CLARE DOES SCIENCE!!!!
There's something new to be learnt everyday as a Biomedical Scientist. This is a record of my continous professional development .
About Me
- Clare
- I'm a busy Mum and a Biomedical Scientist in Haematology. My particular interest is in blood cell morphology and parasitology, where I never stop learning.
Monday 20 November 2023
Which Parameters are truly useful in XbarM analysis and which are a waste of time!
Tuesday 26 September 2023
Unstable Haemoglobin Causing Inaccurate White Cell Differential on Sysmex XN
Look at these white cell differentials analysed on the
Sysmex XN. The first run to the right labelled run 1, gives a neutrophil count of
1.21 x 109/L and Eosinophils of 8.5 x 109/l. The white cell differential on second run
is completely reversed.
This can also be clearly seen on the WDF plots. On run 1, the large orange population of white cells were counted as eosinophils. On the second run, they were counted as
neutrophils represented by the blue population.
Run 1 |
Run 2 |
It is also clear that the white cell populations are
‘squashed’ and restricted to lower portion of the plot. Usually all populations would be
higher up the plot, with the monocytes being the highest . This is because side flouorescent
light (SFL) corresponds to the amount of DNA and RNA in a cell and monocytes have more than other normal white cells.
The inaccuracy of the white cell differential here, was caused by an unstable haemoglobin. These are haemoglobinopathies where the solubility of haemoglobin is altered, leading to precipitates of haemoglobin and haemolytic anaemia due to reduced red cell lifespan.
The reason the unstable haemoglobin leads to inaccuracies in the white cell differential, is because it does not act in the same way as it’s stable
counterparts when the red cells are lysed with Lysercell WDF on the Sysmex XNs.. This is an
important step in the measurement of white blood cells. Reading up on this White_Paper_RBC_Diseases.pdf
(sysmex-europe.com), it is suggested that the unstable haemoglobin
interferes with the fluorescence marker used to differentiate white blood cell
populations, leading to decreased flouorescence and inaccurate differentials. This is why the entire plot is shifted down and appears that all cells have little DNA or RNA content.
Therefore if you encounter an abnormal plot where the
fluorescence is reduced, it is firstly important to perform a manual
differential. Secondly there should be the suspicion of an unstable haemoglobin particularly if haemolysis is present and HPLC and or/genetic sequencing would be indicated.
Tuesday 25 October 2022
Gap analysis on BCSH 2022 Guidelines on the Laboratory Diagnosis of Malaria
Today I used the BCSH audit template to perform a gap analysis on our procedures for the diagnosis of malaria, in comparison to the BCSH 2022 guidelines. British Society for Haematology guidelines for the laboratory diagnosis of malaria - Rogers - 2022 - British Journal of Haematology - Wiley Online Library.
I would recommend that all staff involved in laboratory malaria diagnosis, read the 2022 guidelines and also the following:
WHO information on ‘False Negative RDT results
and p.falciparum histidine-rich protein 2/3 gene deletions MAY 2016 (REV.
SEPTEMBER 2017 AND JULY 2019) https://apps.who.int/iris/bitstream/handle/10665/258972/WHO-HTM-GMP-2017.18-eng.pdf?sequence=1
Learning points- HPR2 deletions in P.Falciparum and how this may lead to a
negative RDT, prozone effect, frequent
negative RDT test for P.Ovale, inclusion of Babesia in the differential
diagnosis when the RDT is negative.
Date of completion |
10/10/22 |
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Name of lead author/ |
Clare Saddington |
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Specialty |
Haematology |
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Title |
An audit of
compliance with the British Society for Haematology guideline on laboratory
diagnosis of malaria |
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Background |
The British
Society for Haematology (BSH) has published guidance on the laboratory
diagnosis of malaria. This audit will review
compliance with some of the recommendations made. |
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Aim & objectives |
To review
whether: 1.
suspected malaria cases
are being appropriately tested 2.
cases of malaria are
being accurately diagnosed and appropriately assessed. |
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Standards & criteria |
If the target (specified as 100% or 0% for each criterion)
is not achieved, there should be documentation in the case notes that
explains the variance. 1.
Both thick and thin films should be routinely
prepared for malaria diagnosis; target 100%. 2.
Thin films should be stained with a Giemsa
stain and thick films with either a Giemsa or Field stain. Giemsa should be
used at pH 7.2; target 100%. 3.
Thick films should be examined by two trained
observers, each viewing a minimum of 200 high power fields; target 100%. 4.
