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Complement System
Ashley Frazer-Able, PhD,
7
and Christine Dorresteyn Stevens, EdD, MT(ASCP)

LEARNING OUTCOMES CHAPTER OUTLINE

After finishing this chapter, you should be able to: PATHWAYS OF THE COMPLEMENT
SYSTEM
1. Describe the roles of the complement system.
The Classical Pathway
2. Differentiate between the classical, alternative, and lectin pathways
and indicate proteins and activators involved in each. The Lectin Pathway
3. Discuss the formation of the three principal units of the classical The Alternative Pathway
pathway: recognition, activation, and membrane attack units. SYSTEM CONTROLS
4. Describe how initiation of the lectin pathway occurs. Regulation of the Classical and Lectin
5. Explain how C3 plays a key role in all pathways. Pathways
6. Describe regulators of the complement system and their role in the Regulation of the Alternative Pathway
complement system. Regulation of Terminal Components
7. Discuss the complement-related kidney disorders and applicable COMPLEMENT RECEPTORS AND THEIR
complement testing. BIOLOGICAL ROLES
8. Relate biological manifestations of complement activation to BIOLOGICAL MANIFESTATIONS
generation of specific complement products. OF COMPLEMENT ACTIVATION
9. Describe the deficiencies of complement components and the diseases COMPLEMENT AND DISEASE STATES
they cause. COMPLEMENT DEFICIENCIES
10. Differentiate tests for functional activity of complement from Major Pathway Components
measurement of individual complement components.
Regulatory Factor Components
11. Analyze laboratory findings and indicate disease implications in
LABORATORY DETECTION
relation to complement abnormalities.
OF COMPLEMENT ABNORMALITIES
Immunologic Assays of Individual
Components
Assays for the Classical Pathway
Alternative Pathway Assays
Interpretation of Laboratory Findings
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

You can go to DavisPlus at [Link] keyword Stevens for the

laboratory exercises that accompany this text.
91
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92 SECTION 1 Nature of the Immune System

KEY TERMS
Activation unit Classical pathway Hemolytic uremic syndrome Membrane cofactor protein
Alternative pathway Complement receptor type (HUS) (MCP)
Anaphylatoxin 1 (CR1) Hereditary angioedema (HAE) Paroxysmal nocturnal
Decay-accelerating factor Immune adherence hemoglobinuria (PNH)
Bystander lysis
(DAF) Lectin pathway Properdin
C1 inhibitor (C1-INH)
Factor H Mannose-binding lectin Recognition unit
C3 glomerulopathies
(C3G) Factor I (MBL) S protein
C4-binding protein Hemolytic titration (CH50) Membrane attack complex
(C4BP) assay (MAC)

As described in Chapter 3, complement is a complex series of cell walls or outer coating of bacteria, viruses, yeast, and protozoa.
more than 30 proteins that play a major part in amplifying the Although each of these pathways will be considered separately,
inflammatory response to destroy and clear foreign antigens. activation seldom involves only one pathway.
These soluble and cell-bound proteins interact in a very spe- Most plasma complement proteins are synthesized in the
cific way and have powerful abilities. They can lyse foreign liver with the exception of C1 components; these are mainly
cells, opsonize and tag the invaders for clearance, and direct produced by intestinal epithelial cells and Factor D, which is
the adaptive immune system to the site of infection.1 Comple- made in adipose tissue.1,6 Other cells, such as monocytes and
ment activation is also proinflammatory in its ability to increase macrophages, are additional sources of early complement com-
vascular permeability, recruit monocytes and neutrophils to the ponents, including C1, C2, C3, and C4.6,7 Most of these pro-
area of antigen concentration, and trigger secretion of im- teins are inactive precursors, or zymogens, which are converted
munoregulatory molecules that amplify the immune response.2 to active enzymes in a very precise order. Table 7–1 lists the
In their proinflammatory role, complement proteins serve as characteristics of the main complement proteins.
an important link between innate and adaptive immunity.
Complement has important “housekeeping” roles as well. The Classical Pathway
Complement recognizes cellular debris such as apoptotic cells
and immune complexes, tagging them for removal by innate The classical pathway, the first activation cascade described, is
immune cells.3 Because of its potential for far reaching effects, the main antibody-directed mechanism for triggering comple-
complement activation needs to be carefully regulated. Chronic ment activation. However, not all immunoglobulins are able
activation can lead to inflammation and tissue damage to the to activate this pathway. The immunoglobulin classes that can
host. Any deficiencies to the complement system can result in activate the classical pathway include IgM, IgG1, IgG2, and
an increased susceptibility to infection or the accumulation of IgG3, but not IgG4, IgA, or IgE. IgM is the most efficient of
immune complexes resulting in possible autoimmune disor- the activating immunoglobulins because it has multiple bind-
ders.4 However, numerous proteins act as controls or regulators ing sites; thus, it takes only one molecule attached to two
of the system. These controls, as well as the major proteins adjacent antigenic determinants to initiate the cascade. Two
involved in activation, will be discussed in detail. IgG molecules must attach to antigen within 30 to 40 nm of
each other before complement can bind; it may take at least
1,000 IgG molecules to ensure that there are two close enough
Pathways of the Complement System to initiate such binding.1,8 Some epitopes, notably the Rh group,

The complement system can be activated in three different ways.

The first pathway described, the classical pathway, involves Connections
nine proteins that are triggered primarily by antigen–antibody
combination. Pillemer and colleagues discovered an antibody- Jules Bordet
independent pathway in the 1950s that plays a major role as a Jules Bordet was awarded the Nobel Prize in 1919 for his role in
natural defense system.5 This second pathway, the alternative elucidating the nature of complement. Complement was origi-
pathway, was originally called the properdin system because the nally recognized in the 1890s; Paul Ehrlich coined the term com-
protein properdin was thought to be the main initiator of this plement because the substance complements the action of
antibody in destroying microorganisms.1 Present as a substance
pathway. Now it is known that properdin’s major function is to
in normal nonimmune serum, complement is part of the innate
stabilize a key enzyme complex formed along the pathway and immune system. Complement is considered an acute phase
that the other forms of activation are more prominent. The third reactant because levels rise during an infection. It also has a
pathway, likely the most ancient of the three, is the lectin path- unique property of being easily inactivated (i.e., heat-labile) by
way, another antibody-independent means of activating comple- heating serum to 56°C for 30 minutes. Refer to Chapter 3 for a
ment proteins. Its prototypic constituent, mannose- (or mannan-) further description of the innate immune system.
binding lectin (MBL), adheres to mannose found mainly in the
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Chapter 7 Complement System 93

Table 7–1 Proteins of the Complement System

SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (μG/ML) FUNCTION
Classical Pathway
C1q 410 150 Binds to Fc region of IgM and IgG
C1r 85 50 Activates C1s
C1s 85 50 Cleaves C4 and C2
C4 205 300–600 Part of C3 convertase (C4b)
C2 102 25 Binds to C4b—forms C3 convertase
C3 190 1,200 Key intermediate in all pathways
C5 190 80 Initiates membrane attack complex
C6 110 45 Binds to C5b in MAC
C7 100 90 Binds to C5bC6 in MAC
C8 150 55 Starts pore formation on membrane
C9 70 60 Polymerizes to cause cell lysis
Alternative Pathway
Factor B 93 200 Binds to C3b to form C3 convertase
Factor D 24 2 Cleaves Factor B
Properdin 55 15–25 Stabilizes C3bBb–C3 convertase
MBL Pathway
MBL 200–600 0.0002–10 Binds to mannose
MASP-1 93 1.5–12 Unknown
MASP-2 76 Unknown Cleaves C4 and C2
Fc = Fragment crystallizable; Ig = immunoglobulin; MAC = membrane attack complex; MBL = mannose-binding lectin; MASP = MBL-associated serine protease.

are too far apart on the cell for this to occur; therefore, they three subunits—C1q, C1r, and C1s—which require the pres-
are unable to fix complement. Within the IgG group, IgG3 is ence of calcium to maintain structure.1,8 The complex is made
the most effective, followed by IgG1 and then IgG2.8 up of one C1q subunit and two each of the C1r and C1s sub-
In addition to antibodies, a few substances can bind com- units (Fig. 7–2). Although the C1q unit is the part that binds
plement directly to initiate the classical cascade. These include to antibody molecules, the C1r and C1s subunits generate
C-reactive protein (CRP), several viruses, mycoplasmas, some enzyme activity to begin the cascade.
protozoa, and certain gram-negative bacteria such as Escherichia C1q has a molecular weight of 410,000 and is composed
coli.8 However, most infectious agents can directly activate only of six strands that form six globular heads with a collagen-like
the alternative or lectin pathways. tail portion. This structure has been likened to a bouquet of
Complement activation can be divided into three main tulips with six blossoms extending outward (see Fig. 7–2). As
stages, each of which is dependent on the grouping of certain long as calcium is present in the serum, C1r and C1s remain
reactants as a unit. The first stage involves the recognition associated with C1q.
unit, which in the case of the classical pathway is C1. Once C1q “recognizes” the fragment crystallizable (Fc) region of
C1 is fixed, the next components activated are C4, C2, and C3, two adjacent antibody molecules, but at least two of the glob-
known collectively as the activation unit of the classical path- ular heads of C1q must be bound to initiate the classical path-
way (and the lectin pathway). C5 through C9 comprise the way. C1r and C1s are serine protease proenzymes, also called
membrane attack complex (MAC); this last unit completes zymogens. As binding of C1q occurs, both are converted into
the lysis of foreign particles. Each of these is discussed in detail active enzymes. Autoactivation of C1r results from a confor-
in the following sections. Figure 7–1 depicts a simplified mational change that takes place as C1q is bound. Once acti-
scheme of the entire pathway. vated, C1r cleaves a thioester bond on C1s which, in turn,
activates it. Activated C1r is extremely specific because its only
The Recognition Unit known substrate is C1s. Likewise, C1s has a limited specificity,
The first complement component of the classical pathway to with its only substrates being C4 and C2. Once C1s is activated
bind is C1, a molecular complex of 740,000 d. It consists of the recognition stage ends.
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94 SECTION 1 Nature of the Immune System

Recognition
C1qrs
C4
C2
C4a Inactive C1qrs
complex
C2b

C4b2a
Activation
C3

C3a

C4b2a3b
Activated C1s
C5 C1q binds Fc

C5a

C5b C1r
Membrane
attack complex C6
C7
C8
C9

C5b6789 FIGURE 7–2 Structure of C1qrs. When two or more globular heads
of C1q attach to bound immunoglobulin molecules, the collagen-
FIGURE 7–1 The classical complement cascade. C1qrs is the recog- like stalks change their configuration. The resulting shape change
nition unit that binds to the FC portion of two antibody molecules. causes C1r to become a serine protease, which cleaves a small frag-
C1s is activated and cleaves C4 and C2 to form C4b2a, also known as ment of C1s, uncovering the C1s protease, whose only targets are
C3 convertase. C3 convertase cleaves C3 to form C4b2a3b, known as C4 and C2.
C5 convertase. The combination of C4b2a3b is the activation unit.
C5 convertase cleaves C5. C5b attracts C6, C7, C8, and C9, which
bind together, forming the membrane attack complex (MAC).
histocompatibility complex (MHC). Each serves a similar
C9 polymerizes to cause lysis of the target cell.
purpose in its particular pathway.8
When combined with C4b in the presence of magnesium
ions, C2 is cleaved by C1s to form C2a (which has a molecular
The Activation Unit weight of 70,000) and C2b (which has a molecular weight of
Phase two, the formation of the activation unit, begins when 34,000) (see Fig. 7–3A). This is the only case for the designa-
C1s cleaves C4 and ends with the production of the enzyme tion “a” to be given to the cleavage piece with enzyme activity,
C5 convertase (Fig. 7–3). C4 is the second most abundant though there are discussions to make the nomenclature of C2
complement protein, with a serum concentration of approx- match that of the other components of complement with the
imately 600 μg/mL.8 C1s cleaves C4 to split off a 77-amino “a” fragment being the smaller fragment. The short life of these
acid fragment called C4a. In the process, it opens a thioester- reactive species serves as a mechanism of control, keeping the
containing active site on the remaining part, C4b. C4b must reaction localized.
bind to protein or carbohydrate within a few seconds or it The combination of C4b and C2a is known as C3 conver-
will react with water molecules to form iC4b, which is rap- tase (see Fig. 7–3B). This is written as C4b2a to indicate that
idly degraded. Thus, C4b binds mainly to antigen in clusters the complex is an active enzyme. This complex is not very
that are within a 40-nm radius of C1. This represents the stable. The half-life is estimated to be between 15 seconds and
first amplification step in the cascade because for every C1 3 minutes, so C3 must be bound quickly. If binding does occur,
attached approximately 30 molecules of C4 are split and C3 is cleaved into two parts, C3a and C3b.
attached.1 C3, the major and central constituent of the complement
C2 is the next component to be activated. Complement pro- system, is present in the plasma at a concentration of 1 mg/mL
teins were named as they were isolated before the sequence of to 1.5 mg/mL.8 It serves as the pivotal point for all three path-
activation was known—hence the irregularity in the number- ways. The cleavage of C3 to C3b represents the most signifi-
ing system. The C2 gene is closely associated with the gene for cant step in the entire process of complement activation.9 The
Factor B (alternative pathway) on chromosome 6 in the major molecule has a molecular weight of 190,000 and consists of
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Chapter 7 Complement System 95

C4b
two polypeptide chains, alpha (α) and beta (β). The α chain
contains a highly reactive thioester group. When C3a is
C2 removed by cleavage of a single bond in the α chain, the
C4a thioester is exposed; the remaining piece, C3b, is then capable
of binding to hydroxyl groups on carbohydrates and proteins
in the immediate vicinity.2,5,9 C3b is estimated to have a half-
life of 60 microseconds if not bound to antigen. Therefore,
C2b only a small percentage of cleaved C3 molecules bind to anti-
C4
gen; most are hydrolyzed by water molecules and decay in the
fluid phase.8,10
The cleavage of C3 represents a second and major amplifi-
cation process because about 200 molecules are split for every
molecule of C4b2a.10 In addition to being required for the for-
mation of the MAC, C3b also serves as a powerful opsonin.
Macrophages have specific receptors for it (discussed later in
C4b2a the chapter) and make a major contribution to the process of
(C3 convertase) phagocytosis. A large number of molecules are needed for this
A
to occur; hence, the need for amplification.
If C3b is bound within 40 nm of the C4b2a, this creates a
new enzyme known as C5 convertase. Figure 7–3C depicts
C3a this last step in the formation of the activation unit. The cleav-
C3
ing of C5 with deposition of C5b at another site on the cell
membrane constitutes the beginning of the MAC.

