IPF) in the Differential Diagnosis of Thrombocytopenia

The Value-Driven Laboratory

The Role of the Immature Platelet Fraction (IPF) in the Differential Diagnosis of Thrombocytopenia

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577 Aptakisic Road • Lincolnshire, IL 60069 USA www.sysmex.com The Value-Driven Laboratory

Immature Platelet Fraction (IPF): An insight into assessment and treatment of platelet disorders

We have previously defined the value-driven laboratory as one that meets market demands by providing more and new types of diagnostic information to support the delivery of high quality and cost-effective patient care. This paper sets forth the value of an automated CBC parameter, the Immature Platelet Fraction (IPF), to help physicians understand the pathophysiological mechanisms leading to low platelet count. The IPF can help physicians determine in a timelier fashion if a thrombocytopenia is due to decreased platelet production by the bone marrow or increased peripheral consumption/destruction of platelets.

Introduction An Overview of Platelet results can show greater imprecision or Parameters be unmeasurable (suppressed by the The Immature Platelet Fraction (IPF) is analyzer) in samples with low platelet Platelet Count (PLT) a measure of thrombopoietic activity counts.5 first described in 1992 by Ault et al.1, A normal platelet count (PLT) who coined the term “reticulated (150,000 – 450,000/μL) is vital for the Immature Platelet Fraction (IPF) platelets” to describe large platelets maintenance of hemostasis. Platelet An automated parameter, the Immature with elevated ribonucleic acid (RNA) counts of <150,000/μL are defined as Platelet Fraction percent (IPF%) indicates content. Reticulated platelets can be thrombocytopenic and spontaneous the ratio of immature platelets to the seen as an analog to the reticulocytes bleeding not associated with injury has total number of platelets in a patient’s in red cell populations. Ault’s study and been associated with platelet counts peripheral blood. These immature others have shown IPF to be a stable and of <20,000/μL. Current hematology platelets, newly released from the bone 2 reproducible parameter and superior to instruments enable platelet counts of marrow, contain increased amounts of the measurement of reticulated platelets <50,000/μL to be accurately measured. cytoplasmic RNA which allows them to be 3,4 using flow cytometry for providing However, such a measurement only differentiated from mature platelets using information to the physician on the reflects the circulating platelet count at a a fluorescence staining method on an rate of thrombopoiesis. The IPF rises as specific point in time. automated analyzer. The Sysmex® production of platelets increases in the XN- and XE-Series Automated Mean Platelet Volume (MPV) bone marrow. Using a peripheral blood Hematology Analyzers are capable of sample, its measurement provides an A normal MPV is approximately 9 – 12 fL. reporting the IPF% result as a direct assessment of bone marrow platelet Historically, some clinicians have used cellular measurement of thrombopoiesis, production in a similar way to how a MPV as a surrogate marker for platelet which can be used with other available reticulocyte count provides a measure production and to assist in the evaluation clinical information to help physicians of red cell production. The IPF result of thrombocytopenia. Platelets newly determine the pathophysiological is useful in monitoring patients with released from the bone marrow are mechanism of thrombocytopenia. thrombocytopenia and helps physicians generally larger and as platelets age, they differentiate thrombocytopenia become smaller. However, interfering Clinicians have used the reticulated caused by platelet destruction versus substances such as schistocytes, platelet information, or IPF%, as a compromised platelet production. As a microcytes or other particles that are measure of thrombopoietic activity of the result, physicians can reach decisions on similar in size to platelets and can be bone marrow, which can be important in appropriate care pathways in a timelier counted along with them can make assessing the likelihood of bleeding. manner. MPV unreliable. Additionally, MPV

2 Absolute Immature Platelet Fraction (IPF#) The absolute-IPF (IPF#), calculated as IPF% x PLT count, reflects the number of immature platelets in circulation and is a measurement of real-time thrombopoiesis. IPF# has been Causes of used in clinical trials to assess the treatment effect of thrombopoietin receptor agonists, Thrombocytopenia such as eltrombopag, and in platelet disorders of production and destruction, more specifically in Immune Thrombocytopenia (ITP).6,7 • Myeloablative therapy for hematologic malignancies In studies assessing the effect of platelet transfusion on IPF% and IPF#, platelet • ITP/TTP transfusions were found to decrease the IPF% due to the increased circulating platelet • Hepatitis C count but did not affect the IPF# result, thus “validating the assay as a reflection of ongoing platelet production by the marrow…”8 • Bone marrow transplant • Acute myelogenous leukemia IPF in the Clinical Setting • PNH/aplastic anemia Normal Ranges • DIC There is remarkably good correlation of reference ranges for IPF% between laboratories • Splenomegaly (Figure 1). A 2006 study9 reported a reference range of 0% to 6% (+/- 2SD) with the mean value of 3.1%. The reproducibility of the assay was good, with coefficients of variation • Autoimmune Disease (CV) ranging from 4.9% to 22.1%. As expected, the lower the IPF%, the higher the CV. • Bacteremia IPF Normal Ranges by Source • HIT

