Platelet Transmission Electron Microscopy and Flow Cytometry 11/15/2015

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1 Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Today our topic looks at hereditary platelet disorders and the role of platelet electron microscopy and platelet surface glycoprotein expression levels in diagnosis. 1

2 Our speaker for this program is Dr. Dong Chen, co-director of the Special Coagulation Laboratory in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota Dr. Chen is also an Associate Professor of Laboratory Medicine and Pathology in the College of Medicine. Dr. Chen thank you for presenting with us today. 2

3 Welcome and thank you for attending this hot topic session. Here are my disclosures. I am a member of the College of American Pathologists Coagulation Resource Committee and North American Specialized Coagulation Laboratory Association platelet proficiency testing group.. 3

4 In today s talk, I will first briefly describe categorization of hereditary platelet disorders, then I will discuss in more detail about platelet electron microscopy and platelet surface glycoprotein assessment by flow cytometry in the diagnosis of hereditary platelet disorders.

5 Platelets are essential for primary hemostasis. They are first released from bone marrow megakaryocytes in circulation. At the damaged vascular site, platelets can bind to exposed subendothelial collagen via glycoprotein IB-von Willebrand factor binding and platelet collagen receptors glycoprotein Ia/IIa/GPVI. Platelets are subsequently activated through signal transduction, and contents of granules are released. The release of ADP, thromboxane A2, and other molecules further activate platelets and cause conformation changes of glycoprotein IIbIIIa, which then bind to fibrinogen and form platelet aggregate. Hereditary platelet disorders are caused by defects in this sequential platelet activation process. We thus can categorize the platelet defects into: Platelet synthetic defect Surface receptor deficiency Signal transduction deficiency and lastly Storage pool deficiency 5

6 In order to accurately diagnose platelet disorders we need a systemic approach, which includes collection of patient s personal and family bleeding histories, CBC and peripheral blood smear review, platelet functional tests and finally more esoteric testing including platelet electron microscopy, flow cytometry, and genotypic studies. Of the routine tests, platelet functional tests, especially the platelet aggregation tests, remain the gold standard laboratory testing to diagnose various platelet disorders. 6

7 The first platelet light transmission aggregometry, as shown here, was invented by Dr. Gustav Born in This invention ignited an explosion of knowledge of platelet biology in the past 50 years and became a reference method for diagnosing platelet disorders.

8 Here is an example of the platelet aggregation tracing: It includes an initial baseline indicated as (A), a spike of increased turbidity due to platelet shape change (B), the first wave of platelet aggregation (C), and second wave of aggregation (E). Sometimes the first wave of aggregation is reversible (D), while the second wave of aggregation is usually irreversible.

9 Using a battery of agonists and their corresponding platelet aggregation patterns, various types of platelet defect can be identified. For instance, the lack of ristocetin induced platelet aggregation and normal response to other agonists are characteristic for Bernard Soulier syndrome. The absence of aggregation response to virtually all agonists except for ristocetin is diagnostic for Glanzmann thrombasthenia. Decreased platelet aggregation response to collagen is likely due to glycoprotein Ia/IIa or glycoprotein VI deficiency. However, platelet aggregation in general is insensitive to platelet storage pool deficiency which is likely the most common platelet disorder based on recent studies. Platelet storage pool deficiency is caused by either granule deficiency or defect in granule release. 9

10 Platelet transmission electron microscopy (or PTEM in short) is the gold standard for assessing platelet ultra-structures such as dense and alpha granules. There are 3 main tests: Platelet whole mount TEM is to quantify dense granules. Platelet thin section TEM is the method to visualize ultrastructures such as alpha granules and inclusions. Buffy coat TEM is to examine aberrant inclusions in white cells.

11 Platelet TEM was first used to study human platelets in 1950s. In the past 50 years, Dr. James White at University of Minnesota devoted his career in establishing platelet TEM as an invaluable tool to diagnose various platelet disorders such as Hermansky- Pudlak syndrome and gray platelet syndrome etc. After his retirement, he continued to perform platelet TEM research and clinical studies and generously helped our validation of platelet EM tests at our institution. 11

12 Now let us first look at the whole mount platelet electron microscopy. Whole blood samples collected in ACD tubes are first centrifuged to prepare platelet rich plasma. Platelet rich plasma are then drop on a coated copper grid. After the grid is air dried, it is then directly examined by TEM. Here is a micrograph of the whole mount image of a platelet. Calcium in the dense granules can block the electron beam and causes an ink-dot like shadow. Therefore, whole mount TEM is a quick and reliable method to evaluate platelet dense granules. 12

13 In contrast, this whole mount TEM image shows no dense granules, a characteristic feature of Hemansky-Pudluck syndrome. However, this feature in not pathognomonic, and can be seen in Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, Jacobsen-Paris Trousseau syndrome, and other severe dense granule deficiencies.

