INTERPRETATION OF THE PERIPHERAL

BLOOD FILM 0272–2712/02 $15.00
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ASSESSMENT OF PLATELET
NUMBERS AND MORPHOLOGY
IN THE PERIPHERAL
BLOOD SMEAR
Alvaro Moreno, MD, and David Menke, MD

The evaluation of the peripheral blood smear (PBS) constitutes a pivotal

tool in the evaluation of patients with hematologic disorders, adding useful
information to that obtained by the automated systems currently in widespread
use for the assessment of patients’ complete blood count. The blood smears are
prepared using capillary or venous nonanticoagulated blood spread onto a clean
glass slide using the wedge smear technique and then dried at room temperature
and stained with a Wright’s or Wright-Giemsa stain. A well-stained platelet
should be purple or purplish pink in color. Anticoagulated blood is also often
used to prepare smears from flagged complete blood counts performed by
automated analyzers. When anticoagulated blood is used, the blood smears
should be prepared and stained between 2 and 4 hours from blood collection to
avoid staining artifacts. Automated instruments often encounter platelet aggre-
gates and clumps, cellular debris, and fragments or microcytic red cells that lead
to falsely abnormal platelet count results and the need for manual platelet count
verification. Frequent indications for a direct evaluation of the PBS include the
evaluation of the number and morphology of the peripheral blood cells, usually
at the request of a clinician looking for clues of hematologic and nonhematologic
disorders in a given patient; to verify abnormal results of an automated analyzer
specimen; or as a quality control to the routine and widespread use of these
analyzers.
A PBS sample from a healthy donor usually demonstrates 15 to 30 platelets

From the Division of Hematology-Oncology and Internal Medicine (AM) and Department of
Pathology (DM), Mayo Clinic; and Mayo Medical School (AM, DM), Jacksonville, Florida

CLINICS IN LABORATORY MEDICINE

VOLUME 22 • NUMBER 1 • MARCH 2002 193

194 MORENO & MENKE

per high-power field under a 400  magnification and 8 to 20 platelets per

high-power field under a 1000  oil magnification. A common method of
verification is to add the number of platelets counted in 8 to 10 fields under
1000  oil magnification (widefield versus standard oculars, respectively) and
multiply it by 2000 to correlate with the total platelet count reported by an
automated analyzer (platelets per ␮L).83 A normal platelet count is usually
established between 150,000 and 400,000/␮L.
Other adjunctive information helpful during the evaluation of abnormal
platelet count includes mean platelet volume (MPV), the platelet distribution
width, and the reticulated platelet count. The MPV is an automated measure-
ment of average platelet volume and correlates inversely with platelet count
when bone marrow function is properly preserved. The clinical usefulness of
the MPV lies in the potential capability of discerning among disorders associated
with increased platelet destruction, such as idiopathic thrombocytopenic pur-
pura (elevated MPV) and hypoproliferative thrombocytopenias (low MPV).
When bone marrow disorders that are associated with decreased platelet produc-
tion are present (aplastic anemia and leukemia), however, this inverse relation-
ship is lost and the patient has decreased platelet count and low MPV. The
platelet distribution width reflects more the uniformity of the platelet size and
is calculated from the platelet volume distribution. The reticulated platelet count
is the reflection of platelet RNA measured by flow cytometry using the thiazole
orange dye and is also considered by some investigators as an aid to help
differentiate between destructive thrombocytopenia (high reticulated platelet)
versus disorders with low platelet production (low reticulated platelet). The
clinical use of these tests has been limited, however, by several factors including
lack of consistent clinical validation (MPV and platelet distribution width) and
technical difficulties for its standardization (reticulated platelet).
Under light microscopy a normal platelet represents non-nucleated frag-
ments of megakaryocyte cytoplasm that usually has a discoid-lentiform shape
and a granular basophilic appearance and measures 1 to 3 ␮m in diameter.
Small numbers of larger platelets measuring up to 5 to 7 ␮m can also be seen.
The average platelet volume is around 5 to 8 fL. The subcellular structure of a
platelet is better evaluated by an electron microscope that allows for examination
of important cytoplasmic organelles, such as granules, mitochondria, dense
bodies, and the so-called open-canalicular system. Lysosomes and peroxisomes
and the dense tubular system, however, are best identified by histochemical
stainings. Electron microscopy is the most effective means of evaluating cyto-
plasmic organelles in the evaluation of inherited platelet disorders.
This section focuses on disorders affecting the number or morphology
of platelets as assessed by evaluation of a PBS. The evaluation of functional
abnormalities of platelets is not covered in this article.