Peer-reviewed Article IPF Normal Range Notation • Sepsis M. Zucker11 1.1% to 7.1% N = 43 healthy adults • Pregnancy C. Briggs3 1.1% to 6.1% N = 50 healthy adults • Drug Induced Thrombocytopenia (DIT). I. Pons4 1.0% to 7.5% N = 336 healthy adults Medications such as quinine, quinidine, sulfa-containing T. Bat8 0.9% to 7.2% N = 107 healthy adults medications, interferon T. Kickler9 0.6% to 6.0 % N = 80 healthy adults

Figure 1: Correlation of reference ranges for IPF% in different studies.

IPF in the Differentiation of Increased Platelet Destruction versus Decreased Platelet Production To determine if they could differentiate peripheral thrombocytopenia due to bone marrow failure versus increased peripheral destruction, Briggs et al.10 followed AITP (Autoimmune Thrombocytopenic Purpura) and TTP (Thrombotic Thrombocytopenic Purpura) patients with platelet counts of <50,000/μL. IPF values were elevated in both patient groups due to excessive platelet consumption. The IPF then decreased as the platelet count recovered.

A study by Kickler9 at Johns Hopkins looked at IPF values as differentiators in cases of thrombocytopenia (Figure 2). High IPF% values were found in patients with increased production, particularly if associated with platelet destruction, while normal values were seen in decreased platelet production.

3 95% Confidence Subject No. of Cases Mean SD Range Level P* Healthy 80 3.1 1.6 1.0 - 7.0 2.8 - 3.5 — Destruction ATP 37 15.0 7.1 3.6 - 35.4 12.7 - 17.2 <.0001 DIC 25 9.5 5.2 2.3 - 18.6 7.5 - 11.5 <.0001 All destruction 62 12.8 2.3 - 35.4 11.0 - 14.5 <.0001 Recovery Chemotherapy 79 4.1 2.0 1.2 - 10.9 3.6 - 4.5 .001 BMT 8 8.0 4.2 4.3 - 15.3 5.1 - 10.8 <.0001 All recovery 87 4.4 2.5 1.2 - 15.3 3.9 - 4.9 <.0001 Suppression AA/PNH 3 6.1 2.5 3.9 - 8.8 3.2 - 8.9 .019 Cancer 16 3.8 2.0 0.8 - 7.1 2.8 - 4.7 NS All auppression 19 4.1 2.2 0.8 - 8.8 3.1 - 5.1 .05 Figure 2: IPF% in Different Populations of Thrombocytopenic Patients.9

IPF in Hepatitis C/Liver Disease Although it is customary to follow engraftment following allogenic stem cell neutrophil recovery after hematologic transplantation, finding that IPF preceded Additional examples of clinical utility of transplantation procedures, IPF may be engraftment. IPF% is evaluation of the mechanism of a more reliable parameter since it is not thrombocytopenia in Chronic Hepatitis affected by graft-versus-host disease. Other uses for IPF have been described. C patients as recently described by 3 Zucker and colleagues12 followed IPF% Briggs et al. have suggested that the Zucker et al.11 The authors concluded in 50 patients undergoing peripheral IPF parameter may be able to alleviate that this mechanism is similar to hematopoietic cell transplants and the need for bone marrow examinations thrombocytopenia associated with liver found that IPF recovered on average 3.1 as well as to determine if platelet disease. days prior to platelet count recovery transfusions are necessary. IPF% was monitored in all patients (Figure 3). Takami et al.14 also reported and found to be normal or increased, the use of IPF as a predictor of platelet supporting the idea that peripheral destruction and sequestration are the 14 major causes of the thrombocytopenia in 13.2 12 12.5 Hepatitis C and liver disease in general. 10 IPF in Bone Marrow Recovery 10.0 8 9.4 Several groups1,4,8,12,13 have studied the 6

clinical utility of the IPF in different Mean Days patient populations: following 4 hematopoietic progenitor cell (HPC) 2 transplantation; myeloablative 0 chemotherapy for hematologic IPF IRF PLT ANC malignancy; Disseminated Intravascular Figure 3: Mean days to recovery for IPF, Immature Coagulation (DIC); non-myeloablative Reticulocyte Fraction (IRF), platelet count (PLT) and therapy in cancer patients and aplastic Absolute Neutrophil Count (ANC) following stem cell anemia; and paroxysmal noctural transplantation12. hemoglobinuria (PNH).