If thick films are positive, the species
should be determined by examination of a thin film, again by two trained observers;
target 100%. 5.
In the case of Plasmodium falciparum or
Plasmodium knowlesi infection, the percentage of parasitised
cells or the number of parasites per microlitre should be estimated and
reported; target 100%. 6.
Rapid diagnostic tests (RDTs) for malarial
antigen should not be used instead of a film at any time including out of
hours; target 0%. 7.
All positive specimens or discrepant results
between RDT and films should be referred to a reference laboratory; target
100%. |
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Method
|
1. Sample
selection ·
All
requests for investigation of possible malaria parasites, either in a period
of one month or to give a total of 30 requests (whichever is more
appropriate).
2. Data to be
collected on proforma (see below). |
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Results
|
The results of this audit show the following compliance
with the standards.
|
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Conclusion |
Laboratory
procedure is compliant with the above standards in BCSH 2022 guidelines on
the laboratory diagnosis of Malaria.
I
would recommend however that all staff involved in Malaria diagnosis read the
following: ·
WHO information on ‘False Negative RDT results
and p.falciparum histidine-rich protein 2/3 gene deletions MAY 2016 (REV.
SEPTEMBER 2017 AND JULY 2019) https://apps.who.int/iris/bitstream/handle/10665/258972/WHO-HTM-GMP-2017.18-eng.pdf?sequence=1
Learning points- HPR2 deletions in P.Falciparum and how this may lead to a
negative RDT, prozone effect, frequent
negative RDT test for P.Ovale, inclusion of Babesia in the differential
diagnosis when the RDT is negative.
·
Also an awareness of guidance from Advisory
Committee for dangerous Pathogens (2015) Management
of Hazard Group 4 viral haemorrhagic fevers and similar human infectious
diseases of high consequence (publishing.service.gov.uk)
These
documents should be read as part of the Parasitology competency reassessment.
Staff where appropriate should
participate in NEQAS Blood Films for Parasites on a regular basis.
|
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Recommendations
for improvement
|
Present the result
with recommendations, actions, and responsibilities for action and a
timescale for implementation. Assign a person(s) responsible to do the work
within a time frame.
Some suggestions: ·
highlight areas of practice that are different ·
present findings. |
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Action plan |
Incorporate
recommendations of BCSH guidelines 2022 guidelines into new draft of
parasitology SOP RWF-BS-HAEM-SOP50 version 1.5. Include section on False
negative RDTs. Target date : October 2022 Responsibility : Clare Saddington
Distribute
link (or a hardcopy) of BCSH guidelines 2022 for the laboratory diagnosis of
Malaria British Society for Haematology
guidelines for the laboratory diagnosis of malaria - Rogers - 2022 - British
Journal of Haematology - Wiley Online Library WHO document on ‘False Negative RDT results
and p.falciparum histidine-rich
protein 2/3 gene deletions MAY 2016 (REV. SEPTEMBER 2017 AND JULY
2019) https://apps.who.int/iris/bitstream/handle/10665/258972/WHO-HTM-GMP-2017.18-eng.pdf?sequence=1 and Advisory Committee for dangerous
Pathogens (2015) Management of Hazard Group 4 viral
haemorrhagic fevers and similar human infectious diseases of high consequence
(publishing.service.gov.uk) Target date November 2022 Responsibility : Clare Saddington
Senior
BMS to provide a training/ review session of the above guidelines. Target date: January 2023 Responsibility : Clare Saddington
BMS
to participate in NEQAS blood films for parasites on a regular basis. Senior
BMS to coordinate this.
Target date: May 2023 Responsibility : Clare Saddington/
Adam Schofield
|
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Re-audit date |
October
2023 |
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Reference |
Rogers CL, Bain BJ, Fernandes S,
Garg M, Mooney C, Chiodini PL et al. British Society for Haematology guidelines
for the laboratory diagnosis of malaria. Br J Haematol 2022 (Epub ahead of print). |
Tuesday 12 July 2022
Cancer cells adapt to store energy like muscle cells, so that they can multiply!
So the first patient’s slide I picked up today was quite
interesting. A known Chronic Lymphocytic Leukaemia (CLL) which had
numerous vacuoles in the cytoplasm of the lymphocytes. Although i've seen this before, I wouldn’t say this is a
feature that is really common and I wasn’t entirely sure why these were present.
So reading up on this, it appears that abnormal cancer cells
have adopted a way of having energy readily available, by storing lipids in the
cytoplasm. These use free fatty acids (FFA)
to produce chemical energy so the abnormal cell can proliferate.