~200x
The Membrane Attack Complex (MAC)
C3b C5 consists of two polypeptide chains, α and β, which are linked
by disulfide bonds to form a molecule with a molecular weight
of about 190,000. C5 convertase, consisting of C4b2b3b, splits
off a 74-amino acid piece known as C5a that is released into cir-
C3 Convertase culation, whereas C5b attaches to the cell membrane, forming
the beginning of the MAC. The splitting of C5 and the cleavage
B of C3 represent the most significant biological consequences of
the complement system as explained in the section on biological
C5a
C5 manifestations of complement activation. However, C5b is
extremely labile and rapidly inactivated unless binding to C6
occurs.1
Once C6 is bound to C5b, subsequent binding involves
C7, C8, and C9. None of these proteins has enzymatic activ-
C5b ity; they are all present in much smaller amounts in serum
than the preceding components. C6 and C7 each have mo-
lecular weights of approximately 110,000 and have similar
physical and chemical properties. C8 is made up of three dis-
C4b2a3b
(C5 convertase)
similar chains joined by disulfide bonds and has a total mo-
lecular weight of about 150,000.6 C9 is a single polypeptide
C chain with a molecular weight of 70,000. The carboxy-
FIGURE 7–3 Formation of the activation unit. (A) Activated C1qrs terminal end is hydrophobic, whereas the amino-terminal
cleaves C4 and C2, with the larger pieces, C4b and C2a, binding to end is hydrophilic. The hydrophobic part serves to anchor
the target cell surface and forming the enzyme C3 convertase. the MAC within the target membrane. Formation of the mem-
(B) Each C3 convertase cleaves ~200 C3 molecules into C3a and C3b. brane attack unit is pictured in Figure 7–4. The complex
C3b is a powerful opsonin that binds to the target in many places. of C5b-C6-C7-C8 and C9 is known as C5b-9 or MAC. If the
(C) Some C3b associates with C4b2a, forming C4b2a3b, also complex is soluble in circulation, it is known as sC5b-9.
known as the C5 convertase. This convertase cleaves C5 into Measurement of the level of sC5b-9 is an indicator of the
the anaphylatoxin C5a and C5b, which binds to the target cell.
amount of terminal pathway activation that is occurring.
When formed, the MAC presents a pore of 70 to 100Å that
allows ions to pass in and out of the membrane.1,10 Destruc-
tion of target cells actually occurs through an influx of water
and a corresponding loss of electrolytes. The presence of C9
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96 SECTION 1 Nature of the Immune System

acute phase protein because it is produced in the liver and is

normally present in the serum but increases during an initial
C8 C9 inflammatory response.13 The enzymatic role played by C1r
and C1s in the classical pathway is played in the lectin pathway
by serine proteases called MBL-associated serine proteases
(MASPs). There are currently three MASPs identified, labeled
C7 MASP-1, MASP-2, and MASP-3. The lectin pathway plays an
important role as a defense mechanism in infancy, during the
interval between the loss of maternal antibody and the acqui-
C6 sition of a full-fledged antibody response to pathogens.15

MAC
C5b The Alternative Pathway
First described by Pillemer and his associates in the early 1950s,
the alternative pathway was originally named for the protein
properdin, a constituent of normal serum with a concentration
of approximately 5 to 15 μg/mL.5 Although the alternative path-
way can be activated on its own, it appears that it functions
mainly as an amplification loop for activation started from the
FIGURE 7–4 Formation of the membrane attack unit. C5b binds classical or lectin pathways. Although properdin has been con-
to the target cell, whereas C6 and C7 attach to it. C8 binds to these firmed to bind and initiate activation, the primary function of
associated molecules and begins (along with C7) to penetrate the properdin is to stabilize the C3 convertase formed from activation
cell membrane. Multiple C9 molecules bind to C5b678 and polymer- of other factors. In addition to properdin, the serum proteins Fac-
ize to form a transmembrane channel, the membrane attack tor B and Factor D are unique to this pathway. C3 is a key com-
complex, which causes lysis of the cell. ponent of this pathway as well as the two other pathways. The
alternative pathway is summarized in Figure 7–5.
Triggering substances for the alternative pathway include
greatly speeds this lysis. However, sufficient perturbation of bacterial cell walls, especially those containing lipopolysaccha-
the membrane can occur in the absence of C9 so that defi- ride, fungal cell walls, yeast, viruses, virally infected cells,
ciencies in C9 appear largely benign. tumor cell lines, and some parasites, especially trypanosomes.1
All of these can serve as sites for binding the complex C3bBb,
one of the end products of this pathway. The conversion of C3
The Lectin Pathway is the first step in this pathway.
The lectin pathway represents another means of activating Native C3 is not stable in plasma. Water is able to hydrolyze
complement. Instead of activation through antibody binding, a thioester bond, thus spontaneously activating a small number
the lectin pathway is activated by recognition of surface moi- of these molecules.16 C3b, sometimes called iC3, is formed by
eties that are found on pathogens.11 This pathway provides an this spontaneous hydrolysis, from activation, or from the clas-
additional link between the innate and acquired immune re- sical or lectin pathways. It acts as the seed of activation of the
sponse because it involves nonspecific recognition of carbohy- alternative pathway. The C3b binds to Factor B, which has a
drates that are common constituents of microbial cell walls and molecular weight of 93,000 and is fairly abundant in the serum,
that are distinct from those found on human cell surfaces.12,13 at a level of 200 μg/ mL.8,17 Once bound to C3b, Factor B
Although this pathway is the most recently described of the can be cleaved by Factor D. The role of Factor B is thus anal-
three activation pathways of complement, it is probably the ogous to that of C2 in the classical pathway because it forms
most ancient. The lectin pathway molecules are structurally an integral part of a C3 convertase.
similar to those of the classical; the classical and lectin path- Factor D is a plasma protein that goes through a conforma-
ways even share the components C4 and C2. Once C4 and C2 tional change when it binds to Factor B.17,18 It is a serine pro-
are cleaved, the rest of the pathway is identical to the classical tease with a molecular weight of 24,000; its only substrate is
pathway. The role C1q serves in the classical pathway is filled bound Factor B. The concentration of Factor D in the plasma
by three classes of recognition molecules in the lectin pathway: is the lowest of all the complement proteins, approximately
lectins, ficolins, and CL-K1.11 The structure of all three classes 2 μg/mL.10 It cleaves Factor B into two pieces: Ba (with a molec-
of recognition molecules is similar to that of C1q because they ular weight of 33,000) and Bb (with a molecular weight of ap-
are all classed as collectins. One key lectin, called mannose- proximately 60,000). Bb remains attached to C3b, forming the
binding, or mannan-binding, lectin (MBL), binds to mannose initial C3 convertase of the alternative pathway. Bb is rapidly
or related sugars in a calcium-dependent manner to initiate inactivated unless it becomes bound to a site on one of the trig-
this pathway.14 These sugars are found in glycoproteins or car- gering cellular antigens.
bohydrates of a wide variety of microorganisms such as bacte- As the alternative pathway convertase, C3bBb is then capable
ria, yeasts, viruses, and some parasites. MBL is considered an of cleaving additional C3 into C3a and C3b. Some C3b attaches
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Chapter 7 Complement System 97

C3a
1 H2O 1. C3 is hydrolyzed by water to produce C3b, which binds
Factor B and together they attach to target cell surface.
C3b B
C3 C3b B 2. B is cleaved by Factor D into the fragments Ba and Bb.
Bb combines with C3b to form C3bBb, an enzyme with
C3 convertase activity.

3. More C3 is cleaved, forming more C3bBb. This enzyme

is stabilized by properdin, and it continues to cleave
additional C3.
2 D Ba 4. If a molecule of C3 remains attached to the C3bBbP
enzyme, the convertase now has the capability to
C3b cleave C5. The C5 convertase thus consists of
C3bBbP3b. After C5 is cleaved, the pathway is identical
Bb to the classical pathway.

C3 C3a
3

C3b Bb P C3b

C5 C5a
4 C5b

C3b Bb P C3b

FIGURE 7–5 The alternative pathway.

to cellular surfaces and acts as a binding site for more Factor B, self-antigens are destroyed and that the reaction remains
resulting in an amplification loop; activation initiated by the clas- localized, several plasma proteins act as system regulators.
sical or lectin pathways is amplified to levels of biological con- In addition, there are specific receptors on certain cells that
sequence. All C3 present in plasma would be rapidly converted also exert a controlling influence on the activation process.
by this method were it not for the fact that the enzyme C3bBb In fact, approximately one-half of the complement compo-
is extremely unstable unless properdin binds to the complex. nents serve as controls for critical steps in the activation
Binding of properdin increases the half-life of C3bBb from process. Because activation of C3 is the pivotal step in all
90 seconds to several minutes.8,17 In this manner, optimal rates pathways, the majority of the control proteins are aimed at
of alternative pathway activation are achieved.14 halting accumulation of C3b. However, there are controls at
C3bBb can also cleave C5, but it is much more efficient at all crucial steps along the way. Regulators will be discussed
cleaving C3.19 If, however, some of the C3b produced remains according to their order of appearance in each of the three
bound to the C3 convertase, the enzyme is altered to form pathways. A brief summary of these is found in Table 7–2.
C3bBb3bP, which has a high affinity for C5 and exhibits C5
convertase activity.16,19 C5 is cleaved to produce C5b, the first
part of the membrane attack unit. From this point on the alter- Regulation of the Classical
native, lectin, and classical pathways are identical. Figure 7–6 and Lectin Pathways
shows the convergence of all three pathways. C1 inhibitor (C1-INH) inhibits activation at the first stages of
both the classical and lectin pathways. Its main role is to inacti-
System Controls vate C1 by binding to the active sites of C1r and C1s. Clr and
Cls become instantly and irreversibly dissociated from C1q.6,10
Activation of complement could cause tissue damage and C1q remains bound to antibody, but all enzymatic activity ceases.
have devastating systemic effects if it were allowed to pro- C1-INH10 also inactivates MASP-2 binding to the MBL-MASP
ceed uncontrolled. To ensure that infectious agents and not complex, thus halting the lectin pathway.12,18 C1-INH is a
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98 SECTION 1 Nature of the Immune System

Classical Pathway Lectin Pathway Alternative Pathway

C1qrs
Teichoic acid
MBL
Zymosan
MASP-1 LPS

MASP-2
MASP-3

C3b ⫹ B

Mannose
Factor D

C4

C2 C3

C4b C2a C3b Bb

C3 convertase
C3 convertase

Properdin stabilizes

C2a

C4b C3b C5 C3b Bb P C3b

C5 convertase
C5 convertase

C5a

C7

C5b
C6 C8

Membrane attack complex

FIGURE 7–6 Convergence of the classical, alternative, and lectin pathways. The binding of C1qrs to two antibody molecules activates
the classical pathway, whereas the alternative pathway is started by hydrolysis of C3. The lectin pathway is triggered by binding of MBP to
mannose on bacterial cell walls. MASP-1, MASP-2, and MASP-3 bind to form an activated C1-like complex. MASP-2 cleaves C2 and C4 and
proceeds like the classical pathway. Factor B and Factor D operate in the alternative pathway. Although C3 convertase is formed differently
in each pathway, C3 is a key component in each one. The C5 convertase in the alternative pathway consists of C3bBb3bP. In the classical
and lectin pathways, C5 convertase is made up of C4b2a3b. After C5 is cleaved, the pathway is common to all.
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Chapter 7 Complement System 99

Table 7–2 Plasma Complement Regulators

SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (MG/ML) FUNCTION
C1 inhibitor 105 240 Dissociates C1r and C1s from C1q
(C1-INH)
Factor I 88 35 Cleaves C3b and C4b
Factor H 150 300–450 Cofactor with I to inactivate C3b;
prevents binding of B to C3b
C4-binding protein 520 250 Acts as a cofactor with I to
(C4BP) inactivate C4b
S protein 84 500 Prevents attachment of the C5b67
(vitronectin) complex to cell membranes

glycoprotein with a molecular weight of 105,000. Like most fibroblasts, and on numerous types of epithelial cells.6–8 DAF
of the other complement proteins, it is mainly synthesized in is capable of dissociating both classical and alternative path-
the liver; however, monocytes also may be involved to some way C3 convertases. It can bind to both C3b and C4b in a
extent in its manufacture. manner similar to CR1.4 It does not prevent initial binding
Further formation of C3 convertase in the classical and of either C2 or Factor B to the cell but can rapidly dissociate
lectin pathways is inhibited by four main regulators: soluble both from their binding sites, thus preventing the assembly
C4-binding protein (C4BP) and three cell-bound receptors, of an active C3 convertase.
complement receptor type 1 (CR1), membrane cofactor The carboxy-terminal portion of DAF is covalently attached
protein (MCP), and decay-accelerating factor (DAF).1,18 All to a glycophospholipid anchor that is inserted into the outer
of these act in concert with Factor I, a serine protease that in- layer of the membrane lipid bilayer. This arrangement allows
activates C3b and C4b when bound to one of these regulators. DAF mobility within the membrane so it can reach C3 conver-
C4BP is abundant in the plasma and has a molecular weight tase sites that are not immediately adjacent to it (Fig. 7–7).2
of about 520,000. It is capable of combining with either fluid- The presence of DAF on host cells protects them from by-
phase or bound C4b; therefore, C4b cannot bind to C2 and is stander lysis. It is one of the main mechanisms used in dis-
made available for degradation by Factor I. If C4BP attaches to crimination of self from nonself because foreign cells do not
cell-bound C4b, it can dissociate it from C4b2a complexes, possess this substance. However, it does not permanently mod-
causing the cessation of the classical pathway. ify C3b or C4b; they are capable of re-forming elsewhere as
CR1, also known as CD35, is a large polymorphic glycopro- active convertases.
tein with a molecular weight between 165,000 and 280,000.7
It is found mainly on peripheral blood cells, including neu-
trophils, monocytes, macrophages, erythrocytes, eosinophils,
Regulation of the Alternative Pathway
B lymphocytes, some T lymphocytes, and follicular dendritic The principal soluble regulator of the alternative pathway is
cells.3 It binds C3b and C4b but has the greatest affinity for Factor H, which has a molecular weight of 160,000.18 It acts
C3b.6,20 Once bound to CR1, both C4b and C3b can then be by binding to C3b, preventing the binding of Factor B. C3b in
degraded by Factor I. the fluid phase has a hundredfold greater affinity for Factor H
A main function of CR1 is as a receptor on platelets and red than for Factor B, but on cell surfaces C3b preferentially binds
blood cells (RBCs), which helps to mediate transport of C3b- to Factor B. Factor H also accelerates the dissociation of the
coated immune complexes to the liver and spleen.7,20 It is there C3bBb complex on cell surfaces. When Factor H binds to
that fixed tissue macrophages strip the immune complexes C3bBb, Bb becomes displaced. In this manner, C3 convertase
from the RBCs, process the complexes, and return the RBCs activity is curtailed in plasma and on cell surfaces.
intact to circulation. The ability of cells to bind complement- Additionally, Factor H acts as a cofactor that allows Factor I
coated particles is referred to as immune adherence. to break down C3b. It appears that only those molecules with
MCP, or CD46, has a molecular weight between 50,000 and tightly bound Factor H acquire high-affinity binding sites for
70,000 and is found on virtually all epithelial and endothelial Factor I.21 When Factor I binds, a conformational change takes
cells except erythrocytes.7 MCP is the most efficient cofactor place that allows it to cleave C3b.21 On cellular surfaces, C3b
for Factor I-mediated cleavage of C3b. It can serve as a cofactor is cleaved into C3f, which is released into the plasma, and iC3b,
for cleavage of C4b, but it is not as effective as C4BP. MCP also which remains attached but is no longer an active enzyme.
helps to control the alternative pathway because binding of iC3b is further broken down to C3c and C3dg by Factor I in con-
Factor B to C3b is inhibited. junction with another cofactor: the CR1 receptor (Fig. 7–8).10
DAF or CD55, a 70,000 d membrane glycoprotein, is the With this key role in complement regulation, it should not be
third main receptor and has a wide tissue distribution. It surprising that Factor H has recently been shown to play a role
is found on peripheral blood cells, on endothelial cells and in a variety of disorders (discussed in the text that follows).
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100 SECTION 1 Nature of the Immune System

DAF DAF

C4b C2a C4b

Classical
pathway

A
DAF DAF

C3b C3b

Bb

Alternative
pathway

B
FIGURE 7–7 A. In the classical pathway, DAF dissociates C2a from C4b. B. Inhibitory effects of DAF. In the alternative pathway, when C3b binds
to cell surfaces that have DAF present, DAF helps dissociate Bb from binding to C3b.

Regulation of Terminal Components include degradation products of C3b, such as C3dg, C3d, and
iC3b. In addition, the Epstein-Barr virus gains entry to B cells
S protein is a soluble control protein that acts at a deeper level by binding to this receptor. CR2 is present only on mature
of complement activation. Also known as vitronectin, S protein B cells and is lost when conversion to plasma cells occurs. CR2
interacts with the C5b-7 complex as it forms in the fluid phase plays an important role as part of the B-cell co-receptor for
and prevents it from binding to cell membranes.8 Binding of antigen. Acting in concert with CD19, it binds complement-
C8 and C9 still proceeds, but polymerization of C9 does not coated antigen and cross-links it to membrane immunoglobu-
occur; therefore, the complex is unable to insert itself into the lin to activate B cells. In this manner, immune complexes are
cell membrane or to produce lysis.4 more effective at enhancing B-cell differentiation and produc-
A receptor, known by various terms, including membrane tion of memory cells than is antigen by itself.2,22
inhibitor of reactive lysis (MIRL) or CD59, also acts to block Another receptor, CR3 (CD11b/CD18), found on mono-
formation of the MAC. MIRL is widely distributed on the cell cytes, macrophages, neutrophils, and natural killer (NK)
membranes of all circulating blood cells, including RBCs, and cells, specifically binds particles opsonized with iC3b, a C3b
on endothelial, epithelial, and many other types of cells.8,10 degradation product.7 It does this in a calcium-dependent
Table 7–3 lists the receptors and indicates the types of cells manner. The CR3 receptor plays a key role in mediating
on which they are found. phagocytosis of particles coated with these complement frag-
ments (Fig. 7–9). These proteins trigger surface adhesion and
increased activity of phagocytic cells.2 Patients whose white
Complement Receptors blood cells (WBCs) lack these receptors fail to exhibit func-
and Their Biological Roles tions such as chemotaxis, surface adherence, and aggregation.
Deficiencies in phagocytosis are also noted. These individuals
Some complement receptors found on host cells amplify and have an impaired capacity to bind iC3b-coated particles and
enhance the immune response by augmenting phagocytosis are subject to recurrent infections.
and stimulating accessory cells rather than acting as regulators The CR4 (CD11c/CD18) receptor is very similar to CR3 in
(see Table 7–3). CR1 has been discussed in the previous sec- that it also binds iC3b fragments in a calcium-dependent fash-
tion. A second receptor, CR2 (or CD21), is found mainly on ion. CR4 proteins are found on neutrophils, monocytes, tissue
B lymphocytes and follicular dendritic cells.22 Ligands for CR2 macrophages, activated T cells, dendritic cells, NK cells, and
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Chapter 7 Complement System 101

1 1. Factor H (FH) competes with factor B (B) for binding

C3 FH
Blocks to spontaneously (hydrolytically) activated C3b.

2. Factor H dissociates any C3bBb complexes that form

C3a C3b B
on self-cell surfaces.

