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Laboratory evaluation of the immune system

Authors: Manish J Butte, MD, PhD, E Richard Stiehm, MD
Section Editor: Luigi D Notarangelo, MD
Deputy Editor: Anna M Feldweg, MD
All topics are updated as new evidence becomes available and our peer review process is complete.
Literature review current through: Jun 2021. | This topic last updated: Nov 01, 2019.
INTRODUCTION
Immunodeficiency most often comes to clinical attention because of an increase in the incidence
or severity of infectious illness beyond what is considered "normal." Patients with
immunodeficiency diseases may also have immune dysregulation that includes unusual or severe
autoimmunity or fevers, lymphoproliferation, and severe inflammation that is unprovoked
(autoinflammation). Some forms of immunodeficiency are also characterized by an increased risk
of malignancies. This topic review will provide a general approach to the laboratory evaluation of
the immune system, beginning with screening tests and progressing through the indications for
more advanced immunologic testing. Indications for referral to a specialist are discussed, and links
to more detailed topics about the different groups of disorders are provided here and throughout
the topic.
● (See "Primary humoral immunodeficiencies: An overview".)

● (See "Combined immunodeficiencies".)
● (See "Primary disorders of phagocyte number and/or function: An overview".)
● (See "Laboratory evaluation of neutrophil disorders".)
● (See "Overview and clinical assessment of the complement system".)
● (See "Autoimmunity in patients with inborn errors of immunity/primary immunodeficiency".)
INITIAL APPROACH TO THE PATIENT
Immunodeficiency disorders should be considered once the more common causes of recurrent
infection, autoimmunity, and inflammation have been excluded. The initial approach to a child or
adult with recurrent infections is described separately. (See "Approach to the child with recurrent
infections" and "Approach to the adult with recurrent infections".)
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Immune dysregulation can result in disorders other than recurrent infections including:
● Autoimmune disorders, such as autoimmune hemolytic anemia

● Inflammatory disorders, such as inflammatory bowel disease or inflammatory arthritis
● Nonmalignant lymphoproliferation (lymphadenopathy, splenomegaly, lymphoid aggregates in
target organs)
● Malignancies, such as lymphoma
● Allergic disease, such as atopic dermatitis, food allergy, allergic rhinosinusitis, and asthma
Before initiating immunologic testing, the clinician should perform a thorough clinical history and
physical examination. In infants and children, height and weight records should be reviewed, as
failure to thrive and poor growth are consistent with immunodeficiency. In patients with possible
immunodeficiency, important historical elements include:
● The nature of the infections – This should include the frequency, chronicity, severity, and
response to therapy. Other considerations include what organ system(s) is involved and what
type of organism has been identified in the past (ie, viral, bacterial, fungal, opportunistic).
Patterns of infections can suggest specific immune defects.
● Age of onset of illness, since different immune problems present in infancy, childhood, and
adulthood.
● The patient's sex, because X-linked defects are mostly or exclusively seen in boys.
● Family history of infections, abnormal inflammatory responses, and autoimmune disorders, as

well as any childhood deaths.
● Family history of lymphoproliferative illnesses, including occurrence of splenectomy.
● Any associated nonimmunologic symptoms and signs, as revealed by a complete review of

systems.
Physical examination — Physical examination findings that suggest immunodeficiency in

children and adults are reviewed separately. (See "Approach to the child with recurrent infections",
section on 'Physical examination' and "Approach to the adult with recurrent infections", section on
'Physical examination'.)
Initial screening laboratory tests — In patients of any age, the laboratory evaluation of the
immune system begins with general studies including:
● Complete blood count (CBC) with differential – Lymphopenia is characteristic of a variety of

combined immunodeficiencies (ie, cellular and antibody deficiency). Lymphopenia is defined
as an absolute lymphocyte count <1500 cells/microL in adults or <2500 cells/microL in
infants. Neutropenia can be found in primary phagocyte disorders, as well as in neutrophil

disorders that lead to secondary immunodeficiency [1]. Leukocytosis is sometimes noted and
suggests chronic infection. Monocytopenia is seen in GATA2 deficiency. Eosinophilia can be
seen in primary atopic disorders and a number of primary immunodeficiencies.
● Chemistry panels – Chemistry panels should be evaluated to assess for metabolic disorders
(diabetes mellitus, renal disease) that might cause secondary immunodeficiency.
Hypoalbuminemia or low serum proteins suggest malnutrition or protein loss. Markedly
elevated globulin levels may be seen in gammopathies or chronic infections.
● Urinalysis – Urinalysis should be obtained for proteinuria, casts, or cells, which suggest
nephritis.
● Tests to evaluate for specific infections, if indicated by the presentation – Tests for specific
infections include appropriate cultures, serologies, and sinus imaging. Sinus films may
uncover extensive chronic rhinosinusitis in patients with immunodeficiency. Children or
adolescents with nasal polyposis should be evaluated for cystic fibrosis, which is a cause of
frequent sinopulmonary infections.
● Chest radiograph – Chest radiographs of an infant showing absence of a thymic shadow

should prompt an emergency evaluation for severe forms of immunodeficiency ( image 1),
though the thymus responds to stress by shrinking in babies and should be followed up by
ultrasound of the anterior chest. In older children and adults, chest radiographs may show
scarring from past infections, interstitial lung disease, or bronchiectasis. Hyperinflated lung
fields suggest chronic obstructive lung disease or chronic asthma.
● Erythrocyte sedimentation rate and/or C-reactive protein – Nonspecific elevations in acute-

phase reactants can be seen with infectious and inflammatory disorders and suggest the
need for further evaluation.
Referral — More advanced immunologic tests require varying degrees of expertise to perform and
interpret, may not be widely available, and are often costly. In addition, knowledge about the
possible diagnoses in question is invaluable in deciding the type of testing to pursue first [2]. Thus,
immunologic testing is best performed in a graded fashion, and referral to an
allergist/immunologist should be sought early in the process, when possible.
Ultimately, definitive diagnosis may require specialized flow cytometric, genetic, and other
advanced tests available in reference laboratories [3,4] or some children's hospitals. The website
of the Immune Deficiency Foundation offers the ability for clinicians to request consultations with
expert immunologists.
CATEGORIES AND PREVALENCE OF IMMUNODEFICIENCIES

Immunodeficiency disorders may be grouped into categories based upon the aspects of the
immune system that are predominantly affected and their approximate frequency [5]:
● Predominantly antibody deficiencies (55 percent)

● Immunodeficiencies affecting cellular and humoral immunity (15 percent)
● Congenital defects of phagocyte number or function (10 percent)
● Diseases of immune dysregulation (5 percent)
● Autoinflammatory disorders (5 percent)
● Complement deficiencies (4 percent)
● Combined immunodeficiencies with associated or syndromic features (3 percent)
● Defects in intrinsic and innate immunity (3 percent)
Each of these categories of disorders has characteristic clinical manifestations, although there are
overlaps among the clinical presentations of the groups. In addition, several conditions have been
described that phenocopy inborn errors of immunity. These include disorders due to somatic
mutations in immune-related genes, as well as diseases associated with autoantibodies against
cytokines, growth factors, and other immune molecules [5].
EVALUATION FOR SPECIFIC TYPES OF DISORDERS
Antibody deficiency and defects — Antibody deficiency most frequently results in recurrent and
severe sinopulmonary infections with encapsulated bacterial strains (eg, Streptococcus
pneumoniae, Haemophilus influenzae). Children commonly present with recurrent otitis media,
rhinosinusitis, and pneumonia. Adults present similarly, although otitis media is less common. Viral
infections of the respiratory tract also occur with greater frequency and severity in these patients.
Clinical presentation is discussed in more detail separately [6]. (See "Primary humoral
immunodeficiencies: An overview".)
The age of the patient can help narrow the differential diagnosis:
● The most common antibody defects that present in infancy are physiologic
hypogammaglobulinemia (to age six months), transient hypogammaglobulinemia of infancy
(after six months, often to the age of six years), selective antibody deficiency (after the age of
two years), and selective immunoglobulin A (IgA) deficiency.
● The most common antibody deficiencies in young children are specific antibody deficiency,
selective IgA deficiency, and immunoglobulin G (IgG) subclass deficiency.
● The most common antibody disorders that present in adulthood include common variable
immunodeficiency, selective IgA deficiency, IgG subclass deficiency, and selective antibody
deficiency.