4 IPF in Acute Myeloid Leukemia groups of patients may be accounted What’s Out? for by the production of younger, and Myelodysplasia (AML/ 1. Inaccurate PLT measurements at the larger platelets in ITP patients versus MDS) Versus Immune very low levels, as can be seen in AML/MDS patients who had lower IPF Thrombocytopenia (ITP) severe thrombocytopenia. values and smaller platelets (Figure 4). It has been difficult to measure platelet 2. Morphologic identification of large/ function in the presence of severe IPF in the Assessment of young platelets. thrombocytopenia, a finding in both Thrombopoietin Agonists AML/MDS and ITP patients. Psaila et Another key role of IPF is in 3. Waiting for Absolute Neutrophil al.15 have noted that “Platelet count determining the efficacy of various Count recovery to determine bone alone does not always predict bleeding treatment protocols for ITP, such as marrow recovery. in severely thrombocytopenic patients”, thrombopoietin receptor agonists i.e., it is necessary but not sufficient. What’s In? (TPO-A). A study by Barsam et al.7 The study found that the difference in showed that patients receiving TPO-A 1. A comprehensive platelet evaluation bleeding tendencies between these two have a higher IPF. beginning with a fluorescent platelet count and simultaneous Platelet parameters in ITP and AML/MDS measurement of the IPF to evaluate A B the maturity of circulating platelets as

-1 an indicator of rate of PLT production L 50,000 Platelet count 250 Platelet size μ (thrombopoiesis), i.e., using IPF 40,000 200 to assist the physician in defining 30,000 150 FLS the best clinical pathway based 20,000 100 *** on destruction/consumption vs.

Platelet count 10,000 50 production issues. 0 0 ITP AML/MDS ITP AML/MDS 2. Use of IPF to predict bone marrow C D recovery and rate of thrombopoiesis. 5 Immature 30 Immature 3. Evaluating TPO medication’s effects platelet platelet 4 on thrombopoiesis.

-1 count (IPC) fraction L μ 3 % 20 (IPC) * 4. Evaluating the use of the IPF%

IPC 2 *** parameter as a reflex test in the 10 1 presence of thrombocytopenia 0 0 for reporting to hematologists/ ITP AML/MDS ITP AML/MDS oncologists. Figure 4: Platelet parameters in ITP and AML/MDS. (A) Platelet count, (B) Platelet size, (C) Immature platelet count (IPC) and (D) Immature platelet fraction (IPF).15

IPF - A Unique Parameter • Automated, easy to perform, standardized results • Inexpensive – utilizes routine CBC reagents and controls • Results available 24/7 for rapid response to clinicians • Could reduce time and cost of diagnosis of thrombocytopenia by the physician • May provide useful information for tailoring treatment regimens by allowing clinicians to link treatment options to mechanisms of low cell counts • Provides the clinician with valuable information in the assessment of the mechanism of newly discovered thrombocytopenias and secondary thrombocytopenias (HIV, Hepatitis C, drugs etc.)

5 Conclusion Immature Platelet Fraction is a parameter that, although new to the clinical setting, has been studied for over a decade. IPF can function as a routine value reported with every platelet count in certain patient populations or as a reflex test based on your laboratory’s platelet count cut-off. It provides guidance to the physician on the etiology of various thrombocytopenic states, and may help anticipate bone marrow and platelet recovery. Regardless of how it is applied, you will be supporting the delivery of high quality, cost effective patient care for a positive impact on patient outcomes.

Clarify the current strategy for thrombocytopenia management 1. How often are you unable to report a low platelet count because of linearity or result vote-out issues with your hematology analyzer?

2. What percentage of your pre-admission CBCs are identified as thrombocytopenic patients? Identify the clinical lab tests that currently trigger follow-up testing by physicians 3. What existing laboratory tests or clinical conditions trigger additional platelet studies in your institution?

4. What are the clinical conditions associated with thrombocytopenia at your organization?

5. What hospital service lines would need to know that a patient is thrombocytopenic?

6. What classes of pharmacologic agents have thrombocytopenia as an adverse event? Identify potential improvements that the clinical laboratory can support 7. Given that the IPF recovers prior to the recovery of the peripheral blood platelet count, are we offering enough information with just a platelet count?