3. Factor H is a cofactor with factor I (FI), enabling

cleavage of C3b. The resuting C3bi loses enzymatic
activity, but is still an opsonin.
2
4. CR1 is a cofactor with FI, enabling cleavage of
FH
Displaces C3bi. The resulting C3dg is an opsonin and a
cofactor in B cell stimulation.
C3b Bb

Self-cell surface

Enables cleavage
3 FI
FH

C3bi C3f

4 Enables
cleavage
FI
CR1

C3dg C3c

FIGURE 7–8 Complement controls. CR1 receptor acts as a cofactor in the inactivation of C3b. Factor I cleaves C3b to form C3dg and C3c.
C3dg is not an effective opsonin and is not capable of further participation in the complement cascade.

Table 7–3 Receptors on Cell Membranes for Complement Components

RECEPTOR LIGAND CELL TYPE FUNCTION
CR1 (CD35) C3b, iC3b, C4b RBCs, neutrophils, monocytes, Cofactor for Factor I;
macrophages, eosinophils, B and T cells, mediates transport of
follicular dendritic cells immune complexes
CR2 (CD21) C3dg, C3d, iC3b B cells, follicular dendritic cells, epithelial B-cell co-receptor for
cells antigen with CD19
CR3 (CD11b/CD18) iC3b, C3d, C3b Monocytes, macrophages, neutrophils, NK Adhesion and increased
cells activity of phagocytic
cells
CR4 (CD11c/CD18) iC3b, C3b Monocytes, macrophages, neutrophils, NK Adhesion and increased
cells, activated T and B cells, dendritic activity of phagocytic
cells cells

Continued
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102 SECTION 1 Nature of the Immune System

Table 7–3 Receptors on Cell Membranes for Complement Components—cont’d

RECEPTOR LIGAND CELL TYPE FUNCTION
DAF (CD55) C3b, C4b RBCs, neutrophils, platelets, monocytes, Dissociates C2b or Bb
endothelial cells, fibroblasts, T cells, from binding sites, thus
B cells, epithelial cells preventing formation of C3
convertase
MIRL (CD59) C8 RBCs, neutrophils, platelets, monocytes, Prevents insertion of C9
endothelial cells, epithelial cells into cell membrane
MCP (CD46) C3b, C4b Neutrophils, monocytes, macrophages, Cofactor for Factor I
platelets, T cells, B cells, endothelial cells cleavage of C3b and C4b

RBC = red blood cell; NK = natural killer.

MAC. Complement proteins also serve as a means of linking

innate and adaptive immunity. They act as opsonins to facil-
itate recognition and subsequent destruction by phagocytic
cells; in addition, they play a major role in the uptake and
Antigen
presentation of antigens so a specific immune response can
occur. They also facilitate B-cell activation. Recent work
demonstrates that complement is necessary for maintaining
immunologic memory.24 Effector molecules generated earlier
in the cascade play a major role in all these areas. Such mol-
FcR ecules can be classified into three main categories: anaphy-
CR3 CRP-R latoxins, chemotaxins, and opsonins.
An anaphylatoxin is a small peptide that causes increased
vascular permeability, contraction of smooth muscle, and re-
lease of histamine from basophils and mast cells. Proteins that
FIGURE 7–9 Role of C3b in opsonization. C3b, antibody, and play such a part are C3a and C5a. Both of these have molecular
C-reactive protein are all important opsonins that coat antigens and weights between 9000 and 11,000 and are formed as cleavage
accelerate phagocytosis. Receptors for C3b (CR3), antibody (FcR), products from larger complement components. Of these mol-
and C-reactive protein (CRP-R) on the phagocytic cell membrane ecules, C5a is the most potent; it is at least 200 times more
bind the opsonins and pull the membrane around to envelop the
powerful than C3a.25
antigen.
C3a and C5a attach to specific receptors on neutrophils, ba-
sophils, mast cells, eosinophils, smooth muscle cells, and vas-
activated B cells.2,7 Neutrophils and monocytes, however, pos- cular endothelium.24,25 C3a attaches to the C3a receptor (C3aR),
sess smaller amounts of CR4 than of CR3. Their function ap- whereas C5a attaches to the C5a receptor (C5aR). When binding
pears to be similar to that of CR3 and they may assist occurs on basophils and mast cells, histamine is released, in-
neutrophil adhesion to the endothelium during inflammation. creasing vascular permeability and causing contraction of
Receptors specific for C1q are found on neutrophils, mono- smooth muscles. C5a causes neutrophils to release hydrolytic
cytes, macrophages, B cells, platelets, and endothelial cells.7,23 enzymes, oxygen radicals, and prostaglandins, which aid in the
These receptors, known as collectin receptors, bind the collagen destruction of foreign antigens.25
portion of C1q and generally enhance the binding of C1q to FC C5a also serves as a chemotaxin for neutrophils, basophils,
receptors. Interacting only with bound C1q, the receptors appear eosinophils, mast cells, monocytes, and dendritic cells. In this
to increase phagocytic cells’ uptake of immune complexes op- manner, these cells are directed to the source of antigen concen-
sonized with C1q. On neutrophils, they may also act to enhance tration. Because of increased vascular permeability, neutrophils
the respiratory burst triggered by IgG binding to FC receptors. migrate from blood vessels to the tissues and tend to aggregate.
Binding of C5a to monocytes causes them to undergo
an oxidative burst that includes increased production of
Biological Manifestations hydrolytic enzymes, neutrophil chemotactic factor, platelet-
of Complement Activation activating factors, interleukin-1 (IL-1), and toxic oxygen
metabolites.13,19 IL-1 is a protein that enhances T-cell acti-
Activation of complement is a very effective means of ampli- vation. The activation may produce fever and lead to an in-
fying the inflammatory response to destroy and clear foreign crease in acute-phase reactants, both of which are characteristic
antigens. The cycle does not always have to proceed to lysis of an inflammatory response.
for this to be accomplished; hence, some of the initiating C3a and C5a are rapidly inactivated by an enzyme in the
proteins are much more plentiful than proteins that form the plasma called carboxypeptidase N to localize and control their
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Chapter 7 Complement System 103

effects. C3a is cleaved in seconds, whereas conversion of C5a B locus is nearby, C2-deficient persons are often reported to have
occurs more slowly. decreases in Factor B also. Other types of complement deficien-
The last major effect of complement-derived peptides is cies are less common.
opsonization. C4b, C3b, iC3b, and C3dg, which accumulate A second deficiency that occurs with some frequency is that
on cell membranes as complement activation proceeds, bind of MBL. Deficiencies and polymorphisms in MBL occur in about
to specific receptors on erythrocytes, neutrophils, monocytes, 30% of the population. The health consequences of such varia-
and macrophages as previously discussed. This binding facili- tion in MBL levels remain unclear, however. Lack of MBL has
tates phagocytosis and clearance of foreign substances or been associated with pneumonia, sepsis, and meningococcal dis-
cellular debris, which is one of the key functions of the com- ease in infants.13,15,31,32 Low MBL has also been associated with
plement system. In addition, attachment of C3 products to an the risk of some cancers, infection during chemotherapy, and
antigen has been found to enhance the B-cell response. certain autoimmune disorders such as systemic lupus erythe-
matosus (SLE), but these connections are not yet well defined.33
The most serious deficiency is that of C3 because it is the
Complement and Disease States key mediator in all pathways. C3 deficiencies are, however, ex-
tremely rare.28 Individuals with a C3 deficiency are prone to
Although complement acts as a powerful weapon to combat
developing severe, recurrent life-threatening infections with en-
infection by amplifying phagocytosis, in some cases it can ac-
capsulated bacteria such as Streptococcus pneumoniae and may
tually contribute to tissue damage or death. Complement can
also be subject to immune complex diseases.16 Such complexes
be harmful if
can lodge in the kidney and result in glomerulonephritis.26,34
• It is activated systemically on a large scale as in gram- It appears that a deficiency of any of the terminal components
negative septicemia of the complement cascade (C5–C8) causes increased suscepti-
• It is activated by tissue necrosis such as myocardial bility to systemic Neisseria infections, including meningococcal
infarction meningitis and disseminated gonorrheal disease.10,28 Table 7–4
• Lysis of red blood cells occurs lists the complement components and the disease states associ-
In the case of septicemia caused by a gram-negative organism, ated with the absence of each individual factor.
large quantities of C3a and C5a are generated, leading to neu-
trophil aggregation and clotting. Damage to the tiny pulmonary
capillaries and interstitial pulmonary edema may result.2
Table 7–4 Deficiencies of Complement
Tissue injury following obstruction of the blood supply,
Components
such as occurs in a myocardial infarction or heart attack, can
cause complement activation and deposition of MACs on cell DEFICIENT
surfaces. Receptors for C3a and C5a have been found in coro- COMPONENT ASSOCIATED DISEASE
nary plaques, indicating that complement components may in- C1 (q, r, or s) Lupuslike syndrome; recurrent infections
crease the damage to heart tissue.25,26 C2 Lupuslike syndrome; recurrent
Lysis may be another end result of complement activation. infections; atherosclerosis
Hemolytic diseases such as cold autoimmune hemolytic anemia
are characterized by the presence of an autoantibody that binds C3 Severe recurrent infections;
at low temperatures. When these cells warm up, complement glomerulonephritis
fixation results in lysis. (See Chapter 15 for a more complete C4 Lupuslike syndrome
discussion of complement-mediated autoimmune diseases.)
C5–C8 Neisseria infections
C9 No known disease association
Complement Deficiencies Hereditary angioedema
C1-INH

Major Pathway Components DAF Paroxysmal nocturnal hemoglobinuria

Although excess activation of the complement system can result MIRL Paroxysmal nocturnal hemoglobinuria
in disease states, lack of individual components also has a dele- Factor H Recurrent pyogenic infections
terious effect. Hereditary deficiency of any complement protein, or Factor I
with the exception of C9, usually manifests itself in increased
MBL Pneumococcal diseases, sepsis,
susceptibility to infection and delayed clearance of immune Neisseria infections
complexes. Most of these conditions are inherited on an auto-
somal recessive gene and are quite rare, occurring in 0.03% of Properdin Neisseria infections
the general population.26 A lack of C2, the most common defi- MASP-2 Pneumococcal diseases
ciency, is found in 1 in 20,000 individuals.4,27–29 Recent evidence
indicates that atherosclerosis may be related to a C2 deficiency.28 C1-INH = C1 inhibitor; DAF = decay-accelerating factor; MASP-2 =
mannose-associated serine protease; MBL = mannose-binding lectin;
C2-deficient individuals may also be more prone to recurrent
MIRL = membrane inhibitor of reactive lysis.
streptococcal and staphylococcal infections.30 Because the Factor
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104 SECTION 1 Nature of the Immune System

Regulatory Factor Components connected to deficiencies, polymorphisms, or autoantibodies

involving one or multiple complement components. Key
A prime example of a disease caused by a missing or defective among these is a set of rare kidney disorders associated with
regulatory component is paroxysmal nocturnal hemoglo- complement. Hemolytic uremic syndrome (HUS) is the
binuria (PNH). Individuals with this disease have RBCs that most common cause of renal failure in children and is char-
are deficient in DAF. Hence, the RBCs are subject to lysis by acterized by hemolytic anemia, low platelet count, and acute
means of the bystander effect once the complement system renal failure.36 The primary cause of HUS is a Shiga toxin re-
has been triggered. These individuals appear to have a defi- lated to infection that is associated with acute diarrhea. The
ciency in the glycophospholipid anchor of the DAF molecule atypical form of HUS (aHUS) occurs because of complement
that prevents its insertion into the cell membrane.10,22 When dysregulation caused by genetic polymorphisms. The atypical
C3b is deposited on erythrocytes through activation of either HUS has a less acute onset and may not be associated with di-
pathway, the result is complement-mediated intravascular arrhea; otherwise, the clinical presentation is similar. The ge-
and extravascular hemolysis, resulting in a chronic hemolytic netic mutations associated with aHUS include those of Factor
anemia. H, MCP, Factor I, Factor B, and thrombomodulin, as well as
Some studies indicate that a DAF deficiency is associated autoantibodies to Factor H and Factor I.36
with a lack of CD59 (MIRL) and both are implicated in PNH.32 Complement has also been implicated in C3 glomeru-
CD59 has the same glycophospholipid anchor found in DAF; lopathies (C3G), which are diseases involving the glomeruli
therefore, the gene deficiency affects both molecules. As men- of the kidneys. Recently several forms of rare glomerulonephri-
tioned previously, CD59 prevents insertion of C9 into the cell tis (GN) were reclassified based on immunofluorescence and
membrane by binding to the C5b-8 complex, inhibiting for- the presence or absence of C3 and immunoglobulins. For the
mation of transmembrane channels.25 Both DAF and CD59 are C3 glomerulopathies, it is only C3 that is found in the deposits
important in protecting RBCs against bystander lysis. on the kidney. Analysis of these patients has shown that 71%
Another complement deficiency disorder that has recently to 100% of these patients have mutation in a complement pro-
received considerable attention is hereditary angioedema tein, specifically C3, Factor B, Factor H, or Factor I. Other pa-
(HAE). HAE involves recurrent attacks of swelling that affect tients have an acquired autoantibody. These autoantibodies are
the extremities, the skin, the gastrointestinal tract, and other known as C3 nephritic factors (C3NeF).
mucosal surfaces. This disease is caused by a deficiency or lack A C3NeF is an antibody that binds the C3-convertase from
of C1-INH, which occurs with a population frequency of 2 in the alternative pathway, C3bBb, holding it together and making
10,000.32 Although C1-INH was named for its role in control- it impervious to the normal control mechanisms. In this way,
ling complement, it is the function of C1-INH in controlling a C3NeF leads to uncontrolled cleavage of C3 with concomi-
the contact pathway of the coagulation system that is critical tant uncontrolled deposition of C3 on the kidneys. C3G caused
in this disease. C1-INH is a serpin (serine protease inhibitor) by C3NeF is clinically indistinguishable from the hereditary
that controls many of the serine proteases on contact. The lack form of the disorder.37 It is only with laboratory measurement
of C1-INH results in localized swelling that can be either sub- of the presence or absence of a C3NeF that the nature of the
cutaneous or found within the bowel or upper-respiratory disorder can be differentiated. In addition, an investigation of
tract.35 Normally, this spontaneously subsides in 48 to 72 hours, a possible complement deficiency can be complicated by de-
but if the edema occurs in the area of the oropharynx, life- pletion of complement components because of consumption
threatening upper-airway obstruction may develop.26 These through activation. For both the autoantibodies and activation-
attacks can be quite debilitating even when they are not life related consumption, laboratory testing is the key way to tell
threatening, so there is a need for proper diagnosis for these the acquired forms from the hereditary forms of complement
patients. disorders.
HAE is separated into two types, type I and type II. Type I
is characterized by a decrease in the C1-INH protein; type II
has normal levels of C1-INH, but the function is decreased. Laboratory Detection
The genetic cause of either type is an autosomal dominant gene of Complement Abnormalities
that codes for either a dysfunctional or an inactive protein. In
addition to the hereditary forms of the disorder, there are ac- Determining the levels of complement components can be use-
quired forms that result from either consumption of C1-INH ful in diagnosing disease. Hereditary deficiencies can be iden-
or from autoantibodies blocking the function of C1-INH. To tified and much can be learned about inflammatory or
differentiate the acquired and hereditary forms, measurement autoimmune states by following the consumption of comple-
of C1q can be helpful; C1q will be low in the acquired forms, ment proteins. Techniques to determine complement abnor-
but not in the hereditary forms. Measurement of C4 can also malities generally fall into two categories: (1) measurement of
be a very helpful screen for HAE, particularly during an attack, components as antigens in serum and (2) measurement of
because patients who do not exhibit a drop in C4 at that time functional activity.8 Many assays that are unavailable in routine
are rare.4,35 clinical laboratories are available in specialized laboratories.
In addition to these well-described diseases of comple- Some of the more common assays will be discussed in the text
ment, there are a growing number of disorders that are being that follows.
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Chapter 7 Complement System 105