The disorders mentioned above are reviewed in detail separately. (See "Transient
hypogammaglobulinemia of infancy" and "Specific antibody deficiency" and "Selective IgA
deficiency: Clinical manifestations, pathophysiology, and diagnosis" and "IgG subclass deficiency"
and "Common variable immunodeficiency in children" and "Clinical manifestations, epidemiology,
and diagnosis of common variable immunodeficiency in adults".)
Measurement of antibody levels — Measurement of serum IgG, IgA, and immunoglobulin M

(IgM) is useful in all cases of suspected antibody deficiency. IgE levels are helpful in the
identification of a number of monogenic causes of antibody deficiency (eg, STAT3, IL21R, IL6R,
and IL6ST deficiencies). Serum immunoglobulin D (IgD) is not used in the diagnosis of
immunodeficiency, though elevated levels of IgD are seen in one of the primary
immunodysregulatory disorders (MVK deficiency).
There are several methods for determining serum immunoglobulin levels, and laboratories use
different systems. Therefore, it is critical that age-adjusted normal reference ranges are used
when making comparisons ( table 1). Hypogammaglobulinemia is defined as an IgG less than
two standard deviations below normal, and agammaglobulinemia is usually considered when IgG
is <100 mg/dL. Panhypogammaglobulinemia (ie, low levels of IgA, IgG, and IgM) is a hallmark of
most forms of severe combined immunodeficiency, although it can be seen infrequently in
common variable immunodeficiency and other less severe combined immunodeficiencies. In other
combined immunodeficiencies, as well as in several predominantly humoral immunodeficiencies,
there are characteristic alterations in the profile of immunoglobulin isotypes that may aid in
diagnosis (eg, selective IgA deficiency, selective IgM deficiency, and hyper-IgM
immunodeficiencies).
In addition to IgG, IgA, and IgM, measurement of IgG subclasses and IgE may be useful:
● Measurement of IgG subclasses may be valuable in children older than one year of age and
in adults, particularly if the IgG level is borderline low and/or there is a weak or absent
antibody response to vaccination. (See "IgG subclass deficiency".)
● Measurement of IgE is helpful in patients with recurrent sinopulmonary infections or scaly or

eczematoid skin disorders, as an elevation is consistent with underlying allergic disease (eg,
>100 international units/mL). A very elevated IgE (eg, >2000 international units/mL) in a
patient with recurrent bacterial infections and dermatitis would raise suspicion for a hyper-IgE
syndrome and several other immunodeficiency disorders ( table 2). (See "Autosomal
dominant hyperimmunoglobulin E syndrome".)
Measurement of antibody function — Antibody function can be assessed by measuring

antibody titers (usually IgG isotype) to specific antigens (also known as specific antibody) in
response to intentional immunization or natural infection. There are two major goals to measuring
vaccine responses: first, to assess if naïve B cells can respond to a new antigen; second, to

assess if memory B cells respond appropriately to an antigen seen in the past. Picking the right
vaccine to test with, thus, is critical for properly assessing B cell function. Measuring antibody
function is difficult if a patient has received immune globulin in the prior six months, as antibodies
to most vaccine-associated antigens are abundant in immunoglobulin preparations. Testing can be
achieved if the patient can be vaccinated with a vaccine antigen that is not routinely used such as
rabies vaccine or Salmonella Typhim M vaccine [7]. (See "Assessing antibody function as part of
an immunologic evaluation", section on 'Other polysaccharide vaccines'.)
● Clinically significant impairment in antibody function can be present even when serum
antibody levels are normal. (See "Specific antibody deficiency".)
● Normal responses to immunization (ie, normal antibody function) can occur with subnormal
levels of total IgG or of IgG subclasses. This pattern is typically seen with secondary causes
of hypogammaglobulinemia, such as that caused by medications, protein loss, or severe
malnutrition. (See "Secondary immunodeficiency induced by biologic therapies".)
Antibody function is assessed by examining the patient's response to the two general types of
antigens: protein antigens and polysaccharide antigens. Routine vaccinations provide examples of
both types:
● Measurement of antibody titers to tetanus, diphtheria, H. influenzae B, and protein-conjugated

pneumococcal vaccines (eg, Prevnar) are used to assess response to protein antigens.
Antibody titers to other vaccines (eg, hepatitis A and hepatitis B, measles, others) can also be
used.
● Measurement of antibody titers to multiple serotypes in pneumococcal polysaccharide

vaccines (eg, Pneumovax 23, Pnu-imune 23) or if the patient is on immunoglobulin therapy:
Salmonella Typhi M vaccine, Typhim Vi are used to assess response to polysaccharide
antigens. This evaluation is useful in adults and children older than two years. This response
is particularly important in making a diagnosis of selective antibody deficiency. (See "Specific
antibody deficiency".)
● Isohemagglutinins are antibodies mostly of the IgM isotype generated in response to

polysaccharides of gut flora that cross-react with A or B blood group erythrocyte antigens.
They generally appear in the blood by six months of age in individuals who have blood types
other than AB. Very low titers in a child suggests poor antibody function. Similarly, antibodies
to streptococcal antigens (eg, Streptolysin O and Anti-DNAase) are normally present in all
subjects after two years of age; very low titers also hint at antibody deficiency. However,
vaccine responsiveness is generally considered to be a more reliable indicator of intact
humoral immune function. Isohemagglutinin testing is reviewed in more detail separately.
(See "Assessing antibody function as part of an immunologic evaluation", section on
'Isohemagglutinin testing'.)

Assessing vaccine responses is reviewed in detail separately. (See "Assessing antibody function
as part of an immunologic evaluation".)
Measurement of immunoglobulin loss — Low levels of immunoglobulins are occasionally due

to loss of immune globulin into the gastrointestinal tract, urine, lung, skin, pleural space, bronchi,
or peritoneal fluid during dialysis in patients with certain chronic diseases. However, there is
usually a loss of other serum proteins, including albumin and alpha-1 antitrypsin, and often
accompanying lymphopenia in these disorders. A method of evaluating the rate of protein loss is
reviewed separately. (See "Protein-losing gastroenteropathy", section on 'Diagnosis'.)
Defects in cellular immunity — Specific cellular immunity is mediated by T cells, and defects

affecting these T cells underlie the most severe immunodeficiencies. However, because antibody
production by B cells requires intact T cell function, most T cell defects lead to combined (cellular
and humoral) immunodeficiency.
Thus, evaluation of T cell numbers and function is critical in patients with "just" antibody
deficiencies, especially in those with certain infections or significant autoimmunity. An evaluation of
cellular immunity is appropriate for patients with severe viral and/or bacterial illnesses or
opportunistic infections. The disorders that can impair cellular immunity are different in various age
groups:
● In children younger than one year of age, primary immunodeficiencies are the most common
cause of impaired cellular immunity, although perinatal cytomegalovirus (CMV) and other
herpes virus infections can cause transient or persistent cellular immunodeficiency [8].
Maternal exposure to immunosuppressive medications (eg, fingolimod, azathioprine) can
cause transient cellular immunodeficiency [9].
● In older children and adults, the major causes are human immunodeficiency virus (HIV)
infection and iatrogenic immune suppression due to therapy for autoimmune disease,
malignancy, or transplantation. Occasionally, mild forms of primary combined
immunodeficiency or DiGeorge syndrome escape diagnosis until adolescence or adulthood.
The most profound combined immunodeficiencies are classified under the heading "severe
combined immunodeficiency" or SCID. SCID disorders usually present in infancy, while less
severe combined immunodeficiencies present in children and occasionally in adolescents or
adults. (See "Severe combined immunodeficiency (SCID): An overview" and "Combined
immunodeficiencies".)
Complete blood count with differential and blood smear — The complete blood count
(CBC) with differential and blood smear provides valuable information about the cellular immune
system. Lymphocyte counts in infants are normally much higher than in older children and adults

