I have been working with Roche products since joining Ahalia Group LLC Abu Dhabi, UAE.

During this period, I have hands on experience including initial validation for cobas 6000 and
cobas b121 and validation verification of elecsys e411 and cobas integra 400 plus. I am well
versed in computer applications and statistical tools necessary for laboratory science.

I always felt the job of an application support crew in Roche Diagnostics as a challenging one
that will definitely push me to my limits to reap success personally and professionally.

Atypical lymphocytes suspected reactive

Cell description:

Size: larger than normal lymphocytes

Nucleus: oval, variable chromatin condensation

Cytoplasm: Diffluent, often around red blood cells

Segmented neutrophil

Cell description:

Size: 12-15 m
Nucleus: clumped chromatin and mostly divided into 2-5 distinct segments connected with
filaments

Cytoplasm: acidophilic with many fine reddish granules spread evenly Function: phagocytosis,
play an important role in the unspecific immune defense, in the tissue they defend the mucosa
against bacteria and fungi

Promonocyte

Cell description:

Size: bigger than monoblast

Nucleus: oval, kidney-shaped or lobulated, diffuse chromatin pattern, sometimes with nucleoli

Cytoplasm: pale basophil with fine azurophil granula.

Cell division is still possible.

Nucleated Red Blood Cell

Cell description: 3 different stages of nucleated red blood cells are known: basophilic,
polychromatic and orthochromatic
Size: around 10 m

Nucleus: round with variable degree of chromatin condensation according to maturation, faint or
absent nucleoli

Cytoplasm: bluish to pink (depending on maturation), no granules

Nucleus decreases in size as the cell matures

Myeloblast

The first microscopically identifiable cell of granulocytic cell line.

Cell description:

Size: 12-20 m

Nucleus: large, round or slightly oval with diffuse chromatin pattern and often 1-5 nucleoli

Cytoplasm: pale blue and usually agranular, sometimes Auer rods visible

Myelocyte
In this maturation stage the separation into the 3 different subpopulations of granulocytes occurs
by development of specific granulation for each (secondary granulation).

Cell description:

Size: 10-18 m

Nucleus: oval or slightly indented with variable degree of chromatin clumping, nucleoli usually
not apparent

Cytoplasm: acidophilic neutrophil: primary azurophilic and secondary neutrophilic granules

Prolymphocyte

Cell description:

Size: 10-18 m, smaller than lymphoblast

Nucleus: round with coarser structure than a lymphoblast, one distinct nucleolus

Cytoplasm: blue without granules

Plasma cell

Cell description:

Size: up to 20 m

Nucleus: eccentric with coarsely clumped chromatin, often clock-face chromatin pattern

Cytoplasm: strongly basophilic cytoplasm with apparent less basophilic Golgi zone adjacent to
the nucleus

Metamyelocyte

Cell description:

Size: 10-12 m

Nucleus: kidney or U-shaped with clumped chromatin

Cytoplasm: acidophilic

neutrophil: fine reddish granulation

Cell division is not possible anymore and protein synthesis has stopped.

Metamyelocyte

Cell description:

Size: 10-12 m

Nucleus: kidney or U-shaped with clumped chromatin

Cytoplasm: acidophilic

neutrophil: fine reddish granulation

Cell division is not possible anymore and protein synthesis has stopped.

Monocyte

Cell description:

Size: 20 m

Nucleus: kidney- to band-shaped

Cytoplasm: grey and clear with fine azurophilic granules

They are shortly located in the peripheral blood and then move into the tissue where they
differentiate into macrophages. Function: Phagocytosis either of harmful pathogens or dead,
dying or damaged cells from the blood.

Eosinophil

Size: 12-17 m

Nucleus: usually bilobed with visible filament

Cytoplasm: weekly basophilic containing coarse reddish-orange granulation packing the

cytoplasm

Function: Phagocytosis, chemotaxis, mortification of parasites, inhibition of mastcell

degranulation, eutralization of histamine

Lymphocyte

Cell description:

Size: 10-16 m
Nucleus: round or slightly indented with condensed and cloddy chromatin, usually invisible
nucleolus

Cytoplasm: scanty, weakly basophilic, may have small numbers of azurophilic granules

Morphologically functional subset of lymphocytes cannot clearly be distinguished.

Function: recognize and eliminate threats to the body. Lymphocytes of the innate immune
system deliver an immediate response to viral attack. Lymphocytes of the adaptive immune
system are specific to a particular antigen.

Hairy cell

Cell description:

Size: larger than normal lymphocytes

Nucleus: round, oval, dumbbell-shaped or bilobed with little chromatin condensation and
sometimes indistinct nucleolus

Cytoplasm: abundant weakly basophilic with irregular hairy margins

Band cell
Cell description:

Size: 12-15 m

Nucleus: curved or coiled band

Cytoplasm: acidophilic containing fine reddish granulation

Basophil

Size: 10-14 m

Nucleus: lobulated but often obscured

Cytoplasm: acidophil with purple-black granulation

Function: basophils can release histamine and heparin to respond to a suspected infection

Blast Cell

It is often difficult to distinguish blast cells of myelocytic, monocytic, or megakaryocytic

lineages from lymphoblasts. Additional techniques, such as immunophenotyping by flow
cytometry, are necessary to determine cell origin.
Cell description:

Size: Variable in sizes usually between 10 and 20 m

Nucleus: round or slightly indented with condensed and cloddy chromatin, nucleoli sometimes
visible

Cytoplasm: scant to moderate, blue, agranular, occasionally few vacuoles might be visible

Hairy cell leukaemia

Peripheral blood (May-Grnwald-Giemsa stain) of a patient with hairy cell leukaemia. A typical
hairy cell (->) can easily be distinguished from a normal lymphocyte (lower right).

Lymphoma cells in peripheral blood

In the peripheral blood (May-Grnwald-Giemsa stain) of this patient lymphoma cells can be
detected. They show a large, deeply indented nucleus (->) and scanty cytoplasm. Further
diagnostics confirmed a mantle cell lymphoma.

Blood film of a patient with polycythaemia

vera (PV)
The automated blood count of this 55-year old man with PV showed a normal haemoglobin
concentration of 15.7 g/dL. Red blood cells were markedly increased to 8.2 million/L, but
appeared very small, as is shown in the peripheral blood film (May-Grnwald-Giemsa stain).
The MCV was only 60 fL, the haematocrit was 61%. White blood cells (22,300/L) and platelets
(612,000/L) were also elevated.

Increase of all three cell lineages in

peripheral blood of a patient with PV
In the peripheral blood (May-Grnwald-Giemsa stain) of this 75-year old woman with PV all
three cell lineages are increased: haemoglobin concentration 16 g/dL, white blood cell count
15,000/L, platelet count 980,000/L. In functional iron deficiency red blood cells often are
microcytic. The MCV is only 75 fL. The haematopoietic cells of the patient show a JAK2
mutation.

