Page 1312 - Clinical Immunology_ Principles and Practice ( PDFDrive )

1274 Part ElEvEn Diagnostic Immunology

KEY COnCEPt KEY COnCEPt
Criteria to Assess Neutrophil Function Estimated Yield from Whole Blood

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Because of (1) the time required to isolate neutrophils and (2) the shortened In general, 1–2 × 10 neutrophils can be isolated per milliliter of whole
life span of neutrophils after isolation, assays of neutrophil function should blood from a normal subject with a normal white blood cell count.
have minimal complexity and enable rapid data collection.

ISOLATION OF NEUTROPHILS (DFP, 1–5 mM). DFP is a volatile, potent neurotoxin that can
irreversibly bind to and inactivate acetylcholinesterase and should
Clinical Indications and Implications be used with extreme caution. The cell suspension is then spun
Assays that avoid neutrophil isolation are preferred because of and the supernatant fluid removed from the cell pellet before
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the artificial priming of neutrophils during isolation. However, freezing. Waste solutions and disposable laboratory items should
most assays require isolated neutrophils to eliminate any possible be flushed with sodium hydroxide to inactivate any residual
contributions of other leukocytes and blood components. In DFP. These frozen neutrophil pellets, though not viable, can also
general, blood should be drawn using either citrate or heparin be a source of DNA for genetic analyses.
as anticoagulant and maintained at 20–25°C in polypropylene
containers. Most isolation protocols require 1–2 hours to obtain HISTOCHEMICAL ANALYSIS OF NEUTROPHILS
purified neutrophils.
Clinical Indications and Implications
Principles and Interpretation of Laboratory Assessment Because of their unique morphology, microscope examination
Most neutrophil isolation protocols use differences in the cell of neutrophil preparations with a differential stain, such as
density as the basis for the separation. The relative densities of Wright stain, or a histochemical stain, such as Kaplow stain, for
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blood cells are as follows: erythrocytes > neutrophils and eosino- myeloperoxidase remains an essential element of neutrophil
phils > monocytes, lymphocytes, and basophils > platelets. study and can provide valuable insight into some genetic
Ficoll-Paque is a solution of sodium diatrizoate (a dense, triiodin- immunodeficiencies.
ated compound) and Ficoll (a polysaccharide) with a density
(1.077 g/cc) that falls between the density of neutrophils and Principle and Interpretation of Laboratory Assessment
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that of the mononuclear cells. To isolate neutrophils, whole In Wright’s stain, the nucleus of a segmented neutrophil is
blood is diluted with saline and underlaid with Ficoll-Paque normally multilobed (usually 2–5 lobes), and each lobe is con-
solution. After centrifugation for 30 minutes at 500 g, the less- nected by a narrow filament (Fig. 94.1A). The nuclear chromatin
dense monocytes, lymphocytes, basophils, and platelets remain is coarsely clumped with purple staining. Nucleoli are generally
at the upper interface of the Ficoll-Paque solution, whereas the not present. In a band neutrophil, the nucleus is horseshoe shaped,
denser erythrocytes and neutrophils pass through the solution with no indication of constriction into lobes. The pink-
and pellet at the bottom. The mononuclear cells are carefully violet staining of the cytosol is associated with numerous,
harvested and the remaining Ficoll-Paque solution aspirated. evenly distributed, specific granules; occasionally a dark-staining
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The erythrocyte/neutrophil pellet is resuspended with saline and primary granule may be present. Kaplow stain identifies
mixed with 3% dextran. Dextran promotes the formation of the myeloperoxidase-containing primary granules as dark blue
rouleaux by the erythrocytes, causing them to sediment more granules uniformly distributed throughout the cytosol (Fig.
rapidly than the neutrophils at 1 g. The neutrophil-enriched 94.1B). Neutrophils (and platelets) from patients with Chédiak-
supernatant fluid is harvested from the bulk of the erythrocytes. Higashi syndrome have giant primary granules that are generally
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Contaminating erythrocytes are removed by a brief (30-second) considered pathognomonic for the disease (see Fig. 94.1C).
hypotonic lysis with 0.2% saline. The isotonicity is quickly restored Myeloperoxidase staining of Chédiak-Higashi neutrophils is very
with an equal volume of 1.6% saline. A second hypotonic lysis distinctive, with staining localized to the discrete giant primary
removes many of the red blood cell (RBC) ghosts. In general, granules (see Fig. 94.1D). Neutrophils from a patient with specific
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1–2 × 10 neutrophils can be isolated per milliliter of whole granule deficiency exhibit primarily bilobed nuclei (pseudo–
blood from a normal subject with a normal white blood cell Pelger-Huët anomaly) with a paucity of specific granule staining
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(WBC) count. All procedures are performed at room temperature, in the cytosol (see Fig. 94.1E). Staining of the myeloperoxidase
and the isolated cells are maintained in a balanced salt solution granules of neutrophils from a patient with specific granule
without divalent cations. The most common cell contaminants deficiency appears normal, since the defect is primarily associated
of the neutrophil preparation are eosinophils. Further purification with the specific granules (see Fig. 94.1F). Neutrophils from a
of a standard neutrophil preparation with anti-CD16 magnetic patient with myeloperoxidase deficiency fail to stain for myelo-
immunobeads results in a neutrophil preparation that is generally peroxidase in the neutrophils. However, the peroxidase in the
≥99% neutrophils. A second neutrophil isolation protocol that eosinophilic granules continues to stain positive (see Fig. 94.1G). 17
uses a discontinuous gradient of plasma/Percoll has often been
used to minimize exposure of neutrophils to trace contamination ANALYSIS OF GRANULE CONSTITUENTS
by bacterial lipopolysaccharide (LPS) and reduce neutrophil
priming. 13 Clinical Indications and Implications
Isolated neutrophils are routinely frozen in aliquots of 5 × The granules of the neutrophils can be distinguished by their
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10 cells/vial. For Western blot studies, neutrophils (1 × 10 cells/ specific contents. Deficiency of only one granule constituent can
mL of buffer) are pretreated for 20 minutes with the cell permeant, be associated with a specific genetic defect, such as myeloper-
irreversible serine protease inhibitor, diisopropylfluorophosphate oxidase deficiency; alternatively, deficiency of multiple constituents