[10,11]. In many primary immunodeficiency disorders, cell populations decline over time. Thus,
normal results in the past cannot be relied upon as a reflection of the patient’s current state.
In patients suspected of having a defect in cellular immunity, the CBC evaluates for lymphopenia
and any associated gross hematologic abnormalities, some of which may greatly assist in
diagnosis. For example, patients with Wiskott-Aldrich syndrome have low numbers of small
platelets. A single finding of lymphopenia should be interpreted with caution, since transient
lymphopenia is frequently found in a variety of common infectious illnesses [12]. However,
significant lymphopenia that does not rapidly correct should not be ignored, since lymphopenia
may be the first indication of cellular immunodeficiency or another serious disease (eg, lymphoma)
( table 3) [13]. In rare situations, a normal total lymphocyte count can be seen in the presence
of a severe immunodeficiency. If the clinical presentation is highly suggestive of an underlying
disorder (eg, a patient with Pneumocystis pneumonia and invasive Candida), then lymphocyte
subsets should be evaluated with flow cytometry, even if the total lymphocyte count is normal.
Evaluation of lymphopenia — Lymphopenia can be caused by an array of disorders (

table 3). Flow cytometry to evaluate lymphocyte populations should be performed in all patients
suspected of cellular immunodeficiency with any significant infection and a total lymphocyte count
<1500 cells/microL (<2500 cells/microL in infants). In the absence of other indicators of immune
dysfunction, there should (at the very least) be subsequent measurement of the lymphocyte count
to document normalization. Persistent lymphopenia requires further investigation of immune
function. (See 'Flow cytometry for cell populations' below.)
Cutaneous delayed-type hypersensitivity — Cutaneous delayed-type hypersensitivity (DTH)

is the classic in vivo test of cellular immunity. This test measures the recall response to an
intradermal injection of an antigen to which an individual has already been exposed over a period
of time [14]. For that reason, skin testing is usually not of much value under the age of one year.
DTH testing has fallen out of common use in most centers but is reviewed briefly here.
A positive response to intracutaneous antigen injection requires uptake and processing of antigen
by antigen-presenting cells, their interaction with CD4+ helper T cells, cytokine production by T
cells, and subsequent recruitment and activation of monocytes and macrophages. Thus, skin
testing is a sensitive indicator of intact cellular immunity, but negative results must be interpreted
with caution, because an impairment in any step of the response pathway will lead to a negative
response. In addition, the DTH response is unreliable in children under one year of age (even with
prior antigen exposure) [15] and is often suppressed during viral and bacterial infection (even with
live-attenuated vaccines as in the combined measles, mumps, and rubella vaccine). DTH skin
reactivity is also suppressed by anti-inflammatory drugs (glucocorticoids) and other
immunosuppressants (cyclosporine, tacrolimus, mycophenolic acid). (See "Glucocorticoid effects
on the immune system" and "Secondary immunodeficiency induced by biologic therapies".)

Method — Delayed hypersensitivity skin responses can be assessed using an intradermal

injection of Candida antigen (Candin) or Trichophyton. These are the only commercially available
reagents intended for use in DTH testing. However, mumps skin test antigen and purified protein
derivative (PPD) of tuberculosis are also available. Tetanus toxoid is no longer available for use as
a skin test reagent. A 0.1 mL dose of undiluted Candin or Trichophyton is injected intradermally in
the volar surface of the forearm. The response is measured 48 to 72 hours after injection.
Induration of more than 5 mm diameter is considered indicative of appropriate cellular immunity in
a patient of any age. In children, induration of more than 2 mm is sometimes accepted as an
appropriate response [16]. Erythema alone without induration is not an appropriate response.
In one author's center (ERS), a panel of three reagents has been used for DTH testing:
● Candida antigen (Candin, as above)

● Tuberculin skin test (TST) (5 units or 0.1 mL)
● Trichophyton (1:500 weight to volume [w/v] ratio)
The main advantages of DTH testing are its ease and economy. It is a useful screening test in
many instances of suspected cellular immunodeficiency. A DTH test that results in no induration
when one is expected should be followed by measurement of lymphocyte populations by flow
cytometry, combined with in vitro assays of T cell function. In vitro assays (particularly mitogen
stimulation) are less sensitive to interference during intercurrent illnesses or by drugs.
Flow cytometry for cell populations — Flow cytometry uses monoclonal antibodies to

identify and quantitate cells of the hematopoietic system that have specific antigens termed
"cluster designations" (CDs). The tables list the fundamental set of markers commonly used and
the lymphocyte populations that they define ( table 4 and table 5). A typical panel of markers
used to identify the major subsets of lymphocytes includes CD3, CD4, CD8, CD19 or CD20, and
CD16 and CD56. Analysis of the expression of markers of naïve (CD45RA in combination with
CD62L or CCR7) and memory (CD45R0) T cells is important in the diagnostic approach to
patients with combined immunodeficiency (CID). The nature and derivation of the nomenclature of
the markers are discussed elsewhere. (See "Normal B and T lymphocyte development".)
A CBC with differential should be performed on a blood specimen obtained at the time of the flow
cytometric analysis, or the cytometer itself should be used to determine the lymphocyte number.
This analysis permits the calculation of the absolute numbers of each lymphocyte subset. It is
possible for the percentage of a particular subset to be abnormal while the total number of cells is
within the normal range and vice versa. For the major subsets of lymphocytes, an absolute
deficiency, rather than a relative (percentage) deficiency, is of much greater clinical significance.
Flow cytometry is invaluable in the assessment of lymphocyte subpopulations in patients with

opportunistic infections or severe or persistent lymphopenia [17,18]. Standard flow cytometry

analysis will be abnormal in almost all cases of SCID, and in many instances of other combined
immunodeficiencies ( table 6).
Note that while total B cell numbers are often normal in common variable immunodeficiency
(CVID), subpopulations of B cells are usually abnormal. In patients with CVID and autoimmunity,
CD21 low B cells are elevated in numbers; these B cells show defective central and peripheral
tolerance to self-antigens. In CVID patients with poor antibody responses, memory B cells
(CD27+) are low, and especially those that have switched their immunoglobulin isotype (IgM-
negative, IgD-negative). (See "Clinical manifestations, epidemiology, and diagnosis of common
variable immunodeficiency in adults".)
T cell numbers are broadly normal in CVID, but when T cell counts are low or patients have
opportunistic infections in what otherwise looks like CVID, one should consider a diagnosis of CID
instead. The analysis of lymphocyte subsets may be diagnostic for various forms of lymphoma as
well. The use of flow cytometry in the diagnosis of specific immunodeficiencies is reviewed in
more detail separately. (See "Flow cytometry for the diagnosis of primary immunodeficiencies".)
Abnormalities in immunodeficiency — The table summarizes anticipated alterations in

the representations of various lymphocyte populations in several immunodeficiency diseases (
table 6). Specific disorders are reviewed separately. (See "Severe combined immunodeficiency
(SCID): An overview".)
While certain immune defects are associated with characteristic patterns of lymphocyte subsets,
lymphocyte populations may appear to be entirely normal even with clinical evidence of significant
immune dysfunction. Conversely, as with total lymphocyte numbers, lymphocyte subsets may be
profoundly altered by common infectious illnesses and other factors [11,12]. Thus, for the purpose
of diagnosis, flow cytometry data must be considered in conjunction with functional tests of the
immune system.
Decreased numbers or absent natural killer CD16/56 cells (≤100 cells/microL), particularly in the
presence of recurrent infection with herpes viruses, should warrant further studies for natural killer
cell deficiencies, including cytotoxicity studies. (See "NK cell deficiency syndromes: Clinical
manifestations and diagnosis".)
Advanced tests — Many advanced tests are available to study specific cellular immune
defects. These tests are best ordered and interpreted by an immunology expert. Advanced tests
include:
● Advanced flow cytometry – Advanced flow cytometry using monoclonal antibodies specific
for activated cells, regulatory T cells, naïve or memory cells, or various stages of B cell
development are of value in further characterizing many antibody deficiencies (eg, CVID) or
cellular immunodeficiencies (eg, immune dysregulation, polyendocrinopathy, enteropathy, X-
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linked or IPEX syndrome). Flow cytometry can be used in the definitive diagnosis of several
genetic immunodeficiencies by assessing the absence or abnormal expression of a specific
protein (eg, X-linked agammaglobulinemia, Wiskott-Aldrich syndrome). Two possible factors
limiting the accuracy of this kind of testing include the existence of missense variants that
result in normal expression of the protein but abnormal function, and the existence of
missense variants that alter the epitope recognized by the antibody during flow cytometry.
(See "Flow cytometry for the diagnosis of primary immunodeficiencies".)
● TREC analysis – Measurement of T cell receptor excision circles (TRECs) by quantitative

polymerase chain reaction (PCR) analysis of peripheral blood provides an assessment of
thymic function by measuring recent thymic emigrant cells. It is primarily used as a screening
test for newborn T cell lymphopenia due to SCID on newborn blood samples. (See "Severe
combined immunodeficiency (SCID): An overview", section on 'Newborn screening'.)
● KREC analysis – Measurement of kappa-element recombination circles (KRECs) by

quantitative PCR analysis of peripheral blood for the assessment of the number of B cells is
often used in conjunction with TRECs in newborn screening procedures. Both TRECs and
KRECs can be done on dried blood spots.
● Cytotoxicity assays – Cytotoxicity assays measure the functional capacity of cytotoxic T

cells or natural killer cells and are critical for assessing effector functions that are often
defective in the familial hemophagocytic lymphohistiocytosis (fHLH) set of monogenic
immunodysregulatory disorders. One such method utilizes antigenic peptides attached to
specific major histocompatibility complex (MHC) molecules labeled with a fluorochrome as
targets. Binding of the cytotoxic cell to the peptide is measured by flow cytometry [19].
• Natural killer cell cytotoxicity is of value in the diagnosis of natural killer cell disorders,
hemophagocytic lymphohistiocytosis, and Chediak-Higashi syndrome. (See "Clinical
features and diagnosis of hemophagocytic lymphohistiocytosis", section on 'Immunologic
profile' and "Chediak-Higashi syndrome", section on 'Laboratory and imaging findings'
and "NK cell deficiency syndromes: Clinical manifestations and diagnosis", section on
'Cytotoxicity testing'.)
• T cell cytotoxicity is measured in the diagnosis of functional T cell deficiencies, where the
number of T cells is normal or near normal. (See "Severe combined immunodeficiency
(SCID): An overview", section on 'Diagnosis'.)
• Defects in T cell cytotoxicity are seen in some patients with herpes virus infections (eg,
cytomegalovirus [CMV], Epstein-Barr virus [EBV]) or HIV, when T cells are challenged
with the patient's own virally-infected cells and found to have deficient function.
● Cytokine assays

