IMMATURE PLATELET FRACTION

About reticulated platelets

1. PRE TEST Prolonged bleeding time is seen in (1M) Hemophilia A Hemophilia B Von willebrand disease Both b and c 1

2. 2. What percentage of body’s platelet does the spleen normally sequester ? (1M) 5% 15% 25% 35% 2

3. 3. Which of the following blood products have the maximum risk of bacterial contamination ? (1M) RBC Platelets FFP Cryoprecipitate 3

4. 4. Which of the following is NOT indicative of Bernard- Soulier ? (1M) It is a Platelet function disorder Giant PLTs with vacuoles and granules Deficiency of GPIIb /IX Deficiency of GPIb /IX 4

5. 5. Which test evaluated the extrinsic pathway ? (1M) APTT PT TT Bleeding time 5

6. 6. IPF’s diagnostic applications include – (1M) Differential diagnosis of thrombocytopenia Clinical evaluation of myelodysplastic syndromes Recovery after marrow/ stem cells transplantation or chemotherapy All of the above 6

7. 7. Drug induced thrombocytopaenia is common following administration of (1M) Heparin vancomycin Quinine All of the above 7

8. 8. Antibodies in ITP are (1M) IgG IgM IgE IgD 8

9. 9. Thrombin agonist used to differentiate heparin induced thrombocytopaenia from afibrinogenemia ? (1M) Reptilase Ristocetin Russel viper venom hyperosmolar urea 9

10. 10. Markers which can be used in early diagnosis of sepsis (1M) Procalcitonin CRP IPF All of the above 10

11. IMMATURE PLATELET FRACTION PRESENTER – DR RUCHIR UTTAM MODERATER – DR SHILPA GUPTA 11

12. CONTENTS INTRODUCTION MEGAKARYOPOESIS CELLULAR PHYSIOLOGY OF THROMBOPOIESIS METHODS FOR IMMATURE PLATELET DETERMINATION REFERENCE RANGES IN HEALTH DIAGNOSTIC APPLICATIONS LIMITATIONS REFERENCES 12

13. INTRODUCTION Platelets newly released from the bone marrow are larger and more reactive than mature and contain larger amounts of RNA. Due to their similarity with reticulocytes, they are called reticulated (RP) or immature platelets (IP). The number of RP in the bone marrow, on average 2- to 3-fold that in the peripheral blood, is correlated with the number of megakaryocytes. 13

14. While in normal conditions, “mature” platelets persist in the circulation for about 7-10 days, RP appear to remain in the blood stream for 24-36 hours, during which time a progressive degradation of RNA and a decrease in volume take place. Immature platelet fraction is the platelet equivalent of the red blood cell’s reticulocyte count. It is used in conjunction with patient diagnosis and platelet count. It is also helpful in determining need for prophylactic transfusions. 14

15. MEGAKARYOPOIESIS Megakaryopoiesis is the process by which bone marrow progenitor cells develop into mature megakaryocyte which in turn produce platelets required for normal haemostasis. Platelets form by fragmentation of megakaryocyte membrane extensions termed proplatelets , in a process that consumes nearly the entire cytoplasmic complement of membranes, organelles, granules and soluble macromolecules. 15

16. 16

17. 17

18. MEGAKARYOPOIESIS- KINETICS The circulatory life span of a platelet is approximately 10 days in humans with normal platelet counts , but somewhat shorter in patients with moderate to severe thrombocytopaenia . Based on a “ normal” level of 200,000 platelet/ ul , blood volume of 5 litres and half life of 10 days , 1x1011 platelets per day are produced. If 1 megakaryocyte produces approximately 1000 platelets, approximately 1x108 megakaryocytes are generated in the marrow each day. 18

19. The transit time from megakaryocyte to progenitor cell to release of platelets into the circulation ranges from 4 to 7 days.’ For example , following platelet apheresis , the platelet count falls, recovers substantially by day 4, and completely recovers by day 7. 19

20. METHODS FOR IMMATURE PLATELET DETERMINATION 1. FLOW CYTOMETRIC METHOD Thiazole orange (TO) has been used extensively for staining and detecting RP as it is excitable at 488 nm, can be used with most flow cytometers, has a high quantum yield when bound to nucleic acid, and readily permeates the cell membrane. TO is marketed as a solution or in solid form dissolving in methanol at 1 mg/mL concentration. 20

