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182 Textbook of Diagnostic and Therapeutic Procedures in Allergy
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Immunodeficiency.” Autoimmune manifestations are not uncommon and include inflammatory
bowel disease, cytopenias, eczematous rash and enteropathy.
Severe Combined Immunodeficiency (SCID) is the most severe manifestation of T cell deficiency.
SCID occurs due to defects in genes responsible for T cell development and is characterized by
< 300 T cells/mcL in peripheral blood. Leaky SCID may occur when the expression of the
defective gene is not fully penetrant, resulting in the development of a few T cells that make it to
peripheral circulation. However, these aberrant T cells then proliferate and become reactive against
self-antigens, therefore leading to autoimmune manifestations including skin rash and enteropathy.
Alternatively, maternal T cells may engraft in a patient with SCID, leading to the expansion of these
T cells and reactivity against the infant’s tissues. Leaky SCID and maternal engraftment can present
with features of autoimmunity including skin rash and enteropathy.
While SCID is the most severe of T cell defects, partial or complete lack of thymic tissue may
also present as SCID, owing to the fact that T cell precursors cannot complete the thymus-specific
final maturation step prior to exiting to the periphery as mature, naïve T cells. Partial or
complete lack of thymic tissue results in 22q11.2 deletion or DiGeorge Syndrome (DGS)
(McDonald-McGinn et al. 2015). DGS is associated with defective development of the pharyngeal
pouch system, caused by chromosomal deletion at 22q11.2. In addition to significant T cell
lymphopenia, these patients present with conotruncal cardiac anomalies and hypocalcemia due to
parathyroid hypoplasia. Complete DGS is a result of the complete absence of the thymus, whereas,
in partial or atypical DGS, a small amount of thymic tissue is present. In such cases, T cells may
develop but are frequently oligoclonal and ineffective not only at mounting adequate responses to
pathogens but also at assisting B cells to produce robust antibody responses. Complete or partial
DGS patients can present clinically similar to patients with SCID, owing to their significantly low
T cell numbers.
Other less-severe T cell-related IEI include defects in genes that encode proteins that drive
T cell development in the thymus, such as major histocompatibility complexes I and II, that are
necessary for the development of CD8 and CD4 T cells respectively (Reith and Mach 2001), or
molecules that are necessary for T cell signaling, such as ZAP70 (Walkovich and Vander Lugt
2021). Rare defects in calcium sensor and calcium-release activated channels due to genetic defects
in STIM1 and ORAI1 are associated with defective T cell activation and T cell deficiency along with
myopathy (Feske et al. 2010).
Recognition of the cause of T cell lymphopenia is critical as it impacts the management of
these patients. If the defect lies in genes that are integral to the T cells, then these patients may
benefit from bone marrow or hematopoietic stem cell transplant to replace defective hematopoietic
precursors (Haddad and Hoenig 2019), or from enzyme replacement therapy (e.g., ADA enzyme
replacement in ADA-deficient SCID) (Booth and Gaspar 2009), or gene therapy, where the patient’s
hematopoietic stem cells are transfected with a viral vector carrying the corrected gene (Cicalese
and Aiuti 2015; Cicalese et al. 2018). However, if the defect lies in the thymus as in DGS, a thymic
implant may be needed depending on the severity of the defect (Davies et al. 2017).
In contrast to inherited defects of T cells which can manifest with severe, life-threatening infections
with opportunistic microorganisms within the first few months of life, B cell developmental defects
tend to present clinically after about 6 months of age (Demirdag and Gupta 2021). This is because
newborns are protected by maternal immunoglobulins that are at their highest level at birth and
wane over the course of the next 5–6 months. If infants are unable to produce adequate levels of
immunoglobulins as maternal immunoglobulin wanes, their susceptibility to infection, particularly
at mucosal surfaces, increases. Transient hypogammaglobulinemia of infancy (THI) may occur in
infants > 6 months of age in whom IgG levels are significantly lower than the expected reference
B Cell Deficiency
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range limit (Dalal et al. 1998). THI generally resolves with age, often within the first 2 years of life,
but has also been known to persist into early childhood.
