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Year : 2015  |  Volume : 1  |  Issue : 1  |  Page : 49-50

Hook effect due to parenteral Vitamin B12 supplementation in Vitamin B12 assay by electrochemiluminescence immunoassay method in a patient with severe anemia

Department of Biochemistry, Pondicherry Institute of Medical Sciences, Affiliated to Pondicherry University, Puducherry, India

Date of Submission25-May-2015
Date of Acceptance11-Jun-2015
Date of Web Publication9-Nov-2015

Correspondence Address:
Sunil Kumar Nanda
Department of Biochemistry, Pondicherry Institute of Medical Sciences, Affiliated to Pondicherry University, Puducherry
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Source of Support: None, Conflict of Interest: None

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Prozone phenomenon or hook effect has been known to occur while performing enzyme-linked immunosorbent assay. Hook effect has been documented for analytes such as prolactin and beta-HCG. We here report a rare case of prozone phenomenon observed in Vitamin B12 estimation by electrochemiluminescence immunoassay in a patient receiving parenteral Vitamin B12 supplementation.

Keywords: Electrochemiluminescence immunoassay, prozone effect, Vitamin B12

How to cite this article:
Nanda SK, Ray L, Dinakaran A. Hook effect due to parenteral Vitamin B12 supplementation in Vitamin B12 assay by electrochemiluminescence immunoassay method in a patient with severe anemia. J Curr Res Sci Med 2015;1:49-50

How to cite this URL:
Nanda SK, Ray L, Dinakaran A. Hook effect due to parenteral Vitamin B12 supplementation in Vitamin B12 assay by electrochemiluminescence immunoassay method in a patient with severe anemia. J Curr Res Sci Med [serial online] 2015 [cited 2021 Jul 30];1:49-50. Available from: https://www.jcrsmed.org/text.asp?2015/1/1/49/168925

  Introduction Top

A request for a repeat sample in the biochemistry laboratory may be due to several reasons such as hemolyzed sample, inadequate sample, inadequate patient preparation, erroneous results, and results not correlating with the clinical condition of the patient.[1] Some of these requests can be avoided if the laboratory staff is well aware about the scientific background of errors related to signal values of parameters. One of the limitations of immunoassays is the hook effect or prozone phenomenon, which has been noted in techniques such as enzyme-linked immunosorbent assay and immunofluorescence.[2],[3] Errors due to prozone phenomenon have been documented for biochemical parameters like prolactin and beta-HCG.[4],[5]

  Case Report Top

A 75-year-old female patient came to our hospital with complaints of breathlessness, easy fatigability, and generalized body ache for 7 days. She was a known hypertensive patient who was not on regular medication. General examination revealed severe pallor and bilateral pitting edema; she had koilonychia and glossitis. Her jugular venous pulse was positive. Systemic examination revealed bilateral crepitations and an early systolic murmur in the aortic area. Routine investigations revealed a low hemoglobin level (2.2 g/dl), low packed cell volume (9.0%), and low red blood cell (RBC) count (2.0 million/ml). Anemia work up was done: Serum iron was 19 µg/dl and total iron binding capacity was 44 µg/dl. Peripheral smear showed marked anisopoikilocytosis with hypochromic RBCs along with elliptocytosis and few pencil shaped macrocytes. WBCs and platelets were within normal limits. The patient was started on iron and vitamin supplementation and her anemia gradually improved, and a sample for Vitamin B12 assay was sent to the biochemistry laboratory.

  Discussion Top

Vitamin B12 assay is performed in our laboratory by Roche Elecsys 2010 (cobas e411) which is based on the principle of electrochemiluminescence immunoassay. The Elecsys Vitamin B12 assay employs a competitive test principle using intrinsic factor (IF) specific for Vitamin B12. Vitamin B12 in the sample competes with the added Vitamin B12 labeled with biotin for the binding sites on the ruthenium-labeled IF complex. The test procedure consists of three phases of incubation. In the first incubation phase, the sample is incubated with dithiothreitol, sodium hydroxide, and sodium cyanide. In the second incubation phase, the pretreated sample is incubated with ruthenium-labeled IF, and in the third incubation phase, sites on the ruthenium-labeled IF become occupied by ruthenium-labeled IF – Vitamin B12 biotin complex. The entire complex becomes bound to the solid phase via the interaction of biotin and streptavidin, which is later aspirated to the measuring cell wherein the microparticles are magnetically captured onto the surface of the electrode. Application of a voltage to the electrode then induces the chemiluminescent emission which is measured by a photomultiplier. The measuring range of Vitamin B12 assay is 30–2000 pg/ml.

The patient sample was processed after performing daily maintenance and running internal quality control samples which were found to be within the acceptable range. The analyzer failed to give a result for the sample and the error message displayed was "signal level below limit." The test when repeated displayed the same error message. A repeat sample was requested which was received by the laboratory the next day. The sample was processed, and it displayed the earlier error message. The test was repeated after a 1:5 dilution of the sample which gave a result of >2000 pg/ml. The patient's medical records were reviewed, and it was observed that the patient was receiving injection optineuron once daily. Optineuron is a medication which contains cyanocobalamin.

In Roche Elecsys 2010 system, the excess Vitamin B12 present in the patient serum saturated all available sites on the IF in the first incubation step. Subsequently, in the second incubation step when biotin-tagged Vitamin B12 was added, there were no vacant sites on the IF for Vitamin B12 to bind. Thus, no signal was generated. This is due to the hook or prozone effect wherein excess antigen in the serum of the patient saturates the signal antibodies which may result in no signal generation, or may lead to falsely low levels of the analyte being measured.[6],[7]

The laboratory staff should be aware of such a phenomenon to avoid unnecessary delay in reporting and unnecessary reagent wastage due to repeat tests. In such situations, the laboratory staff should consider diluting the sample before loading the sample in the analyzer.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Rana SV. No preanalytical errors in laboratory testing: A beneficial aspect for patients. Indian J Clin Biochem 2012;27:319-21.  Back to cited text no. 1
Taborda CP, Rivera J, Zaragoza O, Casadevall A. More is not necessarily better: Prozone-like effects in passive immunization with IgG. J Immunol 2003;170:3621-30.  Back to cited text no. 2
Dubois-Galopin F, Beauvillain C, Dubois D, Pillet A, Renier G, Jeannin P, et al. New markers and an old phenomenon: Prozone effect disturbing detection of filaggrin (keratin) autoantibodies. Ann Rheum Dis 2007;66:1121-2.  Back to cited text no. 3
Agarwal M, Das A, Singh AS. High-dose hook effect in prolactin macroadenomas: A diagnostic concern. J Hum Reprod Sci 2010;3:160-1.  Back to cited text no. 4
[PUBMED]  Medknow Journal  
Hunter CL, Ladde J. Molar pregnancy with false negative ß-hCG urine in the emergency department. West J Emerg Med 2011;12:213-5.  Back to cited text no. 5
Basu S, Chaudhuri S. Vitamin B (12) Immunoassay on roche elecsys 2010: Effects of high excess concentration of serum Vitamin B (12) in CKD patients on parenteral administration. Indian J Clin Biochem 2011;26:413-5.  Back to cited text no. 6
Charrié A, Charriére G, Guerrier A. Hook effect in immunometric assays for prostate-specific antigen. Clin Chem 1995;41:480-1.  Back to cited text no. 7


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