'Rouleaux formation' in blood of a patient

with chronic polyarthritis
'Rouleaux formation' is not specific for multiple myeloma or lymphoma. Also polyclonal
immunoglobulins can cause 'rouleaux formation' (->) in peripheral blood (May-Grnwald-
Giemsa stain), as shown in this patient with chronic polyarthritis.

Rouleaux formation' of red blood cells

In peripheral blood of a patient with multiple myeloma (May-Grnwald-Giemsa stain) no plasma
cells are detectable, because in multiple myeloma these are only rarely found in the periphery.
The monoclonal immunoglobulin causes an increased erythrocyte sedimentation rate (ESR) and
'rouleaux formation' (->) of red blood cells

Macrocytic, hyporegenerative anaemia

Peripheral blood (May-Grnwald-Giemsa stain) of an 82-year old patient with RCMD showing a
macrocytic, hyporegenerative anaemia with clear changes of the red blood cells (micro- and
macrocytosis, poikilocytosis and some teardrop cells (->)).

Peripheral blood of a patient with MDS

Peripheral blood (May-Grnwald-Giemsa stain) of a patient with MDS: on the left a pseudo-
Pelger-Huet cell, on the right a dysplastic metamyelocyte. Anaemia and atypical platelets are
also present. After peripheral blood and bone marrow analysis the diagnosis of refractory
anaemia with excess of blasts (RAEB-1) was made.

Peripheral blood of a patient with CML

The peripheral blood (May-Grnwald-Giemsa stain) of a patient shows as an incidental finding
leukocytosis with left shift up to myelocytes, basophilia and mild thrombocytosis. CML was
suspected and later confirmed by cytogenetic demonstration of the Ph1.

Peripheral blood of a patient with CML

The peripheral blood (May-Grnwald-Giemsa stain) of a 27-year old patient with hearing loss
showed a massive leukocytosis (680,000/L) and a marked left shift with a distinct fraction of
blast cells. Several eosinophilic cells are clearly visible. Platelets are abundant, although not
visible in this part of the blood film. The BCR-ABL fusion gene was detected by FISH analysis
of the peripheral blood and secured the diagnosis of CML. Because of the threat of permanent
loss of hearing chemotherapy was started within hours after venepuncture (without waiting for
the results from bone marrow cytology).

Pancytopenia (tricytopenia) in peripheral

blood
Pancytopenia (tricytopenia) in the peripheral blood (May-Grnwald-Giemsa stain) of a patient
with severe aplastic anaemia: The automated cell count showed granulocytopenia (300/L), mild
lymphocytopenia (800/L), anaemia (haemoglobin 9 g/dL), a reduced reticulocyte count
(18,000/L) and thrombocytopenia (12,000/L).

Isolated congenital reduction of

erythropoiesis in bone marrow
Peripheral blood (May-Grnwald-Giemsa stain) of a patient with isolated congenital reduction of
erythropoiesis in the bone marrow (pure red cell aplasia, PRCA). The haemoglobin concentration
was 9.2 g/dL, white blood cell and platelet counts were normal.

Peripheral blood of a patient showing

isolated neutropenia
The peripheral blood (May-Grnwald-Giemsa stain) of this patient shows only isolated
neutropenia (900/L) with morphologically normal red blood cells and normal red blood cell and
platelet counts. Aplastic anaemia is not present. A lymphocyte can be seen in the middle.

Peripheral blood of a patient with CML

Peripheral blood (May-Grnwald-Giemsa stain) of a 27-year old patient with chronic
myelogenous leukaemia (CML). Normal red blood cells, marked thrombocytosis
(2,750,000/L), increased basophilic granulocytes (->) and a normal white blood cell count are
observed. In this case no JAK2 mutation could be detected; instead the patient was tested
positive for the BCR-ABL fusion gene.

Peripheral blood of a patient with ET

showing an isolated thrombocytosis
Peripheral blood (May-Grnwald-Giemsa stain) of a patient with ET showing an isolated
thrombocytosis. A JAK2 mutation was detected by molecular biological techniques. The BCR-
ABL fusion gene was not found

Giant platelets in peripheral blood

Peripheral blood (May-Grnwald-Giemsa stain) of a patient with essential thrombocythaemia,
ET. Giant platelets (->) are frequently observed in this disease.

Blast cells in peripheral blood

Peripheral blood (May-Grnwald-Giemsa stain) of a 6-year old boy with bone pain and
diarrhoea. In addition to numerous lymphocytes (L) also blast cells (B) are present. The platelet
count is reduced. Bone marrow examination revealed an acute lymphoblastic leukaemia
(childhood ALL, C-ALL).

Acute myeloid leukaemia (AML)

Blasts (33% in total) with Auer rods (->) in the peripheral blood (May-Grnwald-Giemsa stain)
are sufficient proof of an acute myeloid leukaemia (AML).

Peripheral blood of a patient with B-CLL

Peripheral blood (May-Grnwald-Giemsa stain) of a patient with B-CLL. The few platelets are
mostly large (->) (= platelet anisocytosis). Here a secondary immune thrombocytopenia (ITP)
was evoked by immunoglobulin-producing lymphoma cells. The immunoglobulins react with
proteins on the platelet surface, leading to an elimination of these platelets from the peripheral
blood.

Chronic lymphocytic leukaemia /

prolymphocytic leukaemia
Chronic lymphocytic leukaemia with numerous prolymphocytes (->) (CLL/PL) in the peripheral
blood (May-Grnwald-Giemsa stain).

B-CLL with granulocytopenia

Peripheral blood (May-Grnwald-Giemsa stain) showing a case of B-CLL with granulocytopenia
(total 400/L).

Peripheral blood showing a typical B-CLL

Peripheral blood (May-Grnwald-Giemsa stain) showing a typical B-CLL with an elevated
lymphocyte count, normal granulocyte and platelet counts and normal red blood cells.

Peripheral blood of a patient with breast

cancer and bone metastases
The peripheral blood (May-Grnwald-Giemsa stain) of a patient with breast cancer and bone
metastases demonstrates a leucoerythroblastic picture. There are left-shifted granulopoiesis (e.g.
here a myelocyte (M)), erythroblasts (E) and distinct thrombocytopenia.

Primary myelofibrosis (PMF) in fibrotic

stage
PMF (also called CIMF) in the fibrotic stage with a leucoerythroblastic blood picture (May-
Grnwald-Giemsa stain): In this patient immature granulocytes, erythroblasts and many teardrop
cells (->) were found. In the fibrotic stage there is usually anaemia present and a low to normal
or reduced platelet count. Myeloblasts might be present. But more than 10% already indicate
blast cell excess or transition into AML.

