Investigating the presence of human anti-mouse antibodies (HAMA) in the blood of laboratory animal care workers

Mohammad Mahdi Mohammadi; Shahram Bozorgi

doi:10.1515/labmed-2018-0084

Artikel Open Access

Investigating the presence of human anti-mouse antibodies (HAMA) in the blood of laboratory animal care workers

Mohammad Mahdi Mohammadi und Shahram Bozorgi

Veröffentlicht/Copyright: 6. März 2019

Veröffentlicht von

Veröffentlichen auch Sie bei De Gruyter Brill

Manuskript einreichen Informationen für Autor*innen

Aus der Zeitschrift Journal of Laboratory Medicine Band 43 Heft 2

Abstract

Background

In the present study, the researchers evaluated the presence of human anti-mouse antibodies (HAMA) in a normal population and laboratory animal care providers for the first time in the world. Also, the cause of HAMA incidence in the human body through a close contact with mice was identified.

Methods

The study population consisted of 40 laboratory animal care providers aged between 24 and 57 years with a close contact with mice (e.g. taking care of mice, feeding mice, etc.) and 40 individuals of the same age as the above group with no contact with mice. HAMA was measured in both the case and control groups using sandwich enzyme-linked immunosorbent assay (ELISA) method. Data were analyzed using SPSS 18. Univariate and multivariate linear regression and independent t-test were used. The significance of results was measured based on p < 0.05.

Results

The present study revealed that the animal care providers had (p = 000) a higher titer of HAMA (4.95 ng/mL) in their blood than the control group (1.67 ng/mL). Also, the individuals in the case group (exposed to mice) were more allergic (43.6%) than those in the control group (15%) (p = 0.003).

Conclusions

The results of this study revealed that exposure to mice in laboratory care centers can cause production of HAMA in the human body but its titer is possibly lower in Iranian working staff than those in the other parts of the world.

Reviewed Publication:

Sack Ulrich Conrad Karsten Edited by:

Keywords: ELISA; human anti-mouse antibody (HAMA); laboratory animals

Introduction

After frequent contact with mouse antigens, individuals normally produce memory B cells that last for many years and are potential enough to produce human anti-mouse antibodies (HAMA) [1], [2]. HAMA seems to be produced naturally; the high incidence of HAMA may be due to the current use of monoclonal mouse antibodies for diagnostic and therapeutic purposes, the presence of rodents (including mice) and their association with urban and rural living conditions [3], [4], [5]. Numerous studies have been performed about the incidence of HAMA in various countries. In a study by Boscato et al. [6], 40% of those studied were identified to have HAMA. In other studies on normal subjects, this amount varied from 1/9% to 40%, and the patients receiving monoclonal mouse antibodies were estimated to be 74% [7]. Studies about HAMA are important due to two reasons: (a) interfering this type of antibody with a variety of laboratory tests, but still laboratories have not found a simple and practical way to detect the presence of these antibodies, and (b) interfering with the treatment process in patients taking animal antibodies [8], and neutralizing injected drugs which results in the ineffectiveness of treatment. Ledermann et al. [9] showed in their study that favorable results and effective treatments can be achieved by preventing the formation of HAMA in patients with cancer receiving the mouse monoclonal anti-tumor antibodies. Its interference with laboratory tests has been studied frequently and the reports are occasionally published in medical journals [10]. The presence of HAMA causes false-positive results; even in the absence of analysts, it can cause a false increase or decrease in the results [3], [11]. Even in the recipients (e.g. bone marrow recipients) treated with cyclosporine A, HAMA may interfere with the care and treatment of transplanted patients [10].

Determining the prevalence of these antibodies in communities is essential. Medical staff must perceive the interpretation of serological and immunological tests; and having the knowledge of the prevalence of HAMA in normal subjects and consumers of animal products (natural or synthetic), physicians can make a correct decision against the medical interventions and effects of prescribed drugs based on monoclonal animal antibodies.

This study aimed to investigate the presence of HAMA in animal care providers and the incidence of HAMA due to a close contact with mice.