8. How can the laboratory educate physicians and nurses, pharmacists, coding and other ancillary departments about the value of the IPF parameter in the differential diagnosis of thrombocytopenia?

6 Bibliography 1 Ault KA, Rinder HM, Mitchell J, Carmody 7 Barsam SJ, Psaila B, Forestier M, Page LK, 13 Stohlawetz P, Stiegler G, Hocker P et MB, Vary CP, Hillman RS. The significance Sloane PA, Geyer JT, et al. Platelet production al. The rise of reticulated platelets after of platelets with increased RNA content and platelet destruction: assessing intensive chemotherapy for AML reduces (reticulated platelets). A measure of the rate mechanisms of treatment effect in immune the need for platelet transfusions. Ann of of thrombopoiesis. Am J Clin Pathol 1992; thrombocytopenia. Blood 2011 May 26; Hematol 1999;78(6):271-3. 98:637-46. 117(21):5723-32. 14 Takami A, Shibayama M, Orito M, Omote 2 Jung H, Jeon HK, Kim HJ, Kim SH. Immature 8 Bat T, Leitman SF, Calvo KR, Chauvet D, M, Okumura H, Yamashita T, Shimadoi S, platelet fraction: establishment of a Dunbar CE. Measurement of the absolute Yoshida T, Nakao S, Asakura H. Immature reference interval and diagnostic measure for immature platelet number reflects marrow platelet fraction for prediction of platelet thrombocytopenia. Korean J Lab Med 2010 production and is not impacted by platelet engraftment after allogenic stem cell Oct;30(5):451-9. transfusion. Transfusion 2013 June;53(6):1201- transplantation. Bone Marrow Transpl 04. 2007;39:501–7. 3 Briggs C, Hart D, Kunka S, Oguni S, Machin SJ. Immature platelet fraction measurement: 9 Kickler TS, Oguni S, Borowitz MJ. A clinical 15 Psaila B, Bussel JB, Frelinger AL, Babula B, a future guide to platelet transfusion evaluation of high fluorescent platelet Linden MD, Li Y, et al. Differences in platelet requirement after haematopoietic stem fraction percentage in thrombocytopenia. function in patients with acute myeloid cell transplantation. Transfus Med 2006 Am J Clin Pathol 2006 Feb;25(2):282-7. leukemia and myelodysplasia compared to Apr;16(2):101-9. equally thrombocytopenic patients with 10 Briggs C, Kunka S, Hart D, Oguni S, immune thrombocytopenia. J Thromb 4 Pons I, Monteagudo M, Lucchetti G, Munoz Machin SJ. Assessment of an immature Haemost 2011 Nov;9(11):2302-10. L, Perea G, et al. Correlation between platelet fraction (IPF) in peripheral Immature Platelet Fraction and reticulated thrombocytopenia. Br J Haematol 2004 platelets. Usefulness in the etiology diagnosis Jul;126(1):93-9. of thrombocytopenia. Eur J Haematol 2010; 85(2): 158-63. 11 Zucker ML, Hagedorn CH, Murphy CA, Stanley S, Reid KJ, Skikne BS. Mechanism of 5 Yasonuri A, Hideo W, Hiroaki T. A simple thrombocytopenia in chronic hepatitis C as technique to determine thrombopoiesis evaluated by the immature platelet fraction. level using Immature Platelet Fraction (IPF). Int J Lab Hematol 2012 Oct; 34(5):525-32. Thrombosis Research 2006;118(4) 463-69. 12 Zucker ML, Murphy CA, Rachel JM, 6 Psaila B, Bussel JB, Linden MD, Babula Martinez GA, Abhyankar S, McGuirk JP, et B, Li Y, Barnard MR, Tate C, Mathur K, al. Immature platelet fraction as a predictor Frelinger AL, Michelson AD. In vivo effects of of platelet recovery following hematopoietic eltrombopag on platelet function in immune progenitor cell transplantation. Lab Hematol thrombocytopenia: no evidence of platelet 2006;12(3):125-30. activation. Blood 2012; 119:4066-72.

7 Disclaimer Notice of Intended Use The uses or clinical applications described in “The Immature Platelet Fraction (IPF) these publications have not been approved parameter on the XN- and XE-Series Automated or cleared by the FDA. It is the clinician’s Hematology Analyzers is intended for in vitro responsibility to validate any off-label diagnostic use to enumerate the immature applications for use in routine clinical practice. platelet fraction”.