21. The gating strategy for measuring the RP fraction involves the use of a pair of tubes: control and test for each sample. The platelets for each tube are analyzed in a dot plot of side scatter vs TO fluorescence. An area in the upper left corner of the dot plot is obtained with a line starting at the origin and encompassing 1% of platelets in the control tube. 21

22. This fixes the threshold for counting RNA-positive platelets in the subsequent test tube, classified as RP and expressed as a percentage of the total platelet count. A more recently proposed alternative method for staining RP uses the nucleic acid staining dye SYTO 13 ( Thermo Fisher). The main advantages of this dye with respect to TO are its higher quantum yield, its high stability over time that facilitates extended experimental analysis ( eg , sorting), and a more satisfactory correlation with Sysmex analyzers. 22

23. 2. FULLY AUTOMATED METHODS SYSMEX ANALYZERS The Sysmex XE-2100 and 5000 ( Sysmex ) with dedicated software and fluorescent dye ( polymethine ) allow the counting of “RP” together with reticulocytes, calling them “IP fraction” expressed as both a percentage of total platelets (IPF%) and in an absolute count (IPF#). 23

24. 24

25. The most recent XN analyzer has a novel PLT-F channel which uses a new fluorescent dye ( phenoxazine ) for staining RNA. In both the analyzers, the stained cells are passed through a semiconductor diode laser beam and the resulting forward scatter (cell volume) and fluorescence intensity (mainly due to RNA content) are measured A computer algorithm discriminates between the mature and IP on the basis of the intensity of forward light and fluorescence. 25

26. B. ABOTT ANALYSERS The Abbott Cell- Dyn Sapphire (Abbott) measures “RP” ( retPLT ) as a part of reticulocyte analysis, which is based on the fluorescent dyeCD4K530. Scattered light (at a 7° angle) and fluorescence are recorded. An algorithm separates platelets from red blood cells and then defines the gating of retPLT with correction for size-dependent background fluorescence. 26

27. 27

28. C. MINDRAY ANALYSERS The Mindray BC-6800 ( Mindray ) performs IPF% detection together with the reticulocytes by means of proprietary asymmetric cyanine based dye for staining RNA. IPF is derived from forward scatter vs sideward fluorescence scatterplot, results being expressed as a percentage. 28

29. 29

30. CORRELATION BETWEEN MPV AND IPF It is widely believed that reticulated/IP, as well as containing more RNA, are also larger than “mature” and this is in line with the finding that the proportion of RP is higher in the fraction of large platelets than in the fraction of small platelets. In contrast with this opinion, a recent study on healthy adults using the Abbott Sapphire analyzer highlighted a negative association between ret- PLT and MPV, and concluded that the retPLT proportion is higher in small and lower in large platelets. 30

31. With the Sysmex and Mindray analyzers, the results are different and a positive, nonlinear correlation between IPF% and MPV is highlighted. 31

32. 32

33. REFERENCE RANGES IN HEALTH With the use of flow cytometers and fluorescent dyes that bind to RNA, it is possible to count Reticulated platelets with sufficient accuracy. Due to methodological variations and different fluorochromes used, reference intervals specified by different authors vary greatly, ranging from 2% to 17% of the total platelet count. 33

34. When measured as retPLT % or IPF%, the gender difference was statistically significant, but was considered too small for sex-specific intervals to be applied. When measured as IPF% (and IPF#), and subdivided by age, variations were found, values being higher for both males and females <6 months of age. 34

35. In neonates, the mean value was around 4% (value range 2-7) with a progressive increase up to the second week parallel to the increase in total platelets and a return to the base level at the end of the first month. In premature infants, IPF values were found to be higher and negatively correlated with gestational age and birth weight, with a progressive reduction at the close of 32nd gestational week. 35

36. 36

37. This wide range can be explained by the lack of methodology standardization, in particular the different commercial sources and concentration of the fluorochrome , the nonspecific uptake of RNA dyes by the granules present in the cytoplasm of platelets, the time and temperature of incubation, data analysis. 37