Defects of B cell development leading to antibody deficiency commonly present clinically with
increased susceptibility to encapsulated bacteria (e.g., S. pneumoniae, H. influenzae) leading to
recurrent sinopulmonary infections, otitis media and pneumonia or gastrointestinal infections with
organisms, such as Salmonella species, Campylobacter jenuni and rotavirus or with parasites such
as Giardia lamblia. More severe infectious presentations include meningitis, infectious arthritis and
osteomyelitis due to Pseudomonas aeruginosa, Staphylococcus aureus or Mycoplasma (reviewed
in Demirdag and Gupta 2021).
The prototypical humoral or antibody deficiency is X-linked agammaglobulinemia (XLA),
which occurs due to pathogenic variants of the gene encoding Bruton’s Tyrosine Kinase (BTK)
(Bruton 1952). BTK is critical for the early development of B cells in the bone marrow. Therefore,
lack of BTK leads to severely depressed production of B cells and < 1% of B cells are detectable in
the peripheral circulation. As BTK is encoded on the X-chromosome, this disorder manifests in boys
who may present after about 6 months of age with bacterial or viral infections, absent or severely
decreased B cells in peripheral blood and undetectable serum immunoglobulins (IgG, IgA and IgM).
A few other causes of inherited antibody deficiency include (1) selective IgA deficiency,
which is the most common immunodeficiency (Yel 2010); (2) specific antibody deficiency (SAD)
which is characterized by generally normal numbers of B cells and normal immunoglobulin levels,
but impaired responses to vaccines (Ambrosino et al. 1987); (3) hyper IgM syndrome, which is
characterized by high levels of IgM and inability to mount adequate antibody responses to infection
(Notarangelo et al. 1992); (4) Common Variable Immunodeficiency (CVID), which is characterized
by low IgG, often accompanied by low IgA and/or IgM and even though B cells may be present in
normal numbers, they are unable to develop into memory B cells (Bonilla et al. 2016); (5) IgG subclass
deficiency which may be associated with impaired responses to pneumococcal polysaccharides
(IgG2 deficiency) or with a variety of inflammatory diseases (IgG4-related diseases).
The infectious manifestations of antibody deficiency are managed with appropriate antibiotics,
while the underlying immunodeficiency, i.e., low immunoglobulins, is treated with immunoglobulin
replacement therapy either in the form of intravenous immunoglobulin (IVIG) or subcutaneous
immunoglobulin (SCIG). Hyper IgM syndrome, due to pathogenic variants in CD40 ligand, may
require a bone marrow transplant to correct the underlying gene defect.
Neutrophils comprise the body’s initial defense mechanism against infection. Therefore, primary
neutrophil defects typically present early in life when the adaptive immune system (T and
B cells) is still maturing. Patients with neutrophil defects frequently present with severe, often
life-threatening infections with bacteria, such as Staphylococcus aureus, Nocardia species and fungi
such as Aspergillus fumigatus or Candida albicans. Infectious presentations can range from mild
skin infections to septicemia. Patients may present with skin abscesses, lymph node infections and
deep-seated abscesses involving organs, such as the liver or lung infections (reviewed in Dinauer
2020). Secondary neutrophil defects are seen in patients with leukemias, myelodysplastic syndromes
and hematopoietic clonal disorders, such as paroxysmal nocturnal hemoglobinuria (PNH) (Toma
et al. 2012).
Defects of neutrophils are classified into defects of neutrophil numbers (neutropenia) or
defects of neutrophil function. Neutropenia may be mild (1,000–< 1,500 cells/mcL), moderate
(500–< 1,000 cells/mcL), or severe (< 500 cells/mcL) and are either due to pathogenic variants of
genes that are critical to neutrophil development (e.g., Griscelli syndrome and Chediak-Higashi
syndrome) (Donadieu et al. 2017) or due to extrinsic factors such as nutritional deficiency, infections
or autoimmunity (Boxer 2012).
Neutrophil Defects
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Functional neutrophil defects include CGD and LAD, among other, much less frequently
encountered, defects. CGD predominantly occurs as an X-linked disease and therefore manifests in
boys, often within the first year of life, with severe bacterial or fungal infections of the skin, lymph
nodes and lungs. Autosomal recessive (AR) forms of the disease can present in either sex. Infectious
presentations in the AR form of the disease are generally milder and tend to present in older children
or adults. Patients with CGD have severely decreased or absent neutrophil oxidative burst, which
is critical for control of bacterial and fungal infections. Neutrophil oxidative burst is mediated by
the NAPDH oxidase complex, which is composed of six proteins—gp91phox, p22phox, p47phox,
p67phox and p40phox and Rac-2. X-linked CGD occurs due to defects in gp91phox, whereas
AR-CGD occurs due to defects in any one of the other proteins (Yu et al. 2021). LAD is characterized
by the inability of leukocytes to exit the vasculature and migrate into tissue spaces. Three LAD
syndromes, all inherited in an AR manner, have been identified: LAD I, LAD II and LAD III.