Normal cells in the body already adopt this method, such as myocytes (muscle
cells) and adipocytes (fat cells).
The vacuoles in this patient were present in the majority of
lymphocytes and I wonder therefore if these cells have the potential to proliferate
at a higher rate than those patients with no vacuoles. Would the disease
therefore be more aggressive in this patient? It got me thinking that Burkitt’s
Lymphoma is a very aggressive disease, characterised on the blood film by
immature cells with numerous lipid vacuoles in the cytoplasm of the abnormal cells. Does
this mean that these cells have more energy to multiply faster? Could this contribute
to it’s aggressive nature of Burkitt's Lymphoma?
A treatment for CLL, the drug Ibrutinib inhibits
the B Cell Receptor (BCR) And disrupts and eventually stops FFA metabolism in
CLL cells, which will in effect stop the energy source to the abnormal cell. I wonder if the treatment was stopped in this case?
I actually referred this patient to a Consultant Haematologist, as even though a known CLL, the vacuoles had never been commented on and I wondered if the presence of lipids in the majority of lymphocytes could mean accelerated disease progression.
Interesting case!
Ibrutinib inhibits free fatty acid
metabolism in chronic lymphocytic leukemia - PubMed. (n.d.). PubMed. https://pubmed.ncbi.nlm.nih.gov/29465264/
Sunday 22 May 2022
How to tell a patient's sex from their neutrophils!
Look at this neutrophil. Can you see a drumstick (or as I
call it chicken leg!) protruding from the nucleus? This actually represents the inactive
X chromosome and tells us therefore that this patient is female!
Females have two X chromosomes (XX), whilst males have one X
and one Y (XY). FISH analysis has shown the active chromosomes, whether X
or Y to be randomly distributed along the nuclear lobes of the neutrophil, whereas the inactive X
chromosome is usually located in the end lobe of the nucleus . (Karni, Wangh
and Sanchez, 2001).
Sessile nodules which are small bumps protruding from the nucleus, also represent inactive X chromosome and again are found at the terminal end of the neutrophil nucleus.
It is possible for a male to have these drumsticks or sessile
nodules in Kleinfelters syndrome, which makes sense as here the male has an extra X chromosome
(XXY). I've learnt that a female may lack drumsticks in Turner’s Syndrome (XO) as lacking an X
chromosome .
It is easy to mistake the inactive X chromosome for other nuclear projections such as ‘racket shaped’ protrusions, smaller nuclear lobes and other projections. These have a similar appearance to the drumsticks but may be a different size and do not have the same significance. Abnormal nuclear projections can also be seen in Haematological disorders such as MDS and CMML.
This is a smaller lobe in the nucleus, not a drumstick as it's too big. It takes experience recognising a sessile nodule or drumstick from other nuclear projections, but i've learnt that knowing their location on the terminal lobe of the neutrophil nucleus and looking at size are good places to start!
Thursday 19 May 2022
PIN THE CELL ON THE PLOT!
A fun idea I copied from a Sysmex XN course a few years ago.
I asked my colleague to build the XN differential plot and place cells in the
correct location using the knowledge that the more complex a white cell is
(i.e. it has granules and a lobulated nucleus), the further to the right it
will be, and the more DNA/RNA content it has, the higher it will be.
I’ve found it’s a
really good way to reinforce understanding of the plots and allows the correct
thought process to occur when there is a potential abnormality.
As I’ve mentioned in
a previous post, MDS can be picked up as the neutrophil population extends too far
left, suggesting that less complex cells are there i.e. neutrophils lacking
granules or with an abnormally segmented nucleus.
Blasts can be picked up because a population with more
DNA/RNA is present, higher up to the left of the plot, but have few granules so
are located more to the left. Nucleated red cells however have far less DNA/RNA
content and no granules, so will reside in the bottom left.
Even malaria may be seen as a very complex population over
to the right, with lots of pigment.
So Scientists out there, if you see any unusual populations on the plot, ask yourself, what this means by truly understanding how your particular analyser builds the plot.
Sunday 8 May 2022
HELLP - A LIFE THREATENING COMPLICATION OF PREGNANCY
Another weekend shift and another emergency case.!!!
This lady recently came into the hospital at 30 weeks pregnant. The clinical
details I received were ? Pre-eclampsia.