Materials and methods

The study population was divided into case and control groups. The case group included the staff of animal care centers affiliated to Universities of Medical Sciences in Iran (Kerman, Shiraz, Tehran, Razi Serum institution and research centers affiliated to these universities) who have a close contact with mice as their job demands and the control group included hospital employees with no contact with mice. The participants were male. Each group consisted of 40 participants. Having a 6-month contact with rats was the inclusion criterion. These people were matched with respect to their age. The tools of data collection were questionnaires of demographic data and enzyme-linked immunosorbent assay (ELISA) test.

Demographic data

Both groups either in contact with the rats or without any contact with rats were selected from Universities of Medical Sciences and hospitals. Demographic data were collected through questionnaires. Blood samples were used for extracting serum. The level of HAMA for both groups was measured using sandwich ELISA. Possible variables affecting the level of HAMA were considered and measured during the collection of blood samples. Serum HAMA levels in the studied population were measured by sandwich ELISA. The participants were selected based on the mean age. The workers’ resume in animal care centers and their exposure to rats were considered. The education level was also another variable having an effect on HAMA production due to its relationship with standard of living, health and work. It was divided into illiteracy, under diploma, diploma and university degrees. Allergy symptoms including a runny nose, watery eyes, itchy skin, scratchy throat, shortness of breath and a rash appeared in both groups and this kind of allergy was considered positive in the case of having at least one of the above symptoms. After collecting and analyzing data by SPSS 18, mean (standard deviation [SD]) and frequency were used for describing the quantitative and qualitative data, respectively. To investigate the effect of the studied variables on HAMA, and evaluate HAMA in both groups, the univariate and multivariate linear regression and also independent t-test were used, respectively. As there was no reference interval for HAMA in the Iranian population, using the values of these antibodies in the control group and scientific methods, the researchers managed to determine the reference interval, and used the antibody titer to examine the case group (in terms of frequency of positive or negative HAMA). In all cases, p<0.05 was significant. Five milliliters of blood was taken from each patient; serum was isolated and stored at −20° until tests were done. In order to measure the amount of HAMA, IBL (immunoglobulin G [IgG]) ELISA kits (Hamburg, Germany) were used. Three methods including mean±2 SD, geometric SD±2 geometric mean and 95% confidence interval for the median [12] were used to determine the reference interval [12]. However, there are other scientific methods to determine the reference interval including the 97.5^th [13], [14] and 99^th percentiles [15], [16], [17], [18], [19].

Ethical considerations

The subjects were informed about the aims of the study. Oral and written consent was given by each participant before inclusion into the study. Participation was free of charge and voluntary. Due to the confidentiality of information, the ethical codes were taken into consideration.

Results

Frequency of HAMA concentration in the two groups

The mean values of HAMA in the control and case groups were 1.68 ng/mL and 4.95 ng/mL, respectively. The value range of HAMA in the control and case groups was 0.22–4.02 ng/mL and 0.48–20.85 ng/mL, respectively. In the case group, a HAMA value of 20.85 ng/mL was confirmed for one of the participants through repeated tests; except for this person, the HAMA value for the others in the case group was less than 12 ng/mL. To evaluate the possible impact of different variables on the amount of HAMA, common variables such as age, education and allergies were used. Other variables included work experience (in animal care and training medical faculties), the frequency of mice bites, the number of weekly cleaning of mouse nest and taking a bath after cleaning the mouse nest, which were measured only for the case group. All of the variables mentioned above were independent variables and HAMA was the dependent variable; to investigate the relationship between these two variables, univariate and multivariate linear regression model was used. p-Value <0.05 was considered significant.

With regard to the effect of age, education and allergies using multiple regression model, there were not any significant changes in HAMA results. With respect to the effect of mice exposure on HAMA production, the results were significant and it was concluded that HAMA production in individuals who were occupationally exposed to mice was greater (3.273 ng/mL) than those who had no exposure to mice. Therefore, the amount of HAMA was significantly different between the case and control groups (p=0.000) (Table 1).

Table 1:

Effect of common variables on HAMA in both groups.