Primary myelofibrosis (PMF) in prefibrotic

stage
This peripheral blood film (May-Grnwald-Giemsa stain) is from a patient with PMF (also
called CIMF) in the prefibrotic stage. You can see scattered teardrop cells (->). Furthermore, a
few granulocytic precursors and very few erythroblasts were detected (not shown). The
automated cell count showed slight leukocytosis, thrombocytosis and anaemia.

Primary myelofibrosis (PMF)

In primary myelofibrosis (PMF, also called chronic idiopathic myelofibrosis, CIMF) nuclei of
megakaryocytes can be detected in the peripheral blood (May-Grnwald-Giemsa stain) and
rarely, like here, small megakaryocytes. Both derive from extramedullary haematopoiesis.

Thick blood film with high density of malaria

pathogens
A 'thick blood film' is prepared by spreading a drop of blood on a slide in a way that
approximately 20 layers of red blood cells are on top of each other. The slide is then left to dry
and subsequently treated with Giemsa solution to lyse the red blood cells. This process leads to a
higher density of the malaria pathogens (here: trophozoites of Plasmodium falciparum ->) on the
slide so that they can be detected more easily and quickly. The large nuclei are debris from lysed
white blood cells.

Extreme thrombocytosis in chronic

myelogenous leukaemia
Extreme thrombocytosis (3,400,000/L) in chronic myelogenous leukaemia (CML, bcr-abl+); on
the right a basophilic granulocyte.

Diagnosis of AML in bone marrow

Diagnosis of AML in the bone marrow: Characteristic are bundles of Auer rods (->) and many
atypical promyelocytes.

Typical blasts in AML-M3v

Typical blasts (on the right with Auer rods ->) in AML-M3variant.

Atypical promyelocyte in peripheral blood

Atypical promyelocyte in peripheral blood in a patient with AML-M3.

Incidental detection of Borrelia recurrentis

Incidental detection of Borrelia recurrentis (->) in a patient initially suspected to suffer from
malaria. Borrelia recurrentis is transmitted by lice and ticks and is the causative agent of
relapsing fever. Like malaria, relapsing fever often is a travel-related disease that, after an
incubation time of up to 2 weeks, leads to fever attacks.

Streptococci in blood film

Streptococci, extracellular as well as intracellular (->), in a patient with finger gangrene. The
patient died hours later despite intensive care treatment. If bacteria are detectable in a blood film,
a blood film from a different patient should be stained with the same staining solution and be
checked for bacteria to rule out contamination of the staining solution. If none are detectable, the
detection of bacteria has to be reported to the treating physician immediately.

Chronic myelogenous leukaemia (CML)

Blood film of a patient with chronic myelogenous leukaemia (CML). The extremely high white
blood cell count of 680,000/L caused a hyperviscosity syndrome and led to total and only
partially reversible deafness.

Acute T lymphoblastic leukaemia (T-ALL)

White blood cell concentration 180,000/L. Despite intensive search no granulocytes were
detectable. Diagnosis: acute T lymphoblastic leukaemia

Schistocytes and thrombocytopenia

Schistocytes (->) and thrombocytopenia in a patient with haemolytic uraemic syndrome (HUS).
This disease, as well as for example thrombotic thrombocytopenic purpura (TTP), is a so-called
'microangiopathic haemolytic anaemia' (MAHA). Characteristic of MAHA is a responsive
increase in the production of red blood cells and platelets whose immature precursors
(reticulocytes and immature platelet fraction, IPF) can be measured on certain Sysmex analysers.
On a blood film the differential diagnosis of MAHA is not possible.

Leucoerythroblastic condition and

schistocytes
Leucoerythroblastic condition and schistocytes in a patient with colorectal carcinoma. The
occurrence of schistocytes (->) in tumour patients is sometimes an indication of tumour
infiltration of the bone marrow.

Auto-immune haemolytic anaemia

Auto-immune haemolytic anaemia (AIHA) in a case of chronic lymphocytic leukaemia (B-
CLL): Reticulocytes are increased, spherocytes are present, Coombs' test is positive.

Acute haemolysis
Acute haemolysis: fragmented red cells (F), spherocytes (S) and free haemoglobin (= reddish
smears) in a patient with gas gangrene, caused by Clostridium perfringens. (The acanthaceous
appearing cells are no acanthocytes but red blood cells on the verge of disintegration.)

Blood sample reflecting acute intravascular

haemolysis
Serum of a patient at the time of hospitalisation (left) and 12 hours later (right): The serum is
coloured red-brown due to intravascular haemolysis.

Hairy cell leukaemia

Hairy cell from a hairy cell leukaemia. Typical are the monocytic type nuclei with loose
chromatin, and the grey-blue, heterogeneous cytoplasm. The hairy protrusions may be missing in
some cases.

Hairy lymphocyte
This 'hairy' lymphocyte comes from a person suffering from an active infection (without any
malignant disease). The granula identify it as a T or an NK cell.

Cytoplasmic fragments split off a blast

Cytoplasmic fragments split off a blast in a case of acute lymphoblastic leukaemia (ALL). In
certain automated haematology analysers, such fragments might possibly suggest a wrongly high
concentration of platelets.

Cytoplasmic protrusions of a lymphocyte

Cytoplasmic protrusions of a lymphocyte in normal blood. (Although such protrusions are
frequently found with infections, contrary to activated lymphocytes they are of no diagnostic
importance.)

Hairy cell leukaemia-variant

Hairy cell leukaemia-variant (HCL-V).

Thrombocytosis, anisocytosis of platelets and

giant platelets
Thrombocytosis, anisocytosis of the platelets and giant platelets in a patient suffering from
essential thrombocythaemia (ET). (Also, please note the profound poikilocytosis of the red blood
cells.)

Accumulation of platelets on granulocytes

Accumulation of platelets on granulocytes in the presence of EDTA (= platelet satellitosis). In
vitro, this effect can lead to pseudo-thrombocytopenia.

Anisocytosis of platelets
EDTA blood sample after one day of storage, which results in obvious anisocytosis of the
platelets.

Platelets
Inconspicuous number and size of platelets in a healthy individual.

Abnormal platelets
Abnormal platelets of a patient suffering from myelodysplastic syndrome (MDS). (The upper
one is reminiscent of a 'paramecium', the lower of a 'flagellate'.) In this type of disease, very
strange shapes may appear in all cell lines.

Extreme haemolysis
Reddish striations caused by extreme haemolysis in a case of septicaemia caused by Clostridium
perfringens.

Blood film with staining artefact

Blood film with a blue cast as a staining artefact (staining process too long or pH value too high).
(In the centre, a monocyte can be seen.)

Malignant melanoma cells on blood film

Malignant melanoma cells have leaked into the peripheral blood (edge of the blood film).