The Full Blood Count
showed that the platelet count for this lady was low at 64 x 10^9/l and had significantly dropped
from 266 x 10^9/l, two weeks previously. This situation must always
be dealt with immediately. The first question is, is this a genuine result?
After ruling out a clot in the sample, or platelet
clumping/ Fibrin strands, the next question is what is going on in the body to
make the platelets fall like this? The Scientist must then seek the answer by
looking at the white cells and red cells on the blood film, clinical details
and other laboratory results.
The potential cause was revealed on the blood film, by the
presence of red cell fragments in most fields. This is a serious finding in conjunction with a dropping platelet count and the question is, why are
red cells being sheared in half!?
My immediate thought was HELLP Syndrome which stands for Haemolysis, Elevated Liver Enzymes
and Low Platelets. This is a severe, potentially life -threatening form of
pre-eclampsia. Complications include liver haemorrhage or rupture, pulmonary
odema, placental abruption, bleeding and clotting issues.
What is the cause for
red cells being sheared in half ?
It seems that the main cause is a Microangiopathic
Haemolytic anaemia (MAHA). The red cells are sheared off as they pass through
capillaries with damaged endothelium and fibrin strands which leads to the red
cells being fragmented as they pass through.
Another cause for the red cell damage can also be
Disseminated Intravascular Coagulation (DIC).
Why are platelets
reduced in HELLP?
The platelets are aggregating and forming clots due to
endothelial damage.
Further evidence
that this was HELLP Syndrome.
Protein in the urine
A protein: creatinine
ratio or >30mg/mmol suggests significant proteinuria in pregnancy (NICE, 2019). In this case the value was
341.7mg/mmol!
Elevated Liver
enzymes
Nice guidelines ( NICE, 2019) suggest a rise in ALT, twice
the upper limit of the normal range is of concern. The ALT in this case on
presentation was 420 U/L. The normal range is <33 U/L.
What happened next?
The obstetric team and Consultant Haematologist were alerted
to the blood film findings and other laboratory results. The Consultant
Haematologist should be informed as red cell fragments with low platelets could
also be suggestive or other life threatening microangiopathic haemolytic anaemias such as
TTP and HUS, where the course of treatment would be entirely different.
The diagnosis of HELLP was indeed made in this case however and the
decision to deliver the baby prematurely, despite the lady only being 30 weeks pregnant. Delivery
is the cornerstone of treatment for HELLP syndrome (Baha, 2022).
Post delivery we can see quite a quick improvement with an upward trend in the platelet count and downward trend in
ALT. From a haematological point of view,
if the platelet count did not improve, an alternate cause for the
thrombocytopenia such as a primary Microangiopathic Haemolytic anaemia would be
sort.
|
Haemoglobin (g/dl) |
Platelets (x 109/l) |
ALT (U/L) |
PRESENTATION |
118 |
64 |
420 |
DAY 1 (post delivery) |
111 |
114 |
300 |
DAY 2 |
98 |
178 |
212 |
DAY 3 |
99 |
239 |
117 |
DAY 4 |
102 |
337 |
85 |
DAY 8 |
108 |
558 |
32 |
DAY 15 |
119 |
354 |
14 |
Hopefuly mother and baby both had positive outcomes in this
case.
Baha, S., 2022. UpToDate. [online] Uptodate.com. Available at: <https://www.uptodate.com/contents/hellp-syndrome-hemolysis-elevated-liver-enzymes-and-low-platelets?search=hellp%20SYNDROME§ionRank=3&usage_type=default&anchor=H24&source=machineLearnin> [Accessed 8 May 2022].
Petca, A., Miron, B., Pacu,
I., Dumitrașcu, M., Mehedințu, C., Șandru, F., Petca, R. and Rotar, I., 2022.
HELLP Syndrome—Holistic Insight into Pathophysiology. Medicina, 58(2),
p.326.
Nice.org.uk.
2022. Recommendations | Hypertension in
pregnancy: diagnosis and management | Guidance | NICE. [online] Available
at:
<https://www.nice.org.uk/guidance/ng133/chapter/Recommendations#assessment-of-proteinuria-in-hypertensive-disorders-of-pregnancy>
[Accessed 8 May 2022].
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Look at these white cell differentials analysed on the Sysmex XN. The first run to the right labelled run 1, gives a neutrophil count of ...
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Reviewing the XbarM analysis parameters used in our laboratory XbarM control program and looking at whether the current parameters being mon...
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Today I used the BCSH audit template to perform a gap analysis on our procedures for the diagnosis of malaria, in comparison to the BCSH ...