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Platelet Esoteric Testing

A Case Study

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Published: June 2010

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Dr. Chen describes a patient with a history of bleeding and the esoteric platelet studies needed to diagnose the underlying bleeding disorder.

Presenter: Dong Chen, MD, PhD

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Introduction

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Our speaker for this program is Dong Chen, MD, PhD, Assistant Professor of Laboratory Medicine and Pathology and Consultant in the Special Coagulation Laboratory in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. Dr. Chen describes a patient with a history of bleeding and the esoteric platelet studies needed to diagnose the underlying bleeding disorder.

Bleeding and Thrombosing Diseases: 2010 Mayo Update

Mayo Medical Laboratories is sponsoring the 14th annual Bleeding and Thrombosing Diseases Conference which will include a number of case-based presentations.

During these sessions, the faculties will present difficult coagulation cases and review their diagnostic strategies, workup and results. Today’s presentation is one example of these case studies.

Clinical Presentation

A 52-year-old woman came to Mayo Clinic for a clinical consultation for her life long thrombocytopenia, bleeding diathesis and bone marrow infiltration identified by radiology study at an outside institution.

Bleeding History

She had significant bleeding history:

  • At age of 12, she had intermittent hemoptysis, hematuria and she bled for 4 days after a molar tooth extraction.
  • She started to have heavy menstrual periods at age of 16.
  • Later she had multiple episodes of vaginal bleeding which required intervention.
  • She was found to be slightly thrombocytopenic at age of 21, and was suspected to have immune thrombocytopenia purpura.
  • At age of 27, she had tubectomy for ectopic pregnancy which was uneventful.
  • In the following year, she had an uneventful C-section after receiving preoperative platelet transfusion.
  • She had subconjunctival hemorrhage when she was 34.
  • Recently, she had hip pain and radiology studies of her hip were unremarkable except for mild bone marrow infiltration.

Complete Blood Count (CBC)

Except for mild thrombocytopenia and slightly elevated mean platelet volume (MPV), her blood counts were relatively unremarkable.

Peripheral Blood Smear

Her peripheral blood smear showed slightly enlarged platelets with grayish color probably due to decreased purple punctate granules.

Coagulation Studies

Her routine coagulation laboratory test workup showed no evidence of coagulation factor deficiency, von Willebrand Disease(VWD), severe Factor XIII deficiency, lupus anticoagulant(LAC), DIC, aberrant primary fibrinolysis, or dysfibrinogenemia. The only abnormality was the slightly elevated D-dimer.

Platelet Function Studies

Her platelet function studies showed slightly prolonged closure time of PFA epinephrine cartridge. But her platelet light transmission aggregation studies were all normal. Now we finished the routine workup for bleeding diathesis.

Differential Diagnosis ?

Based on these laboratory results, here are some potential differential diagnoses: 1. Her bleeding is most likely caused by thrombocytopenia, a quantitative platelet deficiency, since there is no evidence of a qualitative platelet disorder. 2. Qualitative platelet dysfunction present (detected by prolonged PFA100-Epinephrine closure time). 3. Patient most likely has persistent ITP. 4. In light of patient’s significant bleeding history and abnormal peripheral blood findings, further platelet testing is indicated. We favored number 4, therefore we further pursued additional testing.

Laboratory Approach to Qualitative Platelet Disorders

Laboratory platelet testing includes routine tests and esoteric tests. Routine tests include preanalytical, clinical and family bleeding history collection, platelet count, indices, and morphologic examination and platelet function tests. These we have done for this patient. Esoteric tests include flow cytometry, electron micrographic (EM) study, and molecular testing.

Platelet Qualitative Disorders

Qualitative platelet disorders can be further classified as 4 Ss: Surface deficiency, storage pool deficiency, signal transduction deficiency, syndromic thrombocytopenia.

Laboratory Systemic Approach

Laboratory diagnosis of a hereditary platelet disorder requires a systemic approach. It starts with a clinical finding including patient’s personal and family bleeding history and bleeding pattern. Patients with hereditary platelet disorder commonly present with mild to moderate thrombocytopenia and mucocutaneous bleeding.

Next, the possibility of a plasmatic hemostatic deficiency such as von Willebrand disease (VWD) and acquired platelet deficiencies due to amyloidosis, autoimmune disorder, or medication, etc need to be excluded.

The laboratory platelet testing starts with a platelet count, platelet indices, and morphologic examination. The most important platelet indice is mean platelet volume (MPV), which helps to group platelet disorders into 3 major groups: The small platelets when MPV <5fL, is pathognomonic for Wiskott-Aldrich syndrome (WAS) and its milder form X-linked macrothrombocytopenia.

The large platelets with MPV >9 to10 fl, is typical for so-called giant platelet diseases which include Bernard-Soulier syndrome(BSS), Glanzmann thrombasthenia(GT), Gray platelet syndrome(GP), MYH9-related platelet disorders, platelet type von Willebrand disease(VWD), white platelet syndrome and alpha-delta granule deficiency.

The remaining platelet disorders have normal sized platelets such as ADP, collagen receptor deficiencies, or Hermansky-Pudlak syndrome(HPS). Our patient will fall into the Hermansky-Pudlak group of large platelet group since her MPV was elevated.

Macro-thrombocytopenia

Now let’s further divide our list of diseases based on classification. First, surface/signal transduction deficiency include Bernard-Soulier syndrome(BSS), Glanzmann thrombasthenia(GT) and platelet type von Willebrand disease(VWD); Storage pool deficiency include gray platelet syndrome(GPS), white platelet syndrome, GATA-1 mutation associated platelet disorder and alpha-delta granule deficiency platelet disorder and finally syndromic MYH9-related syndromes.

