Page 500 - Hematology_ Basic Principles and Practice ( PDFDrive )

420 Part IV Disorders of Hematopoietic Cell Development

life-threatening. 13,14 The ability to pharmacologically interfere with GPI Anchor–Based Assays
terminal complement appears to have altered the natural history of
the disease by markedly decreasing the thrombosis rate. 15,16 With Most laboratories use monoclonal antibodies against specific GPI-
appropriate therapy, the natural history of classical PNH is prob- anchored proteins in conjunction with flow cytometry to diagnose
ably no different from age-matched controls. Females and males are PNH. Anti-CD59 is most commonly used because it is widely
equally affected with the median age of diagnosis being 40 years. expressed and is displayed on all hematopoietic lineages. Anti-CD55,
Without therapy, the median survival from time of diagnosis anti-CD14, anti-CD16, anti-CD67, and a variety of other monoclo-
is 10 to 20 years. Thrombosis, severe pancytopenia, evolution to nal antibodies can also be used to establish the diagnosis. Flow
MDS or leukemia, older age, and thrombocytopenia at diagnosis cytometry offers several advantages over complement-based assays for
portend a poor prognosis. Older literature, where patients were diagnosing PNH: it measures the size of the PNH clone in the various
diagnosed with PNH based on the Ham test or sucrose hemolysis cell lineages, it is more sensitive and specific, and it is less affected by
test, reported the occurrence of spontaneous long-term remis- blood transfusions. It is noteworthy that rare congenital deficiencies
sions in up to 10% of PNH cases; however, in patients diagnosed of CD59 and CD55 can lead to a false positive test for PNH if only
by flow cytometry, the spontaneous remission rate is much less one monoclonal antibody is used. This, coupled with the variable
common. expression of GPI-anchored proteins on different hematopoietic
lineages, accounts for the recommendation that at least two different
monoclonal antibodies, directed against two different GPI-anchored
LABORATORY EVALUATION proteins, on at least two different cell lineages, should be used to
diagnose a patient with PNH. Solely screening patients’ red cells for
Blood PNH can lead to false negative tests, especially in the setting of a
recent hemolytic episode or a recent blood transfusion. Granulocytes
Peripheral blood counts in PNH patients vary from severe pancyto- and monocytes have a short half-life and are not affected by blood
penia to nearly normal. Virtually all patients will present with anemia, transfusions; the percentage of PNH cells in these lineages best
frequently with mild macrocytosis. Thrombocytopenia and/or neu- reflects the size of the PNH stem cell pool.
tropenia are also common. A mild to moderate reticulocytosis is
usually present in patients with the classical form of the disease;
however, in patients with hypoplastic PNH (also referred to as aplastic Aerolysin Assays
anemia/PNH overlap), the reticulocyte count can be low for the
degree of anemia. Similarly, in patients with hypoplastic PNH, the A fluorescein-labeled proaerolysin variant, FLAER, is commonly used
biochemical profile can be normal, but in patients with large PNH in conjunction with monoclonal antibodies in a flow cytometric assay
clones the indirect bilirubin and LDH are significantly elevated. In to diagnose PNH. 17,18 Aerolysin is the principal virulence factor of
patients with vigorous hemolysis, it is not uncommon for the labora- the bacterium Aeromonas hydrophila. It is secreted as an inert protoxin
tory to report the specimen as “hemolyzed.” termed proaerolysin, which binds selectively and with high affinity
to the GPI anchor. After binding to its receptor (the glycan portion
of the GPI anchor), the C-terminal peptide of proaerolysin is cleaved
Bone Marrow by cell proteases. This activates the toxin and leads to the formation
of heptameric channels that insert into the membrane and kill the
Bone marrow cellularity can be hypocellular, normocellular, or cell. PNH cells are resistant to aerolysin and proaerolysin because
hypercellular. In patients with classical PNH (not arising from or PNH cells lack GPI-anchored proteins. FLAER binds to the GPI
coinciding with aplastic anemia), the marrow is usually normocel- anchor without forming channels and gives a more accurate assess-
lular to hypercellular with erythroid hyperplasia. Mild to moderate ment of the GPI-anchor deficit in PNH than anti-CD59. Because
dyserythropoiesis is common. Stainable iron is frequently absent the GPI anchor is the major determinant for binding FLAER, it
because of iron loss associated with the intravascular hemolysis. allows for the direct assessment of GPI anchor expression on virtually
Cytogenetic abnormalities can be found in a small number of all cell lineages. Red cells are a notable exception; this may be because
patients. both normal and PNH red cells express large amounts of glycophorin,
a protein shown to bind aerolysin weakly. Nevertheless, in mono-
nuclear cells FLAER eliminates the need for multiple lineage-specific
DIAGNOSIS monoclonal antibodies. These properties make FLAER more reliable
for detecting the small PNH populations often found in patients with
Complement-Based Assays aplastic anemia.
The Ham test and the sucrose hemolysis test (sugar water test) were
two of the first assays used to diagnose PNH. Both assays are per- THERAPY
formed on erythrocytes and discriminate PNH cells from normal cells
based on a differential sensitivity to the hemolytic action of comple- The major indications for therapy in PNH are thrombosis,
ment. In the Ham test, the alternative pathway of complement is transfusion-dependent anemia, severe pancytopenia, disabling
activated by acidification of the serum. This results in lysis of PNH fatigue, and debilitating smooth muscle dystonia. The choice of
erythrocytes but not normal erythrocytes. The Ham test is relatively therapy depends on the underlying cause of these manifestations. If
specific for PNH, but is not very sensitive. the symptoms are from complement-mediated hemolysis or includes
Complement is also activated in a low-ionic-strength sucrose- thrombosis, terminal complement inhibition with eculizumab is
containing medium. Preferential lysis of PNH erythrocytes through indicated; if the symptoms are mainly from underlying bone marrow
the activation of complement in this sucrose-containing medium dysfunction, immunosuppressive therapy or bone marrow transplant
forms the basis of the sugar water test. This assay is easier to should be considered. Some PNH patients can be managed conser-
perform and is more sensitive than the Ham test but not as specific; vatively with watchful waiting.
other hemolytic anemias and even leukemias can produce false
positive results. The complement lysis assay in which complement
is activated with antibody will also detect PNH erythrocytes. These Immunosuppressive Therapy
complement-based red cell assays are important from a historical
perspective, but should no longer be used to establish the diagnosis PNH patients with a hypocellular bone marrow, low reticulocyte
of PNH. count, and pancytopenia (hypoplastic PNH) will frequently respond