Clinical Benefits of Ret-HE and IPF

Parameters with Scattergram

Interpretation
Andrew, MD., ClinPath.
Tugu Koja District Hospital
Outline
• Advance Hematology Parameters: Ret-HE & IPF
• Application of Ret-HE
• Application of IPF
• Summary
• Case Study
Reticulocyte hemoglobin
equivalent (Ret-HE)
Reticulocyte
• The youngest form of RBC
(precursors of mature RBC)
released from bone marrow
into circulating blood.
• Still contain ribosomal
RNA & hemoglobin (HGB).
• Maturation period: 2-4
days.
• Usually used to describe the
erythropoiesis process in
the marrow.
 Reticulocyte
• Manual → BCB
Quantity • Automatic → Ret%

• Ret-He
Quality
Reticulocytes are separated from
more mature RBCs by their
differences in RNA content
Reticulocyte hemoglobin equivalent (Ret-HE)
~ Reticulocyte Hemoglobin Content

Parameter to measure hemoglobin content of reticulocytes

a direct assessment of the incorporation of iron into erythrocyte hemoglobin

Direct estimate of recent functional availability of iron into the erythrocyte

Real-time information on functional iron available (iron supply) for new RBC production
(erythropoiesis).
 Reference Range
• Insert kit: 28 – 35 pg

Age Group 6mo – 5y 6-11 years 12 -18 years

Teixeira et al. 27.5 – 33.4 28.4 – 33.4 28.8 – 35.2
Perchard et al. 27.2 – 34.5 27.2 – 34.5 28.1 – 36.5
Ret-HE: Clinical Application

Assessment of iron deficiency and iron deficiency anemia

Early marker of responsiveness to Iron Supplementation

Determine Iron Availability in CKD Patient with EPO Therapy

Assessment of iron deficiency and iron deficiency
anemia
 Iron status
Classical
(+): Less intervention, cost effective, minimal
Iron storage Serum/plasma Others
haematology
interferences (than biochemistry parameters)
(-): dependent on RBC’s lifetime (120 days) →
late detection

Serum
Iron, TIBC,
Bone marrow
(+): Less intervention. HGB, MCV, transferrin
Ferritin transferrin
puncture
(-): acute phase protein (affected by MCH receptor &
saturation
inflammation) Protoporphyrin
Assessment of iron deficiency and iron deficiency
anemia
Study ID IDA Sen Spe Year

Auerbach M, et al 30.7 pg - 68.2 69.7 2021

Karakas NM, et al 27 pg - 64 96 2022

Neef V, et al 33.5 31.6 ID 90.7 35.8 2021

IDA 90.6 50.4
Perchard M, et al - 29 86 92 2023

Pramantik DN, et al - 27.65 91.7 78.3 2015

Adiwijaja M, et al 27.55 26.85 ID 87.5 75 2018

IDA 100 83
Perchard M, et al: Ret-He is not affected by inflammation, allowing it to be used in the assessment of ID
and severe-ID in patients with chronic disease.
Neef V, et al:
• Ret-He can be used as a single screening parameter for ID and IDA in children without considering other iron parameters
• Economically, the use of Ret-He is highly relevant, as it can save one blood tube per patient and additional costs
Early marker of responsiveness to Iron Supplementation

Iron therapy response: increase appetite, erythroid hyperplasia in BM, increase

reticulocyte, hemoglobin and iron store

Challenges: mature RBC survival in circulation, RBC population remain

constant,

Its take almost 2-3 months elapsing time before changes on MCV clinically
detected

Cellular characteristic of reticulocyte could use as a sensitive response of bone

marrow to exclusive iron therapy
Suega K, et al:
• Ret-He on first week
was found important
and statistically
significant as an early
predictor for
treatment respond (p
= 0.031; 95%CI 0.349-
0.950)
• Clinician also could
use Ret-He at
baseline and during
follow-up of iron
deficiency
erythropoietic
activities to define
iron supplement
response
Pei LX, et al.: the change in RET-He from baseline to 1 week demonstrated to be a significantly stronger predictor of
hemoglobin response
Pei LX, et al.: Single time-point RET-He measures have poor predictive ability; however, change in RET-He after 1 week
was found to be a strong predictor of hemoglobin response among Cambodian women receiving 60 mg elemental iron.
Furthermore, the change in RET-He after 1 week can be measured easily on an automated hematology analyzer and
quickly after only 1 week of iron therapy (as compared with hemoglobin, which requires ~90–120 days for the
incorporation of iron into the red blood cell)
Determine Iron Availability in CKD Patient with EPO Therapy

Renal anemia is a major complication of end-stage renal failure

Recombinant human erythropoietin (rHuEPO) therapy has allowed therapeutic

manipulation of the anemia in dialyzed and non-dialyzed chronic renal failure patients

During the intense use of rHuEPO, the amount of iron immediately available for
erythropoiesis often proves insufficient → ‘functional iron deficiency’

It is essential to determine the status of iron deficiency in CKD patients in order to select
appropriate doses of rHuEPO and iron
Immature Platelet Fraction (IPF)
Thrombocytes/Platelets
Disc-shaped non nucleated cells

Production → From megakaryocyte in bone marrow

4 days on average process

Controlled by hormone thrombopoietin (TPO)

