What is HAMA?

HAMA is an acronym for Human Anti-Mouse Antibodies. Some humans produce HAMA and have it present in their blood. Unfortunately for them, and the in vitro diagnostics industry, the presence of Human anti-mouse Antibodies (HAMA) in patient samples can lead to false positive and false negative results in immunoassays.


Murine MC HAMA

Why do some Humans produce HAMA?

Some people work directly with mice while others might inadvertently encounter mouse proteins or immunoglobulins by coming into contact with mouse urine or contaminated food. These people could develop an immune response against mouse immunoglobulins (antibodies) they encounter such that their immune system produces HAMA.

In the past, mouse monoclonal antibodies were used as therapeutics and could elicit an immune response resulting in the presence of HAMA in human individuals. However, these days monoclonal antibody-based pharmaceuticals are “humanised” to avoid this problem.

Presence of HAMA in individuals is rare, but still needs to be accounted for in immunoassay design.

Could I interfere with your immunoassay?

How can HAMA impact Immunoassays?

When a patient is tested for a condition, the test performed is commonly an immunoassay and the sample that is tested is serum or plasma derived from the patient’s blood. Immunoassays are typically developed using a “matched pair” of mouse monoclonal antibodies to bind to and detect the marker of interest. The ‘marker’ of interest depends on the condition being diagnosed but could be, for example, Troponin I which is a marker of acute myocardial infarction, or HIV p24 antigen which is a marker for the presence of HIV virus. The patient sample (e.g. serum or plasma) is applied to the immunoassay and if the marker being tested for is present the mouse monoclonal antibodies within the assay bind to it and a signal is generated.

However, where HAMA is present in the patient sample the HAMA can bind to the mouse monoclonal antibodies used as immunoassay components and can either i) block the mouse monoclonal antibodies from binding to the marker of interest resulting in a false negative result, or ii) form a bridge between the pair of mouse monoclonal antibodies, generating false positive signal.  Where a patient receives an incorrect diagnosis due to the presence of HAMA, the consequences can be devastating. At least 34 cases of hCG false-positive tests in the United States between 1999-2004 resulted in the patients receiving chemotherapy or surgery, including 10 hysterectomies, for assumed cancer1.

How can HAMA interference be prevented in immunoassays?

Assay manufacturers can develop their assays in such a way as to minimise the interference from HAMA, for example by adding excess mouse immunoglobulin to their assay buffers. When this is done, the HAMA present in the patient sample can bind the excess mouse immunoglobulin rather than the reagents being used in the assay.

Immunoassay developers will need to access patient material in order to design an assay that is not affected by HAMA, and also to show that the assay works in the presence of HAMA. Logical Biological is able to provide HAMA-positive serum and plasma.

Even if blockers are used to control HAMA, the heterogeneous nature of its presence in patients mean that it is difficult to rule out its influence entirely, unless it is measured. If HAMA is suspected, the clinical laboratory can perform serial dilutions with an appropriate buffer to demonstrate nonparallelism (higher recovery of the signal than expected).

An alternative solution to controlling for HAMA interference is to use monoclonal antibodies from an alternative species in the immunoassay, in place of mouse monoclonal antibodies. Rabbit and Sheep monoclonal antibodies are candidates here. However, this is not a great solution as some individuals in the population will be producing human anti-rabbit and anti-sheep antibodies. Synthetic peptide binders and/or recombinant antibodies can theoretically avoid the problem of HAMA when used as alternatives to antibodies raised in animals.

Reference

  1. Human Chorionic Gonadotropin (hCG), By Laurence A. Cole, Stephen A. Butler. Elsevier. 2010.

Logical Biological has added Chagas antibody positive plasma to its range having received clearance from the UK government Health & Safety Executive to handle human biological material containing Trypanosoma cruzi, which is the causative agent of Chagas Disease. T. cruzi is a Hazard Group 3 pathogen according to “The Approved List of Biological Reagents”. Hazard Group 3 is defined as follows: “Can cause severe human disease and may be a serious hazard to employees; it may spread to the community, but there is usually effective prophylaxis or treatment available.”

As part of this process, the UK government has reviewed Logical Biological’s key risk assessments and standard operating procedures. Logical Biological has previously received clearance to handle human biological material infected with other Hazard Group 3 organisms such as HIV, Hepatitis B, Hepatitis C, Malaria (Plasmodium falciparum) and Tapeworm (Taenia solium).

You can see the full list of infectious disease materials offered by Logical Biological here.

