About reticulated platelets

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

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

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

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

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

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

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

8. 8. Antibodies in ITP are (1M)
A. IgG
B. IgM
C. IgE
D. IgD
8

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

10. 10. Markers which can be used in early diagnosis of sepsis (1M)
A. Procalcitonin
B. CRP
C. IPF
D. 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
A. 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
I. the lack of methodology standardization, in particular the different
commercial sources and concentration of the fluorochrome,
II. the nonspecific uptake of RNA dyes by the granules present in the
cytoplasm of platelets,
III. the time and temperature of incubation,
IV. 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. a. 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 130.0109/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.
 There 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* 109 /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
1. Prolonged bleeding time is seen in (1M)
A. Hemophilia A
B. Hemophilia B
C. Von willebrand disease
D. Both b and c
79

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

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

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

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

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

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

86. 8. Antibodies in ITP are (1M)
A. IgG
B. IgM
C. IgE
D. IgD
86

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

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

89. “
”
THANK YOU
89