ISSUE15

ISSUE

July
2021

This issue
Last Month’s Slides P.1
Monthly Case study P.1
Paroxysmal Nocturnal
Haemoglobinuria P.2-3
Cell Quiz P.2
Morphology Tip P.3

Last Month’sSlides
Side 1
Presence of blasts/granularblasts.
Probable AML 1

Slide 2
Oncology Patient.
Monomorphic
hyperlymphocytosis.
Presence of atypical lymphoblasts
with irregular nuclei.

Slide 3
See case study on right

Slide 4
Nothing abnormal noted

Slide 5
Nothing abnormal noted

Slide 6
Nothing abnormal noted

Monthly Digital Case study
June 2021
Presentation
Male 77 years old.

FBC ResultsWBC46.9 (10^3/mm3)RBC
3.13 (10^6/mm3)
HGB
9.9 (g/dL)
HCT
29.4 (%)
MCV
94 (fL)
MCH
31.6 (pg)
MCMH 31.6 (g/dL)
PLT
36 (10^3/mm3)
Neutrophils 22.9 %Lymphocytes 72.0 %Monocytes 1.0 %Eosinophils 0.0 %Basophils 0.0 %Myelocyte

Slide review
Probable lymphoproliferative disorder
Sezary Cell
Immunophenotyping of circulating Lymphocytes.
Sezary cell?
T-cell lymphoproliferative syndrome, presence of granular lymphocytes.
Cancerous T-cells. Called Sezary cells, characterised by an abnormallyshaped nucleus, known as Cerebriform (looks like a brain).

Cell Quiz
From the following choose thedescription to best describe theprominent feature in thedisplayed NeutrophilA) Left ShiftB) Neutro-Toxic Granulation
C) Hypo GranularD) ActivatedLast Month’s Cell
Quiz
Look at the slide & choose thecorrect red cell from themultiple choice below:

Answer:C) Tear drop cell
Teardrop cells, or dacrocytes, are
red blood cells with one round end
and one pointed end. Teardrop cell
formation may be multifactoral
but appears to involve thedistortion of the red cells as they
pass through the marrow orspleen. They are a type ofpoikilocyte which can be seen in
the morphology of patients with:• Beta thalassaemia,• Myelofibrosis,• Leukaemia,• Megaloblastic anaemia,• Haemolytic anaemia

Paroxysmal Nocturnal Haemoglobinuria(PNH)
PNH is a rare haematopoietic stem cell disorder (estimated to affect 1-10 cases
per million) first recognised as a distinct clinical entity in the 1800’s. PNH isclinically heterogenous with some patients having a disease which presents withoverwhelming intravascular haemolysis whilst in other bone marrow failure is
the predominant feature. PNH can occur at any age of life and is a life longcondition. Approximately half of patients with PNH will die as directconsequence of their disease, many others are transfusion dependent fordecades.
In 1882, Paul Strübing recognised PNH as being distinct from Paroxysmal Cold
Haemoglobinuria and March Haemoglobinuria after observing the episodic
occurrence of haemoglobinuria in a 29 year old cartwright. He recognised thatthe red cells had been haemolysed in the blood vessels rather than the kidney
i.e. few or no intact red cells in the urine. He discovered that haemoglobinuria
was only present during the night (apart from upon wakening), therefore theterm PNH was derived. He hypothesized that symptoms of PNH were aconsequence of the abnormal sensitivity of PNH red cells to acidosis resulting forthe accumulation of CO2 during sleep. This hypothesis was used to develop thefirst diagnostic test for PNH – the Ham’s test (acidified serum lysis test) developed by Thomas Ham in the 1930’s and to the pivotal role complement
plays in the lysis if red cells in PNH.
In the 1930 – 1960’s it was discovered that PNH red cells were particularly
sensitive to complement mediated haemolysis which focussed research onto theexact reasons for this. It was also recognised that PNH red cells from the same
person have different susceptibilities to complement mediated haemolysis with
one cohort being many times more sensitive than the other. Investigation of thePNH red cell structure identified that there was a deficiency of multiple cellsurface proteins and these were identified as being GP1 anchored proteins. In
1993 the exact genetic mutation was identified by Dr Kinoshita et al as being amutation of the PIGA gene. The two most important proteins in terms of
inducing complement-mediated intravascular haemolysis are CD55 (decayaccelerating factor [DAF]) and CD59 (membrane inhibitor of reactive lysis –[MIRL]). There is no evidence that GPI anchored proteins other than CD55 andCD59 contribute to the clinical pathophysiology of PNH, therefore can be usedas diagnostic markers.