Endothelial cells on blood film

The endothelial cells are part of the vascular wall, which was damaged during venepuncture.
(Blood coagulation was activated during the process: on the bottom left a fibrin fibre can be
recognised.)

Plasmodium falciparum
Older ring forms of plasmodium falciparum in malaria tropica.

Platelet on top of red blood cell

Platelets can lie on top of red blood cells, and look very much like plasmodia. (By turning the
micrometre screw, one will realize quite easily that the object is on top of the red blood cell and
not inside it, as it would be with plasmodia.)

Platelet aggregates
It is of utmost importance to always report a correct value for the platelet concentration. When
detecting thrombocytopenia the laboratory should therefore double-check that it is not due to
platelet aggregates (->) (pseudo-thrombocytopenia). In a haematology analyser they are usually
automatically identified and flagged. In a blood film they are mostly located at the feather edge
or the lateral edges.

Acute myeloid leukaemia (AML-M4)

White blood cell count of 170,000/L in acute myeloid leukaemia (AML-M4).

Chronic lymphocytic leukaemia /

prolymphocytic leukaemia
Transitional form of chronic lymphocytic leukaemia/prolymphocytic leukaemia with a white
blood cell count of 250,000/L. Prolymphocytes are indicated (->).

Auer rods in cytoplasm of myeloblasts

Detection of Auer rods (->) in the cytoplasm of myeloblasts is characteristic of acute myeloid
leukaemia (AML).

B-ALL / Burkitt lymphoma

B-ALL/Burkitt lymphoma: The blasts typically show a deep blue cytoplasm and several
vacuoles. Tumour cells tend to be very fragile, resulting in 'smudge' cells (->) during blood film
preparation.

Severe aplastic anaemia

In severe anaemia (here severe aplastic anaemia with a haemoglobin concentration of 4.8 g/dL)
or in erythrocytosis with a haematocrit above 50% it is difficult to prepare a proper blood film,
with the blood film being either too thin (anaemia) or too thick. In cases of erythrocytosis it
might be necessary to dilute the blood sample beforehand.

Erythrocytosis
Pronounced erythrocytosis (polyglobulia) can often already be recognised after sedimentation of
the red blood cells. Left tube: haematocrit 82%, right tube: haematocrit 39%.

Iron deficiency anaemia

Patient with established severe iron deficiency anaemia (haemoglobin 5 g/dL). Well recognisable
are anulocytes (->) and unusually small red blood cells compared to a normal lymphocyte. The
MCV was 53 fL

Thrombocytopenia and detection of

cytoplasm fragments from monoblasts
Thrombocytopenia and detection of cytoplasm fragments from monoblasts in AML-M5A. Due
to their similar size the cytoplasm fragments can produce a falsely elevated platelet count in
impedance measurement on certain haematology analysers. Here the initial count was 32,000/L.
The correct platelet value was obtained from the optical channel; it was 7,000/L. Cytoplasm
fragments (C) and a platelet (P) are marked

Platelet satellitosis
Platelet satellitosis: Platelets adhere to granulocytes, induced by the anticoagulant EDTA.

Thrombocytopenia and platelet anisocytosis

Thrombocytopenia (45,000/L) and platelet anisocytosis as an indication of idiopathic
thrombocytopenic purpura (ITP) in a child after viral infection. IPF (immature platelet fraction)
in this case was 18.3%.

Giant platelet
Giant platelet with prominent granulation from a patient with essential thrombocythaemia (ET).
(Defective haematopoiesis causes detectable poikilocytosis.)

Schizont of plasmodium malariae

Schizont of plasmodium malariae in malaria quartana.

Band cells
Storage of normal EDTA blood over a period of two days can falsely elevate the number of band
cells (bottom right).

Granulocyte
Normal granulocyte of a healthy individual.

Frozen blood sample

Blood film of a normal blood sample, which had frozen in the postbox. One can only recognise
red blood cell 'shadows' or 'ghosts'.

HbH cell
Reticulocyte staining. Characteristic HbH cell in a case of -thalassaemia. Today, blood films
are no longer investigated for HbH cells. Instead,

-thalassaemias are diagnosed by molecular genetic tests.

Red blood cell with basophilic stippling and

polychromatic red blood cell
Large red blood cell with basophilic stippling (between the two lymphocytes) in a case of auto-
immune haemolytic anaemia (AIHA). To the right, next to the lower lymphocyte, there is a
polychromatic red blood cell.

Howell-Jolly body
An apparent Howell-Jolly body, caused by contaminated microscope oil, can be identified as an
artefact by the fact that it moves independently over time.

Howell-Jolly bodies
Howell-Jolly bodies in the cell adjacent to the granulocyte, spherocytes, polychromasia, and a
single erythroblast in a case of auto-immune haemolytic anaemia (AIHA). (Howell-Jolly bodies
are DNA remnants. Polychromatic cells still contain RNA.)

Acanthocytes
Acanthocytes in the presence of A--lipoproteinaemia. In cases of dysfunctional lipid
metabolism, the composition of red blood cell membranes is disturbed.

Echinocytes
Large number of echinocytes of a healthy individual resulting from prolonged storage of the
EDTA blood (48 hours).

Echinocytes
Echinocytes ('sea urchin cells') of a healthy individual resulting from prolonged storage of the
EDTA blood (24 hours).

Acanthocytes
Acanthocytes with protrusions of differing lengths and widths as seen in acute hepatic failure.
(This may be caused by modifications of the membrane-bound proteins.)

Red blood cells and lymphocyte

Red blood cells of a healthy individual. Normal red blood cells are a little smaller than the
'standard lymphocyte', which is pictured here.

Fragmented red blood cells and

thrombocytopenia
Fragmented red blood cells and thrombocytopenia in the case of a thrombotic thrombocytopenic
purpura (TTP). In the partially thrombosed capillaries the red blood cells are exposed to a high
degree of shearing force, which causes them to burst.

Auto-immune haemolytic anaemia (AIHA)

Phagocytosis of a red blood cell by a monocyte in a case of auto-immune haemolytic anaemia
(AIHA). The many spherocytes and polychromasia are clearly visible.

Clostridium perfringens
The barrel-shaped bacillus visible in the right granulocyte is Clostridium perfringens from a case
of septic gangrene. (The reddish background is caused by massive red blood cell lysis. The white
blood cells are about to dissolve as well.)

Streptococcal septicaemia
Accumulation of streptococci on a red blood cell of a patient in intensive care suffering from
streptococcal septicaemia (further to the right, a lymphocyte with a cytoplasm vacuole can be
seen).

Bacterial aggregates on blood film

Bacterial aggregates on the blood film caused by contaminated staining solution (dark spots).

Atypical lymphocyte
On the top right a morphologically atypical lymphocyte of a healthy individual with the
abnormality resulting from prolonged storage of the EDTA blood (24 hours).