Univariate regression (n=80)				Multivariate regression (n=80)
Variable	Regression model	SE	p-Value	Regression model	SE	p-Value
Age	−0.024	0.038	0.532	−0.034	0.053	0.516
Education	0.023	0.208	0.911	0.183	0.204	0.374
Allergies	1.329	0.734	0.074	0.129	0.695	0.853
Group	−3.273	0.579	0.000	−3.555	0.691	0.000

SE, standard error.

Using univariate and multivariate regression, work experience, mice bites, weekly cleaning of mouse nest and taking a bath after cleaning the mouse nest were assessed only for the case group and there was no significant effect on the HAMA levels (Table 2). The HAMA’s mean difference between both groups was confirmed using independent sample t-test and it was significant (p=0.00) (Table 3).

Table 2:

Effect of some specific variables on HAMA in the case group.

Univariate regression (n=40)				Multivariate regression (n=40)
Variable	Regression model	SE	p-Value	Regression model	SE	p-Value
Years of work experience	0.042	0.077	0.584	0.067	0.086	0.436
Mice bites	−0.510	1.165	0.664	−0.757	1.311	0.567
Frequency of weekly cleaning	0.009	0.506	0.986	0.027	0.524	0.959
Showering after cleaning the mice nests	−1.023	1.156	0.382	−0.996	1.213	0.417

Table 3:

Different values of HAMA were investigated in both case and control groups.

Group	Mean	Standard deviation	Statistical test	p-Value
Amount of HAMA in the blood, ng/mL
Case	4.95	3.53	Independent sample t-test	0.000
Control	1.67	0.96	Independent sample t-test

With regard to differences in the incidence of allergies between both groups, those who were exposed to mice (control group) were 0.357 U less infected than those who were occupationally exposed (case group) and the results were significantly different between both groups (p=0.003).

Different methods of determining the reference interval for HAMA in Iranian men are shown in Table 4. As the 99^th percentile is used mostly for the determination of reference intervals [15], [16], this method was used to determine the reference interval for HAMA which means that 99% of the control group had a HAMA value of less than or equal to 4 ng/mL. In this study, a 4 ng/mL cut-off seems appropriate. To define a reference interval for HAMA, the study aimed to compare the HAMA levels of the case group with those of the control group and determine the frequency of positive HAMA and negative HAMA. Twenty-one (52.5%) of the participants had negative HAMA and 19 (47.5%) had positive HAMA.

Table 4:

HAMA reference range or cut-off points for Iranian men determined by different methods.

Method	Mean±2 SD	Reference range of HAMA kit	95^th percentile	97.5^th percentile	99^th percentile
Cut-off or reference range	3.59–0	Positive≥25 ng/mL	95^th≤3.56	97.5^th≤3.58	99^th≤4.0

The kit showed that each laboratory determines the normal level. Considering the concentration of about 25 ng/mL in Western societies, it can be argued that 0% of participants in the case group had positive HAMA.

Discussion

Reference range (reference value)

Interpretation of laboratory tests requires the awareness of the limits, normal and expected ranges for test results [12]. Reference values can be defined as a set of values collected from a group of people (or one person alone) in a specific health status [12]. So, it is necessary to collect HAMA reference values from the control group to compare HAMA levels between these groups.

This study aimed to investigate the presence of HAMA in the blood of animal care providers and the production of antibodies in terms of age, education and work experience Also, to evaluate the true value of this antibody in the mice-exposed group (case group), the reference interval for HAMA in the control group was determined. As there are no similar studies, the results could not be compared; so, in this section, the variables are interpreted.

In this study, the participants of both groups were of the same age, hence the difference of age was excluded in calculating the amount of HAMA.

The mean age of the participants in the case and control groups was 40.7 and 40.6 years, respectively. So, the statistical analysis suggest that age did not have a significant effect on HAMA.

The duration of working experience in animal care centers and medical schools was considered as a variable in the case group. Accordingly, those who have many years of working experience in such places may have an increased level of HAMA; however, the researchers of this study did not find its effect on HAMA. Of course, taking safety measures (such as the use of gloves, masks, caps, gowns and goggles, taking a bath after cleaning the mouse nest) can somewhat prevent the staff from being exposed to the mice.

Education is usually helpful in all aspects of life. A poor education, due to the lack of awareness, can increase the exposure to mice both in personal life and working environment which may lead to an increase in the serum levels of HAMA. Again, the effect of education on the amount of HAMA was not found.

