Clinical Chemistry 59:5
746–751 (2013)
A 3-Year-Old Girl with Frequent Nose Bleeds
Peipei Jin,1 Lijun Qiu,1 Siguo Hao,2 Xiangliang Yuan,1 and Lisong Shen1*
CASE DESCRIPTION
A 3-year-old girl presented with petechial hemorrhages
and repeated nosebleeds. Two weeks earlier she had
been admitted to a local hospital with nosebleeds ac-
companied by 2 episodes of vomiting dark red blood.
Results of the laboratory evaluation included: white
blood cell count, 8.2 ⫻ 109/L [reference interval (RI),3
4 ⫻ 109/L to 10 ⫻ 109/L]; hemoglobin, 10.7 g/dL (RI,
11–15 g/dL); platelet count, 142 ⫻ 109/L (RI, 100 ⫻
109/L to 300 ⫻ 109/L), prothrombin time, 11.5 s (RI,
9 –13 s); activated partial thromboplastin time, 31.2 s
(RI, 26 –39 s); and fibrinogen, 2.5 g/L (RI, 2.0 – 4.0 g/L).
The patient was discharged in good condition after in-
sertion of nasal packs.
One day before the current admission, the patient
had nose bleeds once again, this time accompanied by 4
episodes of hematemesis and tarry stool. At presenta-
tion, she had no fever and no diarrhea. She was not on
any medications. The patient had a history of easy
bruising, repeated gum bleeding, but not hemarthro-
sis. There was no family history of abnormal bleeding.
On examination, she appeared pale, with normal vital
signs. Her skin had scattered petechiae. The physical
examination was otherwise unremarkable.
At presentation, laboratory findings included the
following: hemoglobin, 6.2 g/dL (RI, 11–15 g/dL); re-
ticulocyte count, 6.8% (RI, 0.5%–1.5%). Other labora-
tory results are shown in Table 1.
In vitro testing showed that the patient’s platelets
did not aggregate in response to ADP, epinephrine, ar-
achidonic acid, or collagen, but platelets had relatively
normal ristocetin-induced aggregation. These findings
were confirmed on repeat testing. A smear of periph-
eral blood showed no clusters of normal platelets. A
flow cytometry evaluation revealed marked reduction
in glycoprotein IIb/IIIa (GPIIb/IIIa).
1
Departments of Clinical Laboratory and 2 Hematology, Xinhua Hospital, Shang-
hai Jiao Tong University School of Medicine, Shanghai, China.
* Address correspondence to this author at: Xin Hua Hospital, 1665 Kong Jiang
Rd., Yangpu District, Shanghai, NA, China 200092. Fax ⫹86-21-25075173;
e-mail lisongshen@hotmail.com.
Received April 22, 2012; accepted August 16, 2012.
DOI: 10.1373/clinchem.2012.188409
3
Nonstandard abbreviations: RI, reference interval; GPIIb/IIIa, glycoprotein IIb/
IIIa; GT, Glanzmann thrombasthenia; LTA, light transmission aggregometry;
PRP, platelet-rich plasma.
746
Clinical Case Study
QUESTIONS TO CONSIDER
1. What disorders should be considered in the workup of
children with repeated nose bleeds?
2. What are the potential sources of preanalytical variation
in hematologic tests?
3. What is the most likely cause of the results seen in this case?
4. What are the typical symptoms and laboratory test
results associated with various inherited causes of plate-
let dysfunction?
DISCUSSION
DIFFERENTIAL DIAGNOSIS
Childhood epistaxis is a common complaint that usu-
ally abates in adulthood; however, epistaxis can be life
threatening when episodes are frequent and accompa-
nied by substantial blood loss (1 ). A history of muco-
cutaneous hemorrhage, as opposed to hemarthrosis
(bleeding into joint spaces) and muscle hemorrhage,
helps to differentiate disorders of platelet function (in-
cluding von Willebrand disease and afibrinogenemia)
from the hemophilias and related disorders. Hemar-
throsis is rarely seen in disorders of platelet function
and occurs even more rarely in Glanzmann thrombas-
thenia (GT), whereas episodes of hemarthrosis can be
frequent in hemophilia (1 ).
Leukemia, idiopathic thrombocytopenic purpura,
and allergic purpura should be included in the differ-
ential diagnosis of patients with mucocutaneous hem-
orrhage. When mucocutaneous hemorrhage is seen in
leukemia, it is usually accompanied by thrombocyto-
penia in the peripheral blood. Patients with idiopathic
thrombocytopenic purpura have decreased platelet
counts and may have detectable antiplatelet antibodies.
Allergic purpura (also called Henoch–Shönlein pur-
pura) can be recognized by its clinical presentation,
which typically includes joint inflammation, abdomi-
nal pain, and sometimes hematuria and renal damage.
DIAGNOSTIC TESTS FOR PLATELET FUNCTION DISORDERS
Abnormalities of platelet function have been associated
with several acquired and inherited bleeding disorders.
Common inherited causes of platelet-related bleeding in-
clude Bernard–Soulier syndrome, GT, and gray platelet
syndrome. Table 2 summarizes common acquired disor-
 Clinical Case Study