Although PNH is a clonal disease, it is not a malignant disease in that the clone
does not expand exponentially and does not invade sites outside of the bonemarrow. Some patients have only a very small clone of PNH affected cells which
can only be detected by high sensitivity flow cytometry whilst other patients
have large clones which all haematopoiesis is derived from the mutant stem cell.
PIGA is located on the X chromosome, therefore only one mutational event isnecessary to produce PNH cells, as males only have 1 X chromosome andfemales only 1 of the 2 X chromosomes is active in somatic tissues. AplasticAnaemia is associated with PNH and it is possible that PNH is as a consequenceof Aplastic Anaemia. Patients with PNH suffer from a degree of bone marrow
failure which exacerbates anaemia if there is ongoing intravascular haemolysis.

Paroxysmal Nocturnal Haemoglobinuria
As free haemoglobin is released into the serum, it irreversibly binds to Nitric Oxide
which decreases its concentration. Nitric Oxide is a key molecule in homeostasis
and the lower levels led to smooth muscle dysfunction, platelet activation andother consequences. This results in severe disabling abdominal pain, dysphagia,
profound lethargy and in men erectile dysfunction.
Approximately 50% of all PNH patients have thrombotic events the most commonof which are venous thrombosis of the Liver (Budd-Chiari syndrome), abdomen(portal, mesenteric, spleen) and the brain. The risk of developing thrombosis iscorrelated to the proportion of PNH clones and the degree of intravascularhaemolysis.
Diagnosis of PNH
Basic Tests:
FBC, Reticulocytes
Urine Haemoglobinuria
Urine Haemosiderin
Advanced TestFluorescent Aerolysin (FLAER) flow cytometry assay
Flow Cytometry for CD55 and CD59
Treatment:
Treatment depends upon the severity of the disease with the most severe
benefitting from Eculizumab which blocks the activation of complement, thereby
protecting the PNH cells from haemolysis.
Blood transfusion is given to maintain red cell levels in the less severe forms ofPNH along with Folic acid, Iron and Iron chelating treatments.
The only cure for PNH is a Allogenic Bone Marrow Transplant.

MORPHOLOGY TIP
EDTA tubes are routinely used in laboratories to prevent clotting and sample storage.
Prolonged storage of samples in EDTA can, however, cause morphological changes to theblood sample, which are visible on a blood film. Erythrocytes are particularly effected. The
timing between sample collection and analysis is therefore crucial. EDTA effects over 24hours can include crenation, spiculation and the formation of echinocytes or Burr cells.
Artefact can also occur, making a clear examination of the blood film difficult.
Identifying storage related changes is therefore important, so that artefactual changes are
not misinterpreted as true pathological findings.
If in doubt, ALWAYS check the date and time taken on a sample suspected of showing
EDTA changes.

QSP 2.0
Available as a single use
license and a site license
which allows up to 50
concurrent users.
To find out more, contactus.

Bibliography
Prolonged Storage of Bloodin EDTA.
Samual Antin-Balfour
International J.ofEngineering, 2013 1(12) 2023
Update on the diagnosis andmanagement of PNH
Hematology Am Soc
Hematol Edu Program 2016
Dec2; 2016 (1):208-216
Historical Review –Historical Aspects of PNH:
“Defining the Disease”.
British Journal of
Haematology 2002 117, 222
www.Pnhleeds.co.uk

Editorial Team
Kelly Duffy
Andrew Fisher

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Red cells showing EDTA deterioration