Manual immature reticulocyte fraction; a surrogate marker to assess post traumatic blood loss

Abstract

Introduction: Erythropoietic response following chemotherapy and stem cell transplant is assessed by
reticulocytosis and immature reticulocyte fraction (IRF). They are not used to assess erythropoiesis following
acute haemorrhage even though it is known to cause reticulocytosis. This study was performed to evaluate the
use of IRF as a marker of degree of post-traumatic haemorrhage.
Methods: A cross sectional descriptive study was performed using routine anticoagulated blood samples
collected from patients admitted with acute traumatic haemorrhage to an emergency treatment unit. Differential
and total reticulocyte counts were performed manually using standard techniques with bright field microscopy.
The data on severity of blood loss were extracted from clinical records.
Results: There were total of 38 patients. Mean IRF of patients with mild (n = 14), moderate (n = 19) and
severe (n = 05) haemorrhage were 1.86 ± 1.0%, 3.26 ± 1.8% and 10.2 ± 3.6% respectively. Mean manual IRF of
group with severe haemorrhage was significantly higher than those of all the others (p = 0.000). The mean
manual IRF of moderate haemorrhage group was significantly higher than that of mild group (p = 0.01). Manual
IRF and severity of haemorrhage were significantly positively correlated (r = 0.71, p < 0.01, n = 38). Immature
reticulocytes of stage I was observed only in patients with severe haemorrhage.
Conclusion: The manual IRF can be used as a reliable marker of severity of post-traumatic acute haemorrhage.

Full text

Original article
158
Galle Medical Journal, Vol 26: No. 4, December 2021
Manual immature reticulocyte fraction; a surrogate marker to assess
post traumatic blood loss
1 2 3
Wickramaratne KAC , Wijegunawardena JKD , Wijewickrama DC
1 3
Department of Pathology, Department of Physiology, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka.
2Faculty of Allied Health Sciences, University of Ruhuna, Galle, Sri Lanka.
Correspondence: Dr. Chandana Wickramaratne
e-mail: kacw@med.ruh.ac.lk
https://orcid.org/0000-0003-4394-2535
Submitted on 24.10.2021 and accepted for publication on 04.12.2021
ABSTRACT
Introduction: Erythropoietic response following chemotherapy and stem cell transplant is assessed by
reticulocytosis and immature reticulocyte fraction (IRF). They are not used to assess erythropoiesis following
acute haemorrhage even though it is known to cause reticulocytosis. This study was performed to evaluate the
use of IRF as a marker of degree of post-traumatic haemorrhage.
Methods: A cross sectional descriptive study was performed using routine anticoagulated blood samples
collected from patients admitted with acute traumatic haemorrhage to an emergency treatment unit. Differential
and total reticulocyte counts were performed manually using standard techniques with bright field microscopy.
The data on severity of blood loss were extracted from clinical records.
Results: There were total of 38 patients. Mean IRF of patients with mild (n = 14), moderate (n = 19) and
severe (n = 05) haemorrhage were 1.86 ± 1.0%, 3.26 ± 1.8% and 10.2 ± 3.6% respectively. Mean manual IRF of
group with severe haemorrhage was significantly higher than those of all the others (p = 0.000). The mean
manual IRF of moderate haemorrhage group was significantly higher than that of mild group (p = 0.01). Manual
IRF and severity of haemorrhage were significantly positively correlated (r = 0.71, p < 0.01, n = 38). Immature
reticulocytes of stage I was observed only in patients with severe haemorrhage.
Conclusion: The manual IRF can be used as a reliable marker of severity of post-traumatic acute haemorrhage.
Keywords: Acute haemorrhage, surrogate marker, immature reticulocyte fraction, post-traumatic blood loss,
Reticulocytes, manual immature reticulocyte fraction.
Introduction
Post traumatic haemorrhage needs prompt medical
attention as it can cause significant morbidity
and mortality due to acute hypovolaemia and
shock (1). Degree of blood loss is classified based
on clinical assessment in acute trauma care.
Following haemorrhage, erythropoiesis is increased
in the marrow and more immature forms of red
cells (reticulocytes) are rapidly released in to the
peripheral circulation. Reticulocytes can be
classified based on their maturation using both
manual and automated techniques (2-4). The
immature reticulocyte fraction (IRF), is a novel
parameter available in fully automated haematology
analysers. It is used as an early indicator of marrow
erythropoietic activity in stress conditions and
to assess marrow response following therapy or
stem cell transplant (5-7). Automated IRF is
prohibitively costly to use in routine care in limited
resource settings (8-10).
DOI: http://doi.org/10.4038/gmj.v26i4.8112