• Plasma inflammatory cytokines, such as interleukin (IL)-1, TNF-alpha, IL-6, and IL-18,
are elevated in autoinflammatory disorders and in cytokine storms associated with severe
infections, graft-versus-host reactions, and acute flares of primary or secondary
hemophagocytic lymphohistiocytosis (HLH). Serial assays may be of value in tracking the
therapeutic response.
• Cytokine autoantibodies have been associated with immune disorders including anti-IFN-
gamma, (mycobacterial infection), anti-IL-6 (staphylococcal infections), anti-IL-17A, -17F,
and -22 (mucocutaneous candidiasis), and anti-GM-CSF (pulmonary alveolar proteinosis)
[20].
• Cytokine production assays, measured in the supernatants of a patient's cells after

culture with various antigens, may be of value in identifying defects in selected patients
with severe infections or noninfectious inflammatory disorders [21], or for monogenic
disorders where skewing of T cell cytokine production is affected.
• Single cytokine assays are of limited value in the diagnosis of immunodeficiencies and
are mainly used in research.
● Genetic assays – Genetic testing to identify mutated genes are available for many primary
immunodeficiency diseases [6,22-24]. (See "Genetic testing in patients with a suspected
primary immunodeficiency or autoinflammatory syndrome" and "Combined
immunodeficiencies" and "Severe combined immunodeficiency (SCID): An overview".)
● Other advanced tests
• Chromosome analysis is of value in the diagnosis of DiGeorge syndrome. (See

"DiGeorge (22q11.2 deletion) syndrome: Clinical features and diagnosis", section on
'Genetic analysis'.)
• Short tandem repeats (STR) analysis of sorted leukocyte subsets is used to identify
chimerism in newborns with SCID.
• HLA typing is used to identify an appropriate stem cell donor. (See "Hematopoietic cell
transplantation for non-SCID primary immunodeficiencies" and "Hematopoietic cell
transplantation for severe combined immunodeficiencies".)
• CD3/T cell receptor analysis and function. (See "CD3/T cell receptor complex disorders
causing immunodeficiency".)
• X-inactivation tests can analyze the distribution of X-inactivated cells to determine the
influence of X-chromosome variants on hematopoiesis or survival of downstream cell
[25].

T cell function proliferation assays — In vitro studies of T cell function measure peripheral
blood T cell proliferation in response to several different types of stimuli [26]:
● Mitogens (such as the plant lectins phytohemagglutinin, concanavalin A or pokeweed

mitogen, or anti-CD3).
● Specific antigens (such as tetanus and diphtheria toxoids or Candida albicans antigens).
● Allogeneic lymphocytes (ie, mixed lymphocyte culture).
These studies may not be possible in patients with profound lymphopenia. In parallel with (or prior
to) studies of T cell function, flow cytometric measurement of peripheral blood lymphocyte
subpopulations should be performed. (See 'Flow cytometry for cell populations' above.)
In T cell function studies, the patient's purified peripheral blood mononuclear cells (lymphocytes
and monocytes) are incubated in sterile media with the test substance(s) or cells for three to six
days. A control tube with cells and media alone is also incubated. In the most common procedure,
tritiated thymidine is added to the cells during the last 24 hours of culture. Dividing lymphocytes
incorporate the thymidine into their DNA. The extent of proliferation is determined by measuring
the radioactivity taken up by cells. Many laboratories report results as a stimulation index (SI), the
ratio of radioactivity (as counts per minute [CPM]) with stimulation over the CPM in the control
tube without stimulation (background). This method is dependent on the number of lymphocytes in
the sample, and when during the cell cycle the thymidine is added, so as to identify maximal cell
proliferation. Tritiated thymidine incorporation should be interpreted with caution when there are
very low numbers of T cells in the blood.
Suppression of T cell responses to mitogens and antigens may be seen with significant nutritional
deficiencies, moderate-to-severe concurrent illnesses, or with administration of
immunosuppressive drugs. Thus, these tests should be performed when the patient is relatively
well and not receiving glucocorticoids or other immunosuppressive medications, whenever
possible. T cell responses appear to normalize rapidly (ie, within one day or two) after
glucocorticoids are discontinued [27].
Newer assays to assess the proliferative responses of lymphocytes utilize flow cytometry,
eliminating the need for radioactive thymidine. These tests are less susceptible to the effects of
very low numbers of T cells and should exclusively be used when evaluating SCID or severe
lymphopenia. The assays can be performed using mitogens or specific antigens (eg, tetanus or
candida antigen) as stimuli and usually measure the uptake of a thymidine analogue linked to a
fluorescent dye (5-ethynyl-2'deoxyuridine [EdU]). Alternatively, the permeable fluorescent dye
carboxyfluorescein succinimidyl ester (CFSE) can be added to the cells prior to stimulation and its
diminution with each cell division can be measured [28,29]. The "CFSE dilution" test is often
reported as percent of the initial cells that have undergone proliferation.

However the test is performed, it is interpreted as showing low proliferation, partial proliferation, or
normal proliferation by comparing patient results with healthy control lymphocytes assayed
simultaneously, and by comparison with the normal ranges for controls in the laboratory [26].
Response to mitogens — Most mitogens require functional antigen-presenting cells (ie,

monocytes and B cells) for T cell stimulation, although the mechanisms of antigen processing and
presentation are bypassed. Mitogens are powerful stimulants for T cells, and responses may be
assessed in patients of any age, even newborns, regardless of immunization status.
Normal reference ranges are derived from studies on the cells of healthy adults. Newborns
generally have higher mitogen responses compared with adults [30]. When using a tritiated
thymidine incorporation assay, depending upon the mitogens and the laboratory, the range of CPM
reported for normal controls is approximately 50,000 to 300,000, with a SI between 10 and 200.
Phytohemagglutinin (PHA) is the mitogen most commonly used. Results for patients in
comparison with controls are roughly interpreted as normal (>50 percent of control), low (25 to 50
percent), very low (10 to 25 percent), or absent (<10 percent). Results expressed as SI vary
widely between laboratories. In general, a SI <10 may be considered as no response.
Some laboratories use the monoclonal antibody OKT3 as a stimulant of T cell proliferation. OKT3
binds to the epsilon chain of the T cell CD3 complex and stimulates T cells (in the presence of
accessory or antigen-presenting cells) in a manner analogous to T cell mitogens [31]. Cell
response to mitogen or OKT3 is generally similar, although they may sometimes give different
patterns in the context of distinct immune defects [32]. There are no published comparisons in a
large group of normal individuals or immune-deficient patients. Most clinical reference laboratories
use one or more mitogens and one or more antigens, but do not routinely use OKT3.
Diminished or absent proliferation indicates a serious derangement of T cell function. Mitogen

responses will be severely depressed (usually well below the control fifth percentile) in the vast
majority of patients with profound T cell lymphopenia (eg, complete DiGeorge syndrome), SCID
[33], or advanced acquired immunodeficiency syndrome (AIDS). By comparison, the response
may be partial or normal in milder syndromes of combined deficiency (eg, Wiskott-Aldrich
syndrome), in mainly humoral deficiency, or in those with infections (especially CMV and other
herpes viruses) [34].
Response to specific antigens — Specific antigen tests are more sensitive than mitogen
assays as indicators of defects in T cell function, since the mechanisms of antigen-specific
activation go through the T cell receptor and are more complex than those for mitogens. These
tests require antigen processing and presentation by antigen-presenting cells and thus are
affected by states where antigen presenting cells are low (eg, B cell depletion following treatment
with rituximab or with monogenic deficiencies of circulating dendritic cells). As with DTH, in vitro-
specific antigen proliferation tests are not useful in infants or other patients who have not been
vaccinated.
Tetanus and diphtheria toxoids and monilia (Candida) are antigens frequently used for this assay.
Since only a small number of cells respond, the cultures must be maintained longer, and
incorporation of tritiated thymidine is lower than for mitogen stimulation. Proliferation as assessed
by CFSE or EdU incorporation dilution may also be much lower than is seen for mitogen
stimulation. Results expressed as CPM are often reported from 5000 to 50,000; results as SI
generally range from 4 to 50. In general, a SI for specific antigen >4 is usually considered
adequate, although an isolated SI <4 is not a reliable indicator of impaired cellular immunity.
In many forms of combined immunodeficiency, responses to specific antigens are diminished or