Lymphocyte
Normal lymphocyte.

x
Segmented neutrophil

Cell description: Size: 12-15 m Nucleus: clumped chromatin and mostly divided into 2-5
distinct segments connected with filaments Cytoplasm: acidophilic with many fine reddish
granules spread evenly Function: phagocytosis, play an important role in the unspecific immune
defense, in the tissue they defend the mucosa against bacteria and fungi
Technology
A range of technologies to cover all bases

Sysmex diagnostic devices are known around the world for their reliability, accuracy and quality.
Since we were founded over forty years ago, we have been driven by the desire to innovate so
that we can do our job better. To improve the analysis capabilities of our devices so we can help
clinicians help people more effectively.

In those forty years it is fair to say we have changed the haematological analysis landscape.
Nowadays we are the global leader in the haematology market. This is to a large extent due to
our focus on knowledge and technology backed up by service and solution-based thinking.

Modern automated blood cell counters use various technologies to determine the parameters of a
full blood count. When EDTA blood samples are aspirated by the analyser, blood is aliquoted
(separated into small portions before being used in the various channels) and treated with various
reagents to support the cell-specific properties. A defined volume is separated from these reagent
blood mixtures and introduced into different measurement lines for detection. RBC and PLT are
measured simultaneously in one detector using DC sheath flow detection . A cyanide-free
reagent is used to transform the haemoglobin so that it can be measured by absorption
photometry .

Our 5-part differential haematology analysers use fluorescence flow cytometry as the prime
detection method for WBC, differential, reticulocytes and NRBC.

Fluorescence flow cytometry

Fluorescence flow cytometry (FFC) is used to analyse physiological and chemical properties of
cells. It can also be used to analyse other biological particles in urinalysis analysers. It provides:

Information about cell size and structure

Information about a cells interior

In flow cytometry, we examine cells and particles while they are flowing through a very narrow
flow cell.

First a blood sample is aspirated and proportioned, then diluted to a pre-set ratio and labelled
with a proprietary fluorescence marker that binds specifically to nucleic acids.
Next the sample is transported into the flow cell. The sample is illuminated by a semiconductor
laser beam, which can separate the cells using three different signals:

forward-scattered light (forward scatter or FSC)

side-scattered light (side scatter or SSC)
side-fluorescence light (side fluorescence or SFL).

The intensity of the forward scatter indicates the cell volume. The side scatter provides
information about the cell content, such as nucleus and granules. The side fluorescence indicates
the amount of DNA and RNA present in the cell.

Cells with similar physical and chemical properties form a cluster in a graph known as a
scattergram.

The principle of fluorescence flow cytometry is used in different analysers for haematology and
urinalysis. For blood cell counts we use fluorescence flow cytometry, e.g. for the WBC and
differential, for NRBC counting and reticulocyte measurement.

In urinalysis analysers, fluorescence technology is also used for counting bacteria, red blood
cells, white blood cells and other elements.

SLS detection method

Haemoglobin is a routine diagnostic parameter in each blood count. The method recommended
by the ICSH (International Committee for Standardization in Haematology) for measuring
haemoglobin concentration is the cyan-methaemoglobin method.

Our SLS haemoglobin detection method uses cyanide-free sodium lauryl sulphate (SLS). The
reagent lyses red blood cells and white blood cells in the sample. The chemical reaction begins
by altering the globin and then oxidising the haeme group. Now the SLS hydrophilic groups can
bind to the haeme group and form a stable, coloured complex (SLS-HGB), which is analysed
using a photometric method.

An LED sends out monochromatic light and by moving through the mixture light is absorbed by
the SLS-HGB complexes. The absorbance is measured by a photo sensor and is proportional to
the haemoglobin concentration of the sample.

Absorption photometric methods are usually influenced by the turbidity of the sample itself. In
blood samples, turbidity can be caused due to lipaemia or leucocytosis. By using the SLS-HGB
method these interferences can be minimised due to the effect of the reagent.

DC sheath flow detection method

Sysmex analysers use the DC sheath flow detection method to count red blood cells and
platelets, RBC and PLT. A portion of blood is separated from the aspirated whole blood and
mixed with the diluent in a pre-set ratio.

Of this dilution a defined amount is sent to the detection chamber and passed through a small
opening, known as the aperture. There are also electrodes on each side of the aperture and
direct current passes through these electrodes. The direct current resistance between the
electrodes changes as blood cells suspended in the diluent pass through the aperture. This
resistance causes an electrical pulse change proportional to the size of the blood cell.

These electrical data are converted into graphical displays of volume distribution curves, or
histograms.

Once the cells leave the sample nozzle exit they are surrounded by a sheath flow of diluent.
Here, they are aligned and moved to the centre of the orifice. This reduces interference errors and
the possibility of abnormal cell pulse detection, which could be caused by cells passing through
the transducer off-centre. As soon as the cells have passed the orifice, they are seized by another,
inverse flow and immediately led to the drain. This prevents renewed circulation and a change
in the platelet count.

WBC/BASO channel on XE/XT-Series

The WBC/BASO channel records forward scattered light and side scattered light signals.

Forward scattered light, or FSC, provides information about the cell size
Side scattered light, or SSC, describes the internal cell structure.

An acidic reagent (stromatolyser-FB) lyses the red blood cells and shrinks white blood cells to
bare nuclei, with the exception of basophils, which are not affected. The resulting differences
between basophils and other cells are analysed using forward and side scatter information.

The WBC/BASO channel is not available on XS-Series analysers.

The WBC count in CBC mode on XS-Series is performed with the Stromatolyser-4 DL reagent.
All red blood cells are lysed by this reagent in the WBC channel. The forward scatter signal is
used to count the WBC. The results are displayed in a histogram. In the CBC+DIFF mode, the
WBC count is taken from the fluorescence signals from the DIFF channel after treating the cells
with Stromatolyser-4 DS (fluoromarker).

RET/PLT-O Channel
In the RET/PLT-O channel, the surfactant in RET-SEARCH (II) slightly perforates the cell
membranes of red blood cells, white blood cells and platelets and so allows the fluorescence
marker to penetrate the cell. The fluorescence marker then labels the nucleic acids in white blood
cells, NRBC, reticulocytes and platelets. Using the forward scattered light and the fluorescence
signal, the reticulocytes can be separated from mature red blood cells, white blood cells and
NRBCs.

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.

The RET channel also provides the optical platelet count and is available on the XT-2000i, XT-
4000i, XE-2100 and XE-5000 haematology analysers.

IMI channel
The IMI channel selectively detects cells of the immature myeloid series. When exposed to
Stromatolyser-IM reagent, the membranes of mature cells are damaged as they contain a high
lipid concentration, and the intracellular components are eluted.