THROMBOCYTOPENIAS

Pseudothrombocytopenia

A relatively common finding in complete blood counts read by an auto-

mated analyzer is the presence of a low platelet count. The first step on a routine
examination of platelets in a PBS is to rule out the presence of platelet aggregates
and clumps on the smear giving a falsely low platelet count. Aggregates and
clumps are usually seen in smears prepared from poorly anticoagulated speci-
mens leading to platelet activation and formation of small clots or because of
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 195

Figure 1. Pseudothrombocytopenia. Platelet agglutination (Wright-Giemsa, original magni-

fication 600).

the activation of platelets by abnormally present autoantibodies that react with

platelets in anticoagulated blood samples collected using ethylenediaminetetra-
acetic acid (EDTA). The latter occurs secondary to EDTA-induced low calcium
levels in the specimen that leads to a conformational change (divalent cation-
dependent) on the platelet’s surface (most often of the glycoprotein [GP] IIb-IIIa
complex) that induces agglutination (Fig. 1) by these autoantibodies (IgG, IgM,
and IgA). This phenomenon can occur as frequently as 1 in 1000 EDTA-con-
taining samples. Other less common causes of pseudothrombocytopenia include
platelet satellitism (in vitro rosetting of platelets adhering around neutrophils,
bands, and monocytes caused by an EDTA-dependent phenomenon; [Fig. 2]11, 47);
cold agglutinins; and clot formation from a poorly collected sample.

Figure 2. Pseudothrombocytopenia. Platelet satellitism (Wright-Giemsa, original magnifica-

tion  1000).
196 MORENO & MENKE

Dilutional Thrombocytopenia

A common finding in hospitalized patients who have moderate to signifi-

cant active bleeding and are receiving aggressive intravenous fluid replacement
is dilutional thrombocytopenia. The drop in the platelet count can be as signifi-
cant as 40% and larger platelets are seen in the peripheral blood. This finding is
associated with a concomitant fall in the hematocrit.62, 71

Hypoproliferative Thrombocytopenia

Hypoproliferative thrombocytopenias are disorders with inadequate platelet

production by megakaryocytes either caused by a quantitative (decreased mega-
karyocytic mass) or qualitative problem (ineffective thrombopoiesis). The PBS is
a helpful aid because the observation of all the three cell lineages may give clues
to the underlying disorder. Besides thrombocytopenia, the PBS may display
dacryocytes (teardrops); nucleated red cells and immature white cells (leuko-
erythroblastic picture) in marrow infiltration by primary or metastatic malignan-
cies; pancytopenia in aplastic anemia; oval macrocytes; pseudo–Pelger-Hüet
anomaly; and hypogranular neutrophils in myelodysplastic syndromes (MDS).
MDS may also present with isolated thrombocytopenia.73 Other causes of hypo-
proliferative thrombocytopenias include paroxysmal nocturia hemoglobinuria;
folate and vitamin B12 deficiency (oval macrocytes, multilobulated neutrophils,
thrombocytopenia, or leukopenia); acquired or congenital amegakaryocytic
thrombocytopenic purpura (small or normal size platelets, occasional macro-
cytosis, and a bone marrow with severely decreased or absent megakaryocytes
but otherwise normal); cyclic thrombocytopenia; infection-associated thrombocy-
topenia; chemotherapy; and radiation therapy.

Immune Thrombocytopenic Purpura

Idiopathic thrombocytopenic purpura (ITP) refers to a disorder where pre-

mature destruction of platelets occurs by autoantibodies and when no known
etiologic factors are identified. It remains a clinical diagnosis of exclusion and
probably one of the most common autoimmune disorders. ITP manifests clini-
cally as an acute or chronic entity that is associated with different incidences,
prognoses, and responses to therapy.24, 35, 38 The peripheral blood demonstrates
thrombocytopenia with some large platelets, and occasionally megakaryocyte
fragments are seen. The leukocyte and red cell count and morphology should
be normal. Occasionally, eosinophilia has been observed in acute ITP.24, 59 The
investigation of the ultrastructural morphology of normal and ITP platelets has
failed to demonstrate any significant structural differences.51 The need for a bone
marrow examination remains controversial but when done is often useful to
rule out other causes of thrombocytopenia that may mimic ITP. The most
common finding is megakaryocyte hyperplasia and hypertrophy with normal
erythropoiesis and myelopoiesis. Based on the pathophysiologic mechanism of
ITP, a reliable test for its diagnosis has been sought for decades. The observation
of the presence of abnormal levels of antiplatelet IgG (usually targeted against
platelet GPs) in ITP patients has led to the development of platelet antibody
assays. Because of their low specificity (phase II assays: sensitivity 80% to 90%
but variable low specificity) or sensitivity (phase III assays: sensitivity 47% to
60%, specificity 78% to 92%), however, they have not become a reliable and
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 197

clinically useful diagnostic method.17 The findings of an associated hemolytic

anemia with a positive Coombs’ test are diagnostic of Evans’s syndrome.28
Secondary immune thrombocytopenias are disorders of decreased platelet
counts that have an underlying immune-related mechanism of pathogenesis.
Causes include drug-induced thrombocytopenia; pregnancy-induced thrombocy-
topenia; thrombocytopenia related to autoimmune diseases (systemic lupus ery-
thematosus, Hashimoto’s thyroiditis, Graves’ disease, and myasthenia gravis);
hematologic malignancies (chronic lymphocytic leukemia, Hodgkin’s and non-
Hodgkin’s lymphomas, and large granular leukemias); solid tumors (thymoma,
ovarian cancer, and gastric cancer); infections (HIV, hepatitis, and tuberculosis);
neonatal alloimmune thrombocytopenia; and post-transfusion purpura. Some of
these disorders are described next.