Although 43.6% of individuals in the case group and 6% in the control group were diagnosed with different types of allergies, these variables did not have a significant effect on the amount of HAMA. But according to simple linear regression, those who were not exposed to rats were less allergic (0.03) than those in the case group.

The frequency of rat bites was measured only in the case group and 61.5% of individuals in this group were bitten by rats; thus, this variable did not have any significant effect on the amount of HAMA. It seems that no significant amount of antigen enters the blood due to the animal bites.

It was assumed that the more the weekly cleaning of mice nests is, the more frequent the exposure to mouse antigens will be. Thus, the HAMA serum level will increase. This variable had no significant effect on the amount of HAMA levels.

Also, it seems that taking a bath after cleaning the mice nests helps clear the antigens in the body and it leads to a decrease in the serum HAMA level, but these variables had no significant effect on HAMA.

The least amount of HAMA in the case and control groups was 0.48 and 0.22, respectively, which in the case group was almost twice than that in the control group. The maximum amount of HAMA in the case and control groups was 20.85 and 4.02, respectively, which in the case group was almost 2.5 times more than that in the control group. HAMA levels were significantly different between both groups (p=0.00).

Based on the linear regression analysis, the regression coefficient of HAMA for both groups was −3.273 which means that in the control group, the HAMA level was 3.273 ng/mL lower than that in the case group. This difference of variables between both groups was confirmed using independent t-test and p=0.00.

According to the data obtained from the control group and the definition of reference interval, people with a HAMA value over 4 ng/mL had positive HAMA. But further studies on those with high immunoassay test results are required (tests in which the monoclonal mice antibody was used) to investigate the presence of HAMA and measure its serum level in these patients.

Conclusions

This study found that exposure to rats (in animal care staff) showed an increase in serum antibody levels. This increase is statistically significant, but variables such as age, working experience with rats, education level, allergies, animal bites, weekly cleaning of animal nests and taking a bath after each time of cleaning had no significant effect on serum levels of HAMA.

The investigation of HAMA in individuals who have a chance of producing it for various reasons, such as receiving monoclonal mouse antibodies for diagnosis and treatment, excessive exposure to rats, a close contact with mice as their jobs demand, needs to be done for performing immunoassay tests and obtaining unexpected results and thus prevent the losses due to wrong diagnosis and treatments.

Suggestions for future research

Doing a research on large samples in the case and control groups aimed at determining the upper and lower HAMA reference intervals in the Iranian population is suggested. Also, performing a study on HAMA prevalence and titers in patients with cancer, autoimmune diseases and transplanted patients receiving monoclonal mouse antibodies aimed at investigating the possible ineffectiveness of subsequent treatment and, if needed, neutralizing the blood from HAMA through plasmapheresis is suggested.

As collecting and analyzing laboratory serum samples on whom the immunoassay tests have been done and unexpected or discrepant results from the presence of HAMA were obtained, an overview of the prevalence and titers of this antibody and the number of unexpected results from the interference with HAMA is needed.

Correspondence: Mohammad Mahdi Mohammadi, PhD, Department of Immunology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, Tel./Fax: 03433257317

Acknowledgments

All volunteers, teachers and trainers in the Department of Immunology at Kerman’s university of Medical science as well as the participants of this study are highly appreciated.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Sharma SK, Bagshawe KD, Melton RG, Sherwood RF. Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial. Cell Biophys 1992;21:109–20.10.1007/BF02789482Suche in Google Scholar

2. Baum RP, Niesen A, Hertel A, Nancy A, Hess H, Donnerstag B, et al. Activating anti-idiotypic human anti-mouse antibodies for immunotherapy of ovarian carcinoma. Cancer 1994;73:1121–5.10.1002/1097-0142(19940201)73:3+<1121::AID-CNCR2820731353>3.0.CO;2-QSuche in Google Scholar

3. Park S, Wians FH, Jr., Cadeddu JA. Spurious prostate-specific antigen (PSA) recurrence after radical prostatectomy: interference by human antimouse heterophile antibodies. Int J Urol 2007;14:251–3.10.1111/j.1442-2042.2006.01648.xSuche in Google Scholar

