ISSUE

12ISSUEApril
2021

This issue
Last Month’s Slides P.1
Monthly Case study P.1
Reticulocyte measurement P.2
Morphology webinar P.2
Reticulocyte matrix P.3
Breaking news P.3

Last Month’sSlides
March 2021 Slide
Summaries
Side 1
Follow up of CLL
Expert comment – Evolution of
an atypical CLL with 3/5
matures scoring to be comparedwith the rest of the clinicalresults

Slide 2Discreet Eosinophilia

Slide 3
Occasional large platelets

Slide 4Discreet Basophilia

Monthly Digital Case study
Slide 5Presentation
Male (65 years old)

FBC ResultsWBC
17.2*(10^3/mm3)RBC
4.27* (10^6/mm3)
HGB
108* (g/L)
HCT
36.4* (%)
MCV
85*(fL)MCH
25.3* (pg)
MCHC 29.7* (g/dL)
PLT
413*(10^3/mm3)
RDW-SD 87.4 *(fL)RDW-CV 29.2* (%)

Neutrophils 76.2 %Lymphocytes 1.8 %Monocytes 8.8 %Eosinophils 4.4 %Basophils 3.5%

Macrothombocyte

Slide 5Multiple alarms on analyser
Expert Comment – DimorphicRBC, acanthocytes,
Macrothrombocytes, Basophilia
Investigate for
Myeloproliferative disorder

Slide review

Slide 6Diagnosis

Neutrophilia, presence of
myelocytes, metamyelocytes,
promyelocytes, nucleated redblood cells

RBC picture
Multiple alarms on analyser – represented by *Dimorphic RBC, acanthocytes, Howell Jolly Bodies, target cells Basophilia,
Macrothrombocytes (see above).

MyeloproliferativeMorphology
Webinar:
HORIBA recently hosted awebinar on “An Introduction
to Morphology”, which
includes a demonstration on
how to use QSP 2.0 in training.
You can now watch it on
demand here.

Last month’s Quiz:

Slide B shows a microcytic,
hypochromic red cell picture.
Red cells are smaller, with alarger than normal central areaof pallor. This decrease inredness is due to adisproportionate reduction of
haemoglobin in the red cells in
proportion to the red cellvolume. The colour of the redcells can be evaluated by theMCH (mean corpuscularhaemoglobin), and also theMCHC (mean cell haemoglobin
concentration).
The most common causes ofMicrocytic Hypchromic
anaemia are iron deficiency andthalassaemia.

Reticulocyte measurement on HORIBA
analysers
Reticulocytes are immature Red Blood Cells (RBC) that are being constantly released
from the bone marrow (approx. 2.5 billion red cells/Kg/day). The stages of red cell
development within the bone marrow include pronormoblast, basophilic normoblast,
polychromatophilic normoblast, orthochromatic, reticulocyte. Reticulocytes are
released from the bone marrow after the nucleus has been extruded at theorthochromatic stage. In normal adults, the red cell has a lifespan of approx. 120 days,
therefore new red cells have to be constantly produced. Red cell production iscontrolled by the hormone Erythropoietin which increases as the blood oxygen levels
decrease. Reticulocytes can be identified because they still contain ribosomal RNA
required to complete the production of Hb. Reticulocytes mature in the peripheral
circulation for between 1 to 2 days finally becoming mature red cells. Reticulocytes
tend to be larger than mature Red Cells and often show a blue tinge (Polychromasia).
The normal reticulocyte count is 0.5% - 2.5% of Red cells or 20 – 80 x109/L.
It is important to measure Reticulocytes as they give an indication as to the state of
red cell production. An increase in Reticulocytes (Reticulocytosis) indicates that thebone marrow is responding to the bodies need to produce more red cells. A decreasein blood oxygen levels leads to an increased production of the hormone
Erythropoietin, which stimulates the bone marrow to increase red cell production,
thereby increasing the number of Reticulocytes (Reticulocytosis) entering theperipheral blood. Reticulocytosis may be present, even if the patient is not clinicallyanaemic. Causes of reticulocytosis are: haemolysis, blood loss and Iron/B12/Folate
deficiency. A low Reticulocyte count is indicative of the failure of the bone marrow toproduce RBC and could indicate deficiency of Vitamin B12, Folate or iron, anaemia ofchronic disease, Aplastic anaemia etc.

Methodology
HORIBA Medical uses the reagent Fluocyte which contains the fluorochrome Thiazole
Orange to measure reticulocytes. Thiazole Orange which binds to any residual RNA
present within the red cell. Therefore, if RNA is present, fluorescence will be induced
by a laser (488nm) and be detectable.
0.5 uL blood is incubated with 5mL Fluocyte at 350C for 51 sec, then transferred to theflow cell where simultaneous measurement of volume (aperture impedance) andorthogonal fluorescence (laser excitation 488 nm) is performed. Any RNA present on
the cell causes an increase in the degree of fluorescence above a motile threshold. Amatrix (see below) is produced showing the volume and orthogonal fluorescence ofeach cell, a threshold separates the Reticulocytes from the mature RBC. The
Reticulocyte area is split into 3 areas, indicating the degree of fluorescence andtherefore their maturity – the greater the fluorescence the more immature. The
Reticulocyte percentage is calculated from the number of cells in the reticulocyte area
not including any cells in the NRBC or WBC area.

Reticulocyte Matrix as displayed on analyser
FlRet-Huor
Ret-M
Reticulocytesesc
Ret-Len
RBCce
VolumeQSP 2.0
Available as a single
use license and a site
license which allows
up to 10 concurrent
users.
To find out more,contact us.

Bibliography
HORIBA Yumizen H2500
user manual

Editorial Team
Kelly Duffy
Andrew Fisher

Reticulocyte Parameters
Percentage Reticulocyte
Absolute Reticulocyte
Reticulocyte Heamoglobin Cellular Content (RHCc)
Mean Reticulocyte Volume (MRV)
Immature Reticulocyte Fraction (IRF)
Mean Fluorescent Index (MFI)
Corrected Reticulocyte countFor more information, watch the video.

About us

BREAKING NEWSThis month our QSP Newsletter, which is part of HORIBA’s ongoingcommitment to support scientists interested in Blood Film Morphology
has reached the milestone of its first year of publication.
On this occasion, we are delighted to announce that QSP 2.0 multi- site license
now works across a Virtual Private Network (VPN), enabling users to connect
multiple QSP PC (10 concurrent) across different sites, even if they are not on
the same local area network.
If you would like to find out more about QSP 2.0 and discuss your special
requirements, contact us.

HORIBA UK Limited
Kyoto Close
Moulton Park
Northampton, UKNN3 6FL
HORIBA Medical
Parc Euromédecine,
390 Rue du Caducée,
34790, France
www.horiba.com/medical