Heparin- and Drug-Induced Thrombocytopenia

Drugs can lead to thrombocytopenia by either active immune-related plate-

let destruction or marrow suppression. Heparin is by far the most common
cause of drug-related thrombocytopenia and can be seen in as frequently as 1%
to 5% of patients receiving heparin.44 In heparin-induced or heparin-associated
thrombocytopenia the initial sensitization seems to require therapeutic levels of
the drug. In already sensitized patients, however, minimal amounts of heparin
(100 units given intravenously to keep a peripheral intravenous line patent) can
initiate the platelet activation and aggregation. Heparin of bovine origin seems
to be more immunogenic than porcine heparin, but all types of unfractioned
heparin and low-molecular-weight heparin have been reported to induce this
disorder. In the affected patients, the exposure to heparin leads to the release of
platelet factor 4 from the platelet granules that leads to the formation of an
immunogenic factor 4–heparin complex that triggers the development of IgG
antibodies that bind to the factor 4–heparin complex (IgG Fc portion to the
platelet Fc␥IIa receptor).3, 29 This causes platelet activation and aggregation that
activates the procoagulant system leading to intravenous or intra-arterial throm-
bosis in as many as 30% to 50% of symptomatic heparin-induced thrombocyto-
penia cases.108 Not all patients in whom a heparin-induced IgG is detected
develop symptomatic thrombocytopenia, but when it occurs the platelet drop
has its onset between 5 and 14 days after initiating heparin and the mean
platelet count nadir usually reaches 50,000 platelets/␮L.
In drug-induced thrombocytopenia, the degree of thrombocytopenia can
vary widely and frequently can be severe enough to decrease the platelet count
below 20,000/␮L. The onset of it is variable, usually ranging from within a few
days to several weeks after initiating the drug. In some cases it can appear after
the patient has been taking the specific drug for months or even years. In
already sensitized patients it can occur within a few hours after the rechallenge
with the suspected drug. The pathogenesis is platelet opsonization or activation
usually mediated by the formation of drug-dependent IgG antibodies that bind
their Fab portion to platelet GPs, typically the GPIIb-IIIa or GPIb-IX complexes.4, 91
Occasionally, the formation of some of these antibodies is triggered by drug
metabolites. Although screening for these specific platelet antibodies by platelet-
bound antibody assays can be performed in specialized laboratories, they are
cumbersome and many times clinically impractical. The lack of finding these
antibodies does not exclude the diagnosis of this disorder and often the diagno-
sis is based only on clinical grounds. When these drugs also affect the normal
bone marrow hematopoiesis, other findings in the peripheral blood may include
198 MORENO & MENKE

macrocytosis of the red cells, anemia, or leukopenia. Some of these drugs are
also able to produce hemolytic anemia by immune mechanisms and the periph-
eral blood demonstrates spherocytes and schistocytes, increased numbers of
reticulocytes, nucleated red cells, and macrothrombocytes. Drugs that are known
to cause immune-related thrombocytopenia have been summarized in many
detailed reviews and monographs (Table 1).36, 64, 109
A new group of drugs known as GPIIb-IIIa antagonists has made its way
into clinical practice over the last decade. The three most widely used GPIIb-
IIIa antagonists are (1) abciximab (human-murine chimeric monoclonal antibody
against GPIIb/IIIa); (2) tirofiban (tyrosine-derivative nonpeptide mimetic agent);
and (3) eptifibatide (cyclic heptapeptide agent). They all bind to the GPIIb-IIIa
complex and inhibit platelet aggregation in a dose-dependent manner. Tirofiban
and Eptifibatide may induce platelet-leukocyte aggregation.33 The frequency
of clinically significant thrombocytopenia (platelets ⬍100,000/␮L) with these