Original article
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Galle Medical Journal, Vol 26: No. 4, December 2021
Manual reticulocyte counting is performed in
many limited resource laboratories but manual IRF
calculation is not. Therefore, this study was carried
out to evaluate the possibility of using manual
IRF in assessing the degree of haemorrhage in acute
trauma.
Methods
All the patients admitted to an emergency treatment
unit (ETU) of a Teaching Hospital during a period of
one month, with post-traumatic acute haemorrhage
irrespective of the degree of haemorrhage were
recruited to the study. Informed written consent
was taken from the patient when he or she was in
a position to do so and when they were unable to
consent, assent was obtained from a guardian or
relative. Those who had known clinical conditions
that could directly affect haematological results
were excluded from the study.
Clinical assessment of severity of blood loss on
admission and the time taken for admission since
traumatic event were extracted from patient
records which were maintained by acute trauma
physicians.
Venous blood samples (5 ml) collected from
antecubital fossa using standard techniques
{in to tubes containing Dipotassium Ethyle-
-nediaminetetraacetic acid (K2EDTA)} were used
to prepare blood smears to perform reticulocyte
counting. Blood smears were prepared within one
hour of collection of samples and reticulocyte
staining was performed using standard supra vital
staining procedure (9, 10). The quality of the stain
was verified by the presence of stained platelets
and white cells in each smear and it was considered
the positive control. The differential count of
immature reticulocytes and total reticulocyte count
were performed manually using bright field
microscope (X100 power/ oil immersion in
Olympus CX 32) fitted with an ocular graticule.
The IRF (stage I and II reticulocytes) was calculated
as a percentage of all the reticulocytes counted.
Reticulocytes were classified as per the Heilmeier
classification (Figure 1) and the differential count
of each stage of reticulocytes was performed (4). The
reticulocyte counting and manual IRF calculations
were performed in duplicates by the researchers.
A difference of 10% or less in the duplicate
counts was considered acceptable. Reticulocyte
percentages of all the patients were monitored
hourly for four to five hours.
Data were analysed using MS-Excel 2010 and
R-Studio statistical software. According to the
clinical assessment on the patients’ records the
subjects were grouped in to mild, moderate and
severe blood loss. The differences between groups
were assessed using student’s t-Test. The correlation
was assessed with Spearman correlation coefficient.
p < 0.05 was considered significant.
Ethics approval was obtained from the Ethical
Review Committee of Faculty of Allied Health
Sciences, University of Ruhuna, Sri Lanka.
Institutional permission and approval was obtained
to retrieve data and to conduct the research in the
emergency trauma unit, which was mandatory to
the obtain ethical clearance.
Results
There were total of 38 patients who fulfilled the
inclusion criteria and of them 24 (63.1%) were
males and 14 (36.8%) were females. Of the total,
14 had mild, 19 had moderate and five had severe
acute post traumatic haemorrhage. The median age
of the study group was 36 years.
The mean manual IRF were 1.86 ± 1.0%, 3.26 ±
1.8% and 10.2 ± 3.6% in patients with mild,
moderate and severe haemorrhage respectively.
The mean manual IRF of patients with severe
haemorrhage was significantly higher than those
of all the other patients with haemorrhage (mild
and moderate) (p = <0.001). The mean manual
IRF of patients with moderate haemorrhage was
also significantly higher than that of the patients
with mild haemorrhage (p = 0.01). The manual
IRF and the severity of haemorrhage were
significantly positively correlated (r = .71, p < 0.01,
n = 38) (Figure 2).
All four stages of reticulocytes were observed in
patients with severe haemorrhage while only stages
II, III and IV were observed in mild and moderate
haemorrhages (Figure 3).