Table 1. Patient’s laboratory results.

Analyte Day of admission Day 1 Day 2 Day 3 Reference interval

WBC, ⫻10 /L 9 a
20.83 10.33 11.57 10.11 4–10
Hemoglobin, g/dL 6.2 5.8 5.5 10.6 11–15
RBC, ⫻1012/L 2.22 1.93 1.94 3.58 3.5–5
MCV, fL 84.7 85 84.5 84.6 82–95
Hematocrit, % 18.8 16.4 16.4 30.3 34–45
C-reactive protein, mg/L 89 23 33 10 ⬍8
Platelets, ⫻109/L 384 160 117 101 100–300
Reticulocytes, % 6.8 0.5–1.5
Prothrombin time, s 10.3 9–13
Activated partial thromboplastin time, s 25.5 26–39
Fibrinogen, g/L 2.24 2.0–4.0
Factor VIII activity, % 181.8 50–150
Factor IX activity, % 125.1 50–150
VWF antigen, % 212 62–126
Platelet aggregation test, % 44.7–77.8
ADP (2 ␮mol/L) 5.49
Epinephrine (2 ␮mol/L) 3
Arachidonic acid (0.5 mmol/L) 6
Collagen (2 ␮g/mL) 12.3
Ristocetin (1.5 mg/mL) 45
Platelet function–related markers, % 50–100
CD41 (GPIIb) 0
CD61 (GPIIIa) 0
CD42b 100
a
WBC, white blood cells; RBC, red blood cells; MCV, mean corpuscular volume; VWF, von Willebrand factor.

ders of platelet function, including those caused by aspirin
consumption and those due to hematologic and systemic
diseases. It is essential to assess platelet aggregation as part
of the workup of individuals suspected of having an ab-
normal platelet function. Many of the conditions men-
tioned above exhibit characteristic abnormalities in
platelet-aggregation studies. Aggregation testing for the
child highlighted in this case was performed with light
transmission aggregometry (LTA).
The International Society on Thrombosis and
Hemostasis survey on LTA practices (2 ) has helped
identify aspects of practice that lack standardization
worldwide, including what agonists and agonist con-
centrations are used for testing. Guidelines to stan-
dardize and improve diagnostic testing for platelet
function disorders have been published by the CLSI.
When performed and interpreted in accordance with
guidelines, LTA is useful in the evaluation of platelet
function disorders. Laboratories should establish stan-
dard operating procedures to minimize preanalytical
and analytical errors and develop appropriate refer-
ence intervals to guide the interpretation of results.
The first step in the evaluation of an unexpected
platelet function disorder is to rule out preanalytical
causes of imprecision (3 ). It is important to remember
that many drugs (e.g., aspirin and dipyridamole) and
some foods (e.g., garlic, black fungus, green tea, and
red wine) affect platelet function. Patients should be
advised to avoid taking platelet-inhibitory medications
for up to 14 days before the test, and they should be
asked about their current medications and diet before
blood samples are obtained for the test. In addition,
studies should not be conducted after the patient has
consumed a fatty meal, because chylomicrons can in-
terfere with LTA measurement of platelet aggregation.
For aggregation studies, platelets are separated
from red blood cells and white blood cells, and platelet-
rich plasma (PRP) is prepared. PRP quality is influ-
enced by both centrifugation conditions and the num-
ber of platelets in the PRP. Centrifugation of whole