38. Nowdays ,Fully automated hematology analyzers are used which uses dyes suitable for RNA binding, enabling quantification of immature platelets. 38

39. DIAGNOSTIC APPLICATIONS The immature platelet fraction is an index of thrombopoiesis and can help to determine the mechanism of thrombocytopaenia . 1. Increased immature platelet fraction in the presence of thrombocytopaenia is indicative of platelet destruction or consumption. 2. Values at or below the range in combination with thrombocytopaenia are indicative of decreased marrow production. 39

40. Clinically it can be used in – 1. Differential diagnosis of thrombocytopenia 2. Clinical evaluation of myelodysplastic syndromes 3. Recovery after marrow/ stem cells transplantation or chemotherapy 40

41. 4. Increased thrombotic risk in thrombocytosis 5. Cardiovascular risk in renal transplantation 6. Predicting sepsis in critical ill patients 41

42. Immature platelet fraction is typically elevated in 1. DISORDERS OF PLATELET DESTRUCTION Idiopathic thrombocytopenic purpura Thrombotic thrombocytopaenic purpura Disseminated intravascular coagulation 42

43. 2. INDICATOR OF MARROW RECOVERY Immature platelet fraction is used as an early indicator of marrow recovery in post chemotherapy and stem cell transplant patients . 43

44. Differential diagnosis of thrombocytopenia Immature platelet fraction (IPF) is helpful in differentiating hyperdestructive /consumptive thrombocytopenia from hypoproductive thrombocytopenia. 44

45. Platelet indices, including IPF, were measured in 105 healthy individuals, 27 patients with hyperdestructive /consumptive thrombocytopenia and 35 patients with hypoproductive thrombocytopenia using a Sysmex XN-3000 hematology analyzer in a study. 45

46. The platelet distribution width, mean platelet volume, IPF, and absolute immature platelet count (AIPC) were significantly higher in the hyperdestructive /consumptive thrombocytopenia group than in the hypoproductive thrombocytopenia group (P<.001). The IPF decreased 3 to 4 days in advance of platelet count elevation in patients with ITP, whereas the delta AIPC increased 3 days in advance. 46

47. Furthermore, the IPF and delta AIPC increased 5.5 days and 8.5 days, respectively, before platelet counts increased up to 130.010 9/L in cancer patients receiving chemotherapy. 47

48. 48

49. 49

50. 50

51. b. Clinical evaluation of myelodysplastic syndromes IPF% value in MDS patients is higher than in healthy individuals, though there is a great range of IPF% values in MDS patients. Th ere is an inverse correlation between IPF% and the platelet count in healthy individuals. 51

52. All patients with a high IPF% and a platelet count >50* 10 9 /L showed chromosomal abnormalities including chromosome 7 abnormalities. It can also be used as marker for the presence of chromosomal abnormalities (monosomy 7/ chromosome 7 abnormalities) with poor prognosis. 52

53. Patients with high IPF% shows marked high platelet size variation. And also, both MPV( Mean platelet volume ) and PDW ( Platelet distribution width ) in these patients tends to be higher than those in MDS patients without high IPF. 53

54. An increase in IPF% could be the first sign of developing 7 monosomy in patients being followed up for low-risk MDS . Thus, IPF assessment is to be included in routine examination of patients with bone marrow failure as well as in the general check-ups for healthy individuals may be useful for early identification of MDS patients with poor prognosis. 54

55. c. Recovery after marrow/ stem cells transplantation or chemotherapy The recovery phase of thrombocytopaenia in most chemotherapy and transplant patient is preceded by rise in immature platelet fraction percentage several days prior to platelet recovery. 55

56. According to the data , mean days to recovery for IPF was 3.1 days less than for platelet count. In 79% of case , it was revealed that IPF recovered at least 1 day prior to platelet count and was followed by platelet count recovery within 1 to 12 days. 56

57. So ,coming to conclusion that automated immature platelet fraction is a useful parameter in the clinical evaluation of patient and has the potential to allow optimal transfusion of platelet concentrates. Similar study was done in 2021 by Julian Grabek et al showing the importance of IPF as predictor of platelet count recovery following allogenic bone marrow transplantation. 57