The immature cell membrane contains a small amount of lipids and a high concentration of
amino acids. The membranes of these cells are damaged as well, but before elution of
intracellular components can occur, the reagent enters the cell and fixes both the cell membrane
and intracellular components. Using the DC/RF detection method, Sysmex analysers can
distinguish between blast cells, immature granulocytes (myelocytes, metameylocytes,
promyelocytes) and band cells.

The IMI channel is also sensitive in detecting platelet aggregation. This channel is only available
on XE-2100 and XE-5000. Another special application is the enumeration of stem cells in
samples from patients/donors undergoing peripheral blood stem cell harvesting.

WBC differential channel

Analysing differentials consists of a cytochemical reaction of the cells with a reagent, followed
by a fluorescence flow cytometric analysis.
A surfactant in the reagent induces the lysis of red blood cells. At the same time, cell membranes
of white blood cells are perforated with ultramicroscopic pores through which a polymethine-
based fluorescence marker migrates and binds to nucleic acids. This prepared sample is then
analysed using fluorescence flow cytometry.

The measurement signals related to side scatter (SSC) and side fluorescence (SFL) are analysed
and depicted in a scattergram. Cells with similar cytochemical properties fall within the same
area in the scattergram and can be separated using an advanced software algorithm.

The differential channel provides counts of all normal white blood cell subpopulations as well as
flag information in cases of abnormalities.

NRBC channel
In the NRBC channel, a surfactant in the Stromatolyser-NR reagent lyses the red blood cell
membranes, leaving only the nuclei of nucleated red blood cells (NRBC) intact. It also perforates
the cell membrane of white blood cells, in which the polymethine fluorescence marker of
stromatolyser-NR labels the nucleic acids in the nuclei as well as cytoplasmic organelles.

NRBC, on the other hand, have lost their cytoplasm after the reaction so that the fluorescence
marker only labels nucleic acids in the nucleus. This results in two distinct populations based on
differences in fluorescence intensity. NRBC are separated from white blood cells due to their
lower fluorescence intensity.

The white blood cell and lymphocyte count results are automatically corrected if NRBC are
detected in the NRBC channel, prior to reporting.

This channel is only available on XE-2100 and XE-5000.

DIFF Channel
A surfactant in Stromatolyser-4DL causes lysis of red blood cells. At the same time, the cell
membranes of white blood cells are slightly perforated.

The polymethine fluorescence marker Stromatolyser-4DS migrates into the WBC and binds in
particular to nucleic acids and cell organelles. Additionally an organic acid in the Stromatolyser-
4DL binds specifically to the granules in eosinophils as a result of which they can be
differentiated from neutrophils by means of a higher side scatter signal.

Our XE- and XT-Series analysers provide five populations of white blood cells in the DIFF
scattergram, including the Immature Granulocyte population.

The XS analysers scattergrams displays the following five populations: lymphocytes,

monocytes, basophils, eosinophils and neutrophils.
Immature Granulocyte (IG) count
What is an IG count?

With the exception of blood from neonates or pregnant women, the appearance of immature
granulocytes in the peripheral blood indicates an early-stage response to infection, inflammation
or other stimuli of the bone marrow. Being able to detect them quickly and reliably opens doors
to new diagnostic possibilities for related disorders.

Current areas of research regarding the diagnostic significance of circulating immature

granulocytes focus on the early and rapid discrimination of bacterial from viral infections,
particularly in children, recognising bacterial infection in neonates, and the early recognition of
bacterial infection and sepsis in adults, which is of vital importance in particular for intensive
care patients.

Valuable information for immediate action at no additional cost

The high accuracy of our IG counting method provides a valuable tool for physicians in
concluding a diagnosis or requesting further patient investigation. When samples are flagged for
the presence of IG on conventional haematology analysers, they usually require a microscopic
differential count. Our automatic IG counting reduces the review rate significantly. Moreover,
results including the presence and concentration of IGs become available within minutes and
are included in the complete CBC+DIFF analysis.

Many of our devices offer an IG, or immature granulocyte count. They are used especially for
patients who are highly susceptible to infections because of a suppressed immune system and
because the increased IG count reflects an active response by the immune system. In addition to
patients with general infections and inflammations, clinicians will pay particular attention to:

Patients from the intensive care unit,

Patients undergoing chemotherapy,
Patients suffering from HIV/AIDS.

Benefits
Sysmex devices are especially powerful as we provide an actual IG count [%] and not just a
flag for immature granulocytes. We also provide an absolute IG count that considers the ratio
of immature granulocytes and the total of neutrophils.

Automated IG counts generally mean you can reduce the number of blood smears and the
workload by manual counting.

Using IG count
The IG count alone does not let you predict sepsis or infection. However, it can support
diagnosis and prediction together with other parameters such as cytokines, interleukins and
CRP. It is more useful as a monitoring parameter when the patient has already been diagnosed
correctly and is under treatment.

Blood samples of unknown patients with an increased IG count should usually undergo blood
smear preparation and morphological examination to distinguish between malignant and reactive
conditions. In known patients, an automated IG count can avoid the manual review while
therapy monitoring infections or inflammation.

The IG count of paediatric patients, especially premature neonates or neonates younger than
seven days, has to be taken with care due to their immature immune systems and the greater
number of immature cells in the circulating blood

More than error correction

Because nucleated red blood cells (NRBC) have a size and a nucleus similar to that of
lymphocytes, many haematology analysers misclassify NRBC and produce a wrong total
leukocyte and lymphocyte count.

Usually such samples are flagged for microscopic analysis. You then have to count the NRBC in
the blood film manually and mathematically correct the total leukocyte and lymphocyte count.
This is a costly, tedious and error-prone procedure. In addition, if the sample is not flagged, the
NRBC may remain undetected and the total leukocyte and the lymphocyte count may be wrongly
elevated. Many still think automated NRBC measurement is no more than a way of correcting
these counts. Yet the clinical value of measuring NRBC goes far beyond correcting the total
leukocyte and lymphocyte count.

NRBC are seen as a reflection of extreme increases in erythropoietic activity, such as in acute
haemolytic episodes and severe hypoxic stress, or as a result of a haematological malignancy.
This includes many leukaemias and myelodysplastic syndromes, and some kinds of lymphoma.
NRBC can also be present in thalassaemia syndromes, bone marrow metastases of solid tumours,
extramedullary haematopoiesis and other conditions of haematopoietic stress such as sepsis, or
massive haemorrhages. In these situations, their presence is correlated with the severity of the
disease. It has been observed that the entity and duration of the presence of NRBC in peripheral
blood is associated with a poor prognosis in several haematological and non-haematological
diseases.

