Laboratory tests

Testing for an antibody deficiency (or humoral immune function)

The standard screening tests for humoral immune function start with measuring immunoglobulin (Ig), or antibody, levels in the blood serum. These consist of IgG, IgA, IgM, and sometimes IgE levels. The results must be compared to age-matched controls.

There are also tests for specific antibody production. These tests measure how well the immune system can make antibodies against vaccines, as a marker of how well the antibody arm of defense is functioning. There are two main pathways of antibody production that can be tested: 

• The T cell-dependent pathway, measured by the antibody response to protein antigens, such as tetanus and diphtheria toxoids. 
• The T cell-independent pathway, measured by the antibody response to carbohydrate antigens, such as those found in the pneumococcal polysaccharide vaccine (PPSV) known as Pneumovax. 

To test specific antibody production, the person is immunized with these common vaccines, and blood samples are obtained immediately prior to and approximately four weeks after the immunization to evaluate how well the individual forms specific antibodies.

In some instances, the person may have already been immunized with these vaccines as part of their routine care and will already have circulating antibodies (if they make antibodies), while in other instances the individual may have little or no specific antibody prior to the immunization. The use of different types of vaccines is necessary because certain people with recurrent infections (and normal or near normal Ig levels) have been identified with an abnormality in the response to carbohydrate antigens but a normal response to protein antigens. These individuals may have specific antibody deficiency.

It is worth noting that during the maturation of the immune system, the response to carbohydrate antigen vaccines lags behind the response to protein antigen vaccines. This is the reason for having a childhood version of the pneumococcal vaccine, the PCV13, which makes it easier for infants to respond to Streptococcus pneumoniae. The interpretation of vaccine responses is best done by an allergist/immunologist who deals with individuals with PI on a regular basis.

The ability to evaluate the antibody response in a person already receiving Ig replacement therapy is more difficult. This is because Ig products are rich in most of the specific antibodies that are generated following immunizations. When immunized with common vaccines, it is difficult to tell the difference between the antibody provided by the Ig replacement therapy and any that might have been made by the individual. The solution to this is to immunize with vaccines that are not normally encountered by the general population and therefore are unlikely to be present in Ig preparations. Uncommon vaccines, such as typhoid or rabies vaccine, can serve this purpose.

It is important to note that in a person with a previously confirmed antibody deficiency, stopping therapy to recheck for antibody levels and immunization response is unnecessary and may place the individual at risk of acquiring an infection during the period when the Ig replacement therapy is stopped. In someone whose diagnosis of an antibody immunodeficiency is unclear, however, it may be necessary to stop Ig replacement therapy for a period of 4-6 months so that the individual’s humoral immunity can be adequately assessed.

Additional studies used to evaluate people with antibody deficiencies include measuring the different types of lymphocytes in the blood using a test called flow cytometry. The B cell is the lymphocyte that produces antibodies. B cells may be absent in certain antibody deficiencies, such as X-linked agammaglobulinemia (XLA). This test can also evaluate the ability of the B cells to mature.

In addition, analysis of DNA (genetic testing) can be used to confirm a particular diagnosis, such as a variant in the gene encoding Bruton tyrosine kinase (BTK) associated with XLA. Finally, there are studies done in specialized laboratories to assess Ig production by cultured lymphocytes in response to a variety of different kinds of stimuli.

Testing neutrophil function

The laboratory evaluation of neutrophils begins by obtaining a series of white blood cell counts (WBC) with differentials. The WBC and differential will determine if there is a decline in the absolute neutrophil count (ANC), which is known as neutropenia. This is the most common laboratory finding when an individual presents with a clinical history that suggests low neutrophil immunity. Usually, more than a single CBC and differential is necessary to diagnose neutrophil problems.

A careful review of the blood smear is important to rule out certain diseases that are associated with abnormalities in the structure of neutrophils, or the way they look under the microscope. Sometimes, a bone marrow biopsy is needed to see if neutrophils are being made properly. If the initial screening tests of neutrophil numbers were normal, testing then focuses on two possible types of PI: chronic granulomatous disease (CGD) and leukocyte adhesion deficiency (LAD). Both of these disorders have normal or elevated numbers of neutrophils, and each of these disorders has distinctive clinical features that can help to direct the appropriate evaluation.

Laboratory testing to diagnose CGD relies on the evaluation of a critical function of neutrophils that kills certain bacteria and fungi—the creation of reactive oxygen. This process, called the oxidative burst, can be measured using a number of different methods.

Currently, the most commonly used and reliable test uses flow cytometry to measure the oxidative burst of activated neutrophils using a specific dye (dihydrorhodamine 123 or DHR), referred to as the DHR test. The DHR test has been used for more than 20 years, and it is extremely sensitive in making a CGD diagnosis. This test can also be helpful in identifying carriers of CGD. Because of its excellent performance, this test has become the standard in most laboratories supporting clinics that see individuals with CGD regularly. In the past, a dye reduction test called the nitroblue tetrazolium (NBT) test was used, but this test had greater variability in its interpretation. The specific type of CGD is suggested by the results of the DHR test, but it requires confirmation by either specifically evaluating for the defective protein involved or its related gene variant underlying the disease.

Laboratory testing for the most common form of LAD Type 1 involves flow cytometry testing to determine the presence of a specific protein on the surface of neutrophils (and other leukocytes). When this protein is absent or significantly decreased, the movement of neutrophils to sites of infection is hampered. The result is a large increase in the number of neutrophils in the circulation, as well as an increased susceptibility to bacterial skin, oral, and other infections.

Testing innate immunity

Laboratory tests are also available to measure the function of various elements of innate immunity. These include determining the number and activity of lymphocytes, such as natural killer (NK) cells, as well as the function of various cell surface receptors such as the toll-like receptors (TLRs).