even absent while mitogen responses remain intact. In secondary immunodeficiency, specific
antigen responses will also generally be suppressed more easily than mitogen responses.
However, these generalizations are not always true [35]. As with DTH testing, booster
immunization with tetanus followed by a repeat in vitro proliferation assay may detect a significant
response.
Another way to assess function of T cells is to measure cytokine release following antigen
exposure. An example of this type of test that has become widely used in the diagnosis of latent
tuberculosis infection is the interferon release assay, which can be used instead of tuberculin
testing. Cytokine production upon stimulation with mitogens or tetanus antigen is possible. When
low or absent, cytokine production defects indicate a failure of T cell activation that often parallels
proliferative defects.
Chromosomal instability assays for radiation-sensitive patients — Tests for chromosomal

breakage, as are present in Fanconi anemia and Nijmegen breakage syndrome, are commercially
available. (See "Clinical manifestations and diagnosis of Fanconi anemia" and "Nijmegen
breakage syndrome".)
Tests for radiation sensitivity are not widely commercially available. These require radiation of
lymphocyte or fibroblast cell lines followed by analysis of chromosomal breaks and fragility [36]. A
newer generation of radiation sensitivity testing is performed by flow cytometry and relies on the
activation of specific pathways that recognize DNA damage such as gamma-H2AX and SMC1
[37].
Tests for phagocytic abnormalities — Neutrophil defects result in a range of illness from mild
recurrent skin infections to overwhelming, fatal systemic infection. Affected patients are more
susceptible to bacterial and fungal infections, but have a normal resistance to viral infections. Most
are diagnosed in infancy due to the severity of the infection or the unusual presentation of the
organism, but some escape diagnosis until adulthood.
Primary phagocytic deficiencies characteristically lead to recurrent and severe fungal (eg, Candida
and Aspergillus) and bacterial (eg, Staphylococcus aureus, Pseudomonas aeruginosa, Nocardia
asteroides, Salmonella typhi) infections. Response to nontuberculous mycobacteria may also be

abnormal, particularly in patients with chronic granulomatous disease. The most common sites of
infection are the respiratory tract and skin. Tissue and organ abscesses also occur. Other frequent
manifestations include abnormal wound healing, dermatitis/eczema, stomatitis, and delayed
umbilical cord detachment. Many patients have growth failure. Many patients have super-
abundant inflammation in sites of injury or in the colon. (See "Primary disorders of phagocyte
number and/or function: An overview".)
Phagocytic disorders can be caused by extrinsic or intrinsic defects. Extrinsic defects include
opsonic abnormalities secondary to deficiencies of antibody and complement factors, suppression
of granulocyte production, and leukocyte autoantibodies or isoantibodies that decrease the
number of circulating neutrophils. Intrinsic disorders of granulocytes may be divided into defects of
sensing infections, defects in granulocyte killing ability and defects in chemotaxis (cell movement).
The initial test for evaluation of phagocyte disorders is the CBC with differential (see "Laboratory
evaluation of neutrophil disorders"):
● Leukocytosis raises the possibility of a leukocyte adhesion defect (ie, a defect in chemotaxis
or extravasation), and flow cytometry is indicated next to assess for the presence of specific
adhesion molecules. (See "Leukocyte-adhesion deficiency".)
● Severe neutropenia can be seen with congenital agranulocytosis or cyclic neutropenia. (See
"Congenital neutropenia" and "Cyclic neutropenia".)
● Pancytopenia (combined deficiency of all blood cells) can be seen with bone marrow failure
syndromes, such as aplastic anemia and dyskeratosis congenita. (See "Inherited aplastic
anemia in children and adolescents" and "Acquired aplastic anemia in children and
adolescents" and "Aplastic anemia: Pathogenesis, clinical manifestations, and diagnosis".)
● If neutrophil numbers are normal and the patient's history is consistent with a neutrophil
disorder, then an evaluation of neutrophil function is appropriate. (See "Laboratory evaluation
of neutrophil disorders".)
A general approach to the patient with neutropenia, including identification of the cause and
determination of the infectious risk, is presented separately. (See "Overview of neutropenia in
children and adolescents" and "Approach to the adult with unexplained neutropenia".)
Tests for complement defects — Complement disorders may be inherited or acquired. Broadly,

monogenic complement deficiencies can either incur susceptibility to infection or susceptibility to
autoimmunity, with some overlaps. Screening for a classical complement pathway defect is
indicated in patients with any of the following:
● Recurrent, unexplained pyogenic infections in whom the white blood count and
immunoglobulin levels and specific antibody responses are normal

● Recurrent Neisserial infections at any age

● Multiple family members who have experienced Neisserial infections
In addition, it is reasonable to evaluate the complement system in any patient with systemic lupus
erythematosus (SLE) or a familial tendency of SLE. However, this is particularly relevant in those
with familial lupus or subacute cutaneous lupus, in whom C1q or C2 deficiency should be
excluded.
The initial screening test for complement defects is a total hemolytic complement assay (CH50),
which assesses classical pathway function and is widely available. If the CH50 is significantly
reduced or zero, then the levels of individual complement components are measured. Low levels
of multiple components, particularly low C3 and C4, suggest complement consumption, rather
than a primary complement deficiency. If the CH50 is normal and a complement defect is still
suspected, the AH50, a screening test for alternative pathway defects, may be obtained. This test
is largely performed in specialized laboratories [38]. Alternative complement deficiencies include
properdin and factor D deficiencies, both of which are rare. Each specific complement factor and
complement inhibitor can be assessed for its abundance and function in specialized laboratories.
Inherited and acquired defects in complement components are reviewed in more detail elsewhere.
(See "Inherited disorders of the complement system" and "Acquired disorders of the complement
system".)
Deficiencies or defects of C1 esterase inhibitor are associated with hereditary angioedema,

although these disorders are not associated with increased susceptibility to infection. (See
"Hereditary angioedema: Epidemiology, clinical manifestations, exacerbating factors, and
prognosis".)
Tests for innate immune defects — Defects in innate immune mechanisms should be suspected
in patients with chronic mucocutaneous candidiasis, invasive fungal infections, invasive bacterial
infections, and severe mycobacterial disease, when other immunodeficiencies have been
excluded. Many innate immune defects arise in early life (infancy and preschool years), and
should be suspected for example in a newborn with mycobacterial or fungal sepsis. Many are
associated with specific infections (eg, herpes or EBV infections in natural killer cell defects,
atypical mycobacterial infection in interleukin 12/23 (IL-12/23)-interferon-gamma (IFN-gamma)
pathway defects, herpes simplex encephalitis in the toll-like receptor 3 (TLR3) signaling pathway
and other severe infections, often associated with poor inflammatory response).
Natural killer cell defects — Natural killer cell defect can be primary or secondary. Primary
natural killer deficiency should be suspected in patients with low natural killer cells and/or
recurrent severe herpes virus infections. Natural killer defects are also noted in patients with X-
linked lymphoproliferative disorders, hemophagocytic lymphohistiocytosis, and Chediak-Higashi
syndrome. (See "NK cell deficiency syndromes: Clinical manifestations and diagnosis", section on
'Introduction'.)
Defects of IL-12/23-IFN-gamma pathways — Patients with invasive infections caused by low

virulence Mycobacterial and Salmonella species may have defects of the genetic components of
the IL-12/23-IFN-gamma pathways, including the following genes in order of incidence: the IL-12
receptor beta-1 gene (IL12RB1), IFN-gamma receptor 1 gene (IFNGR1), the IL12B gene (IL-
12p40), the IFN-gamma receptor 2 gene (IFNGR2), signal transducer and activator of transcription
1 (STAT1) gene, NEMO, and the ubiquitin-like protein ISG15 gene. Special tests involving western
blotting or flow cytometry can pinpoint the exact molecular defects and must be performed in a
research laboratory at present [23]. (See "Mendelian susceptibility to mycobacterial diseases:
Specific defects".)
Defects of IL-17 pathways — Patients with chronic mucocutaneous candidiasis can have