Where to use NRBC

The NRBC counts provided on many of our analysers are used frequently for new-borns and
young infants. In those patients, NRBC can occur physiologically in high numbers - up to 100
NRBC/100 WBC are possible in premature babies - and are used to correct WBC numbers. It is
useful especially in patients from the neonatology or paediatric department or patients from
paediatric private practices.

NRBC counting is also useful for patients with severe anaemia, especially those with
thalassaemia or sickle cell disease as they usually have high NRBC counts, too. The NRBC
count is important for differential diagnosis and can support patient monitoring to determine
transfusion needs.

NRBC counting is important for all patients from the intensive care unit as detecting NRBC can
indicate an increased mortality risk, and for patients with any condition producing
haematopoietic stress such as severe infection, hypoxia or massive acute haemorrhage. This too
can lead to circulating NRBC.

Benefits
Sysmexs analysers provide a sophisticated NRBC count that is accurate for both high and low
counts. This accuracy is needed because:

In neonate blood samples and others with high NRBC counts the WBC count needs to be
corrected
In adult blood samples even a low NRBC count can be meaningful.

Since we use a separate reagent for NRBC detection, we actually count the cells instead of
estimating them. The NRBC count is fast and inexpensive and on our flagship XN analysers is
included in every measurement. On our X-Class analysers it is performed when needed. NRBC
are reported in % (per 100 WBC) and # (per L).

Using NRBC in routine

In healthy adult patients, the automated NRBC count on a haematology analyser should be zero!
In those who are not in perfect health, the most important benefit of an NRBC count is to
exclude a false high in the WBC count. This could lead to an incorrect diagnosis and treatment,
especially in neonatal patients with sepsis and low WBC counts. Additionally, an NRBC count
should be included routinely for all paediatric and neonatal samples. It is strongly recommended
for severely diseased adult patients as it can indicate whether patients need special attention and
treatment.

Immature Platelet Fraction IPF

What is Immature Platelet Fraction counting?

The immature platelet fraction (%IPF) is a modern parameter that measures young and thereby
reticulated platelets in peripheral blood. The reference range is approximately 1 to 5% of the
total platelet count.

IPF levels rise as bone marrow production of platelets increases. Therefore its measurement
provides an assessment of bone marrow platelet production from a peripheral blood sample, in a
similar way to how a reticulocyte count could provide a measure of red cell production.

There is a high clinical utility of the %IPF in the laboratory diagnosis and treatment of
thrombocytopenia due to the ability to relate raised %IPF levels with increased peripheral
platelet destruction. It is particularly useful for supporting the diagnosis of autoimmune
thrombocytopenic purpura, thrombotic thrombocytopenic purpura and for distinguishing these
from bone marrow suppression or failure. In the case of the latter, the %IPF value would be low.

The %IPF can also be a sensitive measure for evaluating thrombopoietic recovery during aplastic
chemotherapy. In some specialist haematology and cancer centres, for instance, %IPF is taken
into consideration in platelet transfusions. Transfusions may only be considered when the %IPF
values are not rising as this would indicate poor intrinsic thrombopoietic activity.

Where to use IPF?

Since destruction-mediated thrombocytopenic diseases are fairly rare, IPF is most useful in
environments that service a large number of patients. Good examples include laboratories of
large hospitals with haemato-oncologic units, paediatric units / neonatology units for differential
diagnosis of juvenile thrombocytopenia and/or monitoring of the course of thrombocytopenia.

Benefits
The IPF count supports clinicians in differentiating between consumptive versus productive
reasons for thrombocytopenia and helps avoid a bone marrow biopsy with obvious benefits for
the patient. This clinical utility in cases of thrombocytopenia is proven. Its usefulness in
monitoring after chemotherapy and haematopoietic stem cell / bone marrow transplantation has
been suggested.

Reticulocyte haemoglobin equivalent RET-

He
What is Ret-He the Reticulocyte
haemoglobin equivalent?
Measuring the haemoglobin content of reticulocytes, RET-He or reticulocyte haemoglobin
equivalent, is a way of diagnosing and monitoring iron deficiency anaemia. Red blood cells have
a 120-day lifetime. Therefore, detecting iron deficiencies and changes in the iron status of
erythropoiesis is only possible relatively late using classical haematological parameters such as
HGB, MCV, MCH, or by measuring hypochromic red blood cells (%Hypo-He).

Reticulocytes, the precursors of mature red blood cells, are swept into the blood stream from the
bone marrow and usually mature over the course of around two 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.

Conventional biochemical markers for assessing the iron status, such as serum iron, transferrin or
ferritin, are so drastically disturbed e.g. during inflammation in the course of an acute phase
response but also in the presence of many other severe diseases that a clinical interpretation of
the results is difficult or impossible.

So while low ferritin levels, for example, unequivocally indicate a lack of iron, normal or
elevated levels do not let you draw any conclusions as to the bioavailability of the iron. In the
presence of chronic diseases such as rheumatoid arthritis, but also in the presence of liver
damage, tumours or chronic kidney disease, ferritin can also be elevated in the case of functional
iron deficiency. In functional iron deficiency, iron stores can be filled, but the iron is not
sufficiently released to the blood flow and therefore not bioavailable for the erythropoiesis. On
the other hand, measuring the haemoglobin content of the reticulocytes as a direct assessment of
the iron actually used for the biosynthesis of haemoglobin can indicate whether there is enough
iron available for erythropoiesis even in these cases. It lets you take a snapshot of the quality
of erythropoiesis and is an important tool for diagnosing and monitoring iron deficiency diseases.

Where should you use RET-He?

Anaemia is a common symptom of several diseases and one of the most underestimated red
blood cell disorders. As a result, knowing a patients erythropoietic status can be essential. Many
of our analysers offer the RET-He parameter the reticulocyte haemoglobin equivalent. It is
often used for patients with nephrological (kidney) disorders as they frequently suffer from
anaemia in parallel. It is therefore especially important to include patients from the nephrology
department or patients from dialysis centres and practices in analysis.

Ret-He is important for patients with anaemia of chronic disease (ACD). Any patient with a
chronic inflammatory process, chronic infection or malignancy can develop ACD.
Patients with iron deficiency anaemia (IDA) will also benefit. IDA is widespread,
underdiagnosed and can be found in a variety of patients. Some paediatric patients are vulnerable
to developing IDA due to the growth phase.

Benefits
The clinical usefulness of the Ret-He parameter has been proven and it is now an established
parameter in advanced haematological analysis. Reticulocyte haemoglobin content is
recommended in nephrology guidelines such as the European Best Practice Guidelines (EBPG),
National Kidney Foundation Kidney Disease Outcome Quality Initiative (NKF KDOQI).

Ret-He:

Indicates the trend of the current iron status.

RET-He and RET# together 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 and inexpensive!

Using RET-He
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 while low ferritin values together with low RET-He suggest a classic iron deficiency.
Since ferritin is falsely increased during the acute phase of diseases, inflammation should be
checked, e.g. by CRP.