Clinical Chemistry 59:5 (2013) 747

Clinical Case Study

Table 2. Causes of platelet function abnormalities.

Causes Types and associated features Defects

Exogenous
Medications Aspirin, dipyridamole
Foods Garlic, black fungus, green tea, red wine
Endogenous
Bernard–Soulier syndrome Lacks platelet response to ristocetin only and is associated with GPIb/IX or GPVa
thrombocytopenia and giant platelets
von Willebrand disease Reduced or absent ristocetin response VWF
GT Normal platelet-aggregation response to ristocetin only GPIIb/IIIa
Gray platelet syndrome Lacks platelet response to collagen or thrombin Numbers and/or content of
platelet ␣ granules
Hematologic and systemic Abnormalities in platelet aggregation are not specific
diseases
a
GPIb/IX, glycoprotein Ib/IX; GPV, glycoprotein V; VWF, von Willebrand factor.

blood at 200g to 250g for 10 min appears to be the best
condition for preparing PRP for LTA studies (4 ). A
platelet count of the PRP is also required before per-
forming aggregation studies. The number of platelets
in the PRP will influence the aggregation responses.
The best results are obtained when the platelet count
for PRP is between 200 ⫻ 109/L and 600 ⫻ 109/L. Plate-
let aggregation is pH dependent; therefore, the PRP pH
should be maintained between 7.4 and 7.8. PRP sam-
ples should be stored in full, tightly capped tubes, and
testing should be completed within 4 h of preparation.
The choice of platelet-aggregating agents should
be sufficient to allow discrimination between the vari-
ous functional platelet disorders. The aggregating
agents routinely used are ADP, epinephrine, ristocetin,
collagen, and arachidonic acid. Table 1 lists the con-
centrations of agonists according to the CLSI guide-
lines (5 ). Bernard–Soulier syndrome lacks a platelet
response only for ristocetin and is associated with
thrombocytopenia and giant platelets. von Willebrand
disease also has a reduced or absent ristocetin response,
but platelet counts and morphology are normal. GT is
characterized by a normal platelet-aggregation re-
sponse to ristocetin only. Gray platelet syndrome lacks
a platelet response to collagen or thrombin, but it
shows a normal response to other aggregating agents.
Ingestion of aspirin is characterized by absent or mark-
edly reduced platelet aggregation in response to arachi-
donic acid only. Abnormalities in platelet aggregation
in hematologic and systemic diseases are not specific;
these abnormalities are usually recognized by their as-
sociated clinical features.
The second step in evaluating an abnormal platelet
function result should be to repeat the aggregation as-
say and to ensure that potential preanalytical or analyt-
ical factors that might lead to false positives have been
eliminated. If the pattern of aggregation and the clini-
cal features suggest a genetic cause, confirmatory tests
are appropriate (e.g., glycoprotein analysis to confirm
GT or Bernard–Soulier syndrome) (6 ).
OVERVIEW OF GT
GT is a rare autosomal recessive disorder characterized by
mucocutaneous bleeding and absent or severely reduced
platelet aggregation in response to the physiological ago-
nists ADP, epinephrine, and collagen, but relatively nor-
mal aggregation in response to ristocetin (7 ).
Molecular genetic analysis of the ITGA2B4 [in-
tegrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa
complex, antigen CD41)] gene, which encodes
GPIIb, showed 2 heterozygous nonsense mutations
(Gln517Stop and Arg553Stop). Gln517Stop is a novel
mutation that has not previously been reported to the
best of our knowledge. An analysis of family members
indicated that the Gln517Stop and Arg553Stop muta-
tions were located on different alleles and inherited
from the patient’s mother and father, respectively. Her
parents were not consanguineous and had no bleeding
symptoms. The results of laboratory tests for her par-
ents were all normal, including the results of a flow
cytometric analysis of GPIIb and GPIIIa. Nonsense
mutations in humans have been found to reduce the
accumulation of mutant mRNA (8 ).
Molecular testing is considered appropriate for
platelet disorders in pediatric patients with mucocuta-
4
Human genes: ITGA2B, integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex,
antigen CD41); ITGB3, integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61).