Immunologic Assays of Individual Assays for the Classical Pathway

Components Assays that measure lysis, the end point of complement activa-
The methods most frequently used to measure individual com- tion, are functional tests that are frequently run in conjunction
ponents include radial immunodiffusion (RID) and nephelom- with testing of individual components. The hemolytic titration
etry.26,34 C3 and C4 levels are routinely measured in most (CH50) assay is most commonly used for this purpose.26,32 This
clinical laboratories by nephelometry or by turbidity measure- assay measures the amount of patient serum required to lyse
ments that are often automated. Both of these types of meas- 50% of a standardized concentration of antibody-sensitized
urements and the methods used for the other components rely sheep erythrocytes. Because all proteins from C1 to C9 are nec-
on the precipitation of antigen (the complement component essary for this to occur, absence of any one component will result
being measured) and antibody. Nephelometry measures con- in an abnormal CH50, essentially reducing this number to zero.
centration according to the amount of light scattered by a solu- The titer is expressed in CH50 units, which is the reciprocal
tion containing a reagent antibody and a measured patient of the dilution that is able to lyse 50% of the sensitized
sample (refer to Chapter 10 for more details on nephelometry). cells.32,38 The 50% point is used because this is when the
Generally, the more antigen–antibody complexes that are pres- change in lytic activity per unit change in complement is at
ent, the more a beam of light will scatter as it passes through maximum (Fig. 7–11). Most laboratories need to establish
the solution. Such systems have a high degree of accuracy; re- their own normal values.
sults are available quickly and processing is easy because of the An additional CH50 test has also been developed based on
use of automation. However, they involve expensive equipment. lysis of liposomes that release an enzyme when lysed. This lysis
Components for which there are standardized reagents in- can be read on an analyzer and is more accurate than tradi-
clude C1q, C4, C3, C5, Factor B, Factor H, and Factor I. RID tional CH50 testing.25 However, lytic assays in general are com-
uses agarose gel into which specific antibody is incorporated. plicated to perform and lack sensitivity. Individual laboratories
Serum serves as the antigen and is placed in wells that are cut in must establish their own normal values. When the results are
the gel. Diffusion of the antigen from the well occurs in a circular abnormal, the reasons cannot be determined. Such procedures
pattern (Fig. 7–10). The radius of the resulting circle can be re- are useful in establishing functional activity or lack thereof.
lated to antigen concentration (see Chapter 10 for further details Additional testing for individual components should be
on RID). This is a sensitive technique when performed correctly, performed to follow up on any abnormality.
but at least 24 hours are needed before test results are available. Lytic activity can also be measured by radial hemolysis in
None of the assays for individual components are able to agarose plates. Rabbit RBCs that have been sensitized with an-
distinguish whether the molecules are functionally active. tibody are implanted in agarose and patient serum is added to
Thus, although the preceding techniques give quantitative re- wells punched in the gel. Lysis appears as a clear zone around
sults and are relatively easy to perform, test results must be in- each well; if complement standards are run, the size of the zone
terpreted carefully. Nephelometry and RID are both sensitive can be related to complement concentration.26,32,39 Solid-phase
tests.32 Enzyme-linked immunosorbent assay (ELISA) methods IgM attached to the walls of microtiter plates is used to initiate
are available for the measurement of the inhibitor C1-INH. complement activation. Anti-human antibody to C9 conju-
gated to alkaline phosphatase is the indicator of complement
activation. When a substrate is added, if any C9 is present and
the antibody conjugate has attached, a color change will be ev-
Unloaded gel ident. (Refer to Chapter 11 for a complete discussion of the
Calculations principle of ELISA techniques.) This type of testing, which is
Measure blue
precipitation ring very sensitive, is probably the best screen for complement ab-
normalities.25 The same method can detect split products that
Internal result from complement activation. These products include
diameter C4a, C4d, C3a, iC3b, C5a, and the soluble form of the MAC
sC5b-9, all of which are generated only if complement activa-
tion has occurred.
Gel after precipitating and staining

Outer
Alternative Pathway Assays
diameter
Alternative pathway activation can be measured by several
different means. An AH50 can be performed in the same
manner as the CH50, except magnesium chloride and ethyl-
ene glycol tetraacetic acid (EGTA) are added to the buffer and
calcium is left out.32 This buffer chelates calcium, which
Patient with low level blocks classical pathway activation. Rabbit RBCs are used as
FIGURE 7–10 Radioimmunodiffusion for measurement of comple- the indicator because they provide an ideal surface for alter-
ment component C5. native pathway activation.
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106 SECTION 1 Nature of the Immune System

pathway are coated with mannose. Such testing is easy to per-

form and is not dependent upon the use of animal erythro-
cytes, which may be hard to obtain. Deficiencies can be
detected using the combined test results.

Interpretation of Laboratory Findings

Decreased levels of complement components or activity may
be caused by decreased production, consumption, or in vitro
consumption. The third condition must be ruled out before
either of the other two is considered. Specimen handling is
extremely important. Blood should be collected in a clot tube
with no serum separator.39 The tube should be spun down and
Antibody-coated
the serum should be frozen or placed on dry ice if it is not
red blood cells tested within 1 to 2 hours. If a specimen has been inadequately
refrigerated, been subjected to multiple freeze–thaws, or been
Patient serum Complement activation in prolonged storage, the results may be invalid and the test
A Red blood cell lysis needs to be repeated with a fresh specimen. Control serum
Serum to be tested is diluted serially and added to sensitized should also be included with each batch of test sera.
sheep red blood cells. The tubes are incubated at 37°C and then If a complement deficiency is suspected, it is possible to
centrifuged to pellet the unlysed cells.
narrow down the possible candidate components with a CH50
The CH50 is defined as the reciprocal of the dilution that causes and an AH50 assay. If the CH50 is low but the AH50 is normal,
lysis of 50% of the cells used in the assay. the components unique to the classical pathway should be in-
In the example shown, the CH50 would be about 80 U/mL.
vestigated. If the CH50 is normal but the AH50 is low, the al-
ternative pathway components need to be investigated. If both
the CH50 and AH50 are low, suspicion should be on the com-
100% ponents of the terminal pathway because that pathway is
shared by both the classical and alternative pathways. Although
this analysis will be true for most patients, it is also possible, if
there is sufficient activation of complement through any one
pathway, that the activation could consume enough compo-
Percent lysis

nents to lower the function of the other pathways. As previ-

ously stated, complement activation is rarely limited to just one
pathway. Such consumption can result from loss of a control
protein, presence of an autoantibody, an ongoing infection, or
other activation circumstances.
Once a CH50 and AH50 hemolytic assay have been per-
0% formed, it is appropriate to move to testing the levels or func-
1/320 1/160 1/80 1/40 1/20 tion of the components as directed by the relative results of the
B Serial dilutions CH50 and AH50. For many of the components, an antigen
FIGURE 7–11 CH50 testing. (A) Antibody-coated RBCs are added to level is sufficient to determine where the deficiency lies; how-
patient serum. This activates the complement in the sample and the ever, there are a few instances in which measurement of the
RBCs are lysed. Although only C1qrs is shown for simplicity, this test function would be more informative. For C8, it is necessary to
requires C1 through C9 to be present in order for lysis to occur. The measure function because it is a three-subunit protein. The loss
degree of lysis indicates the functional capacity of the complete of one of the three subunits would not put the level out of nor-
classical pathway. (B) CH50 methodology. mal range, but it would render the remaining two subunits
nonfunctional. C2 type II deficiency results from a genetic mu-
tation in C2 that renders the protein nonfunctional but does
An additional means of testing for alternative pathway func- not decrease the level of expression, which is also true for
tion is via ELISA. One such test can detect C3bBbP or C3bP C1-INH in type II HAE.
complexes in very small quantities. Microtiter wells are typi- A typical screening test for complement abnormalities usu-
cally coated with bacterial polysaccharide to trigger activation ally includes determination of the following: C3 and C4, as
of the alternative pathway. well as hemolytic content. Testing for products of complement
One test system has been developed that can determine the activation such as C3a, C4a, C5a, and Ba (as well as breakdown
activity of all three pathways.32,39 Strips used for the classical products including iC3b and C4d) can also be performed as a
pathway are coated with IgM, strips for the alternative pathway means of monitoring inflammatory processes such as rheuma-
are coated with lipopolysaccharide, and strips for the MBL toid arthritis and SLE. Table 7–5 presents some of the possible
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Chapter 7 Complement System 107

Table 7–5 Diagnosis of Complement activation unit consisting of C2, C4, and C3; and the
Abnormalities MAC, consisting of C5, C6, C7, C8, and C9.
IMPAIRED • The lectin pathway is activated by carbohydrates pres-
FUNCTION OR CLASSICAL LECTIN ALTERNATIVE ent in microbial cell walls and serves as an important
DEFICIENCY PATHWAY PATHWAY PATHWAY link between the innate and adaptive immune re-
C1q, C1r, C1s Low Normal Normal sponses. Molecules distinct to the lectin pathway include
C4, C2 Low Low Normal
mannose-binding lectin (MBL), MASP-1, MASP-2, and
MASP-3.
MBL, MASP2 Normal Low Normal • The alternative pathway is triggered by bacterial and fun-
B, D, P Normal Normal Low gal cell walls, yeast, viruses, tumor cells, and certain par-
asites. Factors unique to the alternative pathway include
C3, C5, C6, Low Low Low Factor B, Factor D, and properdin.
C7, C8, C9
• The MAC is common to all three pathways.
C1-INH Low Low Low • Plasma protein regulators of the complement system
Factor H and I Low Low Low
play an extremely important role because if uncon-
trolled, complement activation could have devastating
Improperly Low Low Low systemic effects.
handled • Soluble regulators include C1 inhibitor (C1-INH),
sera C4-binding protein (C4BP), Factor H, Factor I, and
Adapted from Seelen MA, et al. An enzyme-linked immunosorbent assay- S protein.
based method for functional analysis of the three pathways of the comple- • Examples of cell-bound regulators are complement recep-
ment system. In: Detrick B, Hamilton RG, and Folds JD, eds. Manual of tor type 1 (CR1), membrane cofactor protein (MCP), and
Molecular and Clinical Laboratory Immunology. 7th ed. Washington, DC: decay-accelerating factor (DAF).
ASM Press; 2006:124. • Specific complement receptors found on host cells am-
plify the immune response by enhancing phagocytosis
screening results from ELISA testing and correlates these with and stimulating other accessory cells. Some of these
deficiencies of individual factors. An understanding of these receptors include CR1, CR2, CR3, CR4, and collectin
patterns may be helpful in differentiating hereditary deficien- receptors.
cies from activational states that consume available comple- • Effector molecules generated during complement activa-
ment components. Additional testing would be necessary, tion play a major role in recognition and presentation of
however, to actually pinpoint hereditary deficiencies. antigens, activation of B cells, and maintenance of im-
munologic memory. They are classified as anaphylatoxins,
chemotaxins, and opsonins.
SUMMARY • Anaphylatoxins increase vascular permeability, whereas
chemotaxins attract phagocytic cells to a specific area
• The complement system is a series of more than 30 soluble and opsonins coat damaged or foreign cells to enhance
and cell-bound proteins that interact with both the innate phagocytosis.
and adaptive immune systems to enhance host defenses • Deficiencies of complement components can place
against infection. an individual at risk for certain infections. Missing or
• Activities of complement include lysis of foreign or dam- deficient regulators are the cause of diseases such as
aged cells, opsonization, increase in vascular permeability, paroxysmal nocturnal hemoglobinuria and hereditary
and attraction of monocytes and macrophages to areas angioedema.
where needed. • Laboratory assays for individual complement components
• The classical complement pathway is triggered by IgG or include radial immunodiffusion and nephelometry.
IgM binding to the surface of pathogens. Nine major pro- • The hemolytic titration or CH50 assay is a measure of
teins are involved in this pathway. lysis, the end point of complement activation in the clas-
• Three distinct units are involved in the classical pathway. sical pathway. The AH50 assay is a similar test for mea-
They are the recognition unit consisting of C1qrs; the suring the activity of the alternative pathway.
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108 SECTION 1 Nature of the Immune System

CASE STUDIES
1. A 3-year-old child has a history of serious infections and hands. She stated that she has had these symptoms on
is currently hospitalized with meningitis. The doctor sus- several previous occasions. After ruling out appendicitis,
pects that he may have a complement deficiency and or- the physician ordered a battery of tests, including some
ders testing. The following results are obtained: decreased for abnormalities of complement components. The
CH50, decreased AH50, and normal C4 and C3 levels. following results were obtained: red and white blood
cell count normal, total serum protein normal, CH50
Questions
decreased, alternative pathway function normal, C3 level
a. What do the results indicate about the possible normal, and C4 and C2 levels decreased.
pathway(s) affected?
b. Which component(s) are likely to be lacking? Questions
c. What sort of additional follow-up would be a. What symptoms led physicians to consider a possible
recommended? complement abnormality?
2. A 25-year-old female appeared at the local hospital’s b. What are possible reasons for a decrease in both C4
emergency department with symptoms of abdominal pain and C2?
as well as severe vomiting and swelling of the legs and c. What other testing would confirm your suspicions?

REVIEW QUESTIONS
1. The classical complement pathway is activated by 6. Which of the following describes the role of properdin
a. most viruses. in the alternative pathway?
b. antigen–antibody complexes. a. Stabilization of C3/C5 convertase
c. fungal cell walls. b. Conversion of B to Bb
d. mannose in bacterial cell walls. c. Inhibition of C3 convertase formation
d. Binding and cleavage of Factor B
2. Which of the following is characteristic of complement
components? 7. Which best characterizes the membrane attack
a. Normally present in serum complex (MAC)?
b. Mainly synthesized by B cells a. Each pathway uses different factors to form it.
c. Present as active enzymes b. C5 through C9 are not added in any particular
d. Heat stable order.
c. One MAC unit is sufficient to lyse any type
3. All of the following are true of the recognition unit except of cell.
a. it consists of C1q, C1r, and C1s. d. C9 polymerizes to form the transmembrane
b. the subunits require calcium for binding together. channel.
c. binding occurs at the FC region of antibody
molecules. 8. All of the following represent functions of the
d. C1q becomes an active esterase. complement system except
a. decreased clearance of antigen–antibody
4. Which of the following is referred to as C3 convertase? complexes.
a. C1qrs b. lysis of foreign cells.
b. C3bD c. increase in vascular permeability.
c. C3bBb d. migration of neutrophils to the tissues.
d. C4b5a
9. Which of the following is true of the amplification
5. Mannose-binding protein in the lectin pathway is loop in complement activation?
most similar to which classical pathway component? a. It is only found in the alternative pathway.
a. C3 b. The membrane attack unit is amplified.
b. C1rs c. C3b is the product that is increased.
c. C1q d. Increasing amounts of C1qrs are produced.
d. C4
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Chapter 7 Complement System 109

10. Factor H acts by competing with which of the 16. A decreased CH50 level and a normal AH50 level
following for the same binding site? indicate which deficiency?
a. Factor B a. Decrease in components in the lectin pathway only
b. Factor D b. Decrease in components in the alternative pathway
c. C3B only
d. Factor I c. Decrease in components of both classical and
alternative pathways
11. A lack of CR1 receptors on RBCs would result in d. Decrease in components of the classical pathway
which of the following? only
a. Decreased binding of C3b to RBCs
b. Decreased clearance of immune complexes by the 17. Which best describes the role of an anaphylatoxin?
spleen a. Coats cells to increase phagocytosis
c. Decreased breakdown of C1qrs b. Attracts WBCs to the area of antigen concentration
d. Decreased binding of Factor H c. Increases production of interleukin-1
d. Increases permeability of blood vessels
12. Which best describes the role of CR2 on cell
membranes? 18. Which best describes the role of Factor H?
a. Binds C1qrs to inactivate it a. Acts with DAF to break down C3b
b. Acts as co-receptor on B cells for antigen b. Prevents binding of Factor B to C3b
c. Increases clearance of immune complexes c. Binds to the C5C6C7 complex
d. Binds particles opsonized with C3b d. Binds to C1q to shut down the classical pathway

13. Which of the following best characterizes hemolytic 19. A lack of C1-INH might result in which of the
uremic syndrome? following conditions?
a. It is a rare cause of renal failure in children. a. Paroxysmal nocturnal hemoglobinuria
b. It can be associated with deficiencies in Factor H. b. Hemolytic uremic syndrome
c. The major cause is lack of DAF on RBCs. c. Hereditary angioedema
d. It is associated with antibody-to-C3 convertase. d. Increased bacterial infections

14. The CH50 test measures which of the following? 20. Which would be most effective in measuring an
a. Patient serum required to lyse 50% of sensitized individual complement component?
sheep RBCs a. CH50 assay
b. Functioning of both the classical and alternative b. Radial immunodiffusion
pathways c. AH50 assay
c. Genetic deficiencies of any of the complement d. Lytic assay with liposome
components
d. Functioning of the lectin pathway only

15. Which of the following would be most effective in

preventing bystander lysis of RBCs?
a. C1-INH
b. Factor B
c. DAF
d. Factor H
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Basic Immunologic II
Procedures

111
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8 Safety and Quality

Management
Marjorie Schaub Di Lorenzo, MT(ASCP)SH

LEARNING OUTCOMES CHAPTER OUTLINE

After finishing this chapter, you should be able to: LABORATORY HAZARDS
1. List the six components of the chain of infection and the safety pre- Biological Hazards
cautions that break the chain. Sharps Hazards
2. Correctly perform hand hygiene procedures following CDC guidelines. Chemical Hazards
3. Describe the types of personal protective equipment used by laboratory Radioactive Hazards
personnel. Electrical Hazards
4. Differentiate between universal precautions, body substance isolation, Fire and Explosive Hazards
and standard precautions.
Physical Hazards
5. State the acceptable methods for disposal of biological waste and
QUALITY MANAGEMENT
sharp objects in the laboratory.
Procedure Manual
6. Discuss the federal regulations and guidelines for preparing and ship-
ping patient samples from the laboratory. Preexamination Variables
7. Explain the components of the Occupational Exposure to Bloodborne Examination Variables
Pathogens Compliance Directive. Postexamination Variables
8. Describe safety precautions utilized when handling chemicals. REGULATORY ISSUES
9. Discuss the components of Chemical Hygiene Plans and Safety Data Clinical Laboratory Improvement
Sheets. Amendments (CLIA)
10. State and interpret the components of the National Fire Protection Clinical and Laboratory Standards
Association hazardous material labeling system. Institute (CLSI)
11. Describe precautions that laboratory personnel should take with The Joint Commission (TJC)
regard to radioactive, electrical, fire, and physical hazards. College of American Pathologists
12. Explain the RACE and PASS actions to be taken when a fire is discovered. (CAP)
13. Recognize standard hazard warning symbols. QUALITY MANAGEMENT SYSTEMS
14. Define the preexamination (preanalytical), examination (analytical), and Quality System Essentials
postexamination (postanalytical) components of quality management. The Lean System
15. Distinguish between the components of internal quality control, ex- Six Sigma
ternal quality control, electronic quality control, and external quality
SUMMARY
assessment (proficiency testing).
CASE STUDIES
16. Discuss the roles of the Clinical Laboratory Improvement Amend-
ments (CLIA), Clinical and Laboratory Standards Institute (CLSI), the REVIEW QUESTIONS
The Joint Commission (TJC), and the College of American Patholo-
gists (CAP) in the regulation of health care.
17. State and describe the 12 quality essentials used in a quality manage-
ment system.