80% are circulating, 20% held in reserve by spleen

Lifespan : 8-10 days

Immature Platelets= Reticulated platelet

The youngest form of PLT in circulating blood

Contains more granules and mRNA residue

Larger and more reactive on thrombus development compared to mature

platelets

Increased number = activation of megakaryocytes (in bone marrow) as response

to thrombocytopenia due to increased platelet destruction in the peripheral blood
▪ Parameter: IPF%
▪ Provided from the automated
PLT counting with fluorescence
flowcytometry (PLT-F).
▪ Novita, 2012: IPF% is stable for
2 hours at room temperature
in EDTA sample.
▪ Noel MM, et al. 2023:
immature platelet fractions
(IPFs) in the EDTA-
anticoagulated samples for up
to 24 h, and in the citrate-
anticoagulated samples for up
to 6 h, at room temperature
▪ Reference range: 1.1 - 6.1%
(from 50 healthy subjects)
IPF: Clinical Application

Distinguish cause of thrombocytopenia

• Bone marrow failure (intoxication, persistent

infection) vs increased peripheral platelet destruction.
• IPF%: ↓ in hypoplastic thrombocytopenia, ↑ in
autoimmune thrombocytopenia.

Monitoring platelet recovery

• Follow up in dengue-related thrombocytopenia

• Follow up post-chemotherapy
IPF%>7.15% on day 2 of illness and
>7.25% on day 3 of illness are
predictive of a platelet count of >60 ×
109/L on day 7 and day 8 of dengue
illness
Looi KW, et al. 2021: All patients had a decreasing trend in platelet count in the first week of illness concomitant with an
increasing trend in the percentage of immature platelets to total platelets (IPF%) for more than 3 days prior to platelet
recovery.
• This study demonstrates that a cut-off IPF level of
≥10.0% will predict platelet recovery to a
haemostatic level within 72 hours and a safer
situation in the next 72 hours without repeated
determination of platelet counts
• An IPF of ≥10.0% after defervescence predicted
platelet recovery to ≥60 × 109/L within 72 hours in
all patients with dengue infection
 Follow up post-chemotherapy

Platelet Recovery after IPF Cut-off

IPF Cut-off Patient undergoing Patient undergoing
chemotherapy HSCT

IPF ≤ 10% 5 days 6 days

IPF > 10% 2 days 2 days
Summary
• Ret-HE can be used for clinical setting to evaluate patient with iron
deficiency, monitoring effectiveness of iron supplementation and iron
availability in patient with EPO therapy
• Ret-HE relatively cheap and efficient
• IPF can provide insight of bone marrow activity
• IPF can be used to monitoring platelet recovery
Case Study 1
R E T - HE
• Patient Profile
15 year-old, Male, mild fever, cough, fatigue
• Signs and Symptoms:
The patient presented with a chief complaint of mild fever 3 days prior to out-patient clinic,
cough, and easily to get fatigue from daily activity
Laboratory Findings
• Hb 9.8 g/dl
• Ht 30.5 %
• MCV 73.2
• MCH 22.4
• MCHC 29.5
• WBC 16690 (0/0/4/60/26/10)
• PLT 224000
• ESR 48 mm/h
• Pediatrician asked for Iron status work-up, but was cancelled because of inflammation status
• Instead, we checked for Ret-He → 17.5 pg
• Patient get treated for the infection
• Patient get oral iron supplementation
• Follow up after 1 month:
Laboratory Findings
• Hb 10.2 g/dl
• Ht 32.3%
• MCV 75.2
• MCH 22.4
• MCHC 30.5
• WBC 8910 (0/0/1/67/28/4)
• PLT 295000
• ESR 12 mm/h
• Ret-HE 25.4 pg
• The HGB level is elevated due to iron administration for IDA.
• On the Ret Scattergram at the day 30 of treatment, the mature RBC cluster has
shifted towards the area of high forward scattered light intensity (↑)
• Suggesting recovery of the size of RBC.
• The Ret scattergram also shows a shift of the Ret cluster towards the area of high
forward scattered light intensity (↑) which is a bigger shift than the RBC cluster.
• The Ret He level has also increased at day 30 of treatment but is still one the lower
side
• Suggesting persistence of IDA and the need of continued treatment
Case Study 2
IPF
• Patient Profile
43 year-old, Female, high fever
• Signs and Symptoms:
The patient presented with a chief complaint of high fever (up to 40 Celsius degrees) 3 days
before admission. She also was experiencing epigastric pain, nausea, and myalgia.
Laboratory Findings
• Hb 12.9 g/dl
• Ht 37 %
• MCV 80
• MCH 28
• MCHC 35
• WBC 5900 (0/0/1/70/26/3)
• PLT 65000
• ESR 16 mm/h
• IPF 13.2%
• Laboratory Findings day-2 hospitalization (day-4 illness)
• Hb 12.5 g/dl
• Ht 38 %
• WBC 5450
• PLT 32000
• IPF 18.9%
• Laboratory Findings day-3 hospitalization (day-5 illness)
• Hb 12.9 g/dl
• Ht 36 %
• WBC 5500
• PLT 123000 → Delta check!!
• No platelet transfusion, no steroid, no bleeding sign, patient get better
• IPF 22.06%
• Smear confirmation: Platelet >, large platelet (+)
• On day-3 illness when patient come to hospital, she has low plt count corresponding
to dengue infection. Our protocol is to run IPF% if patient has low plt count without
any history (new patient)
• IPF% is >7% indicating peripheral destruction
• On day-2 hospitalization, IPF% >, but plt count <<, we observe for any bleeding sign
• Patient stable, no bleeding sign, no transfusion given
• On day-3, plt count rising → alert delta check, we asked clinician for any change in
treatment and patient condition
• After 2 times rising in IPF% and rising in plt count, patient with stable condition,
clinician discharged patient.