Table 1. DRUGS IMPLICATED IN DRUG-INDUCED THROMBOCYTOPENIA

Abciximab Diflunisal Morphine

Acetaminophen Digitoxin Nalidixic acid
Acetazolamide Digoxin Naphazoline
Acetylsalicylic acid Diltiazem Naproxen
Actinomycin D Diphenylhydantoin Nitroglycerin
Allopurinol Doxepin Novobiocin
Alprenolol Eptifibatide Oxprenolol
Aminoglutethimide Ethambutol Oxyphenbutazone
Aminosalicylic acid Etretinate Oxytetracycline
Amiodarone Fluconazole Papaverine
Amitriptyline Furosemide Penicillin
Amphotericin B Gentamicin Pentamidine
Ampicillin Glyburide Phenylbutazone
Amrinone Gold salts Phenytoin
Antazoline Haloperidol Piperacillin
Captopril Heparin Piroxicam
Carbamazepine Heroin Procainamide
Cefamandole Hydrochlorothiazide Quinidine
Cefotetan Ibuprofen Quinine
Ceftazidime Imipramine Ranitidine
Cephalexin Indomethacin Rifampin
Cephalothin Interferon alpha Sibrafiban
Chlorothiazide Iocetamic acid Spironolactone
Chlorpheniramine Iopanoic acid Stibophen
Chlorpromazine Isoniazid Sulfasalazine
Chlorpropamide Isotretinoin Sulfisoxazole
Chlorthalidone Lamifiban Sulindac
Cimetidine Levamizole Suramin
Ciprofloxacin Lidocaine Tamoxifen
Clarithromycin Lithium Thiothixene
Cocaine Meclofenamate Ticlopidine
Danazol Mefenamic acid Tirofiban
Deferoxamine Meprobamate Tolmentin
Desipramine Methicillin Trimethoprim-
Diazepan Methyldopa sulfamethoxazole
Diazoxide Mezlocillin Valproic acid
Diclofenac Mianserin Vancomycin
Diethylstilbestrol Minoxidil Xemilofiban
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 199

agents ranges from 0.1% to 5.6%, although the exact pathogenic mechanism is
not well understood.34, 41, 42 Abciximab may cause EDTA-induced pseudothrom-
bocytopenia and examination of the PBS has been recommended in thrombocy-
topenic patients receiving this agent.75 The role of these agents will be more
common in the future with the advent of oral GPIIb-IIIa inhibitors (peptido-
mimetics) currently in clinical trials.
Although some chemotherapeutic agents may trigger drug-induced throm-
bocytopenia through an immune-mediated mechanism, most produce thrombo-
cytopenia through nonimmune suppression of megakaryocytes either by direct
destruction of megakaryocytes or their progenitors.
Ethanol has been known to cause blood cytopenias as a result of direct
marrow suppression, shortening of the life span of circulating platelets, folate
deficiency, chronic liver disease, and its associated portal hypertension and
hypersplenism.5, 21, 63, 100 Other findings on a blood smear secondary to alcohol
consumption include red cell macrocytosis (round macrocytes), with or without
anemia; target cells; and leukopenia. The thrombocytopenia is usually mild to
moderate but can be severe when several of these events are superimposed.

Radiotherapy-Induced Thrombocytopenia

Thrombocytopenia is frequently observed after irradiation of areas that

involve normal bone marrow because of hematopoietic failure as a result of the
effects of radiation on marrow progeny cells. It is one of the most frequent
adverse events of low-dose whole-body irradiation given as conditioning regi-
men for autologous or allogeneic marrow transplantation. Platelet production is
very sensitive to radiation damage because megakaryocytes make up only 1%
of the nucleated bone marrow cells and are very radiosensitive cells. Because
radiation damages the megakaryocytes and not the non-nucleated circulating
platelets, the onset of the low platelet count in this setting usually follows 7 to
10 days postradiation (median survival of the platelets in the peripheral blood).

Infection-Associated Thrombocytopenia

Infections are a common cause for thrombocytopenia in hospitalized in-

fected patients. Its pathogenesis is most likely multifactorial and includes among
others bacterial exotoxins and endotoxins, hemagophagocytosis of platelets,
platelet activation by cytokines and other inflammatory mediators, and myelo-
suppression by direct marrow-megakaryocyte infection or mediated by cyto-
kines. The peripheral smear of these patients may show left-shifted differentia-
tion of the white blood cells, toxic granulations of the neutrophils, atypical
lymphocytes, normocytic normochromic anemia, and a microangiopathic picture
in the case of ongoing disseminated intravascular coagulation (DIC).
Almost any infection has the potential to cause thrombocytopenia by these
mechanisms. Well-documented organisms responsible for infection-associated
thrombocytopenia include viruses, such as HIV, parvovirus B19, mumps, mea-
sles, rubella (including cases reported after mumps-measles-rubella vaccination),
herpes simplex, and cytomegalovirus; any gram-negative or gram-positive bac-
teremia; mycoplasma pneumoniae; malaria; syphilis; and most rickettsial dis-
eases.6, 109, 112
200 MORENO & MENKE