You can go to DavisPlus at [Link] keyword Stevens for the

laboratory exercises that accompany this text.
112
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Chapter 8 Safety and Quality Management 113

18. Describe the purpose of quality indicators.

19. List the six areas of the Lean system and describe how it can benefit
the laboratory.
20. State the purpose of the Six Sigma methodology in a management system.

KEY TERMS
Accuracy External quality assessment Postexposure prophylaxis Quality system essentials
Biohazardous material (EQA) (PEP) (QSEs)
Chain of infection Infection control Precision Reliability
Chemical Hygiene Plan The Joint Commission Preexamination variables Safety data sheet (SDS)
(TJC) Proficiency testing Shift
Clinical Laboratory
Improvement Lean system Quality assessment Six Sigma
Amendments (CLIA) Occupational Safety and (QA) Standard deviation (SD)
Clinical and Laboratory Health Administration Quality control (QC) Standard of care
Standards Institute (CLSI) (OSHA)
Quality indicators Standard precautions (SP)
Coefficient of variation (CV) Personal protective
Quality management Trend
equipment (PPE)
Control mean (QM)
Postexamination Turnaround time (TAT)
Delta check Quality management
variables Variable
Examination variables system (QMS)

Laboratory Hazards necessary to prevent infection. The chain of infection requires

a continuous link between six elements: an infectious agent, a
The clinical laboratory contains a wide variety of safety hazards, reservoir, a portal of exit, a means of transmission, a portal of
many capable of producing serious injury or life-threatening dis- entry, and a susceptible source.1
ease. To work safely in this environment, clinical laboratorians Infectious agents consist of bacteria, fungi, parasites, and
must learn what hazards exist and the basic safety precautions viruses. A reservoir is a place where the infectious agent can live
associated with them. They must apply the basic rules of com- and multiply, such as a contaminated clinical specimen or an
mon sense required for everyday safety. Some hazards are unique infected patient. Humans and animals (hosts) or contaminated
to the health-care environment and others are encountered rou- inanimate objects (fomites) that contain blood, urine, or other
tinely throughout life (Table 8–1). It is essential that laboratory body fluids make ideal reservoirs. The infectious agent leaves
personnel know where all safety equipment is located and be the reservoir through a portal of exit such as through the nose,
trained in all aspects of its use on a yearly basis. mouth, and mucous membranes, as well as in blood or other
body fluids, and is transmitted to a susceptible source to con-
tinue the chain of infection.
Biological Hazards Means of transmission include direct contact (the unpro-
In the immunology laboratory, the most significant hazard ex- tected host touches the patient, specimen, or a contaminated
ists in obtaining and testing patient specimens. Understanding object); droplet (the host inhales infected aerosol droplets from
how microorganisms are transmitted (chain of infection) is a patient or specimen); airborne (the host inhales dried aerosol

Table 8–1 Types of Safety Hazards

TYPE SOURCE POSSIBLE INJURY
Biological Infectious agents Bacterial, fungal, viral, or parasitic infections
Sharp Needles, lancets, and broken glass Cuts, punctures, or bloodborne pathogen exposure
Chemical Preservatives and reagents Exposure to toxic, carcinogenic, or caustic agents
Radioactive Equipment and radioisotopes Damage to a fetus or generalized overexposure to radiation
Electrical Ungrounded or wet equipment Burns or shock
and frayed cords
Fire or explosive Open flames and organic chemicals Burns or dismemberment
Physical Wet floors, heavy boxes, and patients Falls, sprains, or strains
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114 SECTION 2 Basic Immunologic Procedures

particles circulating on the air currents or dust particles); vehicle shields. Gloves are worn to protect the health-care worker’s hands
(the host ingests contaminated food or water); or by a vector from contamination by patient body substances and to protect
(from an animal or mosquito bite). The infectious agent now the patient from possible microorganisms on the health-care
must enter a new reservoir through a portal of entry, which can worker’s hands. However, wearing gloves is not a substitute for
be the same as the portal of exit and include mucous membranes hand sanitizing. Hands must always be sanitized when gloves are
of the nose, mouth, and eyes; breaks in the skin; and open removed. A variety of gloves are available, including sterile and
wounds to complete the chain of infection. A susceptible source nonsterile, powdered and unpowdered, and latex and nonlatex.
can be another patient, health-care personnel, or visitors. Patients Allergy to latex is decreasing among health-care workers
receiving chemotherapy, the elderly, and immunocompromised due to the availabliity of other types of gloves. However, labo-
patients are susceptible hosts. The immune system is still devel- ratorians should be alert for symptoms of reactions associated
oping in newborns and infants and begins to weaken as people with latex contact, including irritant contact dermatitis that
age, making these groups of patients more susceptible to infec- produces patches of dry, itchy irritation on the hands; delayed
tion. The immune system also is depressed by stress, fatigue, and hypersensitivity reactions resembling poison ivy that appear
lack of proper nutrition, which contribute to the susceptibility of 24 to 48 hours following exposure; and true immediate hyper-
patients and health-care personnel.1 Once the chain of infection sensitivity reactions often characterized by facial flushing and
is complete, the infected host then becomes another source able respiratory difficulty (see Chapter 14). Hand sanitizing imme-
to transmit the microorganisms to others.1 diately after removal of gloves and avoiding powdered gloves
The most likely source of infection in serological testing is may aid in preventing the development of latex allergy. Any
through contact with patient specimens; the main concern is signs of a latex reaction should be reported to a supervisor
exposure to viruses such as the hepatitis viruses and human because a true latex allergy can be life threatening.4
immunodeficiency virus (HIV). Therefore, safety precautions In the immunology laboratory, fluid-resistant laboratory
are designed to protect health-care workers from exposure to coats with wrist cuffs are worn at all times to protect skin and
potentially harmful infectious agents. The ultimate goal of bi- clothing from contamination by patient specimens. They must
ological safety is to prevent completion of the chain by pre- be completely buttoned with gloves pulled over the cuffs. Both
venting transmission. The infection control team develops gloves and laboratory coats should be changed as soon as pos-
procedures to control and monitor infections occurring within sible if they become visibly soiled and must be removed when
health-care facilities. Figure 8–1 contains the universal symbol leaving the laboratory.
for biohazardous material and illustrates the chain of infec- The mucous membranes of the eyes, nose, and mouth must
tion and how it can be broken by following safety practices.1 be protected from specimen splashes and aerosols. A variety of
Preventing the transmission of microorganisms from infected protective equipment is available, including goggles, full-face
sources to susceptible hosts is critical in controlling the spread plastic shields, and Plexiglas countertop shields (Fig. 8–3).
of infection. Procedures used to prevent microorganism trans- Particular care should be taken to avoid splashes and aerosols
mission include hand hygiene, wearing personal protective when removing container tops and when transferring and cen-
equipment (PPE), isolating highly infective or highly suscep- trifuging specimens. Never centrifuge specimens in uncapped
tible patients, and properly disposing of contaminated materi- tubes or in uncovered centrifuges. When specimens are re-
als. Strict adherence to guidelines published by the Centers for ceived in containers with contaminated exteriors, the exterior
Disease Control and Prevention (CDC) and the Occupational of the container must be disinfected; if necessary, a new spec-
Safety and Health Administration (OSHA) is essential.2 imen may be requested.
Hand Hygiene Standard Precautions
Hand contact represents the number one method of infection The CDC developed standard precautions (SP) by combining
transmission. Hands should always be sanitized before patient recommendations of universal precautions (UP) and body substance
contact, after gloves are removed, before leaving the work area, isolation (BSI) procedures. Under UP, all patients were assumed
whenever the hands have been knowingly contaminated, be- to be potential carriers of bloodborne pathogens. The BSI mod-
fore going to designated break areas, and before and after using ified UP by requiring that gloves be worn when encountering
bathroom facilities. Hand hygiene includes both hand washing blood or any other body substance. The CDC continually mod-
and using alcohol-based antiseptic cleansers. Alcohol-based ifies SP as changes occur in the health-care environment. SP as-
cleansers are not recommended after contact with spore-forming sumes every person in the health-care setting is potentially
bacteria, including Clostridium difficile and Bacillus sp. infected or colonized by an organism that could be transmitted.
The CDC’s guidelines for the correct hand washing technique SP applies to all blood and body fluids, mucous membranes, and
are pictured in Figure 8–2.3 If using alcohol-based cleansers, nonintact skin and stresses hand washing.5
apply the cleanser to the palm of one hand. Rub your hands to- Standard precautions that apply directly to the laboratory
gether and over the entire cleansing area, including between the are as follows:
fingers and thumbs. Continue rubbing until the alcohol dries.
• Hand hygiene—Hand hygiene includes both hand washing
Personal Protective Equipment and the use of alcohol-based antiseptic cleansers. Sanitize
PPE used by laboratorians includes gloves, gowns or laboratory hands after touching blood, body fluids, secretions, excre-
coats, masks, goggles, face shields, and Plexiglas countertop tions, and contaminated items, whether or not gloves are
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Chapter 8 Safety and Quality Management 115

Break the link Break the link

• Immunizations • Disinfection
• Patient isolation Infectious agent • Hand hygiene
• Nursery • Bacteria
precautions • Fungi
• Healthy lifestyle • Parasites
Susceptible • Viruses
host Reservoir
• Patients • Humans
• Elderly • Animals
• Newborns • Insects
• Immuno- • Fomites
compromised • Blood/body
• Health-care fluids
workers

Portal of exit
Portal of
entry • Nose
• Mouth
• Nose
• Mucous
• Mouth
membranes
• Mucous
• Specimen
membranes
collection
• Skin
• Unsterile
equipment

Means of transmission
• Droplet
• Airborne
Break the link • Contact Break the link
• Hand hygiene • Vector • Sealed biohazardous
• Standard precautions • Vehicle waste containers
• PPE • Sealed specimen
• Sterile equipment containers
• Hand hygiene
• Standard precautions
Break the link
• Hand hygiene
• Standard precautions
• PPE
• Patient isolation

FIGURE 8–1 Chain of infection and safety practices related to the biohazard symbol. (From Strasinger SK, DiLorenzo MS. The Phlebotomy Text-
book. 3rd ed. Philadelphia, PA: F.A. Davis, Philadelphia; 2011, with permission.)

worn. Sanitize hands immediately after gloves are removed, patient-care activities that are likely to generate splashes
between patient contacts, and when otherwise indicated to or sprays of blood, body fluids, secretions, and excretions.
avoid transfer of microorganisms to other environments. • Gown—Wear a gown (a clean, nonsterile gown is ade-
• Gloves—Wear gloves (clean, nonsterile gloves are adequate) quate) to protect skin and to prevent soiling of clothing
when touching blood, body fluids, secretions, excretions, during procedures that are likely to generate splashes of
and contaminated items. Remove gloves promptly after use, blood, body fluids, secretions, or excretions. Select a
before touching noncontaminated items and environmental gown that is appropriate for the activity and amount of
surfaces, and before going to another patient. Sanitize hands fluid likely to be encountered (e.g., fluid-resistant in
immediately to avoid transfer of microorganisms to other the laboratory). Remove a soiled gown before leaving the
patients or environments. laboratory environment and sanitize hands to avoid trans-
• Mask, nose, and eye protection—Wear a mask and eye fer of microorganisms to other environments.
protection or a face shield to protect mucous membranes • Respiratory hygiene and cough etiquette—Educate
of the eyes, nose, and mouth during procedures and health-care personnel, patients, and visitors to contain
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116 SECTION 2 Basic Immunologic Procedures

1. Wet hands with warm water. Do not allow any part of the 2. Apply soap.
body to touch the sink.

3. Rub to form a lather, create friction, and loosen debris. Thor- 4. Rinse hands in a downward position to prevent recontamina-
oughly clean between the fingers and under the fingernails for tion of hands and wrists.
at least 20 seconds; include thumbs and wrists in the cleaning.

5. Dry hands with paper towel. 6. Turn off faucets with a clean paper towel to prevent
contamination.
FIGURE 8–2 Hand hygiene. (From Strasinger SK, DiLorenzo MS. Urinalysis and Body Fluids. 6th ed. Philadelphia, PA: F.A. Davis; 2014, with
permission.)

respiratory secretions to prevent droplet and fomite trans- any part of the body; rather, use self-sheathing needles or a
mission of respiratory pathogens. Offer masks to coughing mechanical device designed to conceal the needle. Do not
patients, distance symptomatic patients from others, and remove unsheathed needles from disposable syringes by
practice good hand hygiene to prevent the transmission hand; use a mechanical device. Do not bend, break, or
of respiratory pathogens. otherwise manipulate used needles by hand. Place used
• Needles—Never recap used needles or otherwise manipu- disposable syringes and needles, scalpel blades, and other
late them using both hands; in addition, never use any tech- sharp items in appropriate puncture-resistant containers.
nique that involves directing the point of a needle toward Place reusable syringes and needles in a puncture-resistant
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Chapter 8 Safety and Quality Management 117

In the Laboratory
Components of the OSHA Bloodborne Pathogen
Exposure Control Plan
Engineering Controls
1. Providing sharps disposal containers and needles with safety
devices
2. Requiring discarding of needles with the safety device acti-
vated and the holder attached
3. Labeling all biohazardous materials and containers
Work Practice Controls
4. Requiring all employees to practice standard precautions
5. Prohibiting eating, drinking, smoking, and applying cosmet-
ics in the work area
6. Establishing a daily work surface disinfection protocol
Personal Protective Equipment
FIGURE 8–3 Personal protective equipment using a plastic shield. 7. Providing laboratory coats, gowns, face shields, and gloves
(From Strasinger SK, DiLorenzo MS. Urinalysis and Body Fluids. 6th ed. to employees and laundry facilities for nondisposable pro-
Philadelphia, PA: F.A. Davis; 2014, with permission.) tective clothing
Medical
8. Providing immunization for the hepatitis B virus free of
container for transport to the reprocessing area. (See Sharps charge
Hazards later for additional information.) 9. Providing medical follow-up to employees who have been
accidentally exposed to bloodborne pathogens
Occupational Exposure to Bloodborne Pathogens Documentation
10. Documenting annual training of employees in safety
The federal government has enacted regulations to protect
standards
health-care workers from exposure to bloodborne pathogens. 11. Documenting evaluations and implementation of safer
These regulations are monitored and enforced by OSHA. The needle devices
Occupational Exposure to Bloodborne Pathogens Standard6 12. Involving employees in the selection and evaluation of new
requires all employers to have a written Bloodborne Pathogen devices and maintaining a list of those employees and the
Exposure Control Plan and to provide necessary protection, evaluations
free of charge, for employees. A later compliance directive 13. Maintaining a sharps injury log including the type and brand
called Enforcement Procedures for the Occupational Exposure of safety device, location and description of the incident,
to Bloodborne Pathogens Standard placed more emphasis on and confidential employee follow-up
using engineering controls to prevent accidental exposure to From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia,
bloodborne pathogens.7 The components of the current Blood- PA: F.A. Davis; 2011, with permission.
borne Pathogens Exposure Control Plan that is required of all
institutions are shown in the In the Laboratory: Components
of the OSHA Bloodborne Pathogen Exposure Control Plan
box. Each health-care institution is responsible for designing or yellow color coding. These supplies include alcohol pads,
and implementing its own exposure control plan.8 gauze, bandages, disposable tourniquets, gloves, masks, gowns,
Any accidental exposure to blood through needlestick, mu- and plastic tubes and pipettes. Disposal of needles and other
cous membranes, or nonintact skin must be reported to a sharp objects is discussed in the next section.
supervisor and a confidential medical examination must be Contaminated nondisposable equipment, blood spills, and
immediately started. Evaluation of the incident must begin right blood and body fluid processing areas must be disinfected. The
away to ensure appropriate postexposure prophylaxis (PEP) most commonly used disinfectant is a 1:10 dilution of sodium
is initiated within 24 hours. Needlesticks are the most fre- hypochlorite (household bleach) prepared daily and stored in
quently encountered exposure and place the laboratorian in a plastic, not a glass, bottle. The bleach should be allowed to
danger of contracting HIV, HBV, and hepatitis C virus (HCV). air-dry on the contaminated area before being wiped off.9
The CDC has recommended procedures to prevent these (see Transporting Patient Specimens
In the Laboratory: Postexposure Prophylaxis).
If a laboratory accepts specimens from other health-care insti-
Biological Waste Disposal tutions, then it is important to know the regulations for pack-
All biological waste, except urine, must be placed in appropri- aging, transporting, and receiving these specimens. The U.S.
ate containers labeled with the biohazard symbol. This waste Department of Transportation (DOT), the International Air
includes not only specimens but also the materials with which Transport Association (IATA), and the United Nations (UN)
the specimens come in contact. Any supplies contaminated have stringent regulations that must be followed if a laboratory
with blood and body fluids must also be disposed of in con- is going to be involved in transporting or receiving patient
tainers clearly marked with the biohazard symbol or with red specimens from another institution.10,11
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118 SECTION 2 Basic Immunologic Procedures

