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A 3-year-old girl with frequent nose bleeds.

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 accompanied by 2 episodes of vomiting dark red blood. Results of the laboratory evaluation included: white blood cell count, 8.2 x [10.sup.9]/L [reference interval (RI), [3] 4 x [10.sup.9]/L to 10 x [10.sup.9]/L]; hemoglobin, 10.7 g/dL (RI, 11-15 g/dL); platelet count, 142 x [10.sup.9]/L (RI, 100 x [10.sup.9]/L to 300 x [10.sup.9]/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 insertion 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 presentation, 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 hemarthrosis. 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); reticulocyte count, 6.8% (RI, 0.5%-1.5%). Other laboratory results are shown in Table 1.

In vitro testing showed that the patient's platelets did not aggregate in response to ADP, epinephrine, arachidonic acid, or collagen, but platelets had relatively normal ristocetin-induced aggregation. These findings were confirmed on repeat testing. A smear of peripheral blood showed no clusters of normal platelets. A flow cytometry evaluation revealed marked reduction in glycoprotein IIb/IIIa (GPIIb/IIIa).

DISCUSSION

DIFFERENTIAL DIAGNOSIS

Childhood epistaxis is a common complaint that usually abates in adulthood; however, epistaxis can be life threatening when episodes are frequent and accompanied by substantial blood loss (1). A history of mucocutaneous hemorrhage, as opposed to hemarthrosis (bleeding into joint spaces) and muscle hemorrhage, helps to differentiate disorders of platelet function (including von Willebrand disease and afibrinogenemia) from the hemophilias and related disorders. Hemarthrosis is rarely seen in disorders of platelet function and occurs even more rarely in Glanzmann thrombasthenia (GT), whereas episodes of hemarthrosis can be frequent in hemophilia (1).

Leukemia, idiopathic thrombocytopenic purpura, and allergic purpura should be included in the differential diagnosis of patients with mucocutaneous hemorrhage. When mucocutaneous hemorrhage is seen in leukemia, it is usually accompanied by thrombocytopenia in the peripheral blood. Patients with idiopathic thrombocytopenic purpura have decreased platelet counts and may have detectable antiplatelet antibodies. Allergic purpura (also called Henoch-Shonlein purpura) can be recognized by its clinical presentation, which typically includes joint inflammation, abdominal 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 include Bernard-Soulier syndrome, GT, and gray platelet syndrome. Table 2 summarizes common acquired disorders 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 abnormal platelet function. Many of the conditions mentioned 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 concentrations are used for testing. Guidelines to standardize 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 standard operating procedures to minimize preanalytical and analytical errors and develop appropriate reference 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 interfere 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 influenced by both centrifugation conditions and the number of platelets in the PRP. Centrifugation of whole blood at 200g to 250 g 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 performing 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 x [10.sup.9]/ Land 600 x [10.sup.9]/L. Platelet aggregation is pH dependent; therefore, the PRP pH should be maintained between 7.4 and 7.8. PRP samples 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 various functional platelet disorders. The aggregating agents routinely used are ADP, epinephrine, ristocetin, collagen, and arachidonic acid. Table 1 lists the concentrations of agonists according to the CLSI guidelines (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 response 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 markedly reduced platelet aggregation in response to arachidonic acid only. Abnormalities in platelet aggregation in hematologic and systemic diseases are not specific; these abnormalities are usually recognized by their associated clinical features.

The second step in evaluating an abnormal platelet function result should be to repeat the aggregation assay and to ensure that potential preanalytical or analytical factors that might lead to false positives have been eliminated. If the pattern of aggregation and the clinical 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 agonists ADP, epinephrine, and collagen, but relatively normal aggregation in response to ristocetin (7).

Molecular genetic analysis of the ITGA2B [4] [integrin, alpha 2b (platelet glycoprotein lib 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 mutations 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 parents 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 mucocutaneous 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 development of molecular diagnosis and widespread use of gene chips, prenatal diagnosis has become commonplace throughout the world (9).