Logical Biological has received clearance from the UK government Health & Safety Executive to handle human biological material containing Trypanosoma cruzi, which is the causative agent of Chagas Disease. T. cruzi is a Hazard Group 3 pathogen according to “The Approved List of Biological Reagents”. Hazard Group 3 is defined as follows: “Can cause severe human disease and may be a serious hazard to employees; it may spread to the community, but there is usually effective prophylaxis or treatment available.”

As part of this process, the UK government has reviewed Logical Biological’s key risk assessments and standard operating procedures. Logical Biological has previously received clearance to handle human biological material infected with other Hazard Group 3 organisms such as HIV, Hepatitis B, Hepatitis C, Malaria (Plasmodium falciparum) and Tapeworm (Taenia solium).

You can see the full list of infectious disease patient material offered by Logical Biological here. Please contact us for further information on any of our plasma/serum, antigen and monoclonal antibody products for IVD.

Irregular antibodies are antibodies found in the blood of transfusion donors that have the potential to cause hemolysis of the recipient blood. Screening using an indirect Coombs Test should be performed to ensure that the donor blood is compatible with the recipient. “Irregular Antibodies” refers to all antibodies, other than those detecting ABO blood group antigens, that can cause incompatibility in blood transfusions and between mother & child.

The ABO Blood Group System

Most people are aware of the ABO blood group system. There are 4 major blood types in humans – A, B, O and AB. Some of the blood groups are incompatible with others; if a blood group is transfused into a patient with an incompatible blood group, hemolysis results. The incompatibility can result in death.

Example of ABO Incompatibility

Terence (recipient) is Blood Group A. He has A antigens on his red blood cells and anti-B antibodies in his plasma.
Theresa (donor) is Blood Group B. She has B antigens on her red blood cells and anti-A antibodies in her plasma.

If Theresa’s Group B blood is given to Terence, Terence’s anti-B antibodies will attack Theresa’s blood and cause it to hemolyse.

Since blood group incompatibility is life-threatening it is essential to confirm donor and recipient compatibility before a transfusion occurs. The ABO and Rhesus blood group systems are the most well-known but there are other less well known factors that can cause hemolysis in transfusion patients and new-borns – these are the Irregular Antibodies  

Anti-Kell – an example of an Irregular Antibody

Anti-Kell antibodies may develop in individuals which lack the Kell antigen upon:

  1. Receipt of a blood transfusion containing Kell antigen
  2. At childbirth following transplacental hemorrhage

In these cases the individual’s immune system will recognise the Kell antigen as a foreign molecule and elicit an immune-response, becoming sensitized to it.

Testing for donor compatibility – the Indirect Coombs Test

The indirect Coombs Test, also known as Indirect Antiglobulin Test, detects irregular antibodies. Again, taking anti-Kell as an example (See Figure 1):

  1. Recipient serum may contain irregular antibodies (e.g. anti-Kell)
  2. Donor blood sample is added to recipient serum
  3. Recipient irregular antibodies, if present, bind to donor red blood cells where the corresponding antigen is present
  4. Anti-human Immunoglobulins (Coombs reagent) is added. The antibodies within Coombs reagent bind the Fc region of any irregular antibodies and, where those irregular antibodies have bound the donor red blood cells, form bridges between immune complexes on red blood cells, resulting in agglutination
Positive Coombs Test result for Irregular Antibodies

There are many other irregular antibodies, such as those listed below, available from Logical Biological.

Irregular Antibody Product Identifier
anti-c H191
anti-Cw H193
anti-D H189
anti-E H190
anti-Fya H195
anti-Jka H194
anti-Kell H192
anti-Kpb H205
anti-Lea H200
anti-Leb (Lewis) H201
anti-Lua H203
anti-Lub H204
anti-M H196
anti-N H197
anti-P1 H202
anti-Public H207
anti-S H198
anti-s H199
auto-Pap H206

All over the planet, women are getting pregnant, over 200 million of them each year. Most of these pregnancies are unintended1. This means two things: a lot of babies, and a lot of surprises. Despite this, some 8% of couples are unable to conceive within two years and are considered infertile. Risk factors for infertility include age, previous medical history (sexually-transmitted diseases, cancer treatment) and obesity. 

Fortunately, a wide-range of fertility treatments are now available, although sadly the cost of such treatment is prohibitive for many. Despite this, for those who can access it fertility treatment does work; in the UK the Human Fertility and Embryology Authority reported ~20,000 births in 2016 from ~68,000 treatment cycles.