14 Not every opaque object on whole mount TEM is a dense granule. A collaboration with Dr. White we first presented our dense granule calling criteria in the 2013 annual meeting of American Society of Hematology. Dense granules should have uniformly dark texture, perfectly round and sharp contour, and greater than 100 nanometer in diameter. The larger, pale and frequently irregular-shaped granules are likely alpha granules. There are also background chains and other unspecific opaque bodies which should not be counted as dense granules. 14

15 We provide these criteria to all 9 participants of the NASCOLA electron microscopy dense granule interpretation challenges. Each laboratory was asked to count the number of dense granules in the provided image as shown on the left. We observed very good agreement among different laboratories. 15

16 After establishing the dense granule calling criteria, we studied normal range of the mean dense granule count per platelet. In this study, we enrolled 111 healthy donors with balanced gender. We counted at least 100 platelets of each donor sample. The lower normal cutoff is 1.2 dense granules/platelet. We also found that dense granule counts were not associated with age or gender. This normal range will be critical to identify mild to moderate dense granule deficiencies. Finally, stability studies showed that whole blood collected in ACD tubes and stored at room temperate gave stable dense granule counts for up to 4 days. Therefore, TEM study can be actually performed on properly transported samples. 16

17 Next, we examine platelet ultrastructure by thin section electron microscopy, which will allow us to examine platelet size, shape, alpha granules, canalicular system, Golgi complex, aberrant inclusions. Here is example of a so called gray platelet which lacks alpha granules as shown on the right.

18 Sometimes we need to look at white cells. For example, among the differential diagnoses of marked dense granule deficiency, we can diagnose Chediak-Higashi syndrome by examining abnormal lysosomal inclusions in neutrophils by buffy coat electron microscopy.

19 Here is an example. Besides dense granule deficiency, abnormal inclusions are present in neutrophils and platelets. This case was a confirmed case of Chediak- Higashi syndrome. 19

20 Now let us switch gears to talk about platelet flow cytometry. Platelet surface receptors are essential for platelet function. Glycoprotein IIb/IIIa is the fibrinogen receptor, its deficiency causes Glanzmann thrombasthenia. Both glycoprotein IalphaIIalpha and glycoprotein VI are collagen receptors, and their deficiency will cause abnormal platelet aggregation response to collagen. Glycoprotein Ib-V-IX binds von Willebrand factor, their abnormality causes Bernard- Soulier syndrome.

21 We developed a quantitative flow cytometry panel to measure these 6 platelet surface glycoproteins. Platelets collected in ACD tube are stained with fluorescent-labeled specific antibodies. Platelets are first gated by light scatter and then the mean fluorescent intensity (MFI) of each antibody is measured. The raw MFI is divided by the median of normal donor MFI and give a percentage of expression level. 21

22 We also established normal ranges of all 6 glycoproteins as shown in this table. Expression levels of these glycoproteins are all above 60 to70 percent. 22

23 Whole blood collected in ACD tube and stored at room temperature give stable results of all glycoprotein levels for up to 4 days. There are not overt changes in MFI of the 6 markers. 23

24 Here is an example of normal donor platelet flow cytometry histogram. Qualitatively, the platelets have normal expression level of glycoproteins. Here are histograms of a case of Glanzmann thrombasthenia. You may notice that the platelets have decreased CD41 and CD61 expression. 24

25 Here are the final results and interpretation of the case. The expression level of each glycoproteins are measured and converted to percentage of median normal expression. Expression of GPIIb is at 1.2% and GPIIIa is at 6.1%. These findings are consistent with Glanzmann thrombasthenia. 25

26 Finally, I would like to emphasize that the diagnosis of hereditary platelet disorder requires systemic approach which includes clinical history collection, CBC and peripheral blood smear review, platelet function testing, and esoteric testing. Today, I introduced to you 2 new tests, platelet electron microscopy and flow cytometry. This battery of phenotyping tests will also assist future evaluation of genotypic testing of hereditary platelet disorders. 26

27 Finally, thank you for your attention. Please do not hesitate to contact us if you have any questions regarding these tests. 27

28 28

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