In the Laboratory then all DOT rules apply. The transport vehicle should be
used exclusively for transport of specimens and should be
Postexposure Prophylaxis equipped to secure the transport containers.12 Minimum
1. Draw a baseline blood sample from the employee and test it shipping standards for this type of transportation include13
for HBV, HCV, and HIV. • Leakproof, watertight specimen containers
2. If possible, identify the source patient, collect a blood sample, • Tightly capped tubes placed in a rack to maintain an up-
and test it for HBV, HCV, and HIV. Patients must usually give right position
informed consent for these tests and they do not become
• Leakproof inner packaging surrounded by enough ab-
part of the patient’s record. In some states, a physician’s order
sorbent material to completely absorb all the liquid present
or court order can replace patient consent because a needle-
stick is considered a significant exposure. • A leakproof plastic or metal transport box with a secure,
3. Testing must be completed within 24 hours for maximum tight-fitting cover
benefit from PEP. • Properly labeled transport boxes accompanied by speci-
Source patient tests positive for HIV: men data and identification forms
1. Employee is counseled about receiving PEP using zidovudine Specimens picked up by a courier that are to be shipped to an
(ZDV) and one or two additional anti-HIV medications. out-of-the-area laboratory, such as a reference laboratory, must
2. Medications are started within 24 hours.
follow DOT regulations. Many of these laboratories supply
3. Employee is retested at intervals of 6 weeks, 12 weeks, and
6 months.
shipping containers to their clients.
4. Additional evaluation and counseling is needed if the source
patient is unidentified or untested. Sharps Hazards
Source patient tests positive for HBV: Sharp objects in the laboratory, including needles, lancets, and
1. Unvaccinated employees can be given hepatitis B immune broken glassware, present a serious biological hazard for pos-
globulin (HBIG) and HBV vaccine.
sible exposure to bloodborne pathogens caused by accidental
2. Vaccinated employees are tested for immunity and receive
PEP, if necessary.
puncture. Although bloodborne pathogens are also transmitted
Source patient tests positive for HCV: through contact with mucous membranes and nonintact skin,
1. No PEP is available. a needle or lancet used to collect blood has the capability to
2. Employee is monitored for early detection of HCV infection produce a very significant exposure to bloodborne pathogens.
and treated appropriately. It is essential that safety precautions be followed at all times
Any exposed employee should be counseled to report any when sharp hazards are present.
symptoms related to viral infection that occur within 12 weeks The number one personal safety rule when handling needles
of the exposure. is to never recap a needle. Many safety devices are available for
From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia,
needle disposal that provide a variety of safeguards. These include
PA: F.A. Davis; 2011, with permission. needle holders that become a sheath, needles that automatically
resheath or become blunt, and needles with attached sheaths. All
sharps must be disposed of in puncture-resistant, leakproof con-
tainers labeled with the biohazard symbol (Fig. 8–5). Containers
DOT and IATA Specimen Transport. Under DOT and IATA should be located in close proximity to the work area and must
regulations, all diagnostic specimens require triple packaging always be replaced when the safe capacity mark is reached. Never
(Fig. 8–4). This includes the following: use any technique that involves directing the point of a needle
• The primary container (glass, metal, or plastic) must be toward any part of the body.
watertight with a positive (screw-on) cap. The Needlestick Safety and Prevention Act was signed into
• The primary container must be wrapped with enough ab- law in 2001.14 In June 2002, OSHA issued a revision to the
sorbent material to be capable of absorbing all of its con- Bloodborne Pathogens Standard compliance directive men-
tents. Multiple specimens must be wrapped individually tioned previously.15 In the revised directive, the agency requires
before placing them in the leakproof secondary container. that all blood holders with needles attached be immediately dis-
• The secondary container must be placed in a sturdy outer carded into a sharps container after the device’s safety feature is
container made of corrugated fiberboard, wood, metal, or activated. The rationale for the new directive was based on the
rigid plastic. An itemized list of contents in a sealed plastic exposure of workers to the unprotected stopper-puncturing end
bag is also placed in the outer container. Ice packs are of evacuated tube needles, the increased needle manipulation
placed between the secondary and the outer container. required to remove it from the holder, and the possible worker
Additional measures must be taken when using ice and exposure from the use of contaminated holders.
dry ice.
Courier-Delivered Specimen Transport. Specimens trans-
Chemical Hazards
ported by a hospital courier among clinics, physicians’ offices, Serological testing may involve use of chemical reagents that
and the hospital laboratory are exempt from most DOT must be handled in a safe manner to avoid injury. The general
rules, unless they are suspected of containing an infectious rules for safe handling of chemicals include taking precautions
substance. If specimens may contain an infectious substance, to avoid getting chemicals on the body, clothes, and work
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Chapter 8 Safety and Quality Management 119

Infectious substance
Absorbent packing
Primary receptacle material (for liquids)
leakproof or siftproof
Cross section of closed package

Secondary packing
leakproof or siftproof Primary receptacle
(e.g., sealed plastic bag) leakproof or siftproof

Secondary packing
leakproof or siftproof
Bio
(e.g., sealed plastic bag
Su logica or other intermediate
Catbstanc l
erg
ory e packaging)
B Rigid outer
Rigid outer packaging packaging
UN3
373 Absorbent material
Cushioning
Package mark material

Name and telephone number of a

person responsible. (This information
may instead be provided on a written
document such as an air waybill.)
FIGURE 8–4 Packing and labeling of category B infectious substances. If multiple fragile primary receptacles are placed in a single secondary
package, they must be either individually wrapped or separated to prevent contact. (From U.S. Department of Transportation. Pipeline and
Hazardous Materials Safety Administration.)

then seek medical attention. Laboratorians must know the lo-

cation of the emergency shower and eyewash station in the lab-
oratory. Do not try to neutralize chemicals spilled on the skin.
Safety Data Sheets (SDS)
All chemicals and reagents containing hazardous ingredients in
a concentration greater than 1% are required by OSHA to have a
safety data sheet (SDS) on file in the work area. By law, vendors
must provide these sheets to purchasers; however, it is the re-
sponsibility of the facility to obtain and keep them available to
employees. A SDS contains information on physical and chemical
characteristics, fire, explosion reactivity, health hazards, primary
routes of entry, exposure limits and carcinogenic potential, pre-
cautions for safe handling, spill cleanup, and emergency first aid.
FIGURE 8–5 Examples of puncture–resistant containers. (From Containers of chemicals that pose a high risk must be labeled
Strasinger SK, DiLorenzo MS. The Phlebotomy Textbook. 3rd ed. with a chemical hazard symbol representing the possible hazard,
Philadelphia, PA: F.A. Davis; 2011, with permission.) such as flammable, poisonous, corrosive, and so on. State and
federal regulations should be consulted for the disposal of chem-
area; wearing PPE such as safety goggles when pouring chem- icals. Containers of chemicals that pose a high risk must be
icals; observing strict labeling practices; and following instruc- labeled with a chemical hazard symbol representing the possible
tions carefully. Preparing reagents under a fume hood is also a hazard, such as flammable, poison, or corrosive (Fig. 8–6).
recommended safety precaution. Chemicals should never be
Chemical Hygiene Plan
mixed together unless specific instructions are followed; in
addition, they must be added in the order specified. This is OSHA requires that all facilities that use hazardous chemicals
particularly important when combining acid and water because have a written Chemical Hygiene Plan available to employ-
acid should always be added to water, rather than adding water ees.16,17 The purpose of the plan is to detail the following:
to acid, to avoid the possibility of sudden splashing. • Appropriate work practices
When skin or eye contact occurs, the best first aid is to im- • Standard operating procedures
mediately flush the area with water for at least 15 minutes and • Personal protective equipment
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120 SECTION 2 Basic Immunologic Procedures

FIGURE 8–6 Chemical hazard symbols. (From Strasinger SK,

DiLorenzo MS. The Phlebotomy Textbook. 3rd ed. Philadelphia,
PA: F.A. Davis; 2011, with permission.)

• Engineering controls, such as fume hoods and safety cab-

inets for flammables
• Employee training requirements RADIATION
• Medical consultation guidelines
FIGURE 8–7 Radioactive symbol. (From Strasinger SK, DiLorenzo MS.
Each facility must appoint a chemical hygiene officer who is The Phlebotomy Textbook. 3rd ed. Philadelphia, PA: F.A. Davis; 2011,
responsible for implementing and documenting compliance with permission.)
with the plan.
Chemical Waste Disposal
Any hazardous chemical waste should be disposed of per cur- laboratories use radioactivity in testing anymore, mainly be-
rent EPA regulations. Most reagents used in the laboratory cause of the problem of disposing of waste. If radioactivity is
come with a SDS, mentioned previously. The SDS gives specific used, the laboratory typically contracts with a waste disposal
information for disposal of particular chemicals. All chemicals service that picks up the radioactive waste material.
used should be disposed of by following SDS directions. Many
kits used in immunologic testing often contain sodium azide Electrical Hazards
as a preservative, which can be disposed of by flushing down
The laboratory setting contains electrical equipment with
the drain with plenty of water. Using large amounts of water
which laboratorians have frequent contact. The same general
helps to avoid buildup in plumbing.
rules of electrical safety observed outside the workplace apply
in the laboratory, such as checking for frayed cords or over-
Radioactive Hazards loaded circuits. Laboratorians also have frequent contact with
Laboratorians can be exposed to radioactivity in the clinical water, fluids, and chemical agents; therefore, the danger of
laboratory when performing procedures using radioisotopes, water or fluid coming in contact with equipment is greater in
such as radioimmunoassay. The amount of radioactivity pres- the laboratory setting. Equipment should not be operated with
ent in most medical situations is very small and represents little wet hands. Designated hospital personnel closely monitor elec-
danger. However, the effects of radiation are related to the trical equipment. However, laboratory personnel should be ob-
length of exposure and are cumulative. Exposure to radiation servant for any dangerous conditions and report them to the
is dependent on the combination of time, distance, and shield- appropriate persons.
ing. Persons working in a radioactive environment are required When an accident involving electrical shock occurs, the
to wear measuring devices to determine the amount of radia- electrical source must be removed immediately without touch-
tion they are accumulating. ing the person or the equipment involved. Persons responding
Laboratorians should be familiar with the radioactive sym- to the accident must avoid transferring the current to them-
bol shown in Figure 8–7. This symbol must be displayed on selves by turning off the circuit breaker before unplugging the
the doors of all areas where radioactive material is present. Ex- equipment or moving the equipment using a nonconductive
posure to radiation during pregnancy presents a danger to the glass or wood object. The victim should receive immediate
fetus; personnel who are or who think they may be pregnant medical assistance following discontinuation of the electricity.
should avoid areas with this symbol. Cardiopulmonary resuscitation (CPR) may be necessary.
Medical Radioactive Waste Disposal
Disposal of medical radioactive waste is regulated by the Nu-
Fire and Explosive Hazards
clear Regulatory Commission (NRC) and is also subject to local Clinical laboratory work involves the use of potentially volatile
regulations. Such waste must be separated from other waste or explosive chemicals that require special procedures for han-
materials in the laboratory and placed in containers marked dling and storage. Flammable chemicals should be stored in
with the radioactive symbol. Disposal varies with the type safety cabinets and explosion–proof refrigerators. Cylinders of
of material (solid, liquid, or volatile chemical) and depends compressed gas should be located away from heat and securely
upon the amount of radioactivity present. Very few immunology fastened to a stationary device to prevent accidental tipping.
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Chapter 8 Safety and Quality Management 121

The Joint Commission (TJC), an independent body that cer- HAZARDOUS MATERIALS
tifies and accredits health-care organizations in the United States, CLASSIFICATION
requires that all health-care facilities post evacuation routes and
detailed plans to follow in the event of a fire. Laboratory person- HEALTH HAZARD FIRE HAZARD
nel should be familiar with these routes. When a fire is discov- Flash Point
4 Deadly 4 Below 73 F
ered, all employees are expected to take the actions described by 3 Extreme Danger 3 Below 100 F
the acronym RACE: 2 Hazardous 2 Below 200 F

2
Rescue—rescue anyone in immediate danger 1 Slightly Hazardous 1 Above 200 F
0 Normal Material 0 Will not burn
Alarm—activate the institutional fire alarm system
Contain—close all doors to potentially affected areas
Extinguish or Evacuate—attempt to extinguish the fire if

3 1
possible, or evacuate, closing the door
Fire blankets should be present in the laboratory. Persons
whose clothes are on fire should be wrapped in the blanket to
smother the flames. The acronym PASS can be used to remem-

W
ber the steps in operating a fire extinguisher:
1. Pull pin
2. Aim at the base of the fire SPECIFIC REACTIVITY
3. Squeeze handles HAZARD
4. Sweep nozzle side to side 4 May deteriorate
3 Shock and heat
The Standard System for the Identification of the Fire Hazard Oxidizer OXY may deteriorate
of Materials, NFPA 704, is a symbol system used to inform fire- Acid ACID 2 Violent chemical
Alkali ALK change
fighters of the hazards they may encounter when fighting a fire Corrosive COR 1 Unstable if
in a particular area. The color-coded areas contain information Use No Water W heated
relating to health hazards, flammability, reactivity, use of water, Radiation 0 Stable
and personal protection. These symbols are placed on doors,
cabinets, and reagent bottles. An example of the hazardous
material symbol and information is shown in Figure 8–8. FIGURE 8–8 NFPA hazardous material symbol and classification.
(From Strasinger SK, DiLorenzo MS. The Phlebotomy Textbook. 3rd ed.
Physical Hazards Philadelphia, PA: F.A. Davis; 2014.)

