large ketones in the urine (>80 mg/dL). She was admitted for observation and discharged home the following day with a presumed diagnosis of idiopathic ketotic hypoglycemia.

She had a similar episode 2 weeks later, which parents treated with apple juice before calling an ambulance. The emergency personnel measured her glucose, which was normal at 120 mg/dL. Following this second episode, she was admitted to the local hospital for a diagnostic fast. After fasting for 12 hours, her plasma glucose was 52 mg/dL and a critical sample showed: lactate 2 mmol/L, beta-hydroxybutyrate 3.59 mmol/L, cortisol 10.5 mcg/dL, growth hormone 6.66 ng/mL, IGFI 94 ng/mL, ammonia 33 umol/L, insulin 48.1 uIU/mL (334 pmol/L) and C-peptide 0.2 ng/mL (0.07 nmol/L). The high insulin/C-peptide (4.77) raised concern for exogenous insulin. The family was questioned but no potential source of exogenous insulin was identified.

Another fasting test was performed, after approximately 24 hours of fasting, her plasma glucose was 41 mg/dL, plasma insulin was 42 uIU/mL (292 pmol/L) and C-peptide was 0.4 ng/mL (0.13 nmol/L), thus, the insulin/C-peptide ratio was again elevated at 2.2. At this point, Child Protective Services was contacted for the suspicion of Munchausen by proxy. She was transferred to the Congenital Hyperinsulinism Center at Children’s Hospital of Philadelphia (CHOP) for further evaluation.  

Diagnostic findings

We performed a diagnostic fast under 1:1 observation and without the parents present. Her glucose remained above 70 mg/dL for 19 hours and the fast was terminated at 23 hours because of elevated point-of-care betahydroxybutyrate of 3.4 mmol/L. A critical sample was obtained at the end of fast which showed: plasma glucose 67 mg/dL, HCO3 16 mmol/L, lactate 2.1 mmol/L, BOHB 3.59 mmol/L, ammonia 11 μmol/L, insulin 3.8 uIU/mL, and C-peptide 0.3 ng/mL (ratio 0.3). After analyzing all the diagnostic data, we suspected insulin assay interference because the level of ketosis was inconsistent with the level of insulinemia in both critical samples.

We requested that the critical samples were sent to an outside laboratory to measure insulin by liquid chromatography-mass spectrometry (LC/MS). For the first critical sample, when the plasma insulin was measured at 42 uIU/mL by immunoassay, the insulin concentration was 2 by LC/MS. For the second critical sample, when the plasma insulin was measured at 3.8 uIU/mL, the insulin concentration was 2 by LC/MS. Additional testing demonstrated that she had high titers of human anti-mouse antibodies at 181 ng/mL.

Outcome

We concluded that the falsely elevated plasma insulin concentrations were the result of assay interference by the anti-mouse antibodies. She was discharged with the diagnosis of ketotic hypoglycemia, and the family was cleared of suspicion for exogeneous insulin administration. She has not had further episodes of hypoglycemia.

Discussion

Human heterophilic antibodies are antibodies that are formed due to exposure to external antigens and can bind to immunoglobulins of other species. Human anti-mouse antibodies are one of these antibodies that formed against murine monoclonal immunoglobulin. These antibodies may interfere with immunoassay measurements as most immunoassay reagents used for hormone measurements utilize antisera derived from mice and other species. Falsely elevated hormone levels occur when nonspecific human anti-mouse antibodies cross-link isotypic determinants expressed on the Fc portions of the capture and the signal antibodies of “sandwich” immunoassays. As it occurred with our patient’s samples, this can lead to forming more “sandwiches” and a falsely elevated signal.

This case illustrates the importance of performing a comprehensive evaluation and considering the whole picture when making diagnostic conclusions. In hyperinsulinemic hypoglycemia, ketone levels are low because insulin suppresses lipolysis and ketogenesis, thus, the finding of elevated ketones with elevated insulin in the setting of hypoglycemia is incongruent. While it is important to always consider the possibility of exogenous insulin administration in a child who presents with late onset hyperinsulinemic hypoglycemia and in whom the insulin/C-peptide ratio is greater than 1, the wrong conclusion was made by the referring team because of lack of consideration of all the laboratory results.

Key points

• The diagnosis of hyperinsulinemic hypoglycemia relies on the demonstration that insulin secretion and actions are inappropriate during hypoglycemia, this includes not only finding an elevated plasma insulin concentration, but also evidence that lipolysis, ketogenesis, and glycogenolysis are suppressed, as inferred by finding low concentration of plasma free fatty acids (< 1.7 mmol/L) and betahydroxybutyrate (< 1.8 mmol/L), and a glycemic response to glucagon (> 30 mg/dL).  
• The presence of heterophile antibodies can result in falsely elevated hormone levels and wrong diagnoses when an immunoassay is used.
• Erroneous results should be considered when the clinical presentation and laboratory evaluation are inconsistent.

To read more about this case, see Insulin Immunoassay Interference Due to Human Antimouse Antibodies in a Patient With Ketotic Hypoglycemia. Craven M, Lord K, Leavens KF, De Leon DD. JCEM Case Rep. 2023:;1(2):luad029.