Hemolytic Uremic Syndrome and Thrombotic

Thrombocytopenic Purpura

Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic pur-

pura (TTP) are two overlapping thrombotic microangiopathies that can occur
either as an acquired disorder or as part of a familial syndrome. Clinically, TTP
patients present with several or all of the classic pentad of manifestations
characterized by thrombocytopenia, microangiopathic hemolytic anemia, fever,
renal dysfunction, and neurologic symptoms. In HUS, patients present with
abdominal pain, diarrhea, gastrointestinal bleeding, and renal failure, usually
following symptoms of a respiratory tract infection or gastroenteritis. Nonfamil-
ial TTP is caused by an acquired inhibitor of von Willebrand factor (vWF)–
cleaving protease, whereas the familial form seems to be caused by a constitu-
tional deficiency of the protease.32 Familial forms of HUS and TTP have been
described in association with decreased serum levels of the third complement
component (C3). The reduced levels of C3 in familial HUS and TTP are likely
related to a genetically determined deficiency in factor H (a modulator of the
alternative pathway of complement activation).81 Recent investigations reported
five mutations in the factor H gene. Genetically determined deficiencies in factor
H are involved in both autosomal-dominant and autosomal-recessive HUS.16
The observation that patients with TTP have a deficiency of vWF-cleaving
protease, whereas patients with HUS do not, has led to the development of a
new assay that can distinguish these two disorders based on the preferential
binding of high-molecular-weight forms of vWF to collagen.39 The peripheral
smear of these patients shows consumptive thrombocytopenia, polychromasia
and stippling of red cells, increased number of nucleated red cells, schistocytes,
and left-shifted white blood cells. Affected organs show microthrombi (formed
by platelets, fibrin, and vWF) of the terminal arterioles and capillaries with
hyaline deposits in the subendothelium.90

Disseminated Intravascular Coagulation

Disseminated intravascular coagulation is a consumption coagulopathy trig-

gered by a variety of systemic disorders that induce the activation of the soluble
coagulation system resulting in intravascular thrombosis; coagulation factor
consumption; and the activation of platelets resulting in thromboembolism (10%
to 40%), bleeding (70% to 90%), and thrombocytopenia (90% to 100%).94, 98
Thrombocytopenia, elevated fibrin-fibrinogen degradation products, prolonged
protime, prolonged activated partial thromboplastin time, and low levels of
fibrinogen are the most common laboratory findings. Besides thrombocytopenia,
the PBS may show anemia, reticulocytosis, schistocytes (Fig. 3), microsphero-
cytes, and leukocytosis with or without left-shifted myeloid differentiation.
Infections, malignancies, obstetric complications, shock, massive tissue damage
(extensive trauma, burns, and fat embolism), and vascular malformations are
systemic disorders that cause DIC.

Post-Transfusion Purpura

Post-transfusion purpura occurs abruptly 5 to 15 days following red cell

transfusion, usually of a multiparous woman or previously transfused patient,
whose platelets lack the HPA-1a platelet antigen (3% of the population). When
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 201

Figure 3. Disseminated intravascular coagulation. Schistocytes and low platelets (Wright-

Giemsa, original magnification 600).

patients who lack the HPA-1a receive a transfusion (usually red cells, but other
blood products have also been reported to cause this syndrome) from an HPA-
1a–positive donor, endogenous anti–HPA-1a antibodies destroy not only the
allogeneic platelets but also the autogeneic platelets by a mechanism that is not
well understood.107 This rare but potentially fatal disorder is usually self-limited
and resolves within 2 to 4 weeks, although the use of plasmapheresis or immu-
noglobulin infusions may shorten this period.7, 18, 61

Von Willebrand’s Disease

Two types of von Willebrand’s disease are associated with low platelet
counts. Type IIB comprises 5% of all von Willebrand’s disease and is character-
ized by thrombocytopenia, normal or decreased levels of factor VIII and von
Willebrand’s disease antigen, decreased ristocetin cofactor, high von Wille-
brand’s multimers, and increased ristocetin-induced platelet aggregation.1, 60 Al-
though platelet morphology is usually normal, several reports have described
the presence of giant platelets in patients with type 2B von Willebrand’s dis-
ease.84 Platelet-type von Willebrand’s disease or pseudo–von Willebrand’s dis-
ease occurs because of a mutation in GPIb alpha that leads to a normal expres-
sion of GPIb-V-IX but an abnormal predisposition to bind von Willebrand’s
disease spontaneously, which in consequence leads to platelet aggregation and
thrombocytopenia.19

Hypersplenism

The causes of thrombocytopenia in hypersplenism are multiple and multi-

factorial. The examination of the PBS frequently shows thrombocytopenia or
pancytopenia and a bone marrow with normal to increased numbers of megakar-
yocytes. Other potential findings are usually associated with the underlying
disorder causing the enlarged spleen. Causes include congestive splenomegaly,
202 MORENO & MENKE

storage diseases, myeloproliferative disorders, lymphomas, leukemias, amy-

loidosis, infections, hemolytic anemias, and autoimmune diseases.

Congenital and Neonatal Thrombocytopenias

The number of disorders that can cause thrombocytopenia during this

period of life is vast. The underlying pathogenic mechanisms and the findings
observed in the PBS are similar to adults. Consumption and platelet activation
(infections, neonatal DIC, Kasabach-Merritt syndrome, massive thrombosis, se-
vere cold stress, lipid-containing parenteral nutrition, and congenital cyanotic
heart disease); immune (neonatal alloimmune thrombocytopenia, maternal sys-
temic lupus erythematosus, post-transfusion purpura, drugs, and maternal ITP);
hypoproliferative (thrombocytopenia with absent radius, congenital pure amega-
karyocytic thrombocytopenic purpura, and osteopetrosis); and hereditary throm-
bocytopenias (see later) are examples.