defects in a number of specific pathways, including the IL-17 receptor chains IL-17RA and Il-
17RC, in the IL-17F cytokine, in the STAT1 gene that lead to gain-of-function defects, and in the
protein ACT1 (gene name TRAF3IP2) that allows for responses to the IL-17 cytokines. Defects in
making T helper 17 cells due to deficiencies of the IL-23 receptor and in the master transcription
factor RORC lead to susceptibility to candidiasis as well. Finally, autoantibodies that block IL-17 or
its receptor have been described as the possible cause of chronic mucocutaneous candidiasis in
AIRE deficiency (APECED or APS1). (See "Chronic mucocutaneous candidiasis".)
Toll-like receptor pathway defects — Pattern recognition receptors (PRRs) are receptors

specific for molecular components of micro-organisms widely expressed on phagocytic cells
(neutrophils, macrophages, and monocytes and several other cells). Defects in PRRs can cause
increased susceptibility to infections that is most severe in infancy, before the adaptive (ie,
humoral and cellular) immune system has developed. The frequency and severity of infections
generally lessen as the patient matures, in contrast to most other types of immunodeficiency [39].
(See "An overview of the innate immune system".)
The toll-like receptors (TLRs) are one group of PRRs that, after ligation with specific micro-
organisms, initiate a series of steps that eventually activate the nucleus to initiate cytokine
synthesis. Several genetic disorders of the signaling pathways have been identified, including an
IL-1 receptor-associated kinase 4 defect (IRAK4), a myeloid differentiation primary response gene
88 defect (MYD88), a nuclear factor (NF)-kappa-B essential modulator defect (NEMO), a TLR3
receptor defect, and a UNC93B defect. Screening tests to evaluate these defects are available,
including a flow cytometric assay [6,23,24]. (See "Toll-like receptors: Roles in disease and
therapy", section on 'TLR signaling defects in primary immunodeficiency' and "Flow cytometry for
the diagnosis of primary immunodeficiencies".)
Advanced genomic studies for all forms of immunodeficiencies — Primary immunodeficiency

diseases are monogenic diseases and are named after the genes that undergo either loss- or
gain-of-function due to pathogenic variants in the gene. Thus, a central step in making the
diagnosis of PID includes genetic testing. Next-generation sequencing (NGS) tools include whole

exome sequencing (WES), whole genome sequencing (WGS), and target enrichment NGS ("gene
panels"). Genetic testing techniques had previously been performed at research centers, but
availability has expanded through a number of academic and commercial laboratories. Due to the
decrease in cost and increased availability, every patient with suspected primary
immunodeficiency should have a genetic diagnosis as part of their workup [40-50]. Increasingly,
genetic testing is performed earlier in the workup, and results of genetic tests can inform the need
for other laboratory tests. Older techniques such as linkage analysis and homozygosity mapping
worked well for identifying monogenic traits, particularly in patients with syndromic features,
although a large pedigree or several smaller pedigrees were needed. Microarray methods of
comparative genomic hybridization can detect relatively large areas of gain or loss of copy number
and may identify clinically relevant change. (See "Next-generation DNA sequencing (NGS):
Principles and clinical applications".)
Targeted NGS is a cost-effective method to screen for defects in known primary

immunodeficiency-associated genes. It can identify patients with atypical presentations of known
genetic defects and can also be used to identify the defect in patients with a primary
immunodeficiency that has multiple associated candidate genes. Most panels allow for
identification of copy number variation because they use high numbers of NGS reads. WES is
usually performed next if targeted NGS screening is uninformative. As the cost of WES and WGS
decreases, the role of gene panels may become less clear.
WES assesses about 2 percent of the genome that encompasses most of the coding genes (the
exons) and the few nucleotides that surround each exon (regions of splicing). WGS includes the
entire genome including the introns. Thus, WES is less laborious and expensive, and easier to
analyze. Mutations found by WGS in the deep intronic regions or in the promoter regions of genes
are difficult to interpret by themselves. Thus WGS is almost always performed in the research
setting and is often coupled with RNA sequencing to allow for interpretations of alternative splicing
and transcriptional levels. Both WES and WGS are used for identifying rare genetic defects,
particularly in patients with early-onset severe disease. Validating a new variant either in a newly-
described or a known gene is extraordinarily difficult, as the underlying biology needs to be
thoroughly understood, and the putatively pathogenic variants often have to be recreated in the
lab to understand their impact on signaling or other aspects of the immune response.
These techniques also work well if several genes are involved (polygenic traits), although analysis
can be challenging and laborious in that circumstance. Mutations identified by NGS methods still
require further study to determine the biologic effects of the mutations identified.
SOCIETY GUIDELINE LINKS
Links to society and government-sponsored guidelines from selected countries and regions
around the world are provided separately. (See "Society guideline links: Primary
SUMMARY AND RECOMMENDATIONS
● Immunodeficiency disorders should be considered once the more common causes of

recurrent infection have been excluded. (See "Approach to the child with recurrent infections"
and "Approach to the adult with recurrent infections".)
● Before initiating immunologic testing, the clinician should perform a thorough clinical history
and physical examination. In infants and children, height and weight records should be
reviewed to detect failure to thrive and poor growth. Routine laboratories can provide clues
about other causes of infections and help focus further immunologic evaluation. Screening
laboratories include complete blood count (CBC) with differential, chemistry panels, urinalysis,
test to diagnose specific organ involvement (eg, sinus computed tomography [CT] or chest
radiograph), and C-reactive protein or erythrocyte sedimentation rate to look for elevations
that may accompany chronic infections. (See 'Initial approach to the patient' above.)
● Every patient with a clinical phenotype compatible with a primary immunodeficiency disease
should be referred to an immunology specialist early in the evaluation, when possible.
Immunologic testing is best performed in a graded fashion, because many tests require
varying degrees of expertise to perform and interpret, may not be widely available, and are
often costly. (See 'Referral' above.)
● Defects in humoral immunity, which account for about 65 percent of primary

immunodeficiencies, usually result in recurrent and severe sinopulmonary infections with
encapsulated bacterial strains. Immunoglobulin G (IgG), immunoglobulin A (IgA), and
immunoglobulin M (IgM) should be measured initially. The next step in evaluation is the
measurement of serum-specific antibody titers (IgG) in response to intentional immunization
(vaccine response) or natural infection. (See 'Antibody deficiency and defects' above and
"Assessing antibody function as part of an immunologic evaluation".)
● Defects in cellular immune function (T cell disorders) are often accompanied by antibody
defects, yielding combined immunodeficiencies. Isolated cellular defects and combined
defects together account for about 20 percent of primary immunodeficiencies. These
disorders should be considered in any child presenting with recurrent or severe viral and/or
bacterial illnesses or opportunistic infections in the first year of life. Presentation in adulthood
is uncommon, although it does occur. (See 'Defects in cellular immunity' above.)
• Lymphopenia is an early indicator of severe combined immunodeficiency (SCID) in

infants and of combined immunodeficiencies in older children and occasionally adults.
Lymphopenic newborns are usually identified by TREC analysis of heel stick blood. The

presence of lymphopenia should be followed by flow cytometry to determine which

lymphocyte subsets are deficient ( table 4 and table 5). In most cases, the
evaluation should be repeated at several points in time, as viral infections and other
factors can influence lymphocyte counts. (See 'Complete blood count with differential and
blood smear' above and 'Evaluation of lymphopenia' above.)
• An overview of the anticipated alterations in various lymphocyte populations observed in

several immunodeficiency diseases is provided ( table 6). (See 'Abnormalities in
immunodeficiency' above and "Flow cytometry for the diagnosis of primary
• Cutaneous delayed-type hypersensitivity (DTH) testing is a sensitive indicator of intact

cellular immunity, because a normal recall response to an antigen requires that multiple
cell types and cellular interactions are functional. For these reasons, DTH testing can be
used as a simple and economical means to exclude a defect in cellular immunity in adults
and older children. However, negative results must be interpreted with caution, because
there are multiple reasons for nonresponse. (See 'Cutaneous delayed-type
hypersensitivity' above.)
• In vitro studies of T cell function measure peripheral blood T cell proliferation in response
to mitogens or antigens. Mitogen responses may be assessed in patients of any age,
regardless of immunization status. Specific antigen tests are only useful in patients who
have been previously immunized to that antigen, but may be more sensitive than mitogen
assays as indicators of defects in T cell function. (See 'T cell function proliferation assays'
above.)
• Advanced tests for T cell deficiencies may include cytokine levels, cytokine release
assays, autoimmune anti-cytokine antibodies, radiosensitivity assays, and cytotoxic
assays using human leukocyte antigen (HLA)-matched target cells.
● Primary phagocytic cell deficiencies, which account for about 10 percent of primary
immunodeficiencies, characteristically lead to recurrent and severe fungal (eg, Candida and
Aspergillus) and bacterial infections. The most common sites of infection are the respiratory
tract and skin, but deep tissue and organ abscesses are also seen. Initial testing for these
disorders is the CBC with differential, followed by various specific tests of phagocyte function,
depending upon the disorder suspected. (See 'Tests for phagocytic abnormalities' above.)
● Complement defects should be considered in patients with recurrent pyogenic infections with
normal white blood cell counts and immunoglobulins and in patients with a personal or family
history of recurrent neisserial infections. A total hemolytic complement (CH50) is the initial
screening test. (See 'Tests for complement defects' above.)