Leukocyte Adhesion Deficiency I (LAD I) occurs due to defects in neutrophil surface proteins
known as β2-integrins. This family of surface receptors is essential for enabling neutrophils to arrest
the vascular endothelium in response to an infectious stimulus and through diapedesis, enter tissue
spaces to clear the infection. Pathogenic mutations of CD18, the beta-chain of the β2 integrin family,
lead to severely decreased or loss of β2 integrin expression, leading to an inability of neutrophils
to migrate to sites of infection. The classical clinical presentation in LAD I is omphalitis (delayed
separation of the umbilical stump). Patients also can present with skin, respiratory tract, perianal or
bowel infections. Erosive gingivitis is another feature. Characteristic findings in LAD I include the
absence of pus at sites of infection as neutrophils are unable to migrate to these sites and cause a
dramatic increase in neutrophils in the peripheral circulation (leukocytosis) (Etzioni 2009).
LAD II occurs due to defective fucosylation of macromolecules. Pathogenic variants of the
guanosine diphosphate (GDP)-fucose transporter gene (SLC35C1) result in impaired transport
of fucose to the Golgi complex, thereby affecting fucosylation of macromolecules. This defect
impacts sialyl Lewis X or CD15s, which is the ligand for selectins that are expressed on vascular
endothelium. The interaction of CD15s and selectins is essential for neutrophil rolling and tethering
to the vascular endothelium, prior to migration into tissue spaces. Thus, LAD II patients’ neutrophils
are functionally impaired, therefore leading to increased susceptibility to infection. However, these
patients tend to have fewer and milder infections compared with LAD I patients. Additionally,
defective fucosylation affects H antigen expression. Therefore, LAD II is associated with the rare
Bombay (hh) blood type. Non-immune clinical findings include short stature, severe intellectual
impairment, microcephaly and distinctive facial features that include a depressed nasal bridge. As
with LAD I, neutrophilia is a consistent finding.
LAD III, previously described as the LAD I variant, is characterized by severe infections,
leukocytosis and severe bleeding disorder. The underlying defect is in the protein, kindlin-3,
encoded by the gene FERMT3. Kindlin-3 is an adapter protein that binds the intracellular portions
of β-1, β-2 and β-3 integrins and increases their activation and binding to their cognate ligands.
As these integrins are found on both neutrophils and platelets, the function of both of these blood
components is affected. Clinical features of LAD III included delayed umbilical cord separation,
increased susceptibility to infection and severe bleeding episodes that are similar to those seen in
patients with Glanzmann thrombasthenia. LAD III is also characterized by marked leukocytosis.
The complement system comprises serum as well as cell membrane-associated proteins that
interact with both innate and adaptive immune systems. By efficiently transporting potential
self-reactive nuclear products of apoptotic cells as well as inflammatory immune complexes to
the reticuloendothelial system, wherein they are phagocytosed and eliminated, complement
proteins and complement receptors maintain immune homeostasis (Shih and Murali 2015). The
tightly regulated complement pathways recognize the biochemical divergence of self-structures
Complement Defects
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from structural pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide of
gram-negative bacteria (alternative pathway or AP) or mannose present on fungi and ficolins of some
gram-positive bacteria (lectin or mannose pathway or LP). Antigen-antibody complexes activate the
Classical Pathway (CP). These events result in activation of distinct early complement proteins and
generation of convertases that converge on C3 (C3 convertases), giving rise to the anaphylatoxins
C4a and C3a as well as C3b. Besides its opsonic function, C3b functions as an enzyme in the
generation of C5a, a potent chemoattract for phagocytic cells. The anaphylatoxins (C3a, C4a and
C5a) mediate the vascular phase of inflammation while C5a recruits phagocytic cells. These events
integrate the vascular and cellular phases of immune defense and inflammation. Derangements in
this cascade of activating and regulatory proteins results in either failure of complement mediated
defenses and hence recurrent or severe infections or autoactivation of complement proteins leading
to immune inflammation, while defects in complement mediated clearance of immune complexes
and apoptotic cells cause autoimmune diseases.