58. In this study , 32 patients who underwent allogenic bone marrow transplantation were evaluated. The outcomes analysed were platelet count versus time and IPF versus time to establish the predicitive ability of IPF to determine platelet count recovery. 58

59. The IPF was shown to rise 5 days prior to platelet count increase. An IPF rise was also shown to correlate with higher average platelet counts at day 30 of transplant. So , in conclusion , IPF is a reliable predictor of platelet recovery in allogenic bone marrow transplantation. 59

60. d . Increased thrombotic risk in thrombocytosis Platelet kinetics is evaluated to show that increased percentages and absolute numbers of RP are highly associated with thrombosis in patients with thrombocytosis. All thrombocytosis patients with thrombosis had significantly higher RP% compared with patients without thrombosis. 60

61. Reticulated platelet measurements may prove useful for monitoring therapeutic responses in patients with thrombocytosis and perhaps in stratifying those patients at risk for thrombotic complications. 61

62. e,f . Cardiovascular risk in renal transplantation Renal transplant recipients (RTRs) patients are at increased risk of cardiovascular morbidity and mortality. Cardiovascular disease is the major cause of death among RTRs and various factors can contribute to induce its susceptibility. 62

63. A study was done by Francesca Cesari et al in 2010 in to assess reticulated platelet(RP). Reticulated platelet was measured by a Sysmex XE-2100 and were expressed as the percentage. RP were expressed as a percentage of RP of the total optical platelet count (immature platelet fraction; IPF), as the percentage of RP highly fluorescent (H-IPF) and as the absolute number of RP (IPF#). 63

64. There is presence of a high platelet turn-over in RTRs patients and increased platelet reactivity in RTRs patients not on anti-aggregating therapy ( patients on acetyl salicylic acid ). This higher platelet turn-over in groups of RTRs patients and increased platelet reactivity in RTRs patients who are not on acetyl salicylic acid therapy was depicted by IPF Value. 64

65. 65

66. g. Predicting sepsis in critical ill patients IPF serves as a great indicator for indicating impending sepsis in patients admitted to ICU. This easily measured cellular variable reflecting the thrombopoietic rate, which is rarely used in general ICUs, has relatively low diagnostic sensitivity (56.2 %) but high specificity (90.0 %) as an early marker predicting the onset of sepsis. 66

67. During a study , a cut off value of < 4.7 % was measured at ICU admission and was considered normal IPF value. Patients having value more than 4.7% , were considered for impending sepsis . Out of 64 patients , 31 patients had increased IPF Value above 4.7% , 3 days before onset of sepsis. 67

68. Unlike the commonly used markers related to infection or inflammation (PCT, CRP), IPF% is unrelated to manifest infection or clinical sepsis criteria but is significantly related to the development of sepsis . Hence, IPF% has a specifically predictive value indicating the compensatory platelet production that precedes clinically manifest sepsis. 68

69. Measuring IPF% through advanced but easily available technology can therefore give clinicians additional information, thus providing an early cellular marker predicting sepsis within 3 days. 69

70. LIMITATIONS The IPF value may not be reliable when the patient’s platelet count is less than 10 x 10 3 / μL due to the higher imprecision of the IPF at low counts. Drugs used in patient therapies could also impact the accuracy of the IPF result. The IPF is probably most informative upon initial presentation in patients who are not being treated. 70

71. CONCLUSION In conclusion, Immature platelet fraction is the platelet equivalent of the red blood cell’s reticulocyte count. These immature platelets, newly released from the bone marrow, may contain increased amounts of cytoplasmic RNA which allows them to be differentiated from mature platelets. 71

72. It is a rapid, inexpensive automated method with high accuracy with few limitations. It should be considered as standard parameter in evaluating the thrombocytopenic patient and in cases of impending sepsis. The reference ranges in health depends on many factors including the automated analyser used. 72

73. REFERENCES 1. Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. British journal of haematology . 2004 Jul;126(1):93-9. 2. Meintker L, Haimerl M, Ringwald J, Krause SW. Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients. Clinical chemistry and laboratory medicine. 2013 Nov 1;51(11):2125-31. 73

