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Laboratory tests

Laboratory tests that measure different parts of the immune system are important for diagnosing an individual with primary immunodeficiency (PI) and determining which of over 450 different conditions they may have.

Laboratory tests are necessary to determine if an individual has a primary immunodeficiency (PI). These tests are usually prompted by the individual experiencing clinical symptoms, particularly recurrent and/or chronic infections.

An accurate medical history, family history, and physical examination are critical in developing the best strategy for laboratory evaluation. This typically begins with screening tests, followed by more sophisticated (and costly) tests chosen based on the screening test results. If the clinical problems include infections, information regarding the types of microorganisms causing infection, the sites of infection, and the therapies required to treat the infections often help focus the laboratory studies.

The most common laboratory tests used to evaluate immune disorders identify:

  1. Antibody deficiencies.
  2. Cellular (T cell) defects.
  3. Neutrophil disorders.
  4. Complement deficiencies.

Another emerging laboratory test for the diagnosis of PI is genetic testing. In the past, genetic tests were available mainly in research settings, but now, several commercial labs offer genetic sequencing for the identification of known mutations causing various types of PI.

Normal versus abnormal laboratory values

Proper interpretation of any laboratory value depends on comparing the result to the appropriate established normal reference ranges, which are age-specific in some cases. To determine what is normal, the laboratory test is performed on a group of healthy individuals, usually adults and equally divided between males and females. These results are used to determine what the normal range is using a variety of statistical approaches.

A common statistical measurement is called a 95% confidence interval (95% CI), which is the range that includes 95% of the results from healthy tested subjects (as in a bell curve). It is important to note that when the definition of the normal range is set as a 95% CI, 2.5% of healthy individuals will be below the range, and 2.5% will be above, even though they were originally selected as being healthy. This is one of the challenges with using statistical methods to define a normal range and must be remembered when evaluating a test result falling near either end of the reference range.

Using the measurement of height as an example, healthy individuals can be just above or just below a normal range (or 95% CI). Someone one inch taller than the 95% CI is not necessarily a giant, and someone one inch shorter does not necessarily have a growth issue. The fact that 5% of otherwise healthy individuals will fall outside the normal range is important when looking at laboratory results—finding a value outside of the reference range does not automatically represent an abnormality. The clinical relevance of an abnormal laboratory finding must be based on the clinical history, as well as the size of the difference from the reference range.

Characteristics of the group, such as the age of the group, used to determine the reference range are crucial since the immune system undergoes substantial development during infancy and childhood. The range of test values that are typical in infancy will be quite different when the child is 2 or 20 years old.

Consequently, all tests in children must be compared with age-matched controls. If the laboratory does not provide age-specific information, it is important to consult with a specialist who knows the age-specific reference ranges. Optimally, the laboratory doing the test should provide this, but if unavailable, there are published age-specific reference ranges.

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 cellular (T cell) immunity

The laboratory evaluation of cellular, or T cell, immunity focuses on determining the numbers of different types of T cells and evaluating the function of these cells.

The first test to evaluate T cell immunity occurs without most people knowing about it, as all states in the U.S. now screen for very low T cell numbers at birth. This occurs through the newborn screening program, which detects severe, treatable genetic defects in otherwise healthy-looking infants.

Outside of the newborn period, the simplest test to evaluate possible decreased or absent T cells is a complete blood count (CBC) with differential to establish the total blood (absolute) lymphocyte count (ALC). This is a reasonable method to assess for diminished T cell numbers, since normally about 75% of circulating lymphocytes are T cells and a reduction in T cells will usually cause a reduction in the total number of lymphocytes, or ALC. One must be careful, however, as individuals can have fairly typical ALC but still have a low T cell count. The actual T cell count can be confirmed by using flow cytometry with markers specific for different types of T cells.

The measurement of the number of T cells is often accompanied by cell culture studies that evaluate T cell function. This is done by measuring the ability of the T cells to respond to different types of stimuli, including mitogens (plant-derived chemicals such as phytohemaglutinin [PHA]) and antigens (such as tetanus toxoid or Candida antigen). The T cell response to these various stimuli is measured by observing whether the T cells divide and grow (called proliferation) and/or whether they produce various proteins important in immune responses called cytokines (such as interferon). There is an increasing variety of functional tests that are available to evaluate T cells. An immunologist is the best person to undertake this interpretation.

Many types of PI affecting cellular immunity are associated with specific genetic variants. This is particularly true of SCID, in which more than 20 different genetic causes have been identified. These can all be evaluated using genetic testing, which is the most accurate means to establish a definitive diagnosis.

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 for complement deficiencies

The standard screening test for deficiencies in the classical complement pathway is the total hemolytic complement assay or CH50. In situations with a defect in one complement component, the CH50 will be almost completely negative. Specialized complement laboratories can provide additional testing that will identify the specific complement component that is defective.

There are some extremely rare conditions in which there are defects in another complement pathway (the alternate pathway). These can be screened for by using a functional test directed specifically at this pathway, the AH50 test.

The complement cascade can also be initiated by the mannose-binding lectin pathway, and there are some individuals with a deficiency in mannose-binding lectin, although the clinical relevance of the laboratory finding is inconclusive.

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).

This page contains general medical and/or legal information that cannot be applied safely to any individual case. Medical and/or legal knowledge and practice can change rapidly. Therefore, this page should not be used as a substitute for professional medical and/or legal advice.

Adapted from the IDF Patient & Family Handbook for Primary Immunodeficiency Diseases, Sixth Edition 
Copyright ©2019 by Immune Deficiency Foundation, USA