Complement deficiencies account for approximately 0.06% of inherited immune deficiencies
in the general population. Complement deficiencies may manifest with primarily autoimmune
phenomena (e.g., Systemic Lupus Erythematosus), primarily infectious manifestations
(e.g., Neisserial meningitis) or immune dysregulatory defects (e.g., Hereditary Angioedema (HAE),
Hemolytic Uremic Syndrome (HUS), or Paroxysmal Nocturnal Hemoglobinuria (PNH)). Fulminant
infections with Neisseria meningitidis, including septicemia and meningitis, almost exclusively
occur in deficiencies of the terminal complement pathway proteins (C5, C6, C7, C8 and C9). HAE is
a genetically inherited disease that occurs due to low levels or abnormal function of the complement
control protein C-1 esterase inhibitor (C1-INH). C1-INH is responsible for not only regulating
activation of the complement pathway but also the regulation of the Hageman, bradykinin and
fibrinolytic pathways. This deficiency manifests as spontaneous episodes of angioedema (mediated
by bradykinin), which may be localized (hands or feet), involve mucosa of the gut resulting in
severe abdominal pain or may manifest as life-threatening laryngeal edema. The trigger for these
episodes has not been well defined (Schroder-Braunstein and Kirschfink 2019).
Table 4 depicts some of the congenital complement deficiency diseases and the resulting
infections and/or autoimmune manifestations.
Table 4. Congenital complement deficiency states and disease association.
Complement Deciency Consequences Disease Association
C1q, C1r, C1s, C4 and C2 Inability to generate/activate classical C3
C3 Defective C3b opsonic activity and
Late Components – C5, C6, C7,
C8 and C9
Factor D and Properdin Inability to generate/activate the AP C3
Mannose Binding Lectin (MBL)
and MASP-2
Factor H, I, MCP and C3
Nephritic Factors
C1-Inhibitor (C1-INH) Loss of regulation of C1 and bradykinin
DAF (CD55), Homologous
Restriction Factor (HRF) or CD59
Systemic lupus erythematosus (SLE)
convertase or CP
Recurrent childhood infections.
MAC activity, no activation of AP and
decreased inammation (no C3a activity)
Inability to form the MAC Recurrent, disseminated Neisserial
convertase
Decreased or absent activity to activate
the LP
Lack of regulation of C3 convertases by
all pathways of complement activation
activation
Failure to regulate complement activation
on autologous cells (particularly red cells)
N. meningitidis, S. pneumoniae and
other encapsulated bacteria
infections
Recurrent, disseminated Neisserial
infections
Recurrent childhood infections,
pyogenic bacteria
Membranoproliferative
glomerulonephritis, atypical hemolytic
uremic syndrome, age-related macular
degeneration.
Hereditary angioedema
PNH
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Laboratory Workup for Suspected Immunodeficiency
Detailed clinical history of recurrent, difficult-to-treat or unusual infections, family history of similar
clinical symptoms and/or early death and a thorough physical examination are crucial when suspicion
for immunodeficiency is high. While genetic analysis is becoming increasingly cheaper and more
widely available and oftentimes used as the first line of laboratory investigation, it should not replace
routine laboratory tests as they provide clinically significant and useful information when working
up a patient as well as when monitoring clinical outcomes in patients with immunodeficiency.
General laboratory workup includes complete blood count (CBC) with differential
(see Chapter 2), and serum immunoglobulin levels to assess whether the distribution of RBC and
RBC parameters, WBC (neutrophils, lymphocytes, monocytes, basophils and eosinophils), platelets
and serum immunoglobulins (IgG, IgA and IgM) are within the expected range. It is important to note
that these parameters vary considerably with age, therefore interpretation of patients’ test results in
the context of age-matched reference ranges is critical. Additionally, maternal immunoglobulin G is
transferred transplacentally to the fetus and reaches maximal levels at birth. Maternal IgG declines over
the first 6 months of life and infant IgG increases, as the newborn begins to synthesize it along with the
other immunoglobulin isotypes (IgA, IgM and IgE). Thus, assessment of serum immunoglobulins for
evaluation of antibody deficiencies is not reliable within the first 6 months of age.