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Stage I Stage II
Stage III Stage IV
Figure 1: Maturation stages of reticulocytes according to Heilmeier classification. Stage I: non-nucleated
red cells appearing with a dense clumped reticulum; Stage II: extended network of loose
reticulum; Stage III; scattered granules with residual reticulum network; Stage IV: scattered
granules
Figure 2: Correlation of immature reticulocyte fraction (IRF) and severity of haemorrhage

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1hr 2hrs 3hrs 4hrs
Mild
Stage 2
Stage 3
Stage 4
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1hr 2hrs 5hrs
Severe
Stage 1
Stage 2
Stage 3
Stage 4
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1hr 2hrs 3hrs 4hrs
Moderate
Stage 1
Stage 2
Stage 3
Stage 4
Reticulocyte %
Reticulocyte %
Reticulocyte %
Figure 3: Percentages of reticulocyte stages and time duration since trauma in mild,
moderate and severe haemorrhages and their trend lines
Discussion
Assessment of trauma-related blood loss is
subjective and is supported mainly by clinical
criteria (1). The subjective nature of this assessment
is improved by the use of non-laboratory parameters
such as blood pressure, pulse rate, pulse pressure,
urine output and level of consciousness. The
assessment of haemorrhagic shock is aided by
laboratory parameters such as complete blood count
and arterial blood gas analysis (10-14). Yet, accurate
estimation of blood loss is subjective and
challenging.
The automated IRF is considered one of the best
parameters to detect early marrow response
following chemotherapy or stem cell transplant
(1-8). The data on use of automated or manual IRF
to assess degree of haemorrhage in routine trauma
care is not found in literature.
The peripheral blood reticulocyte count of healthy
adults is 1% - 2%. All the reticulocytes in peripheral
blood of healthy individuals belong to Heilmeier
stages III and IV and are considered the mature
forms (4, 8, 9). The reticulocytes stages I and II
(immature forms) are not normally found in
peripheral circulation of healthy individuals (15).
In this study, both stages I and II of immature
reticulocytes were seen in peripheral blood of all
the patients with severe haemorrhage. The patients
with mild and moderate haemorrhage showed
only the stage II of immature reticulocytes.
Therefore, increased IRF (both stages I & II) in
peripheral blood appears to be an objective marker
which differentiate severe haemorrhage from mild
and moderate. Increased IRF of only stage II could
be considered a marker of mild and moderate
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haemorrhage. In addition, stage IV reticulocytes
showed progressive decline over five hours of study
period in all the patients with severe haemorrhage.
None of the acute blood loss assessment tools
have incorporated either automated or manual IRF
for estimation of blood loss. The findings of this
study show the value of estimation of either
manual or possibly automated IRF as an objective
surrogate indicator of degree of haemorrhage.
Since, patients with moderate and severe bleeding
are inevitably managed with crystalloids and/or
blood transfusions; one can argue the reliability of
IRF following an intervention. The details of
standard care given to the patients before collection
of blood samples for IRF were not collected in
this study. However, irrespective of whether volume
expanders were given or not, the patients with
moderate and severe haemorrhages showed high
IRF proving its applicability in detecting the degree
of post traumatic acute haemorrhage. In addition,
decrement of percentages of mature forms (stage IV)
and increment of immature forms (stages I and II)
appeared to be unique for severe haemorrhage.
Further studies are needed to assess the effects of
volume resuscitation and other comorbid factors
on IRF in post-traumatic acute haemorrhage and
to demonstrate the reliability of IRF in estimation
of degree of haemorrhage in non-traumatic or
trauma related concealed (retroperitoneal, intra-
abdominal or intra muscular) haemorrhage.
Limitations
This study was carried out using manual method
available for reticulocyte counting and it has its
own limitations related to distribution of cells in
smear, counting procedure and the limited number
of cells counted. If the automated method was
used, highly accurate and precise information on
reticulocytes could have been obtained as large
number of cells are counted in that method thus,
it would have provided more accurate objective
information on the relationship between IRF and
the degree of acute blood loss in trauma. In this
study, the grading of blood loss was performed
by acute care physicians and surgeons and not by
the researchers. If a universal grading scale for
classifying degree of blood loss was incorporated
in the assessment of blood loss, highly standardized
information could have been gathered.
Manual IRF and progressive increment of IRF while
decrement of percentages of mature reticulocytes
reliably indicate severity of post traumatic acute
haemorrhage and would be potentially useful even
in assessment of acute concealed haemorrhage.
Acknowledgements
We acknowledge the administrative team of
emergency treatment unit, Teaching Hospital,
Karapitiya, Galle, Sri Lanka and staff of Medical
Laboratory Science Department of Faculty of Allied
Health Sciences for the provision of laboratory
facilities to conduct this research project.
Competing Interests:
The authors have no competing interests.
Funding:
Authors received no funding from any institution
or organization to carry out this study.
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