Much of the success in fertility treatment stems from the use of medications that seek to address fertility issues in individual patients. While this is welcome news, as the number of fertility treatments increases and their use becomes more widespread there is a need for diagnostic test manufacturers to ensure that the tests they develop give the right results in patients taking fertility drugs.

For example, low levels of Progesterone early in pregnancy can be suggestive of increased risk of miscarriage and ectopic pregnancy. A patient with a low result for Progesterone levels may be treated in order to manage this situation and reduce the likelihood of miscarriage or to confirm and manage an ectopic pregnancy. If such patients are also being treated with Clom1fene – not an unlikely scenario for a patient in the early stages of pregnancy – then it would be important to know that the Progesterone test result is not interfered with by the presence of Clom1fene in the patient. Therefore, there is a need within the disease diagnosis industry for material from patients treated with fertility drugs to ensure that diagnostic tests being developed are not interfered with.

During immunoassay development, diagnostic test manufacturers typically test their assays for specificity. They construct a list of potential cross-reacting molecules, and then spike these on an individual basis into human specimens that do not contain the analyte of interest (e.g. Progesterone). They would then test the specimens in the assay to see if any “Progesterone” was detected, expecting a value of zero if the cross-reactant does not interfere with the assay.

Let’s now review some of the drugs used in fertility, all of which have the potential to interfere with diagnostic assays. Logical Biological can provide plasma and serum from individuals in every stage of pregnancy (0-40 gestation weeks) and those treated with medications covered in this article. Do let us know if this would be of interest to your organisation.

Drugs which stimulate ovulation

Clom1fene* (brand name: Clom1d) – a commonly used drug which stimulates ovulation by causing the pituitary gland to release more Follicle-Stimulating Hormone (FSH) and Luteinising Hormone (LH), which it does by blocking estrogen receptors. This stimulates the growth of an ovarian follicle containing an egg.

Gonadotropins – FSH, LH or human Chorionic Gonadotrophin (hCG) strongly stimulate the ovary directly to mature and release eggs. These hormones can be used individually or in combination and are administered by injection. Brand names of this type of drug include Gonal-F, Follistim AQ, Bravelle, Menopur, Ovidrel and Pregnyl.

Metformin (brand names include Glucophage) is used when insulin is the suspected underlying cause of infertility. Metformin helps improve insulin resistance and thus increases the likelihood of ovulation.

Letrozole (brand name: Fermara) is an aromatase inhibitor, developed for use in treating breast cancer, which also serves to stimulate ovulation by decreasing the amount of testosterone converted to estradiol. This reduces estrogen activity and leads to more FSH secretion.  

Bromocriptine and cabergoline (brand names: Parlodol and Dostinex) are dopamine agonists which can be used where ovulation problems, such as irregular or absent ovulation, are caused by excess production of prolactin by the pituitary gland (hyperprolactinemia).

Drugs which suppress ovulation

Some drugs are actually used by fertility clinics to suppress ovulation. This could be to prevent ovulation before the eggs can be retrieved surgically for IVF use. Gonadotropin-releasing hormone (GnRH) agonists achieve this and examples include ganirelix acetate and cetrorelix acetate. Brand names include Antagon, Ganirelix, Cetrotide and Orgalutran.

Other fertility medications are GnRH agonists. These stimulate FSH and LH production but then cause the body to stop producing them, allowing control of ovulation.  Examples are leuprolide acetate, nafarelin acetate, buserelin and goserelin. These are manufactured under brand names including Lupron, Synarel, Suprecur and Zoladex, which variously may be delivered to the patient by nasal spray, implant or injection.

Other Drugs

Birth control pills (contraceptive pills) may also be used by fertility clinics to enhance response to Clom1d of women with polycystic ovary syndrome. Progesterone may be prescribed for luteal phase defects or recurrent miscarriages. Estrogen may be prescribed to alleviate a symptom of Clom1d, which is a thicker endometrial lining that can interfere with conception.

Conclusions

As technology advances there is a strong trend towards increased uptake of fertility treatments. Diagnostics immunoassay developers should continue to include fertility drugs in their cross-reactivity studies to determine the specificity of their assays.

References

  1. Global, regional, and subregional trends in unintended pregnancy and its outcomes from 1990 to 2014: estimates from a Bayesian hierarchical model. Bearak J, Popinchalk A, Alkema L, Sedgh G. Lancet Glob Health. 2018 Apr;6(4):e380-e389.

This article should be viewed for general interest purposes only, may not be fully accurate or comprehensive, and should not be considered as medical advice.
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