Pregnancy-Related Thrombocytopenias

Similar to the anemia of pregnancy, the most common cause of thrombocyto-

penia in pregnancy is a dilutional decrease in the platelet count in about 10% of
uncomplicated pregnancies.106 Hypertension-preeclampsia is the second most
common cause of thrombocytopenia during pregnancy followed by the immune-
related thrombocytopenias (ITP of pregnancy; systemic lupus erythematosus;
and hemolysis, elevated liver enzymes, and low platelets syndrome).40, 79 The
PBS of patients with incidental thrombocytopenia of pregnancy and ITP of
pregnancy shows low platelet count and the hemodilutional  iron deficiency
anemia typical of pregnancy but should reflect no other hematologic abnormal-
ity. The hemolysis, elevated liver enzymes, and low platelet syndrome may
display a DIC-type picture and is often associated with preeclampsia.105

INHERITED DISORDERS OF PLATELET NUMBERS AND

MORPHOLOGY

Bernard-Soulier Syndrome

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive disorder of

defective platelet adhesion manifested by mild thrombocytopenia (rarely platelet
counts are below 50,000/␮L); giant platelets on peripheral smear (greater than
or equal to the size of erythrocytes); and moderate-to-severe mucosal hemor-
rhage out of proportion to the degree of decrease in platelet count.8, 37 The
mean platelet volume is increased and the platelet surface GPIb-V-IX complex
(responsible for binding the vWF to the platelet surface) is either missing or
defective because of deficiencies of the polypeptides Iba, Ib␤, V, and IX.19, 20, 66
Determination of the platelet levels of these polypeptides by immunologic tech-
niques is a reliable method of diagnosis.76, 102 All four genes encoding the
complex have been cloned (the GPIb mapped to 22q11.2)110 and 17 variants of
BSS have been described, some of those associated with anatomic malformations.
Laboratory findings include a prolonged bleeding time and abnormal ristocetin-
induced aggregation, but normal platelet aggregation by adenosine diphosphate,
epinephrine, and collagen.10, 50 The clot retraction test is normal.
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 203

An autosomal-dominant variant has also been described as heterozygous

BSS.93 These patients have a reduced platelet count (from 22 to 178  109/L);
recognizable defect of GPIb-IX-V complex; increased MPV (range from 10.4 to
17.2 fL); or any combination of these. In vitro platelet aggregation is usually
normal. Clinical manifestations are frequently absent or mild but when present
manifest as bleeding diathesis typical of platelet dysfunction. The frequency of
heterozygote BSS may be as frequent as 1 in 500, and the diagnosis is usually
missed, particularly when there are no homozygous BSS patients in the family.

Gray Platelet Syndrome

Gray platelet syndrome is a very rare congenital disorder characterized by
large platelets; mild to moderate thrombocytopenia; mild hemorrhagic tendency;
prolonged bleeding time; and abnormal platelet function (deficient aggregation
by collagen or thrombin).52, 65, 69, 89 Classic findings include large agranular plate-
lets with a gray to gray-blue coloration when observed under light microscopy
(Fig. 4), and the severe reduction or complete absence of alpha granules on
electron microscopy. There is not only a decreased number of alpha granules but
also decreased synthesis of their contents (platelet factor 4, ␤-thromboglobulin,
fibrinogen, fibronectin, vWF, platelet-derived growth factor, and throm-
bospondin) within platelets and megakaryocytes. Less common, synthesis of
several of these proteins may be normal but the syndrome can be caused by the
defect in the storage mechanism of these proteins into the alpha granules;
elevated levels of them may be found in plasma; and some are possibly linked
to the increased frequency of mild, nonprogressive myelofibrosis reported in this
disorder.9, 13–15, 52, 55

Familial Platelet Disorder With Predisposition to Acute

Myelogenous Leukemia
An autosomal dominant disorder characterized by quantitative and qualita-
tive platelet defects and propensity to develop acute myelogenous leukemia

Figure 4. Gray platelet syndrome. Gray platelet (Wright-Giemsa, original magnification

1000).
204 MORENO & MENKE

(30% lifetime risk) has been described. This disorder was most recently shown
to be associated with nonsense mutations or intragenic deletion of one allele
of the hematopoietic transcription factor CBFA2 (formerly acute myelogenous
leukemia-1).49, 97 Patients have mild thrombocytopenia and some may have ana-
tomic defects, such as hypospadias and umbilical hernia.2