● Defects in innate immune mechanisms are rare disorders that should be suspected in
patients with recurrent infections, often of a specific type (eg, herpes viruses), in which other
immunodeficiencies have been excluded. (See 'Tests for innate immune defects' above.)
● PID diseases are monogenetic diseases, and with the advent of low cost next-generation
sequencing, we recommend that all patients with PID be offered a genetic diagnosis. Whole
exome sequencing (WES) or next-generation sequencing (NGS) by "gene panels" are well-
established approaches to identify pathogenic variants underlying primary
immunodeficiencies. Whole genome sequencing (WGS) and RNA sequencing offer even
more advanced approaches but are typically performed in the research setting due to the
difficulty in analysis. (See 'Advanced genomic studies for all forms of immunodeficiencies'
above.)
ACKNOWLEDGMENT
The editorial staff at UpToDate would like to acknowledge Francisco A Bonilla, MD, PhD, who
contributed to an earlier version of this topic review.
Use of UpToDate is subject to the Subscription and License Agreement.
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48. Al-Mousa H, Abouelhoda M, Monies DM, et al. Unbiased targeted next-generation

sequencing molecular approach for primary immunodeficiency diseases. J Allergy Clin
Immunol 2016; 137:1780.
49. Kojima D, Wang X, Muramatsu H, et al. Application of extensively targeted next-generation
sequencing for the diagnosis of primary immunodeficiencies. J Allergy Clin Immunol 2016;
138:303.
50. Meyts I, Bosch B, Bolze A, et al. Exome and genome sequencing for inborn errors of
immunity. J Allergy Clin Immunol 2016; 138:957.
Topic 3903 Version 23.0

GRAPHICS
Chest radiographs with and without thymic shadows
The top chest radiograph (A) shows a normal thymic shadow. The bottom
radiograph (B) shows absence of the thymic shadow in an infant with
severe combined immunodeficiency (SCID).
Courtesy of J Pierre Sasson, MD.
Graphic 67690 Version 5.0

Levels of immunoglobulins in sera of normal subjects by age*

Total immunoglobulin
Age IgG (mg/dL) IgM (mg/dL) IgA (mg/dL)
(mg/dL)
Newborn 1031±200¶ 11±5 2±3 1044±201
1 to 3 months 430±119 30±11 21±13 481±127
4 to 6 months 427±186 43±17 28±18 498±204
7 to 12 months 661±219 54±23 37±18 752±242
13 to 24 months 762±209 58±23 50±24 870±258
25 to 36 months 892±183 61±19 71±37 1024±205
3 to 5 years 929±228 56±18 93±27 1078±245
6 to 8 years 923±256 65±25 124±45 1112±293
9 to 11 years 1124±235 79±33 131±60 1334±254
12 to 16 years 946±124 59±20 148±63 1153±169
Adults 1158±305 99±27 200±61 1457±353
IgG: immunoglobulin G; IgM: immunoglobulin M; IgA: immunoglobulin.
* The values were divided from measurements made in 296 healthy children and adults. Levels were
determined by the radial diffusion technique using specific rabbit antisera to human immunoglobulins.
¶ One standard deviation.

Reproduced with permission from Pediatrics, Vol. 37, 715-27, Copyright © 1966 by the AAP.

Conditions associated with elevated serum IgE*

Parasitic:
Ascariasis
Schistosomiasis
Strongyloidiasis
Infectious diseases Human immunodeficiency virus (HIV) infection

Mycobacterium tuberculosis
Cytomegalovirus
Epstein-Barr virus
Leprosy
Candidiasis
Allergic bronchopulmonary aspergillosis
Allergic fungal rhinosinusitis
Atopic diseases Atopic dermatitis
Allergic asthma
Allergic rhinitis
Hyperimmunoglobulin E syndrome (multiple mutations)
Wiskott-Aldrich syndrome
Netherton disease
Nezelof syndrome
Immunodeficiencies
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome
(IPEX)
Omenn syndrome
Atypical complete DiGeorge syndrome
Eosinophilic granulomatosis with polyangiitis (Churg-Strauss)
Inflammatory diseases Kawasaki disease
Kimura disease
Hodgkin lymphoma
Neoplasms
IgE myeloma
Tobacco smokers
Cystic fibrosis
Nephrotic syndrome
Others
Bone marrow transplantation
Graft versus host disease
Bullous pemphigoid
Drug effect Aztreonam, penicillin G
IgE: immunoglobulin E.
* Allergic disease is the most common etiology of elevated IgE in industrialized countries, whereas parasitic
infection is the most common cause in developing countries.

Causes of lymphocytopenia
Infection
Viral (eg, HIV, SARS, SARS-CoV-2, influenza, measles, hepatitis)
Bacterial (eg, tuberculosis, typhoid fever, brucellosis)
Fungal (eg, histoplasmosis)
Parasitic (eg, malaria)
Congenital immunodeficiency disorders
Iatrogenic
Immunosuppressive agents (eg, glucocorticoids, antilymphocyte globulin, alemtuzumab, rituximab)
Chemotherapy (eg, fludarabine, cladribine), hematopoietic cell transplantation
Radiation therapy (eg, total body irradiation, radiation injury)
Systemic disease
Autoimmune disorders (eg, systemic lupus erythematosus, rheumatoid arthritis, Sjögren syndrome)
Lymphoma
Other malignancies
Sarcoidosis
Renal failure
Aplastic anemia, pancytopenia
Cushing's syndrome
Other causes
Alcohol abuse
Zinc deficiency
Malnutrition, stress, exercise, trauma
Thoracic duct leak, rupture, diversion
Protein-losing enteropathy
Idiopathic CD4+ lymphocytopenia
HIV: Human immunodeficiency virus; SARS: Severe acute respiratory syndrome; SARS-CoV-2: SARS
coronavirus 2.

Markers commonly used for assessment of lymphocyte subsets by flow cytometry

Marker name Cell type Comment
CD3 T cells Expressed on all T cells and no other cell type
CD4 T cell subset Predominantly helper/inducer T cells
CD8 T cell subset Predominantly cytotoxic T cells; expressed by up to one-third of NK cells
CD19 or CD20 B cells
CD16 NK cells Some NK cells may not express CD16
CD56 NK cells Expressed on the majority of NK cells
Expressed on the majority of NK cells; combinations of CD16, CD56, and
CD57 NK cells
CD57 will more reliably evaluate NK cell number than any marker alone
CD45RA Naïve T cells
CD45RO Memory T cells
CD: cluster of differentiation; NK: natural killer.

