• Moderator : Dr.

Jyoti Shukla
• Speaker : Dr. Shashikant C.U. Patne
 HFPF% or IPF%

• Automated method for counting reticulated

platelets.
• Automated method uses XE-2100 automated
blood cell counter equipped with a laser and
upgraded software.
• Extension of a standard method used to
measure retic and retic fraction on the XE-
2100 automated haematology analyzer.
THROMBOCYTOPENIA IN PBS -
WHY??

• Increased platelet consumption

OR

• Decreased platelet production

 ↑Platelet consumption
↓
↑Thromobopoietin production
↓
↑Platelet production by megakaryocytes
↓
Residual RNA present in early platelets
(Reticulated platelets analogous to Retic)
↓
RNA can be detected by RNA binding dyes
↓
Reticulated platelets-measure of adequate
functionality of bone marrow
 METHODS OF DETECTING
RETICULATED PLATELETS
• Easily detected with supravital dye
staining (e.g. new methylene blue) on
blood films,
• More commonly with fluorescent dyes
(e.g thiazole orange) and flow
cytometry.
• Automated haematology analyzer with
fluorescent dye and flow cytometry.
 CLINICAL APPLICATIONS
• A measure of bone marrow platelet
production and is sensitive and specific
enough to discriminate between
consumptive and aplastic causes of
thrombocytopenia.
• Recovery of platelet production/kinetics
prior to normalization of the platelet count
during various therapeutic regimes, e.g. the
recovery of platelet count during treatment
of thrombocytopenia or bone marrow
transplantation.
• In patients receiving growth factor therapy
 CLINICAL APPLICATIONS…

• Early indicator of increased platelet

destruction as seen in patients with pre-
eclampsia or thrombotic thrombocytopenic
purpura (TTP).
• Analysis of the reticulocyte fraction in
platelet concentrates for transfusion may
improve the viability, function and storage
conditions of these products, and could
lead to the preparation of a reticulocyte
enriched product for refractory patients.
SYSMEX XE-2100
 PRINCIPLE
• The analyzer uses optical properties and
proprietary fluorescent nucleic acid dyes
which stain residual RNA of the platelets.
• Fluorescent flow cytometer separates
reticulated platelets and reticulocytes from
other cells and from each other.
• The analyzer arbitrarily but reproducibly
defines HFP as those with highest 3%
intensity of fluorescence.
 METHOD

• The blood is collected in K2-EDTA vial.

• In the fully automated haematology analyzer
(Sysmex XE-2100), HFPF% is reliably
quantified in the optical (fluorescence)
reticulocyte/ platelet channel using
upgraded software.
• The blood cells are stained with proprietary
fluorescent dye containing ploymethine and
oxazine.
 METHOD…
• The dyes penetrate the cell membrane and
stain the RNA of RBC and platelets.
• The stained cells are passed through a
semiconductor diode laser beam.
• The resulting forward scatter light (cell
volume) and fluorescence intensity (RNA
content) is measured.
• A computer algorithm discriminates the
mature platelet and immature platelet .
 METHOD…
• Mature platelets appear as blue dots and the
immature platelets are displayed as green
dots.
• Immature platelet fraction (IPF) or High
fluorescent platelet fraction is usually
expressed as a propotional value of the total
optical platelet count.
 RESULTS & INTERPRETATIONS
Subjects HFPF% value Interpretation

1. Healthy 1.1-6.1% (3.1) Normal

2. AITP 9.2-33% (22.3) ↑↑↑

3. Acute TTP 11.2-30%(17.2) ↑↑↑
4. BM Recovery 1.2-15.3%(4.4) Normal /↑

5. BM Suppr. 0.8-8.8%(4.1) Normal /↑

 RESULTS & INTERPRETATIONS…

• Inverse correlation between platelet count

and HFPF% is demonstrated in patients with
autoimmune thrombocytopenia.

• Low platelet count → High HFPF% value and

vice versa.
 ADVANTAGES OF HFPF%
• Non invasive.
• Fully automated, rapid method.
• Staining of platelet RNA and its analysis in
whole blood without preparatory steps.
• Reproducibility/precision of results – no
intraobserver or interobserver variation.
• Stability of the sample up to 48 hrs.
• Reference range is available for healthy
subjects.