4. Azrin MA. The use of antibodies in clinical cardiology. Am Heart J 1992;124:753–68.10.1016/0002-8703(92)90287-6Suche in Google Scholar

5. Ballow M, Nelson R. Immunopharmacology: immunomodulation and immunotherapy. JAMA 1997;278:2008–17.10.1001/jama.1997.03550220214027Suche in Google Scholar

6. Boscato LM, Stuart MC. Incidence and specificity of interference in two-site immunoassays. Clin Chem 1986;32:1491–5.10.1093/clinchem/32.8.1491Suche in Google Scholar

7. Vaidya HC, Beatty BG. Eliminating interference from heterophilic antibodies in a two-site immunoassay for creatine kinase MB by using F(ab’)2 conjugate and polyclonal mouse IgG. Clin Chem 1992;38:1737–42.10.1093/clinchem/38.9.1737Suche in Google Scholar

8. Fleetwood MK. Interference in clinical laboratory tests by human antibodies to specific and non-specific immunogens. Laboratory Medicine 2003;34:292–4.10.1309/V1F89FAQ2G13D26LSuche in Google Scholar

9. Ledermann JA, Begent RH, Bagshawe KD, Riggs SJ, Searle F, Glaser MG, et al. Repeated antitumour antibody therapy in man with suppression of the host response by cyclosporin A. Br J Cancer 1988;58:654–7.10.1038/bjc.1988.279Suche in Google Scholar

10. Peter A, Shipkova M, Wieland E, Schleicher E, Muller I. Increased cyclosporine concentrations in the absence of cyclosporine administration. Clin Chem 2011;57:670–3.10.1373/clinchem.2010.148718Suche in Google Scholar

11. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem 1998;44:440–54.10.1093/clinchem/44.3.440Suche in Google Scholar

12. McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods, 22nd ed. Philadelphia, PA, USA: Elsevier Health Sciences, 2016.Suche in Google Scholar

13. Boer DP, de Rijke YB, Hop WC, Cransberg K, Dorresteijn EM. Reference values for serum creatinine in children younger than 1 year of age. Pediatr Nephrol 2010;25:2107–13.10.1007/s00467-010-1533-ySuche in Google Scholar PubMed PubMed Central

14. Al-Marzooq F, Mohd Yusof MY, Tay ST. Molecular analysis of antibiotic resistance determinants and plasmids in Malaysian isolates of multidrug resistant Klebsiella pneumoniae. PLoS One 2015;10:e0133654.10.1371/journal.pone.0133654Suche in Google Scholar PubMed PubMed Central

15. Ortel TL. Antiphospholipid syndrome: laboratory testing and diagnostic strategies. Am J Hematol 2012;87:S75–81.10.1002/ajh.23196Suche in Google Scholar PubMed PubMed Central

16. Pengo V, Ruffatti A. Critical update of the antiphospholipid syndrome criteria. Open Autoimmun J 2010;2:18–20.10.2174/1876894601002020018Suche in Google Scholar

17. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, et al. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2009;7:1737–40.10.1111/j.1538-7836.2009.03555.xSuche in Google Scholar PubMed

18. Apple FS, Ler R, Murakami MM. Determination of 19 cardiac troponin I and T assay 99th percentile values from a common presumably healthy population. Clin Chem 2012;58:1574–81.10.1373/clinchem.2012.192716Suche in Google Scholar PubMed

19. Hammarsten O, Fu ML, Sigurjonsdottir R, Petzold M, Said L, Landin-Wilhelmsen K, et al. Troponin T percentiles from a random population sample, emergency room patients and patients with myocardial infarction. Clin Chem 2012;58:628–37.10.1373/clinchem.2011.171496Suche in Google Scholar PubMed

Received: 2018-07-02

Accepted: 2018-10-20

Published Online: 2019-03-06

Published in Print: 2019-04-24

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Artikel in diesem Heft

https://doi.org/10.1515/labmed-2018-0084

Schlagwörter für diesen Artikel

ELISA; human anti-mouse antibody (HAMA); laboratory animals

Creative Commons

BY-NC-ND 3.0