Responses to the routine vaccine (antibody titers) may be assessed to determine whether the
patient is capable of responding to vaccination. The distribution of lymphocyte populations (T cells,
B cells and Natural Killer (NK) cells) may be assessed by flow cytometry. Functional analysis of
neutrophils and lymphocytes is performed in specialized laboratories when a functional deficit is
suspected. Some of the more commonly used laboratory tests for immunodeficiency work up are
presented in Table 5 and are discussed in further detail below.
Table 5. Commonly used immunological tests for workup of immunodeficiency.
Test Utility for Evaluation of Immunodeciency
TREC analysis by PCR Newborn screening for SCID
CBC with dierential Evaluation of cytopenias (e.g., anemia, neutropenia and lymphopenia), abnormal
cellular morphology (e.g., giant granules in Chediak-Higashi Syndrome)
Serum immunoglobulin
levels and serum protein
electrophoresis
Vaccine response Specic antibody titers to routinely administered vaccines (e.g., diphtheria, tetanus and
Lymphocyte subset analysis Abnormal decreases or increases in T cells (CD3, CD4 and CD8), B cells (CD19
Neutrophil oxidative burst Evaluates the ability of neutrophils to produce reactive oxygen intermediates that are
Neutrophil adherence markers Flow cytometry evaluation of expression of b2-integrins and sialyl Lewis X or CD15s
Lymphocyte proliferation Flow cytometry evaluation of the ability of lymphocytes to divide following
Complement analysis Functional testing for the integrity of the classical and alternative complement
Abbreviations: TREC; T Cell Receptor Excision Circle; Severe Combined Immunodeficiency (SCID).
Hypogammaglobulinemia, selective IgA-deciency, hyper IgM and hyper IgE
syndromes and immunodeciency due to monoclonal B cell abnormalities
pertussis (DTvP2 and DTaP), pneumococcal vaccine (PCV13 and PCV23)
and CD20) and NK cells (CD16/CD56). Markers for naïve (CD45RA) and memory
(CD45RO) T cells, naïve (IgD+CD27-) and memory (CD27+) B cells, as well as classswitched memory (IgD-CD27+) B cells are useful
critical for clearance of pathogens
on the surface of neutrophils
stimulation with a mitogen or an antigen or after T cell receptor (anti-CD3) and CD28
activation
pathways. Analysis of specic complement proteins, both level and function and
complement pathway inhibitors
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T Cell Defects
Newborn Screening for SCID
Newborn screening (NBS) for SCID was developed by the states of Wisconsin and California
in 2008. Since then, screening for SCID at birth has been implemented across the United States
and several countries across the world. NBS for SCID involves polymerase chain reaction (PCR)
amplification of a piece of DNA known as TREC or T Cell Receptor Excision Circle (Puck 2012).
TREC is generated in newly developed T cells as they re-arrange their T cell receptor genes in the
thymus and emerge as naïve T cells in the peripheral circulation. The starting sample for TREC
analysis is DNA extracted from dried blood spots following a heel prick that is routinely collected
in newborn babies to screen for a variety of metabolic diseases. Abnormally low TREC levels are
indicative of low T cell production and are followed up by analysis of T cells in peripheral circulation
by flow cytometry (Figure 1A–C and described under “Lymphocyte Subset Phenotyping”).
Newborns who test abnormal on the NBS–SCID screen but have detectable T cells in peripheral
circulation, occasionally up to 1,500 T cells/mcL, may have leaky SCID (hypomorphic mutations of
SCID-related genes), Omenn Syndrome (hypomorphic gene defects, erythroderma and elevated IgE)
or maternal T cell engraftment (Shearer et al. 2014). In these patients, analysis of the proportions of
naïve and memory T cells is invaluable in order to clinch the diagnosis (Knight et al. 2020). At birth,
the vast majority of a newborn’s T cells are antigen-inexperienced, naïve T cells which express the
glycoprotein, CD45RA. Over the course of life, as naïve T cells encounter antigen, a percentage turn
into CD45RO-expressing memory T cells. The relative proportion of memory T cells increases from
infancy to adulthood. Thus, analysis of CD45RA and CD45RO on T cells enables quantification of
the relative proportions of naïve and memory T cells. In leaky SCID, Omenn Syndrome or maternal
engraftment, the few T cells that are present are reactive memory cells and express CD45RO
(Figure 2).