The hemorrhagic diathesis in GT is notable for its variability and for the lack of a correlation between the biochemical platelet abnormalities and clinical severity (1). Thus, factors other than the platelet defect itself appear to play an important role in determining the risk of bleeding.

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 platelet dysfunction?

POINTS TO REMEMBER

* Platelet-aggregation tests should be performed with a standardized procedure and with validated reference intervals.

* Many drugs, such as aspirin, and some foods are important 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 characterized by absent or severely reduced platelet aggregation in response to the physiological agonists ADP, epinephrine, 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.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paperandhave metthe 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 intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript 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.

References

(1.) George JN, Caen JP, Nurden AT. Glanzmann's thrombasthenia: the spectrum of clinical disease. Blood 1990;75:1383-95.

(2.) Cattaneo M, Hayward CP, Moffat KA, Puqliano MT, Liu Y, Michelson AD. Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH. J Thromb Haemost 2009;7:1029.

(3.) Kamal AH, Tefferi A, Pruthi RK. How to interpret and pursue an abnormal prothrombin time, activated partial thromboplastin time, and bleeding time in adults. Mayo Clin Proc 2007;82:864-73.

(4.) Femia EA, Puqliano M, Podda G, Cattaneo M. Comparison of different procedures to prepare platelet-rich plasma for studies of platelet aggregation by light transmission aggregometry. Platelets 2012;23:7-10.

(5.) CLSI. Platelet function testing by aggregometry; approved guideline. Wayne (PA): CLSI; 2008. Section 6.3.2, Agonists used; p. 12-3. CLSI document H58-A.

(6.) Hayward CP, Moffat KA, Raby A, Israels S, Plumhoff E, Flynn G, Zehnder JL. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol 2010;134:955-63.

(7.) Nurden AT. Glanzmann thrombasthenia. Orphanet J Rare Dis 2006;1:10.

(8.) Nurden AT, Fiore M, Nurden P, Pillois X. Glanzmann thrombasthenia; a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models. Blood 2011;118:5996-6005.

(9.) Peyvandi F, Garaqiola I, Mortarino M. Prenatal diagnosis and preimplantation genetic diagnosis: novel technologies and state of the art of PGD in different regions of the world. Haemophilia 2011;17 Suppl 1:14-7.

Peipei Jin, [1] Lijun Qiu, [1] Siguo Hao, [2] Xiangliang Yuan, [1] and Lisong Shen [1] *

[1] Departments of Clinical Laboratory and [2] Hematology, Xinhua Hospital, Shanghai 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.

[4] Human genes: ITGA2B, integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41); ITGBB3, integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61).

Commentary

Marco Cattaneo *

This report highlights the case of a 3-year-old girl affected by Glanzmann thrombasthenia, an inherited platelet function disorder (PFD), characterized by abnormalities of glycoprotein complex IIb/IIIa (integrin [[alpha].sub.IIb][[beta].sub.3]), the platelet receptor for adhesion proteins that plays an essential role in platelet aggregation. PFDs are associated with a heightened risk for mucocutaneous bleeding and excessive hemorrhage of early onset after surgery or trauma. In addition to the severe and relatively rare forms mentioned in this report (Glanzmann thrombasthenia, Bernard-Soulier syndrome, and gray platelet syndrome), other PFDs [including abnormalities of receptors for platelet agonists (e.g., the [P2Y.sub.12] receptor for ADP), platelet granules (e.g., storage pool deficiency), signal transduction, and membrane phospholipids (e.g., Scott syndrome) (1)] occur commonly and are generally milder. The diagnostic laboratory assessment of a suspected PFD should be based on a 2-step diagnostic strategy: The first step is the application of screening tests to help generate a diagnostic hypothesis, which then can be tested with specific tests in the second step. The first step should include a complete blood count, examination of the peripheral blood smear, and assessment of platelet aggregation. Although light transmission aggregometry (LTA) is the most widely used platelet function test, it is relatively insensitive to defects ofplatelet secretion, which are the most common PFDs. For this reason, laboratory tests that measure platelet aggregation and secretion simultaneously, such as lumiaggregometry, should be preferred to traditional LTA. The second step includes specific tests (e.g., flow cytometry, Western blotting, DNA analysis, and so on). Platelet transfusions should be used only to treat severe bleeding episodes. Recombinant factor VIIa can be used in patients with severe bleeding episodes who do not respond to platelet transfusion because of alloimmunization. Fibrinolytic inhibitors or desmopressin should be used to treat all other bleeding episodes that require pharmacologic intervention (1).