Fechtner Syndrome and Epstein’s Syndrome

Fechtner syndrome is an autosomal-dominant disorder characterized by

macrothrombocytopenia, high-tone sensorineural hearing loss, nephritis, cata-
racts, and neutrophilic inclusions.88 The 1- to 2-␮m inclusions seen in neutrophils
and eosinophils are cytoplasmic, small, and pale blue, resembling the toxic
Döhle’s bodies. Ultrastructural analysis revealed that these are clusters of ribo-
some and small segments of rough endoplasmic reticulum but they lack the
parallel strands of rough endoplasmic reticulum characteristics of Döhle’s bodies
or the 10-nm filaments seen in May-Hegglin anomaly.88, 104 Thrombocytopenia is
mild to moderate (range from 30,000 to 90,000/␮L) with a MPV as high as 20
fL. Aggregation studies are normal.72, 88 The disease shows a gene mutation
within chromosome 22q11-13 similar to Sebastian syndrome and May-Hegglin
anomaly (see later).77, 103 Epstein’s syndrome was initially described in 1972 with
a clinical picture and inheritance trait similar to Fechtner syndrome.26 Contrary
to Fechtner syndrome, however, the leukocyte inclusions are not a part of
Epstein’s syndrome. Although Epstein’s syndrome is considered a variant of
Alport’s syndrome, it does not have the typical mutations in collagen IV genes
seen in typical Alport’s syndrome.78 Both illnesses can progress to end-stage
renal disease.

May-Hegglin Anomaly

May-Hegglin anomaly is an autosomal-dominant disorder linked to chro-

mosome 22q12-13 and involving the gene MYH9.53, 54, 58 Patients present with
basophilic spindle-shaped Döhle’s-like inclusion bodies in the granulocyte series
(neutrophils, eosinophils, monocytes, and basophils), giant platelets (Fig. 5), and
variable thrombocytopenia, but bleeding time, platelet aggregation studies, and
platelet ultrastructure are normal.45, 68 In some cases of associated bleeding
diathesis, desmopressin has been found to be helpful.82 The granulocyte inclu-
sions are larger than true Döhle’s bodies and seem to be formed by RNA,
ribosomes, and small segments of rough endoplasmic reticulum. Granulocyte
function is normal.45, 68

Montreal Giant Platelet Syndrome

Montreal giant platelet syndrome is another rare hereditary giant platelet

syndrome where the large volume attained by these platelets seems related to a
defect in the mechanism that regulates platelet size and shape during platelet
activation.31, 74 The platelet count is moderately to severely reduced. The bleeding
time is prolonged but the clot retraction is normal. There is spontaneous platelet
aggregation, which may be caused by the reduction of calpain activity observed
in these platelets.87
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 205

Figure 5. May-Hegglin anomaly. Giant platelet with granulocyte inclusions (arrows).

Paris-Trousseau Thrombocytopenia

Paris-Trousseau thrombocytopenia is the only alpha-granule disorder associ-

ated with a cytogenetic abnormality (del[11]q23.3).12, 95 This is a congenital throm-
bocytopenic disorder associated with mild hemorrhagic tendency apparently
caused by the inability of the alpha granules to release their contents after
stimulation by thrombin. About 15% of platelets in the peripheral smear have
giant alpha granules that stain red with the Giemsa stain. There is an increased
number of bone marrow microkaryocytes and megakaryocytes that in vitro
show an increased cell lysis at the end of their maturation. This phenomenon is
considered responsible for the thrombocytopenia observed in these patients. The
circulating platelets have a normal life span.12

Sebastian Syndrome

Sebastian syndrome is an autosomal dominant macrothrombocytopenic dis-

order. The neutrophils have inclusions (Döhle’s body–like) similar to the ones
observed in Fechtner syndrome but different from May-Hegglin anomaly, Chéd-
iak-Higashi syndrome, or the toxic Döhle’s bodies of septicemic patients.46 This
disorder is not associated with sensorineural hearing loss, nephritis, or cataracts
as in Fechtner syndrome. Recent studies suggest, however, that Sebastian syn-
drome, May-Hegglin anomaly, and Fechtner syndrome are allelic-related disor-
ders of chromosome 22 (22q12.3-q13.2). The mutated gene is possibly the one
encoding the nonmuscle myosin heavy chain-A, which is expressed in platelets
and upregulated during granulocyte differentiation.54, 57, 77, 104 Patients with these
three disorders are either asymptomatic or show only a mild bleeding tendency.
Platelet counts vary but range usually between 20,000 and 120,000/␮L and the
mean platelet volume is around 15 to 20 fL.46
206 MORENO & MENKE

Swiss Cheese Platelets

This is a congenital disorder with accompanied bleeding diathesis. Some

investigators have considered this not a true entity but an ultrastructural platelet
defect shared by some of the hereditary giant platelet syndromes. Patients
initially described with this anomaly have thrombocytopenia, prolonged bleed-
ing time, and giant platelets with a complex and dilated tortuous surface-
connected canalicular system that gives the appearance of Swiss cheese on
electron microscopy.43, 96 Patients may have impaired calcium mobilization from
this abnormal canalicular system leading to abnormal platelet aggregation stud-
ies in response to epinephrine, collagen, and adenosine diphosphate.43

Upshaw-Schulman Syndrome

Upshaw-Schulman syndrome is a congenital thrombocytopenic disorder

associated with recurrent episodes of microangiopathic hemolytic anemia, pro-
teinuria, and hematuria. Large vWF multimers have been observed in these
cases during remission and relapses. There are reported cases of transient im-
provement of the platelet count when plasma or various plasma components
are infused,56, 92 raising the question of this disorder being a variant of familial
HUS-TTP syndrome.