Lymphocyte Subset Phenotyping
Lymphocytes in peripheral blood can be identified by tagging them with fluorescently labeled
monoclonal antibodies that are specific for certain cell surface proteins that define specific subsets
of lymphocytes. These fluorescently labeled cells can by analyzed by flow cytometry, whereby
cells are interrogated by a laser beam that activate the fluorescent molecules. Activation of these
fluorescent molecules leads to release of photons whose wavelength is converted into digital signals
that are read by specialized software. The technique enables high throughput and rapid analysis of
complex mixtures of cells, such as those found in peripheral blood (Figure 3).
Lymphocyte subset phenotyping is performed by flow cytometry analysis of peripheral blood.
In brief, whole blood (50–100 mcL) is incubated with monoclonal antibodies that are specific for
leukocyte and lymphocyte surface proteins (Table 6). Following a 15–20-minute incubation, blood
is washed by centrifugation, RBCs are lysed and the sample is analyzed by flow cytometry.
Leukocytes, which include lymphocytes, monocytes and neutrophils are identified by their
expression of CD45 and the variation in their complexity (i.e., smaller, less complex leukocytes
are lymphocytes and larger, more complex leukocytes are neutrophils). The lymphocyte population
is further analyzed for the percentages of T cells (CD3+, CD4+ and CD8+), B cells (CD19+), NK
and CD16+CD56+ cells. Flow cytometry analysis yields relative percentages of these lymphocyte
subpopulations. Absolute numbers can be calculated using the absolute lymphocyte count from the
corresponding CBC and the percentages from flow cytometry analysis and are useful parameters for
clinical follow up rather than relying on just percentages
Lymphocyte subset phenotyping is routinely performed to assess percentages and absolute
numbers of T, B and NK cells. A variation of this test is used for CD4 T cell counts for diagnosis
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SCID testing and confirmation
A B
C
Primers
TREC
TREC
Abnormal
Confirm with
flow cytometry
repeat
Indeterminate
Normal
no action
Figure 1. Screening and Confirmatory Testing for Severe Combined Immunodeficiency. (A) Newborn screening for SCID: Blood spots are collected via heel sticks from newborns. A 3
mm punch is taken from a dried blood spot and DNA is extracted. Primers specific for TREC and a housekeeping gene (generally β-actin) are used to amplify a portion of TREC and the β-actin
gene, which is used to assess the quality of the extracted DNA. If TREC levels are normal and β-actin is adequate, the result of the screen is “normal,” and no further action is required. If TREC
levels are low and β-actin amplification is inadequate, the result is “indeterminate” and a repeat PCR and/or DNA extraction is needed. If TREC levels are low and β-actin is adequate, the result
is “abnormal” and must be followed by confirmatory flow cytometry. (B) Flow cytometry analysis of peripheral blood for CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD19+ B
cells and CD16/56+ NK cells. The flow cytometry plots show analysis of a normal, healthy adult donor. (C) Flow cytometry analysis of peripheral blood collected from a newborn who had an
abnormal SCID screening test result. Note that T cells are barely detectable, whereas B cells and NK cells appear adequate. This flow cytometry result is typical of a T-B+NK+ SCID and may
also occur in 22q11 deletion syndrome (DiGeorge syndrome).
Laboratory Analysis of Primary Immunodeficiency 189
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analysis of leaky SCID, Omenn Syndrome, or maternal T-cell engraftment. CD45RO+ memory T cells are generally higher in adults (A) and CD45RA+ naïve T cells are higher in infants and
young children (B). In patients with leaky SCID, the few T cells that get to the periphery expand, develop a memory phenotype and may react against self-tissues. Panel C shows an analysis
Figure 2. Analysis of Naïve and Memory T Cells. Follow-up testing for SCID includes analysis of naïve (CD45RA+) and memory (CD45RO+) T cells. This is particularly important for the
of T cells in leaky SCID, where close to 100% of T cells are CD45RO+.