Physical hazards are not unique to the laboratory; routine pre-

cautions observed outside the workplace apply. Maintaining a
clean and organized work area is essential for minimizing the test results, relationship of patient information to patient test re-
hazards. General precautions to consider include not running sults, patient confidentiality, specimen identification and integrity,
in rooms and hallways, watching for wet floors, bending the personnel competency, personnel qualifications and evaluations,
knees when lifting heavy objects, keeping long hair pulled back, communication protocols, complaint investigations, QA review
and avoiding dangling jewelry. Closed-toed shoes that provide with staff, and maintenance of QA records for 2 years.18
maximum support are essential for safety and comfort. Documentation of QA procedures is required by all labora-
tory accreditation agencies, including TJC, College of American
Pathologists (CAP), American Association of Blood Banks
Quality Management (AABB), American Osteopathic Association (AOA), American
Society of Histocompatibility and Immunogenetics (ASHI), and
The term quality management (QM) refers to the overall the Commission on Laboratory Assessment (COLA); it is also
process of guaranteeing quality patient care. As it relates to the required for Medicare and Medicaid reimbursement. Guide-
clinical laboratory, QM is the continual monitoring of the entire lines published by CAP and the Clinical and Laboratory
test process from test ordering and specimen collection Standards Institute (CLSI) provide very complete instruc-
through reporting and interpreting results. Written policies and tions for documentation and are used as a reference for the
documented actions as they pertain to the patient, the labora- ensuing discussion of the specific areas of immunology QA.1
tory, ancillary personnel, and the health-care provider are re- Documentation in the form of a procedure manual is required
quired. In addition, written remedial actions mandating the in all laboratories; this format is used as the basis for the
steps to take when any part of the system fails is essential to a following discussion.
QA program.
The Clinical Laboratory Improvements Amendments
(CLIA) are regulations that specify required components for QA
Procedure Manual
to include patient test management assessment, quality control A procedure manual (paper or digital) containing all the pro-
(QC) assessment, proficiency testing assessment, comparison of cedures performed in the immunology section of the laboratory
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122 SECTION 2 Basic Immunologic Procedures

must be available for reference in the working area and must Preexamination Variables
comply with the CLSI guidelines. For each test performed, the
procedure manual provides In a clinical laboratory, a QM program encompasses preex-
amination variables (e.g., specimen collection, handling, and
• The principle or purpose of the test
storage), examination variables (e.g., reagent and test per-
• Clinical significance
formance, instrument calibration and maintenance, personnel
• Patient identification and preparation
requirements, and technical competence), postexamination
• Specimen type
variables (e.g., reporting of results and interpretations), and
• Method of collection
documentation that the program is being meticulously followed.
• Specimen labeling
A variable is defined as anything that can be changed or al-
• Specimen preservation
tered. Identification of variables throughout the testing process
• Conditions of transport and storage before testing
provides the basis for development of procedures and policies
• Specimen acceptability and criteria for rejection
within the immunology department that are located in the
• Reagents
procedure manual.
• Standards and controls acceptability and expiration policy
Preexamination variables occur before the actual testing of
• Instrument calibration and maintenance protocols and
the specimen and include test requests, patient preparation,
schedules
timing, specimen collection, handling, and storage. Health-
• Step-by-step procedure
care personnel outside the immunology department control
• Calculations
many of these factors, such as ordering tests and collecting
• Frequency and tolerance limits for controls and corrective
specimens; however, communication between departments
actions
and adequate training on the correct procedures for ordering
• Reference values and critical values
a test, collecting a specimen, and transporting the specimen
• Interpretation of results
improves the turnaround time (TAT) of results, avoids dupli-
• Common interferences
cation of test orders, and ensures a high-quality specimen. TAT
• Specific procedure notes
is defined as the amount of time required between the point at
• Limitations of the method
which a test is ordered by the health-care provider and the re-
• Method validation
sults are reported to the health-care provider. The laboratory
• Confirmatory testing
can monitor the TATs for both stat and routine tests to deter-
• Recording of results
mine areas in the process that need improvement.
• References
• Effective date Specimen Collection and Documentation
• Author Specific guidelines for specimen collection and handling should
• Review schedule19 be stated at the beginning of each procedure listed in the manual.
Current package inserts for all test kits used should be re- In addition to following the guidelines for specimen collection
viewed and included in the manual. The laboratory must also for each specific procedure, requisition forms and electronic entry
have a documented procedure for the correction of erroneous forms should be used to document the type of specimen to be
results.19 collected and the time and date of collection. The form should
The printed procedural manuals and electronic procedural have space for documenting (1) the patient’s first and last name,
manuals are subjected to proper document control. Only au- (2) the patient’s gender, (3) the patient’s age or date of birth,
thorized persons may make changes; these are dated and (4) the name of the person requesting the test, (5) the name of
signed (manually or digitally). The manuals must undergo the person to contact with critical results, (6) the name of the test
periodic review; documentation (i.e., proof) of the review is ordered, (7) any special handling requirements, (8) the time and
included in the manual. date of specimen collection, (9) the time the specimen was
Evaluating procedures and adopting new methodologies is delivered to the laboratory, and (10) any additional information
an ongoing process in the clinical laboratory. Whenever pertinent to laboratory interpretation.20 Information regarding
changes are made, the written procedure in the manual should patient preparation (e.g., fasting or elimination of interfering
be reviewed, referenced, and signed by a person with desig- medications) and the type and volume of specimen required must
nated authority, such as the laboratory director or section su- be included in the specific procedure.
pervisor, and personnel should be notified of the changes. An The criteria for specimen rejection for both physical char-
annual review of all procedures by the designated authority acteristics and labeling errors must be present. If a specimen
must also be documented. is rejected, the criteria for rejecting that specimen must be doc-
The procedure manual provides the basis for all testing in umented and available to the health-care provider and nursing
the immunology laboratory. Quality care in testing relies on staff. Laboratory personnel must determine the suitability of a
strictly following procedures as written. Documentation in- specimen and document any problems and corrective actions
cludes every step from specimen collection to the reporting of taken using an internal laboratory quality improvement form
results. A well-documented QM program ensures quality test (see In the Laboratory: An Example of an Internal Labora-
results and patient care. tory Quality Improvement Form). This report enables the
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Chapter 8 Safety and Quality Management 123

In the Laboratory Reagents

The name and chemical formula of each reagent used, any
An Example of an Internal Laboratory Quality
necessary instructions for preparation or company source of
Improvement Form
prepared materials, storage requirements, and procedures for
Quality Improvement Follow-Up Report
CONFIDENTIAL
reagent QC are all found in the procedure manual. The type
Instructions: Section I should be completed by the individual identifying the event. of water used for preparing reagents and controls must be spec-
ified. Distilled or deionized water or clinical laboratory reagent
Date of report: Reported by:
Date of incident: Date/time of discovery: water (CLRW) must be available. A bold-type statement of any
Patient MR#: Patient accession #: safety or health precautions associated with reagents should be
Section I. Summary of Incident present.
Describe what happened: All reagents must be properly labeled with the date of prepa-
ration or opening, purchase and received date, expiration date,
What immediate corrective action was taken? and appropriate safety information. Reagents should be checked
against two levels of commercial control solutions on each shift,
or at a minimum once a day and whenever a new reagent is
Provide the ORIGINAL to team leader or technical specialist within 24 hours of inci- opened. Results of all reagent checks are properly recorded.
dent discovery.
Date: Instrumentation and Equipment
To:
The procedure manual must clearly provide instructions regard-
Forwarded for follow-up: ing the operation, performance, frequency of calibration, and
Date:
To: limitations of the instrumentation and equipment. The proce-
dures to follow when limitations or linearity are exceeded, such
Section II. Management Investigation: Tracking #:
Instructions: Section II should be completed by laboratory management within as dilution procedures, must be included in the manual, as well
72 hours. as instructions detailing the appropriate recording procedures.
Check the appropriate problem category.
䡺 Unacceptable patient samples 䡺 Wrong tube type
Two levels of commercial controls must be run and
(Caused by hemolysis, QNS, or contamination) recorded. Evidence of corrective action for any failed QC tests
䡺 Equipment-related event 䡺 Misidentified sample must be documented. No patient’s testing may be performed
䡺 Standard operating procedure deviation 䡺 Wrong location
䡺 Communication problem or complaint 䡺 Other (explain) until QC is acceptable. A routine PM schedule for instruments
䡺 Accident and equipment should be prepared as mandated by the TJC
Explain answers: or CAP guidelines and records kept of all routine and nonrou-
tine maintenance performed.
Preventive or corrective action recommendations:
Deionized water used for reagent preparation is quality con-
trolled by checking its pH and purity meter resistance on a
weekly basis, as well as the bacterial count on a monthly sched-
Technical specialist or team leader: Date: ule. All results must be recorded on the appropriate forms.
Medical director review: Date:
Quality assurance review: Date: Testing Procedures
FDA reportable: Yes or no Date reported:
Detailed and concise testing instructions are written in a step-
Adapted from Danville Regional Medical Center Laboratory, Danville, VA, with by-step manner. Instructions should begin with specimen
permission. preparation, such as time and speed of centrifugation, and in-
clude types of glassware needed, time limitations and stability
of specimens and reagents, calculation formulas and a sample
laboratory director to capture the information to determine the calculation, health and safety precautions, and procedures. Ad-
root cause and develop a preventive or corrective action plan. ditional procedure information including reasons for special
Laboratory information systems have the capability to electron- precautions, sources of error and interfering substances, helpful
ically generate these forms for review. An acceptable specimen hints, clinical situations that influence the test, alternative pro-
requires verification of the patient’s identification information on cedures, and acceptable TATs for stat tests are listed under the
the requisition form and the tube label, proper collection and title of Procedure Notes following the step-by-step procedure.
processing procedures, and timely transport to the laboratory. Reference sources should be listed. The manufacturer’s
package inserts may be included but cannot replace the writ-
Examination Variables ten procedure. The laboratory director must sign and date
new procedures and all modifications of procedures before
The examination variables are the processes that directly affect they are used.21
the testing of specimens. They include reagents, instrumentation
and equipment, testing procedure, QC, preventive maintenance Quality Control
(PM), access to procedure manuals, and the competency of per- Quality control (QC) refers to the materials, procedures, and
sonnel performing the tests. techniques that monitor the accuracy, precision, and reliability
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124 SECTION 2 Basic Immunologic Procedures

of a laboratory test. QC procedures are performed to ensure that

Day
acceptable standards are met during the process of patient testing.
2 4 6 8 10 12 14 16 18 20
Specific QC information regarding the type of control specimen,
preparation and handling, frequency of use, tolerance levels, and + 2 S.D.
methods of recording should be included in the step-by-step + 1 S.D.
instructions in the procedures manual for each test. X
– 1 S.D.
QC is performed at scheduled times, such as at the begin-
– 2 S.D.
ning of each shift or before testing patient samples, and it must
In control
always be performed if reagents are changed, an instrument
malfunction has occurred, or if test results are questioned by
the health-care provider. Control results must be recorded in a + 2 S.D.
paper or electronic log. Patient test results may not be reported + 1 S.D.
X
until the QC is verified.
– 1 S.D.
External Controls. External controls are used to verify the ac- – 2 S.D.
curacy (ability to obtain the expected result) and precision Shift
(ability to obtain the same result on the same specimen) of a
test. The control material is exposed to the same conditions as + 2 S.D.
the patient samples. Reliability is the ability to maintain both + 1 S.D.
precision and accuracy. Analysis of two levels of control mate- X
rial is required. One of these is a high level control and the – 1 S.D.
– 2 S.D.
other is a low level control. The concentration of controls
Trend
should be at medically significant levels and should be as much
like the human specimen as possible. Documentation of QC
FIGURE 8–9 Levey–Jennings charts showing in–control, shift, and
includes dating and initialing the material when it is first trend results. (From Strasinger SK, DiLorenzo MS. Urinalysis and Body
opened and recording the manufacturer’s lot number and the Fluids. 6th ed. Philadelphia, PA: F.A. Davis; 2014.)
expiration date each time a control is run and the test result is
obtained. Food and Drug Administration (FDA) standards
require that control material test negative for HIV and HBV.
External controls are tested and interpreted in the laboratory
of scatter about the mean and an uneven distribution above
by the same person performing the patient testing.
and below the mean that are most often caused by errors in
The control data are evaluated before releasing patient
technique.
results. Data obtained from repeated measurements have a
When control values are outside the tolerance limits, cor-
Gaussian distribution or spread in the values that indicate the
rective action—including the use of new reagents, or controls,
ability to repeat the analysis and obtain the same value. The
and the verification of lot numbers and expiration dates—must
laboratory, after repeated testing, establishes the value for each
be taken and documented. A protocol for corrective action is
analyte and calculates the control mean (the average of all data
shown in Figure 8–10. A designated supervisor reviews all of
points) and the standard deviation (SD) (a measurement sta-
the QC results.
tistic that describes the average distance each data point in a
Some laboratories may participate in a commercial QC
normal distribution is from the mean). The coefficient of vari-
program run by the manufacturer of the QC material. The
ation (CV) is the SD expressed as a percentage of the mean.
results from the same lot of QC material are returned to the
The CV indicates whether the distribution of values about the
manufacturer for statistical analysis and comparison with
mean is in a narrow versus broad range and should be less than
other laboratories using the same methodology.
5%. Confidence intervals are the limits between which the spec-
ified proportion or percentage of results will lie. Control ranges Internal Controls. Internal controls, also called procedural
are determined by setting confidence limits that are within controls, consists of internal monitoring systems built into the
±2 SD or ±3 SD of the mean, which indicates that 95.5% to test system. Internal controls monitor the sufficient addition
99.7% of the values are expected to be within that range. of a patient specimen or reagent, the instrument’s and reagent’s
Values are plotted on Levey-Jennings control charts to visu- interaction, and, for lateral flow test methods, whether the
ally monitor control values. Immediate decisions about patient sample migrated through the test strip properly.22
results are based on the ability of control values to remain Electronic Controls. Electronic controls use a mechanical or
within a preestablished limit. Changes in accuracy of results electrical device in place of a liquid QC specimen. This type of
are indicated by either a trend, a gradual changing in the mean QC can be an internal or an external component inserted into
in one direction that may be caused by a gradual deterioration a point-of-care (POC) instrument. Electronic controls verify
of reagents or deterioration of instrument performance, or a the functional ability of a testing device, but it does not verify
shift, an abrupt change in the mean that may be caused by a the integrity of the testing supplies. Many test systems use a
malfunction of the instrument or a new lot number of reagents combination of external and internal controls to verify that the
(Fig. 8–9). Changes in precision are shown by a large amount entire test system is working properly.
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Chapter 8 Safety and Quality Management 125

1. Run control Personnel and Facilities

In control Out of control
QC is only as good as the personnel performing and monitor-
ing it. Personnel assessment includes education and training,
continuing education, competency assessment, and perfor-
Proceed with Go to step 2
testing mance appraisals. Each new employee must have documentation
of training during his or her orientation to the laboratory. This
documentation is a checklist of procedures and must include
2. Inspect control for: Outdate (age), proper storage, the date and initials of the person doing the training as well as
correct lot number, signs of contamination
the employee being trained. Up-to-date reference materials and
Yes there is a problem No obvious explanation atlases should be readily available and documentation of con-
tinuing education must be maintained.22
Make new control Retest
An adequate, uncluttered, safe working area is also essen-
and retest tial for both quality work and personnel morale. Standard
In control Out of control precautions for handling body fluids must be followed at
all times.
Proceed with Go to step 3
testing
Postexamination Variables
Postexamination variables are processes that affect the report-
3. Make up new bottle of control
ing of results and correct interpretation of data.
In control Out of control Reporting Results
Standardized reporting methods minimize health-care provider
Discard old control Go to step 4 confusion when interpreting results. The forms for reporting
and proceed with testing
results should be designed so that they present the information
in a logical sequence and provide adequate space for writing.
4. Open new can of reagent strips and test with new control A standardized reporting format and, when applicable, refer-
ence ranges should be included with each procedure in the
In control Out of control
procedure manual.
Electronic transmission is now the most common method
Discard bad reagent strips Switch lot numbers for reporting results. Many automated instruments have the
and proceed with testing and retest
capability for the laboratorian to transmit results directly from
In control Out of control the instrument to the designated health-care provider. It is
essential that the laboratorian carefully review results before
Discard entire lot, Notify supervisor or transmittal. Results may also be manually entered into the
notify manufacturer, resource person: do not
and proceed with testing proceed with testing laboratory computer system and then transmitted to the
health-care providers.
FIGURE 8–10 Procedure for “out of control” results. (Adapted from Documentation of the reporting of results is essential and
Schweitzer SC, Schumann JL, Schumann GB. Quality assurance guide-
required by accrediting agencies. In addition, permanent
lines for the urinalysis laboratory. J Med Technol. 1986; 3(11):567–572.
records of all reported results must be available. A method to
verify the actual reporting of results also must be available and
used by all employees.
Proficiency Testing (External Quality Assessment). Profi- The telephone is frequently used to transmit results of stat
ciency testing, or external quality assessment (EQA), is the tests and critical values. Personnel on hospital units and from
testing of unknown samples received from an outside agency. health-care providers often call requesting additional results.
It provides unbiased validation of the accuracy, and thus qual- When telephoning results, confirm that the results are being
ity, of patient test results. Several commercial vendors, such reported to the appropriate person. The time of the call and
as the CAP, provide proficiency testing. Laboratories subscrib- the name of the person receiving the results must be docu-
ing to these programs receive lyophilized or ready-to-use spec- mented according to the facility’s policy. The Joint Commis-
imens. The results are returned to the proficiency testing sion Patient Safety Goals require that when verbally reporting
vendors, where they are statistically analyzed with those from test results the information must be repeated by the person
all participating laboratories. The laboratory director receives receiving the information and documented by the person giv-
a report from the vendor, which enables the director to eval- ing the report. Written procedures should be available for the
uate the laboratory’s accuracy and compare it with other lab- reporting of critical values.
oratories using the same method of analysis. The director
ensures that the laboratory takes action to correct unaccept- Result Errors
able results.16 The CLIA mandate comparison testing for lab- Errors may be discovered in the laboratory through a QM proce-
oratory accreditation.23 dure known as the delta check that compares a patient’s test results
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126 SECTION 2 Basic Immunologic Procedures

with the previous results. Variation outside the established param- Regulatory Issues
eters alerts laboratory personnel to the possibility of an error
that occurred during the testing procedure or in patient identifica- QM is regulated throughout the total testing system. The
tion. Autoverification is often programmed into many laboratory health-care regulation systems include both governmental and
analyzers.20 public agencies. All agencies have the same goal, which is to
Erroneous results must be corrected in a timely manner provide safe and effective health care.
to assure that the patient does not receive treatment based
on incorrect results. Errors can occur in patient identifica-
tion, specimen labeling, or result transcription. The patient’s
Clinical Laboratory Improvement
record should be corrected as soon as the error is detected. Amendments (CLIA)
However, the original result must not be erased in the event The CLIA is a governmental regulatory agency administered
that the health-care provider treated the patient based on the by the Centers for Medicare and Medicaid Services (CMS) and
erroneous results. Appropriate documentation of erroneous the Food and Drug Administration (FDA). CLIA stipulates that
results should follow institutional protocol. The manual all laboratories that perform testing on human specimens for
must contain a written procedure for reporting, reviewing, the purposes of diagnosis, treatment, monitoring, or screening
and correcting errors. must be licensed and obtain a certificate from the CMS. Labo-
The In the Laboratory: Summary of Quality Manage- ratories with CLIA certification are inspected to document
ment Errors box summarizes QM errors in each phase of lab- compliance with the regulations. The inspections may be
oratory testing. performed by CMS personnel or an accrediting agency recog-
Interpreting Results nized by CMS such as the CAP, the TJC, or the Commission
The specificity and the sensitivity for each test should be in- on Laboratory Assessment (COLA).
cluded in the procedure manual for correct interpretation of CLIA classifies laboratory tests into three categories:
results. Sensitivity and specificity vary among manufacturers. waived, provider-performed microscopy procedures (PPMP),
All known interfering substances should be listed for evalua- and nonwaived testing (See In the Laboratory: CLIA Test
tion of patient test data. Classifications). Nonwaived testing is separated into the
categories of moderate and high complexity with regard to
requirements for personnel performing the tests. Laboratories
must obtain the correct certification for the level of testing
In the Laboratory complexity performed. For each category, there is a descrip-
tion of the educational level necessary for personnel who
Summary of Quality Management Errors
Preexamination
• Patient misidentification
• Wrong test ordered In the Laboratory
• Incorrect specimen type collected
• Insufficient specimen volume CLIA Test Classifications
• Delayed transport of specimen to the laboratory Waived Testing
• Inadequate processing of specimen • Tests considered easy to perform by following the manufac-
• Delayed separation of serum or plasma from cells turer’s instructions that have little risk of error. No special train-
• Incorrect storage of specimen ing or education is required.
Examination • Example: Urine pregnancy test
• Sample misidentification Provider-Performed Microscopy Procedures (PPMP)
• Erroneous instrument calibration • Microscopy tests performed by a physician, midlevel practi-
• Reagent deterioration tioner, or a dentist.
• Poor testing technique • Example: Microscopic urinalysis
• Instrument malfunction Nonwaived Tests
• Interfering substances present • Moderate complexity tests
• Misinterpretation of quality control data Tests that require documentation of training in test princi-
Postexamination ples, instrument calibration, periodic proficiency testing, and on-
• Patient misidentification site inspections.
• Poor handwriting Example: Automated complete blood count (CBC)
• Transcription error • High complexity tests
• Poor quality of instrument printer Tests that require sophisticated instrumentation and a high
• Failure to send report degree of interpretation.
• Failure to call critical values • Proficiency testing and on-site inspections are required.
• Inability to identify interfering substances Example: Urine culture and susceptibility