Wiskott-Aldrich Syndrome and X-Linked Thrombocytopenia

Wiskott-Aldrich syndrome is an X-linked disorder caused by mutations of

the Wiskott-Aldrich syndrome protein gene (chromosome Xp11.22)23 in hemato-
poietic cells and manifested by immunodeficiency with associated recurrent
infections, eczema, high incidence of malignancies, autoimmune disorders, and
microthrombocytopenia with bleeding diathesis. Mean platelet volume is de-
creased and it improves or normalizes along with the platelet count after sple-
nectomy.67 X-linked thrombocytopenia is considered a milder form of Wiskott-
Aldrich syndrome.85, 86, 99
A novel X-linked thrombocytopenia syndrome has been described over the
last 2 years in association with mutations in the GATA-1 gene (a transcription
factor protein considered essential for normal erythropoiesis and megakaryocyte
differentiation). Different than the Wiskott-Aldrich syndrome–related X-linked
thrombocytopenia, these patients have macrothrombocytopenia, no immunode-
ficiency, increased number of small dysplastic marrow megakaryocytes, and
variable degrees of dyserythropoietic anemia. The mutations described are a
substitution of methionine for valine at amino acid 205,80 glycine for aspartate
in codon 218,30 or glycine to serine in codon 208 of the GATA-1 gene.25

THROMBOCYTOSIS

Thrombocytosis is defined as the elevation of the platelet count above the

upper limit of the normal range (usually ⬎ 400,000/␮L). It can be caused by a
primary clonal bone marrow disorder or secondary to an underlying acute or
chronic inflammatory process.
 ASSESSMENT OF PLATELET NUMBERS AND MORPHOLOGY 207

Figure 6. Essential thrombocythemia. Platelet drifts and a basophil (Wright-Giemsa, original

magnification 600).

Primary

This group encompasses the clonal bone marrow disorders that are part of
the chronic myeloproliferative diseases. The most representative is essential
thrombocythemia. The peripheral smear shows an elevated platelet count (often
⬎ 1,000,000/␮L); platelet drifts (Fig. 6); some degree of anemia and abnormal
red cell morphology depending on the degree of myelofibrosis; and leukocytosis
with or without left-shift immature myeloid cells (leukoerythroblastic picture).
Some ultrastructural defects observed have been a decrease in the number of
alpha granules and excessive dilatation of the open canalicular system.48, 70

Secondary

Multiple disorders have been associated with secondary thrombocytosis as

listed next:
Primary clonal bone marrow disorders
Essential thrombocythemia
Polycythemia rubra vera
Chronic myelogenous leukemia
Myeloid metaplasia with myelofibrosis
Reactive or secondary thrombocytosis
Postsplenectomy and functional asplenia
Iron deficiency anemia
Infectious diseases
Tuberculosis
Bacterial meningitis
Osteomyelitis
Subacute bacterial endocarditis
Malignant diseases
208 MORENO & MENKE

Hodgkin’s disease
Non-Hodgkin’s lymphoma
Prostate cancer
Bladder cancer
Ovarian cancer
Mesothelioma
Pancreatic cancer
Lung cancer
Inflammatory disorders
Rheumatoid arthritis
Polyarteritis nodosa
Giant cell arteritis
Wagener’s granulomatosis
Kawasaki disease
Ankylosing spondylitis
Still’s disease
Polymyalgia rheumatica
Psoriasis
Ulcerative colitis
Crohn’s disease
Tropical sprue
Whipple’s disease
Acute and chronic hemorrhages
Hemolytic anemia
Postsurgery (coronary artery bypass graft)
Postpartum
Vigorous exercise
Drugs
Epinephrine
Vincristine
5-Fluoracil
Postwithdrawal syndrome from severe alcohol use
Acetaminophen overdose
Recovery phase of acute thrombocytopenia
The most common pathogenic mechanism is a cytokine-mediated increase
in interleukin-6, interleukin 1-beta, or tumor necrosis factor-alpha that stimulates
thrombopoiesis, which mobilizes extravascular or intrasplenic platelet pools.22,
27, 101, 111
Elevated serum C-reactive protein levels are common in secondary
thrombocytosis, which may correlate with elevated interleukin-6 levels.101 Al-
though there are no pathognomic findings in the PBS of reactive thrombocytosis,
the PBS may show abnormalities. Patients with anatomic or functional asplenia
demonstrate Howell-Jolly bodies and target cells. Iron deficiency may reveal
microcytosis and hypochromic red cells. Red cell morphology is also altered in
hemolytic anemias (agglutination, reticulocytosis, spherocytosis, and fragmented
red cells) and neutrophilia and Döhle’s bodies are frequent with an underlying
systemic infection.

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