The reference range for RET-He is approximately 28-35 pg [~1.77-2.22 fmol], below 28 pg [1.77
fmol] is considered iron deficient.

RET-He is used for monitoring erythropoietin (EPO) and/or IV iron therapy. If the value
increases it indicates the therapy is having a positive effect.

Delta-He
While RET-He is a measure of the haemoglobin content in reticulocytes, the research parameter
RBC-He gives information about the haemoglobin content of mature red blood cells. The
research parameter Delta-He is derived from the difference between these two parameters.
Delta-He values above the normal range may indicate an improvement in erythropoiesis, for
example after EPO and/or iron therapy. In contrast, when Delta-He values are below the normal
range for longer periods, this may indicate the onset of an anaemia.
The microcytic (MicroR*) and macrocytic
(MacroR*) red blood cell populations
Red blood cells (RBC) and platelets (PLT) are counted in the RBC/PLT channel using the sheath
flow DC (direct current) detection method. Hydrodynamic focussing is used so that only single
cells pass through the detector, and the resulting RBC size distribution shows a nearly Gaussian
distribution. Values for the MicroR and MacroR parameters are obtained from either end of the
RBC histogram. The RBC histograms of samples with microcytic RBC are shifted to the left and
often exhibit a shoulder on the left due to an increase in small RBC. In contrast, samples with
macrocytic RBC generate histograms exhibiting a longer slope on the right. By applying two
distinct discriminators at the lower and upper area of the histogram, a microcytic and a
macrocytic population of red blood cells can be determined, and the resulting parameters reflect
the microcytic (MicroR) and macrocytic RBC (MacroR) as a percentage of all red blood cells.
In certain diseases (for example, in myelodysplastic syndrome), patients may have an MCV
value that is within the reference range despite having an increased MicroR or MacroR. The
parameters MicroR and MacroR are therefore useful to narrow down the possible causes of
anaemia.

Figure: Microcytic RBC (MicroR, upper panel) and macrocytic RBC (MacroR, lower panel) in
the RBC histogram

*The parameters MicroR and MacroR are research parameters on XN-Series analysers and part
of the complete blood count (CBC). On the X-Class analysers XT-4000i and XE-5000 they are
named %MicroR and %MacroR. Research parameters should not be used for in vitro
diagnostics.
Percentage of hypo-haemoglobinised red cells
(HYPO-He*) and hyper-haemoglobinised red
cells (HYPER-He*)
HYPO-He and HYPER-He are parameters analysed in the reticulocyte (RET) channel. They are
derived from the haemoglobin content of all mature RBC (RBC-He), which can be calculated
based on the high-angle forward scatter (FSC). The FSC signal is converted into picograms (pg)
using a proprietary algorithm and in healthy individuals the resulting RBC-He value is
comparable to the mean corpuscular haemoglobin (MCH) value, which is calculated using the
HGB and RBC parameters that are measured as part of a complete blood count (CBC) by the
HGB and RBC/PLT channels in Sysmex instruments. Subsequently, this RBC-He value is used
to determine HYPO-He and HYPER-He: 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. While mature RBC are separated from immature
cells in the RET scattergram based on fluorescence intensity (which is directly proportional to
RNA content), hypo-haemoglobinised and hyper-haemoglobinised red cells are classified based
on their high-angle forward-scattered light signal (which, as explained above, is directly
proportional to haemoglobin content).

Figure: 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. The left panel shows a sample from a healthy individual with
HYPO-He less than 1% and the right panel shows a sample with HYPO-He more than 60%.

* HYPO-He and HYPER-He are research parameters on XN-Series analysers that have the RET
channel. In the XT-4000i and XE-5000 analysers these research parameters are measured with
the RET channel and named %HYPO-He and %HYPER-He. Research parameters should not be
used for in vitro diagnostics.

Fragmented red blood cells (FRC*)

Sysmex analysers use the fluorescence flow cytometry method in the reticulocyte channel to
measure fragmented red blood cells (FRC% and FRC#) as research parameters.

A specific area below the RBC area in the RET scattergram is used for identification of
fragmented red blood cells. Due to the absence of nucleic acids in red blood cells the intensity of
the measured side fluorescence signals (SFL) is extremely low. In addition, the high-angle
forward scatter (FSC) is lower than that of intact red blood cells.

Figure 1: each cell is plotted in the RET scattergram based on its fluorescence intensity (SFL on
x-axis) and its high-angle forward scatter (FSC on y-axis), which reflects characteristics of both
cell size and cellular content. Fragmented red blood cells (FRC) are visible below the RBC
population.

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 destroy the
integrity of the red blood cells, producing cell remnants that appear as helmets (cells with two
tapered and hornlike projections on either end) and other odd shapes when viewed under a
microscope.

Figure 2: peripheral blood smear with helmet cells (arrows).

* FRC% and FRC# are research parameters obtained in the RET channel on XN-Series and X-
Class analysers (XT-4000i and XE-5000). Research parameters should not be used for in vitro
diagnostics.

Neutrophil granulation (NEUT-SSC*)

The 90 degree side-scattered light (SSC) of the WBC differential channel provides information
about cell density or complexity, which represents the granularity of the cells. Neutrophils and
eosinophils exhibit the highest SSC of all normal cell populations, especially when compared to
mononuclear cells. The parameter NEUT-SSC is a measure of the granularity of the neutrophil
population. Hypogranular neutrophils have a low NEUT-SSC value whereas a high NEUT-SSC
value is indicative of hypergranularity.

Hypogranularity, identified by a reduced NEUT-SSC value, is a feature of neutrophil dysplasia,

which is commonly observed in myelodysplastic syndromes (MDS). The automated detection of
hypogranular neutrophils is of great diagnostic value in differentiating between MDS and
reactive and benign idiopathic and hereditary causes of neutrophilia. Benign conditions can be
ruled out if dysplasia is present, and therefore a neutrophilia in conjunction with a low NEUT-
SSC value would suggest an underlying myeloproliferative disorder. Myeloproliferative
disorders that show a dysplasia include chronic myelomonocytic leukaemia (CMML) and
atypical chronic myeloid leukaemia (aCML). These two conditions can be distinguished by
considering the relative prominence of monocytes in relation to neutrophils, as well as immature
granulocytes (IG).

Figure: the SSC signal of the neutrophil population, which is plotted on the x-axis of the
scattergram, is an indication of the granularity and internal structure of the cells. Fluorescence
intensity, which corresponds to RNA/DNA cell content, is plotted on the y-axis.
 *The NEUT-SSC is a research parameter from the WDF channel on XN-Series analysers. In X-
Class analysers the parameter is named NEUT-X. Research parameters should not be used for in
vitro diagnostics.