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, o analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Reference

(1.) Cattaneo M. Inherited platelet-based bleeding disorders. J Thromb Haemost 2003;1:1628-36.

Unita di Medicina 3, Ospedale San Paolo, Dipartimento di Scienze della Salute, University degli Studi di Milano, Milan, Italy.

* Address correspondence to the author at: Unita di Medicina 3, Ospedale San Paolo, Universita di Milano, Via di Rudinl 8, 20142 Milano, Italy. Fax +390250323089; e-mail marco.cattaneo@unimi.it.

Received September 19, 2012; accepted October 24, 2012.

DOI: 10.1373/clinchem.2012.195248

Commentary

Rajiv K. Pruthi [1,2] *

The hemostatic system consists of vascular, platelet, and plasma coagulation factors, and a defect in any one of these factors typically can cause a bleeding disorder. The costs associated with laboratory evaluation of all components of the hemostatic system in every patient would be prohibitive, however. Typically, the initial evaluation involves screening the patient for a history of hemostasis disorders. A mucocutaneous bleeding pattern (e.g., epistaxis, ease of bruising, gastrointestinal bleeding, and hematuria) suggests a primary hemostatic defect (vascular, platelet, or von Willebrand factor), whereas a history of soft tissue hemorrhage and/or hemarthrosis suggests a secondary defect (coagulation factor deficiencies). Age of onset of bleeding and family history can be used to distinguish between a congenital and an acquired bleeding disorder.

Typical initial laboratory testing includes such screening tests as the prothrombin time, the activated partial thromboplastin time, and the thrombin time. Additional diagnostic assays include testing for von Willebrand disease (the most common hereditary bleeding disorder) and factor assays to evaluate prolonged prothrombin and/or activated partial thromboplastin times. If the results of these studies are negative, pursuit of rare bleeding disorders, such as hereditary defects in platelet function and factor XIII deficiency, may be warranted.

Jin et al. present a child with persistent severe epistaxis for whom the results of platelet-aggregation testing were consistent with Glanzmann thrombasthenia, a rare bleeding disorder. They also elucidated the molecular basis of Glanzmann thrombasthenia in this family. A consideration of the pre-analytical variables and analytical variables, which were thoroughly reviewed in their report, is critical to avoid erroneous diagnoses, and adherence to published laboratory testing guidelines is important. In selected patients, however, platelet aggregation may be normal or borderline abnormal. For such patients, platelet electron microscopy, an evolving tool, is especially useful for evaluating dense granule-deficiency disorders. Having the necessary expertise, establishment of a reference interval, standardization of methods, and regulatory agency certification are equally important for laboratories offering electron microscopy.

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 intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared anypotential conflicts of interest.

[1] Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN; [2] Division of Hematopathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

* Address correspondence to the author at: Division of Hematology, Department of Internal Medicine, Mayo Clinic, 200 First St. SW, Rochester, MN 55902. Fax 507-266-4972; e-mail Pruthi.rajiv@mayo.edu.

Received November 5, 2012; accepted November 12, 2012.

DOI: 10.1373/clinchem.2012.195255
Table 1. Patient's laboratory results.

