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Welcome to:

Clinical Application of Ret-He in Anemia

Heri Fadjari
Hasan Sadikin General Hospital
Bandung - Indonesia

Saturday, 24 March 2018 Fairmont Hotel, Jakarta

Anemia
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• Anemia has been defined as a reduction in one or more of the major

red blood cell (RBC) measurements obtained as a part of the complete
blood count: hemoglobin concentration, hematocrit, or RBC count.
• In practice, however, a low hemoglobin concentration or a low
hematocrit is most widely employed for this purpose.
• A Hb concentration <13.5 g/dL or a HCT <41.0 percent represents
anemia in men; a value <12.0 g/dL or <36.0 percent represents anemia
in women.
• Differences may also exist between races, in older adults, in
pregnancy, and in athletes.

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Anemia
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Kinetic approach
• Decreased red cell production
- Decrease in effective erythropoiesis: B12 and Iron deficiency, bone
marrow infiltration, bone marrow suppression, Epo deficiency,
hypothyroidism, hypogonadism, anemia of inflammation
- Ineffective erythropoiesis: Thalassemia, MDS, Sideroblastic anemia
• Increased red cell destruction
- Inherit: hereditary spherocytosis, thalassemia
- Acquired: AIHA, TTP, Malaria, PNH
- Hypersplenism
• Blood loss
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Anemia
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Morphological approach
• Microcytic anemias are associated with an MCV <80 fL.
The most commonly seen causes are iron deficiency, thalassemia, and
the anemia of inflammation (ACD)
• Macrocytic anemias are characterized by an MCV >100 fL
The most common causes include alcoholism, liver disease, folate and
vitamin B12 deficiency, and myelodysplasia.
• Normocytic anemia. The MCV is between 80 and 100 fL

Examination of the blood smear is important in order to determine

if there is a small abnormal population of red cells.
However ……………..

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However…
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The traditional microscopic method based on the count of 100 cells has 3
types of error:

1. Statistical error, because it is invariably related to the total number of

cells analyzed.
2. Distributional error owing to unequal distribution of cells in the
smear, and
3. Error in identifying cells related to the subjective interpretation of the
examiner.

This method, therefore, suffers from imprecision, poor accuracy, and

reduced clinical sensitivity

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 Automated
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Counters

• During the last 3 decades, automated blood cell counters have undergone
a formidable technological evolution owing to the introduction of new
physical principles for cellular analysis and the progressive evolution of
software.

• These analyses are highly automated, and the correct interpretation of

results requires extensive knowledge of the analytic performance of the
instruments and the clinical significance of the results they provide.

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Reticulocyte Haemoglobin
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• Since red blood cells have a 120-day lifetime, detecting iron deficiencies and
changes in the iron status of erythropoiesis is only possible relatively late
using classical haematological parameters such as HGB, MCV, or MCH.
• Reticulocytes, are swept into the blood stream from the bone marrow and
usually mature over the course of 2-4 days. Measuring the number of
reticulocytes is therefore a quick measure of ‘quantity’ in erythropoiesis in
the marrow.
• Measuring the haemoglobin content of the reticulocytes means you can look
at the current iron supply to erythropoiesis and judge the ‘quality’ of the
cells.
• This lets you detect changes in iron status far earlier than through the
haemoglobin content of mature red blood cells.

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Why
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• In general, functional iron deficiency refers to the failure to release iron

rapidly enough to keep pace with the demands of the bone marrow for
erythropoiesis. It may occur even when the body has adequate iron stores.
• Conventional biochemical markers for assessing iron status, such as serum
iron, transferrin or ferritin, are disturbed during inflammation with an acute
phase response, or in the presence of chronic diseases.
• Normal or elevated Ferritin levels alone do not let you draw any conclusions
as to the bioavailability of the iron. In the presence of chronic diseases such
as rheumatoid arthritis, liver damage, tumours or chronic kidney disease,
ferritin can also be elevated in the case of functional iron deficiency.

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BENEFITS
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• Considered the most sensitive indicator of current iron availability

for erythropoiesis regardless of disease status.

• RET-He let clinicians draw conclusions on both the quality and

quantity of the young RBC fraction.

• Is an early marker for disease - earlier than clinical chemistry

markers!

• Fast, inexpensive and practical to perform!

Available at https://www.sysmex-europe.com/academy/knowledge-centre/
sysmex-parameters/reticulocyte-haemoglobin-equivalent-ret-he.html
Using Ret-He
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• RET-He alone gives information on the current bioavailability of iron – a low

value means iron is lacking or iron is not bioavailable for erythropoiesis. It is
often used together with ferritin.
• A high or normal ferritin value together with a low RET-He value can suggest
functional iron deficiency.
• Low ferritin values together with low RET-He suggest a classic iron deficiency.
• RET-He is used for monitoring erythropoietin (EPO) and/or IV iron therapy.
• The reference range for RET-He is approximately 28-35 pg, below 28 pg is
considered iron deficient.

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Reticulocytes Channel
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• According to their fluorescence intensity,

reticulocytes are fractionated into three categories,
representing different stages of maturity:
- LFR (low fluorescence reticulocytes)
- MFR (medium fluorescence reticulocytes)
- HFR (high fluorescence reticulocytes)
• The IRF (immature reticulocyte fraction) reflects the
proportion of immature reticulocytes and is
calculated from the sum of MFR plus HFR.
• RET-He is a diagnostic advanced clinical parameter
that is derived from the RET channel.

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Reticulocytes Channel
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• According to their fluorescence intensity,

reticulocytes are fractionated into three categories,
RBC LFR MFR HFR representing different stages of maturity:
- LFR (low fluorescence reticulocytes)
- MFR (medium fluorescence reticulocytes)
- HFR (high fluorescence reticulocytes)
• The IRF (immature reticulocyte fraction) reflects the
Plt-O
proportion of immature reticulocytes and is
calculated from the sum of MFR plus HFR.
• RET-He is a diagnostic advanced clinical parameter
that is derived from the RET channel.

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Erythrocytes Histogram
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RL RU RL RU

The histogram curve should start and end at the base line within the
discriminators

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Erythrocytes Histogram
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RL RU The curve does not start from base line

Possible causes:
• Giant platelets
• Microerythrocytes
• Fragmented RBC’s
• Platelet clumps
25-75 fl 200-250 fl

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Erythrocytes Histogram
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The curve does not end at base line

Possible causes:
• Cold agglutinins
• RBC agglutination
• Rouleaux formation

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Hypo-HE andMaster
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• Each cell is plotted in the RET scattergram based
on its fluorescence intensity (x-axis) and its high-
angle forward-scattered light signal (y-axis), which
reflects characteristics of both cell size and
cellular content.
• HYPO-He is the percentage of RBC with cellular
haemoglobin content lower than 17 pg, whereas
HYPER-He is the percentage of RBC with cellular
haemoglobin content higher than 49 pg.
• The upper panel shows a sample from a healthy
individual with HYPO-He less than 1% and the
lower panel shows a sample with HYPO-He more
than 60%

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Fragmented
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• A specific area below the RBC area in the RET
scattergram is used for identification of
fragmented red blood cells.
• Fragmented red blood cells are generally the
consequence of mechanical damage, usually
in the context of turbulent blood flow or
contact with a pathologically altered
endothelium, the latter occurring most
commonly in the microvasculature.
• These abnormal shear forces producing cell
remnants that appear as ‘helmets’

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Clinical
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Master
Immature
reticulocytes
Acute leukemia Haemolysis
High

Polycyth.
Dyserythro- verae
poiesis
Normal Normal

Low Aplasia

Low Normal High Reticulocytes

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