Normal human blood lymphocyte subpopulations at various ages

Age groups
Subpopulations 2 to 3 4 to 8 12 to 23
Core blood 2 to 5 years 7 to 17 years Adult
months months months
Total lymphocytes
Median
cells/uL (%
5400 (41%) 5680 (66%) 5990 (64%) 5160 (59%) 4060 (50%) 2400 (40%) 2100 (32%)
total
leukocytes)
4200 (35%) 2920 (55%) 3610 (45%) 2180 (44%) 2400 (38%) 2000 (36%) 1600 (28%)
Confidence
to 6900 to 8840 to 8840 to 8270 to 5810 to 2700 to 2400
intervals
(47%)* (78%) (79%) (72%) (64%) (43%)* (39%)*
CD3 T cells
Median
cells/uL (%
3100 (55%) 4030 (72%) 4270 (71%) 3300 (66%) 3040 (72%) 1800 (70%) 1600 (73%)
total
lymphocytes)
2400 (49%) 2070 (55%) 2280 (45%) 1460 (53%) 1610 (62%) 1400 (66%)
Confidence 960 (61%) to
to 3700 to 6540 to 6450 to 5440 to 4230 to 2000
intervals 2600 (84%)*
(62%)* (78%) (79%) (81%) (80%) (76%)*
CD4 T cells
Median
cells/uL (%
1900 (35%) 2830 (52%) 2950 (49%) 2070 (43%) 1800 (42%) 800 (37%) 940 (46%)
total
lymphocytes)
1500 (28%) 1460 (41%) 1690 (36%) 1020 (31%)
Confidence 900 (35%) to 700 (33%) to 540 (32%) to
to 2400 to 5116 to 4600 to 3600
intervals 2860 (51%) 1100 (41%)* 1660 (60%)*
(42%)* (64%) (61%) (54%)
CD8 T cells
Median
cells/uL (%
1500 (29%) 1410 (25%) 1450 (24%) 1320 (25%) 1180 (30%) 800 (30%) 520 (27%)
total
lymphocytes)
1200 (26%)
Confidence 650 (16%) to 720 (16%) to 570 (16%) to 630 (22%) to 600 (27%) to 270 (13%) to
to 2000
intervals 2450 (35%) 2490 (34%) 2230 (38%) 1910 (38%) 900 (35%)* 930 (40%)*
(33%)*
B cells¶
Median
cells/uL (%
1000 (20%)¶ 900 (23%) 900 (23%) 900 (23%) 900 (24%) 400 (16%)¶ 246 (13%)¶
total
lymphocytes)
Confidence 200 (14%) to 500 (19%) to 500 (19%) to 500 (19%) to 700 (21%) to 300 (12%) to 122 (10%) to
intervals 1500 (23%)* 1500 (31%) 1500 (31%) 1500 (31%) 1300 (28%) 500 (22%)* 632 (31%)*
CD4:CD8 ratio
Median 1.2 2.2 2.1 1.6 1.4 1.3 1.7
Confidence
0.8 to 1.8 1.3 to 3.5 1.2 to 3.5 1.0 to 3.0 1.0 to 2.1 1.1 to 1.4 0.9 to 4.5
intervals
Confidence intervals given are the 5th to 95th percentiles except where indicated (*). B cells use the CD20
antigen except where indicated (¶).
Each subgroup contains at least 22 health subjects.

* These are the 25th to 75th percentiles.
¶ These use the CD19 antigen.

This book chapter was published in Immunologic Disorders in Infants and Children, 5th ed, Stiehm ER, Ochs
HD, Winkelstein JA, Immunodeficiency disorders: General considerations, 311, Copyright Elsevier (2004).

Alterations of lymphocyte populations in some defined immunodeficiencies

Disease CD3 CD4 CD8 CD19/20 CD16/56/57
X-linked SCID ↓↓↓ ↓↓↓ ↓↓↓ nl, ↑ ↓
JAK3 SCID ↓↓↓ ↓↓↓ ↓↓↓ nl, ↑ ↓
IL-7R alpha SCID ↓↓↓ ↓↓↓ ↓↓↓ nl, ↑ nl, ↑*
SCID due to recombination defects ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ nl, ↑*
ADA deficiency ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
PNP deficiency ↓ ↓p ↓p nl nl, ↑*
MHC class II deficiency ↓ ↓↓ ↓↓ nl nl
Reticular dysgenesis ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
CD3 deficiency ↓↓↓ ↓↓↓ ↓↓↓ nl nl
ZAP-70 deficiency nl nl ↓↓ nl nl
NK deficiency nl nl nl nl ↓↓↓
XLA nl nl nl ↓↓↓ nl
WAS ↓ ↓p ↓p nl nl
AT ↓ ↓ ↓ ↓ nl
DiGeorge anomaly nl, ↓ nl, ↓ nl, ↓ nl nl
CVID nl, ↓ nl, ↓ nl, ↓ nl, ↓ nl
SCID: severe combined immunodeficiency; JAK-3: Janus kinase 3; IL-7 R alpha: interleukin-7 receptor alpha
chain (CD127) defect; SCID due to recombination defects: these include recombinase-activating genes 1 and
2 (RAG1 and RAG2), Artemis, DNA protein kinase catalytic subunit; ADA deficiency: adenosine deaminase
deficiency; PNP deficiency: purine nucleoside phosphorylase deficiency; MHC: major histocompatibility
complex; CD3 deficiency: deficiency of a component of CD3 (gamma, epsilon); ZAP-70 deficiency: deficiency
of the CD3 zeta-associated 70 kd tyrosine kinase; NK deficiency: primary natural killer cell deficiency; XLA:
X-linked agammaglobulinemia; WAS: Wiskott-Aldrich syndrome; AT: ataxia-telangiectasia; CVID: common
variable immunodeficiency; nl: normal; p: the decrease is progressive over time.
* The percentage of natural killer (NK) cells may be increased due to the absence of the subset principally
affected in the disease. The absolute NK cell number is generally normal.

Contributor Disclosures
Manish J Butte, MD, PhD Grant/Research/Clinical Trial Support: Shire [Immunoglobulin].
Consultant/Advisory Boards: Horizon Pharma [Interferon-gamma]. Speaker's Bureau: CSL, Grifols
[Immunoglobulin]. E Richard Stiehm, MD Consultant/Advisory Boards: ADMA Biologics [Infections of
respiratory syncytial virus in organ transplants]. Luigi D Notarangelo, MD Nothing to disclose Anna M
Feldweg, MD Nothing to disclose
Contributor disclosures are reviewed for conflicts of interest by the editorial group. When found, these are
addressed by vetting through a multi-level review process, and through requirements for references to be
provided to support the content. Appropriately referenced content is required of all authors and must conform
to UpToDate standards of evidence.
Conflict of interest policy

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7/9/2021 Laboratory evaluation of the immune system - UpToDate

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Laboratory evaluation of the immune system

● (See "Primary humoral immunodeficiencies: An overview".)

INITIAL APPROACH TO THE PATIENT

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● Autoimmune disorders, such as autoimmune hemolytic anemia

● Family history of infections, abnormal inflammatory responses, and autoimmune disorders, as

● Family history of lymphoproliferative illnesses, including occurrence of splenectomy.

● Any associated nonimmunologic symptoms and signs, as revealed by a complete review of

Physical examination — Physical examination findings that suggest immunodeficiency in

● Complete blood count (CBC) with differential – Lymphopenia is characteristic of a variety of

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● Chest radiograph – Chest radiographs of an infant showing absence of a thymic shadow

● Erythrocyte sedimentation rate and/or C-reactive protein – Nonspecific elevations in acute-

CATEGORIES AND PREVALENCE OF IMMUNODEFICIENCIES

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● Predominantly antibody deficiencies (55 percent)

EVALUATION FOR SPECIFIC TYPES OF DISORDERS

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Measurement of antibody levels — Measurement of serum IgG, IgA, and immunoglobulin M

● Measurement of IgE is helpful in patients with recurrent sinopulmonary infections or scaly or

Measurement of antibody function — Antibody function can be assessed by measuring

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● Measurement of antibody titers to tetanus, diphtheria, H. influenzae B, and protein-conjugated

● Measurement of antibody titers to multiple serotypes in pneumococcal polysaccharide

● Isohemagglutinins are antibodies mostly of the IgM isotype generated in response to

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Measurement of immunoglobulin loss — Low levels of immunoglobulins are occasionally due

Defects in cellular immunity — Specific cellular immunity is mediated by T cells, and defects

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Evaluation of lymphopenia — Lymphopenia can be caused by an array of disorders (

Cutaneous delayed-type hypersensitivity — Cutaneous delayed-type hypersensitivity (DTH)

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Method — Delayed hypersensitivity skin responses can be assessed using an intradermal

● Candida antigen (Candin, as above)

Flow cytometry for cell populations — Flow cytometry uses monoclonal antibodies to

Flow cytometry is invaluable in the assessment of lymphocyte subpopulations in patients with

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Abnormalities in immunodeficiency — The table summarizes anticipated alterations in

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● TREC analysis – Measurement of T cell receptor excision circles (TRECs) by quantitative

● KREC analysis – Measurement of kappa-element recombination circles (KRECs) by

● Cytotoxicity assays – Cytotoxicity assays measure the functional capacity of cytotoxic T

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• Cytokine production assays, measured in the supernatants of a patient's cells after

● Other advanced tests

• Chromosome analysis is of value in the diagnosis of DiGeorge syndrome. (See

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● Mitogens (such as the plant lectins phytohemagglutinin, concanavalin A or pokeweed

● Allogeneic lymphocytes (ie, mixed lymphocyte culture).

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Response to mitogens — Most mitogens require functional antigen-presenting cells (ie,

Diminished or absent proliferation indicates a serious derangement of T cell function. Mitogen

In many forms of combined immunodeficiency, responses to specific antigens are diminished or

Chromosomal instability assays for radiation-sensitive patients — Tests for chromosomal

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Tests for complement defects — Complement disorders may be inherited or acquired. Broadly,