74. 3. Ibarra AL, Marín LM, Durán GP, Puig MT. Evaluation of immature platelet fraction in patients with myelodysplastic syndromes. Association with poor prognosis factors. Clinical Chemistry and Laboratory Medicine (CCLM). 2019 Jun 1;57(6):e128-30. 4. Sugimori N, Kondo Y, Shibayama M, Omote M, Takami A, Sugimori C, Ishiyama K, Yamazaki H, Nakao S. Aberrant increase in the immature platelet fraction in patients with myelodysplastic syndrome: a marker of karyotypic abnormalities associated with poor prognosis. European journal of haematology . 2009 Jan;82(1):54-60. 74

75. 5. Cesari F, Marcucci R, Gori AM, Caporale R, Fanelli A, Casola G, Balzi D, Barchielli A, Valente S, Giglioli C, Gensini GF. Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Thrombosis and haemostasis . 2013;109(05):846-53. 6. Stratz C, Nührenberg T, Amann M, Cederqvist M, Kleiner P, Valina CM, Trenk D, Neumann FJ, Hochholzer W. Impact of reticulated platelets on antiplatelet response to thienopyridines is independent of platelet turnover. Thrombosis and haemostasis . 2016 Nov;116(11):941-8. 75

76. 7. Robinson MS, Harrison C, Mackie IJ, Machin SJ, Harrison P. Reticulated platelets in primary and reactive thrombocytosis. British journal of haematology . 1998 May 1;101(2):388-9. 8. De Blasi RA, Cardelli P, Costante A, Sandri M, Mercieri M, Arcioni R. Immature platelet fraction in predicting sepsis in critically ill patients. Intensive care medicine. 2013 Apr;39(4):636-43. 76

77. 9. Ko YJ, Hur M, Kim H, Choi SG, Moon HW, Yun YM. Reference interval for immature platelet fraction on Sysmex XN hematology analyzer: a comparison study with Sysmex XE-2100. Clinical Chemistry and Laboratory Medicine (CCLM). 2015 Jun 1;53(7):1091-7. 10. Cybulska A, Meintker L, Ringwald J, Krause SW. Measurements of immature platelets with haematology analysers are of limited value to separate immune thrombocytopenia from bone marrow failure. British journal of haematology . 2017 May;177(4):612-9. 77

78. 11. Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. 12. Takami A, Shibayama M, Orito M, Omote M, Okumura H, Yamashita T, Shimadoi S, Yoshida T, Nakao S, Asakura H. Immature platelet fraction for prediction of platelet engraftment after allogeneic stem cell transplantation. Bone marrow transplantation. 2007 Apr;39(8):501-7. 78

79. POST TEST Prolonged bleeding time is seen in (1M) Hemophilia A Hemophilia B Von willebrand disease Both b and c 79

80. 2. What percentage of body’s platelet does the spleen normally sequester ? (1M) 5% 15% 25% 35% 80

81. 3. Which of the following blood products have the maximum risk of bacterial contamination ? (1M) RBC Platelets FFP Cryoprecipitate 81

82. 4. Which of the following is NOT indicative of Bernard- Soulier ? (1M) It is a Platelet function disorder Giant PLTs with vacuoles and granules Deficiency of GPIIb /IX Deficiency of GPIb /IX 82

83. 5. Which test evaluated the extrinsic pathway ? (1M) APTT PT TT Bleeding time 83

84. 6. IPF’s diagnostic applications include – (1M) Differential diagnosis of thrombocytopenia Clinical evaluation of myelodysplastic syndromes Recovery after marrow/ stem cells transplantation or chemotherapy All of the above 84

85. 7. Drug induced thrombocytopaenia is common following administration of (1M) Heparin vancomycin Quinine All of the above 85

86. 8. Antibodies in ITP are (1M) IgG IgM IgE IgD 86

87. 9. Thrombin agonist used to differentiate heparin induced thrombocytopaenia from afibrinogenemia ? (1M) Reptilase Ristocetin Russel viper venom hyperosmolar urea 87

88. 10. Markers which can be used in early diagnosis of sepsis (1M) Procalcitonin CRP IPF All of the above 88

89. THANK YOU 89