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A B
D
Figure 3. Overview of flow cytometry analysis of peripheral blood. Flow Cytometry is a technique to measure the physical properties of particles, including cells in suspension. Individual
cell types in heterogeneous samples, such as peripheral blood can be analyzed and quantified by flow cytometry. (A) Cells in suspension are injected into the flow cell of the flow cytometer. As
the cells pass through the flow cell, they are interrogated by a laser beam. Light scattered incident to the laser beam measures the size of the cell (also known as forward scatter; FSC), and light
scatter at right angles to the laser beam measures the internal complexity of the cells (side scatter; SSC). (B) The combination of forward scatter and side scatter enables the separation of cells
according to these physical properties. (C) Specific molecules on the surface of cells can be tagged with fluorescently labeled monoclonal antibodies such as anti-CD3, anti-CD4 and anti-CD8
antibodies for the detection of T cells, or anti-CD19 for recognition of B cells. The signals from the activation of these fluorescent molecules as they are interrogated by the laser beam can be
analyzed by special software to provide relative percentages of the various cell subsets in the sample. (D) Flow cytometry analysis of whole blood: whole blood separates into lymphocytes (low
FSC and low SSC), monocytes (moderate FSC and SSC) and granulocytes (high FSC and SSC). Analysis of lymphocytes tagged with anti-CD3 and anti-CD19 enables quantification of CD3+
T cells and CD19+ B cells.
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Table 6. Cell surface markers used for routine analysis of peripheral blood lymphocytes.
Cell Surface Marker Description
CD45 Expressed in all nucleated cells of the hematopoietic lineage.
CD3 Expressed on all T cells. CD3 in association with the T cell receptor is essential for T cell
signaling and function.
CD4 Denes “helper” T cells. These cells recognize antigens presented by MHC Class II.
CD8 Denes “cytotoxic” T cells. These cells recognize antigens presented by MHC Class I.
CD19 Highly expressed on B cells at all stages of development.
CD20 Highly expressed on B cells at all stages of development with the exception of antibody-
producing plasmablasts and plasma cells.
CD16 Strongly expressed in NK cells. Used along with CD56 to identify NK cells.
CD56 Strongly expressed in NK cells.
CD45RA Isoform of CD45 that is expressed chiey on naïve T cells. A subset of terminally dierentiated
T cells may express CD45RA as well.
CD45RO Isoform of CD45 that is expressed on memory T cells.
IgD Expressed on naïve B cells and non-class-switched memory B cells.
CD27 Expressed on memory B cells. Note: Memory B cells are either non-class switched (i.e.,
express CD27 and IgD) or class-switched (i.e., express CD27 and lose expression of IgD).
Class-switched memory B cells express surface IgG, IgM, IgA or IgE and secrete the
corresponding antibody isotype).
Abbreviations: Major Histocompatibility Complex (MHC); Natural Killer (NK)
and management of patients with HIV and for HIV staging. T, B and NK lymphocyte analysis
form part of the routine workup for suspected cellular immunodeficiency and as a follow-up to an
abnormal NBS for SCID. Flow cytometry analysis for abnormal NBS-SCID may yield T-B+NK+,
T-B-NK+ or T-B-NK- results, whereby severe T cell deficiency may be accompanied by a deficiency
in B and/or NK cells. These results can often provide clues to the underlying genetic defect,
i.e., whether T cell development-specific genes alone are affected, whether genes specific to T and B
cell development are affected or whether genes specific to the development of all three lymphocyte
lineages are affected. B cells are low or absent in inherited defects involving B cell development, a
classic example of which is XLA.
It should be noted that lymphocyte subset analyses by flow cytometry provide a quantitative
profile and often needs corroborative assessment of their functional state, and this is achieved by
proliferation studies as indicated below.
Lymphocyte and/or T Cell Proliferation
The ability of lymphocytes or T cells to proliferate to specific stimuli can be assessed by several
methods. The most widely used method to date is the incorporation of tritiated thymidine (3H-Tdr)
into the DNA of proliferating cells, and the detection of incorporated radioactivity as a measure of
the extent of proliferation. The major caveat to this method is the inability of the assay to discriminate
among the various cell populations that proliferate in response to stimulation. Therefore, with this
method, an overall reduction in T cell numbers and compromised T cell function yield a similar
result, i.e., diminished overall lymphocyte proliferation. Newer methods that use DNA binding
dyes or fluorescently tagged nucleotides followed by flow cytometry enable analyses of specific
lymphocyte populations, such as T cells.
To assess lymphocyte or T cell proliferation, peripheral blood mononuclear cells are separated
from whole blood through density gradient centrifugation. The cells are resuspended in a cell
culture medium and stimulated either with mitogens (e.g., phytohemagglutinin (PHA), which is
a pan-T cell stimulator) or antigens (e.g., tetanus toxoid, which specifically stimulates memory
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