Analyte                       Day of       Day 1     Day 2
                             admission

WBC, x [10.sup.9]/L (a)        20.83       10.33     11.57
Hemoglobin, g/dL                6.2         5.8       5.5
RBC, x [10.sup.12]/L           2.22        1.93      1.94
MCV, fL                        84.7         85       84.5
Hematocrit, %                  18.8        16.4      16.4
C-reactive protein, mg/L        89          23        33
Platelets, X [10.sup.9]/L       384         160       117
Reticulocytes, %                6.8
Prothrombin time, s                        10.3
Activated partial
  thromboplastin time, s                   25.5
Fibrinogen, g/L                            2.24
Factor VIII activity, %                    181.8
Factor IX activity, %                      125.1
VWF antigen, %                              212
Platelet aggregation
     test, %
  ADP (2 [micro]mol/L)                     5.49
  Epinephrine
    (2 [micro]mol/L)                         3
  Arachidonic acid
    (0.5 mmol/L)                             6
  Collagen (2 [micro]g/mL)                 12.3
  Ristocetin (1.5 mg/mL)                    45
Platelet function-
related markers, %
  CD41 (GPIIb)                               0
  CD61 (GPIIIa)                              0
  CD42b                                     100

Analyte                        Day 3     Reference
                                         interval

WBC, x [10.sup.9]/L (a)        10.11       4-10
Hemoglobin, g/dL               10.6        11-15
RBC, x [10.sup.12]/L           3.58        3.5-5
MCV, fL                        84.6        82-95
Hematocrit, %                  30.3        34-45
C-reactive protein, mg/L        10          <8
Platelets, X [10.sup.9]/L       101       100-300
Reticulocytes, %                          0.5-1.5
Prothrombin time, s                        9-13
Activated partial
  thromboplastin time, s                   26-39
Fibrinogen, g/L                           2.0-4.0
Factor VIII activity, %                   50-150
Factor IX activity, %                     50-150
VWF antigen, %                            62-126
Platelet aggregation
     test, %                             44.7-77.8
  ADP (2 [micro]mol/L)
  Epinephrine
    (2 [micro]mol/L)
  Arachidonic acid
    (0.5 mmol/L)
  Collagen (2 [micro]g/mL)
  Ristocetin (1.5 mg/mL)
Platelet function-
related markers, %                        50-100
  CD41 (GPIIb)
  CD61 (GPIIIa)
  CD42b

(a) WBC, white blood cells; RBC, red blood cells;
MCV, mean corpuscular volume; VWF, von Willebrand
factor.

Table 2. Causes of platelet function abnormalities.

Causes               Types and associated             Defects
                     features

Exogenous
  Medications        Aspirin, dipyridamole
  Foods              Garlic, black fungus,
                       green tea, red wine
Endogenous
  Bernard-Soulier    Lacks platelet response to       GPIb/IX
    syndrome           ristocetin only and is           or GPV (a)
                       associated with
                       thrombocytopenia and
                       giant platelets
  von Willebrand     Reduced or absent                VWF
    disease            ristocetin response

  GT                 Normal platelet-aggregation      GPIIb/IIIa
                       response to
                       ristocetin only
  Gray platelet      Lacks platelet response          Numbers and/or
    syndrome           to collagen or thrombin          content
                                                        of platelet
  Hematologic and    Abnormalities in platelet          [alpha]
    systemic           aggregation                      granules
    diseases           are not specific

(a) GPIb/IX, glycoprotein Ib/IX; GPV, glycoprotein
V; VWF, von Willebrand factor.
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Article Details
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Title Annotation:Clinical Case Study
Author:Jin, Peipei; Qiu, Lijun; Hao, Siguo; Yuan, Xiangliang; Shen, Lisong
Publication:Clinical Chemistry
Article Type:Clinical report
Geographic Code:1USA
Date:May 1, 2013
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