748 Clinical Chemistry 59:5 (2013)


neous bleeding and abnormal platelet aggregation. In
addition, some patients with atypical laboratory results
can be evaluated with molecular testing. Molecular
testing can identify the pathogenic gene quickly and
accurately. Thus, the disease can be diagnosed earlier,
thereby allowing proper treatment. With the develop-
ment of molecular diagnosis and widespread use of
gene chips, prenatal diagnosis has become common-
place throughout the world (9 ).
The hemorrhagic diathesis in GT is notable for its vari-
ability and for the lack of a correlation between the biochem-
ical platelet abnormalities and clinical severity (1). Thus, fac-
tors other than the platelet defect itself appear to play an
important role in determining the risk of bleeding.
POINTS TO REMEMBER
• Platelet-aggregation tests should be performed with a stan-
dardized procedure and with validated reference intervals.
• Many drugs, such as aspirin, and some foods are im-
portant preanalytical variables that can affect platelet
function. Patients should be advised to avoid taking
these medications and ingesting such foods for up to 14
days before the test.
• GT is a rare autosomal recessive disorder. It is usually char-
acterized by absent or severely reduced platelet aggregation
in response to the physiological agonists ADP, epi-
nephrine, and collagen, but platelets show relatively
normal aggregation in response to ristocetin.
• Although the GT phenotype is well defined, bleeding
severity varies considerably among affected individuals,
even within the same family or ethnic group. Many
factors other than the platelet defect itself play an
important role in determining the risk of bleeding.
• Molecular analysis is available for identifying mutations
in the ITGA2B and ITGB3 [integrin, beta 3 (platelet
glycoprotein IIIa, antigen CD61)] genes and is important
for genetic counseling.
Commentary
Marco Cattaneo*
This report highlights the case of a 3-year-old girl af-
fected by Glanzmann thrombasthenia, an inherited
Unità di Medicina 3, Ospedale San Paolo, Dipartimento di Scienze della Salute,
Università degli Studi di Milano, Milan, Italy.
* Address correspondence to the author at: Unità di Medicina 3, Ospedale San
Paolo, Università di Milano, Via di Rudinı̀ 8, 20142 Milano, Italy. Fax ⫹39-
0250323089; e-mail marco.cattaneo@unimi.it.
Clinical Case Study
Author Contributions: All authors confirmed they have contributed to the
intellectual content of this paper and have met the following 3 requirements: (a)
significant contributions to the conception and design, acquisition of data, or
analysis and interpretation of data; (b) drafting or revising the article for intel-
lectual content; and (c) final approval of the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: L. Shen, ASIC research project of the Shanghai
Science and Technology Commission (11jc1408300).
Expert Testimony: None declared.
Patents: None declared.
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Received September 19, 2012; accepted October 24, 2012.
DOI: 10.1373/clinchem.2012.195248
Clinical Chemistry 59:5 (2013) 749