From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia, From Strasinger SK, DiLorenzo MA. The Phlebotomy Textbook. 3rd ed. Philadelphia,
PA: F.A. Davis; 2011, with permission. PA: F.A. Davis; 2011, with permission.
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Chapter 8 Safety and Quality Management 127

may perform the test, as well as the type of quality assurance Quality Management Systems
procedures that must be in place.
A quality management system (QMS) incorporates many of
Clinical and Laboratory Standards the objectives of total quality management and continuous
Institute (CLSI) quality improvement to ensure quality results, staff compe-
tence, and efficiency within an organization. In addition, QMS
The CLSI is a nonprofit organization that publishes recommen- also utilizes the concepts of the International Organization for
dations by nationally recognized experts for the performance Standardization (ISO 151189) and the Lean and Six sigma
of laboratory testing. CLSI standards are considered the stan- methods. The requirements of TJC and the CAP accreditation
dard of care for laboratory procedures. The standard of care organizations are included in QMS.
is the attention, caution, and prudence that a reasonable person A QMS is designed to coordinate activities to direct and
in the same circumstances would exercise. In a legal situation, control an organization with regard to quality and the reduc-
the CLSI standards would be considered the standard of care tion of medical errors. The first step in a laboratory QMS is to
that should have been met. determine the pathway of workflow through the laboratory as
discussed previously under the preexamination, examination,
The Joint Commission (TJC) and postexamination phases of testing. In each area of the path-
way, all the processes and procedures that occur are deter-
TJC is an independent, not-for-profit organization that accred-
mined and analyzed so everyone knows what they are
its and certifies more than 15,000 health-care organizations
supposed to do, how they are supposed to do it, and when
and programs in the United States. The mission of TJC is to
they are supposed to do it.
continuously improve the safety and quality of care provided
to the public through the provision of health-care accreditation
and related services that support performance improvement in Quality System Essentials
health-care organizations. Quality system essentials (QSEs) form the basis of a QMS.
TJC has recently published the Joint Commission Patient The 12 QSEs contain the management information needed
Safety Goals. It is essential that health-care organizations adhere for a laboratory to perform quality work (Table 8–2). They
to these goals to maintain their accreditation. Goals pertaining were developed by the former National Committee for Clinical
to the laboratory are: Laboratory Standards and the current CLSI and include the
Goal 1: Improving the accuracy of patient identification methods to meet the requirements of regulatory, accreditation,
Goal 2: Improving the effectiveness of communication and standard setting organizations. Quality indicators are the
among health-care givers measurements developed by each laboratory to determine if
Goal 7: Reduce the risk of health care-associated infections the quality system essentials are being met. They may include
(HAIs) such items as appropriateness of the testing, correct patient
Goal 13: Encourage patients’ active involvement in their identification, timely reporting of laboratory results, and correct
own care as a patient safety strategy proficiency testing results.

College of American Pathologists (CAP)

The Lean System
The CAP is an organization of board-certified pathologists that
advocates high-quality and cost-effective medical care. CAP The Lean system originated with the automobile manufactur-
provides laboratory accreditation and proficiency testing for ing industry in Japan. Its concepts have been adopted by many
laboratories. American industries, including the health-care industry. Lean
For accreditation purposes, CAP-trained pathologists and utilizes a tool called “6S,” which stands for: sort, straighten,
laboratory managers and technologists perform on-site labo- scrub, safety, standardize, and sustain. The focus is on the elim-
ratory inspections on a biennial basis. Inspectors examine the ination of waste to allow a facility to do more with less and at
laboratory’s records and QC of procedures for the preceding the same time increase customer and employee satisfaction. In
2 years. Also examined are the qualifications of the laboratory the health-care environment, the ability to decrease costs while
staff including continuing education attendance, the labora- providing quality health care is of primary importance.
tory’s equipment, facilities, safety program, and laboratory
management. CAP accreditation is accepted by both the CMS
and the TJC and fulfills Medicare and Medicaid requirements.
Six Sigma
As previously described, laboratories that subscribe to this Six Sigma is a statistical modification of the original Plan-Do-
proficiency program receive periodic samples to analyze and Check-Act (PDCA) method adopted by the TJC as a guideline
return their results to the CAP. The laboratory receives a report for health-care organizations. The primary goal of Six Sigma is
on how its results compared with other laboratories performing to reduce variables and decrease errors to a level of 3.4 defects
the procedures in a similar manner. Failure to perform satis- per 1 million opportunities. Attaining this goal indicates that
factorily on a proficiency test can result in a laboratory losing the laboratory is addressing factors critical to customer satis-
its CLIA certificate to perform the failed test. faction and quality care.
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128 SECTION 2 Basic Immunologic Procedures

Table 8–2 The 12 Laboratory Quality System Essentials

QUALITY SYSTEM ESSENTIALS PROCESSES AND PROCEDURES
The Laboratory QSEs
1. Organization Personnel roles, responsibilities, and reporting relationships
Quality planning and risk assessment
Allocation of personnel and material resources
Review and assessment of meeting goals
2. Facilities and safety Space designed for efficiency
Adequate storage space
Required safety precautions and equipment availability
Housekeeping
Safety training
3. Personnel Qualifications
Current job descriptions
Orientation of new employees
Competency assessment
Continuing education
4. Equipment Selection criteria
Space needed and special instrument requirements
Ongoing preventive maintenance
Service and repair records
5. Purchasing and inventory Inventory of initial materials and reagents
Service contracts
Availability of reagents, supplies, and service
The Work System QSEs
6. Process control Identification of all laboratory processes
Procedure manuals and instructions for tasks
Test method verification
Verification that manufacturer specifications are in procedure manuals
Quality control and statistics
7. Documents and records Availability of all process and procedure documents
Periodic review of all process and procedure documents
Access to quality control records
Monitoring of record storage and retention
8. Information management Availability of patient records
Security of patient records
Methods for providing patient information
Processes to prevent Medicare and Medicaid fraud
The Measurement QSEs
9. Occurrence management Identification and reporting of all events
and nonconforming event Remedial actions taken
management Plans to eliminate future events
Initiation of changes
10. Assessments: external Obtaining external licensing and accreditation
and internal Participation in external proficiency testing
Periodic on-site auditing by accrediting agencies
Development of quality indicators for each phase of testing
11. Customer service Feedback from customers including patients, patients’ families, and health-care
providers
Feedback from employees
Feedback from offsite referral laboratories and health-care providers
12. Process improvement Monitoring of the above QSEs results
Determination of the root cause of problems
Utilize the Lean system tools
Utilize Six Sigma methodology
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Chapter 8 Safety and Quality Management 129

The Six Sigma methodology is represented by the acronym

DMAIC: • Dispose of radioactive material following NRC guidelines.
• Be observant for frayed cords, overloaded circuits, and im-
Define goals and current processes properly grounded equipment. Avoid working with electri-
Measure current processes and collect data cal equipment when you, or the equipment, is wet.
Analyze the data for cause-and-effect information • Follow routine safety protocols and maintain a clean,
Improve the process using the data collected organized work area to avoid physical hazards.
Control the correction of concerns displayed in the data • The acronym RACE outlines the steps to follow when a
By instituting quality improvement methodologies, a health- fire is discovered: (R) rescue anyone in danger, (A) activate
care institution can develop a structured standardized format the fire alarm, (C) contain the fire, (E) extinguish the fire
to systematically assess and document the quality of services if possible or evacuate, closing the door.
to the customer. • Quality management is the overall process of guaranteeing
quality throughout the entire testing system.
• Quality control involves performing individual procedures
SUMMARY using acceptable standards and control material at various
medically significant levels.
• Transmission of biological hazards that are encountered • Documentation includes a procedure manual, policies to
when testing patient specimens requires a chain of infec- control and monitor procedure variables, and records of
tion, which consists of an infectious agent, reservoir, por- competency assessment and continuing education.
tal of exit, mode of transmission, portal of entry, and a • Preexamination variables occur before sample testing.
susceptible host. Examination variables occur during the specimen testing.
• Hand hygiene and wearing PPE are essential actions to Postexamination variables occur during interpretation and
prevent transmission of infectious organisms. Standard reporting of test results.
precautions should be followed at all times. • Agencies regulating the laboratory include:
• Specimens, except urine, and contaminated supplies must • CLIA—provides requirements for persons performing
be disposed of in a biohazard container. waived, provider-performed microscopy, moderate-
• All sharps, including needles and holders, must be disposed complexity, and high-complexity testing
of in puncture-proof containers. Recapping of needles is • TJC—provides accreditation and certification of health-
prohibited. care organizations
• The Occupational Exposure to Bloodborne Pathogens Stan- • CAP—provides laboratory accreditation and provision
dards are a means of providing protection from accidental of proficiency testing
exposure to bloodborne pathogens through the use of engi- • CLSI—develops written standards and guidelines for
neering controls, work practice controls, and use of PPE. sample collection, handling and processing, and labo-
• When transporting biological specimens, Department of ratory testing and reporting
Transportation and International Air Transit Association • The 12 quality essentials provide the management docu-
regulations must be followed. They include placing spec- mentation needed to demonstrate quality work. Quality
imens in screw cap containers, wrapping them in ab- indicators are developed to monitor each phase of testing.
sorbent material, and placing them in a sturdy leakproof • The Lean system utilizes the “6S” tools (sort, straighten,
container. scrub, safety, standardize, and sustain) to enhance effi-
• Follow specific directions when mixing chemicals and ciency and proficiency.
always add acid to water, rather than water to acid. • The goal of the statistical Six Sigma method is to reduce
• When chemical contact with the skin or eyes occurs, variables and decrease errors to a level of 3.4 defects per
immediately flush the area with water for 15 minutes. 1 million opportunities.
• A SDS and a Chemical Hygiene Plan must be available to
employees. Dispose of chemicals per EPA guidelines.
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130 SECTION 2 Basic Immunologic Procedures

CASE STUDIES
1. The immunology supervisor who has been working for the 2. As the supervisor of the immunology section, you en-
last 20 years in a small rural hospital is training a new em- counter the following situations. Explain whether you
ployee. A dilution of a patient’s serum must be made to run would accept them or take corrective action.
a particular test. The supervisor is having difficulty using a
Questions
serological pipette so she removes one glove. In uncapping
the serum tube, a small amount of serum splashes onto the a. You are told that only the supervisor performs the CAP
workbench. She cleans this up with a paper towel, which proficiency survey.
she discards in the regular paper trash. She also spills a b. QC is not performed daily on the Centaur instrument.
small amount onto her disposable lab coat. She tells the c. The Streptozyme test reporting procedure has been re-
new employee that, because it is such a small amount, she cently revised.
isn’t going to worry about it and continues on to pipette d. Opened, unlabeled commercial quality control bottles
the specimen. She then replaces the glove onto her un- are in the refrigerator.
gloved hand and says that, because it is almost break time,
she will wait to wash her hands until then.
Questions
a. Please identify all the safety violations involved.

REVIEW QUESTIONS
1. A technologist who observes a red rash on her hands 5. A technician places tightly capped noninfectious
after removing her gloves serum tubes in a rack and places the rack and the
a. should apply antimicrobial lotion to the hands. specimen data in a labeled leakproof metal courier
b. may be washing the hands too frequently. box. Is there anything wrong with this scenario?
c. may have developed a latex allergy. a. Yes, DOT requirements are not met.
d. should not create friction when washing the hands. b. No, the tubes are placed in a rack.
c. Yes, absorbent material is missing.
2. In the chain of infection, a contaminated work area d. No, the box contains the specimen data.
would serve as which of the following?
a. Reservoir 6. The Occupational Exposure to Bloodborne Pathogens
b. Means of transmission Standard developed by OSHA requires employers to
c. Portal of entry provide all of the following except
d. Portal of exit a. hepatitis B immunization.
b. safety training.
3. The only biological waste that does not have to be dis- c. hepatitis C immunization.
carded in a container with a biohazard symbol is d. laundry facilities for nondisposable lab coats.
a. urine.
b. serum. 7. An employee who receives an accidental needlestick
c. feces. should immediately
d. serum tubes. a. apply sodium hypochlorite to the area.
b. notify a supervisor.
4. Patient specimens transported by the Department of c. receive HIV prophylaxis.
Transportation must be labeled as a d. receive a hepatitis B booster shot.
a. diagnostic specimen.
b. clinical specimen.
c. biological specimen, category b.
d. laboratory specimen.
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Chapter 8 Safety and Quality Management 131

8. The first thing to do when acid is spilled on the skin is to 15. When external quality control is run, what informa-
a. notify a supervisor. tion must be documented?
b. neutralize the area with a base. a. The lot number
c. apply burn ointment. b. Expiration date of the control
d. flush the area with water. c. The test results
d. All of the above
9. When combining acid and water,
a. acid is added to water. 16. What steps are taken when the results of the quality
b. water is added to acid. control testing are outside of the stated confidence
c. water is slowly added to acid. limits?
d. both solutions are combined simultaneously. a. Check the expiration date of the control material
b. Run a new control
10. To determine the chemical characteristics of sodium c. Open a new control bottle
azide, an employee would consult the d. All of the above
a. Chemical Hygiene Plan.
b. Merck manual. 17. When a new bottle of QC material is opened, what in-
c. SDS. formation is placed on the label?
d. NRC guidelines. a. The time the bottle was opened
b. The supervisor’s initials
11. A technician who is pregnant should avoid working c. The lot number
with d. The date and the laboratory worker’s initials
a. organic chemicals.
b. radioisotopes. 18. What is the primary goal of TQM?
c. HIV-positive serum. a. Precise test results
d. needles and lancets. b. Increased laboratory productivity
c. Improved patient outcomes
12. Which of the following laboratory regulatory agencies d. Reproducible test results
classifies laboratory tests by their complexity?
a. OSHA 19. Would a control sample that has accidentally
b. CAP become diluted produce a trend or a shift in the
c. TJC Levey-Jennings plot?
d. CMS a. Trend
b. Shift
13. Which of the following organizations publishes guide-
lines that are considered the standard of care for labo- Fill in the Blank
ratory procedures? 20. Indicate whether each of the following would be
a. CLIA considered a (1) preexamination, (2) examination, or
b. CLSI (3) postexamination variable by placing the appropri-
c. TJC ate number in the space.
d. CAP Reagent expiration date
Rejection of a hemolyzed specimen
14. Quality managment refers to Construction of a Levey-Jennings chart
a. performance of two levels of testing controls. Telephoning a critical result to the nurse
b. reliable control results. Calibrating the centrifuge
c. increased productivity. Pipetting the diluent
d. quality of specimens and patient care.