Flow Cytometry Studies

We first look at the possibility of a surface deficiency. Platelet flow cytometry study is the gold stand test for platelet surface deficiency. In this case, the patient had no deficiency of glycoprotein IIb IIIa, Ib-V-IX or collagen receptor deficiency. When platelets are activated by either thrombin analog sTRAP or ADP, platelets had normal glycoprotein IIb IIIa response, but slightly decreased surface P-selectin expression.

Platelet Activity By Flow Cytometry

Here is the histogram of P-selectin expression upon platelet activation. The upper panel was normal control. The lower panel was the patient’s. You can see that a subset of patient’s platelets had decreased P-selectin expression indicated by the pink curve and red arrow. This finding may suggest possible alpha-granule deficiency.

Macro-thrombocytopenia

From both platelet function tests and flow cytometry studies, patient unlikely had a surface/signal transduction deficiency. In addition, her clinical history and unremarkable peripheral blood morphology do not suggest a syndromic deficiency. Now let’s look at storage pool deficiencies.

Ultrastructure Studies Whole Mount

The best way to study platelet storage pool is platelet electron microscopy. The first method is the whole mount EM. Patient’s platelet rich plasma was applied directly onto the EM grid and examined. The dense bodies contain calcium which blocks electron beam and consequently creates an ink dot shadow. By counting these ink dots we can estimate dense body count per platelet. The normal range is more than 2 dense bodies per platelets. And this case, the mean dense body count is 10 per platelet, so there is no evidence of dense body deficiency.

Ultrastructure Studies Thin Sections

Thin section EM is used to visualize platelet ultrastructures such as alpha granules and inclusions. For this case, the alpha granules are markedly decreased, the empty space was taken up by increased dense tubular network. There are no aberrant inclusions, structures or increased golgi complex.

Macro-thrombocytopenia

The absence of golgi complex excluded white platelet syndrome and the normal dense body count excluded alpha/delta granule deficiency. The lack of alpha granules narrows our diagnosis to either a gray platelet syndrome or GATA-1 associated platelet disorder.

Molecular Studies

GATA-1 gene was sequenced in Dr. Steensma’s lab at Mayo Clinic and revealed no mutations.

Final Diagnosis

The final diagnosis is gray platelet syndrome(GPS) for this patient.

Global Prevalence of GPS

Gray platelet syndrome is a rare hereditary platelet disorder with scattered case reports through out all continents.

GPS-Clinical Presentation

It is an autosomal dominant or recessive disorder with mild to moderate bleeding tendency.

Laboratory Findings

Patients usually present with mild to moderate thrombocytopenia with slightly enlarged platelets; platelets on peripheral blood smear look grayish due to the lack of purple granules. Platelet function tests by either PFA-100 or platelet aggregation may be normal.

The hallmark is the lack of or markedly decreased alpha granules by thin section electron microscopy. Flow cytometry study may show decreased P-section expression upon platelet activation.

Differential Diagnoses of Gray Platelets

However, hypogranular or gray platelets can be seen in other acquired or congenital conditions. The acquired conditions include myelodysplastic syndrome(MDS) or myocardial infarction, and hypersplenism. The congenital conditions include alpha-delta granule deficiency, GATA-1 mutation associated platelet disorder, gray platelet syndrome, white platelet syndrome, Medich platelet disorder and Quebec platelet syndrome. Therefore, a systemic approach is needed to reach a final diagnosis.

Unique Bone Marrow Findings in Patients with GPS

Patients with gray platelet syndrome(GPS) may also develop bone marrow reticulin fibrosis, possibly due to premature release of alpha-granule contents. In bone marrow, megakaryocytes may show emperipolesis which stands for engulfing other blood cells into their cytoplasm of megakaryocytes.

Clinical Presentation

Indeed, our patient also had a bone marrow infiltration identified by radiology study.

Myelofibrosis

Her bone marrow biopsy interestingly demonstrated mild reticulin fibrosis indicated by the dark lines.

Emperipolesis

And megakaryocytes showed evident emperipolesis. The upper 2 panels show the lymphocytes in cytoplasm of megakaryocytes on a bone marrow aspirate smear. The lower panels show neutrophils are present in the cytoplasm of megakaryocytes, which is better appreciated when the cytoplasm of megakaryocytes is highlighted by factor VIII immunohistochemical stain.

GPS-Treatment

The treatment options for gray platelet syndrome are platelet transfusion, DDAVP and recombinant factor VII administration. Myelofibrosis usually does not progress. However, sometimes patient may develop secondary pulmonary fibrosis similar to Hermansky-Pudlak syndrome.

Summary

In summary, this case demonstrated the importance of clinical history and underscored the value of a systemic approach by effectively utilizing both routine and esoteric laboratory tests to tease out the nature of a platelet disorder. And finally, given the fact that hereditary platelet disorders are rare, clinical and laboratory consultation sometimes could be very helpful.

Acknowledgment

Acknowledgment: The routine and esoteric platelet tests that I described in this case were performed in our special coagulation and EM core laboratories.

Bleeding and Thrombosing Diseases: 2010 Mayo Update

Finally, I would like to remind you again that more cases and clinical and laboratory issues will be discussed in our upcoming annual Bleeding and Thrombosing Diseases Conference. We are looking forward to your participation. We hope to see you soon in August.


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