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LO1: Basics of purine metabolism

Purines are broken down to form hypo-xanthine and sequentially oxidised by xanthine oxidase to form urate.


Clinical features of acute gout

* Rapid build up of paint
* “Exquisite pain”
* Affected joint red, hot, swollen
* Most common joint = 1st MTP joint of the big toe
* Management = Reduce inflammation (vs managing hyperuricaemia more for chronic gout) - impt differentiator


Gout aetiology + epidemiology

* Crystal arthropathy - monosodium urate crystals
* Needle shaped crystals - sets off intensive inflammatory reactions

* Epidemiology
* Males 3% (post puberty except Lesch Nyhan)
* Females 0.5% (more common post menopause)


Why do humans have issues with urate

In other animals, urate is then broken down to form allantoin by urease.

Allantoin is highly water soluble and freely excreted in the urine.

we do not have functional urease (while we do have the rudimentary gene)

* Urate is relatively insoluble
* Urate circulates in the bloodstream at concentrations just below it max solubility, meaning that it is constantly on the brink of precipitating out to form urate crystals.

Plasma concentrations of monosodium urate
* Men: 0.12-0.42 mmol/l
* Women: 0.12-0.36 mmol/l (one reason why women get less gout than men)

* Solubility at 37oC = 0.40mmol/l
* Solubility drops at cooler temperature and lower pH (more acidic)


3 Key Principles of purine metabolism

1. You can make purines by 2 ways: de novo synthesis (green box) or salvage mechanism aka recycling (blue box)
* De novo = difficult, inefficient, highly metabolically expensive, body only uses this if there is extremely high requirements of purines e.g. bone marrow due to high cellular division whereby salvage alone is insufficient
* Salvage = highly energy efficient, body predominantly uses this pathway

2. Rate limiting step in de novo purine synthesis is catalysed by enzyme PAT (PPRP Amido Transferase)
* PAT under feedback inhibition by outputs GMP and AMP (the left and right dotted arrows)
* PAT is also under feed forward effect whereby activity is stimulated by higher levels of PPRP (substrate)
* Break and accelerator example

3. HPRT/HGPRT = hypoxanthine-guanine phosphoribosyltransferase is the main enzyme that recycles partially catabolised purine and bring them back up the metabolic pathway (salvage pathway)
* Absolute deficient = Lesh Nyhan
* No HGPRT = no recycling of purines to GMP or IMP
* Lack of GMP/IMP = reduced negative inhibition of PAT
* De novo synthesis pathway goes into overdrive, excessive purine synthesis
* The massive loads of purines has no where else to go (because there is not corresponding need) and is broken down the purine pathway (IMP → Inosine → hypoxanthine → Xanthine → Urate)
* Additionally, lack of guanine recycling causes accumulation of PPRP (forward accelerator)


Two types of gout

* Acute - Podagra (pod/foot aggravation)
* Chronic - Tophaceous (not acute pain but chronic deposition of urate crystals in soft tissue, tophi in earlobes and adjacent to joints- chalky, cottage cheese)


Treatment acute gout (reduce inflammation)

* NSAIDS (1st line)
* Colchicine (2nd line, inhibit polymerisation of tubulin which in contact of gout 1. reduce cell turnover and 2. reduce motility of neutrophils to prevent neutrophils from participating in inflammation)
* Glucocorticoids
* DO NOT ATTEMPT TO MODIFY PLASMA URATE CONCENTRATION (reducing can paradoxically increase urate crystal formation)


Treatment chronic gout or interval non-acute attacks (managing hyperuricaemia)

* Drink plenty water
* Reverse factors putting up urate (diuretics especially thiazide diuretics)
* Reduce synthesis with allopurinol (inhibit xanthine oxidase)
* Increase renal excretion with probenecid (uricosuric)


Allopurinol cannot be given with ____ + why

* Do not give together with AZATHIOPRINE - steroid sparing agent, used to gently suppress bone marrow to act as immune suppressor, prodrug for 6-mercaptourine → thioinosinate which interfere with purine metabolism
* If given with allopurinol which is inhibitor of xanthine oxidase and given azathioprine (which makes purine and adds to purine pathway), you amplify the effects of azathioprine to toxic levels = shut down bone marrow and result in pancytopenia (cardinal medical sin - also do not give trimethoprim with methotrexate)


Diagnosis of gout (3)

* Tap effusion
* View under polarised light
* Use red filter

Study birefringence
* birefringence = property of the crystal to rotate the axis of the polarised light
* Urate crystals = negatively birefringence (urate crystal = needle light = negative)
* Negative means it rotates 90 degrees to the filter (blue needle)
* Pyrophosphate (psuedogout) is blue in the axis of the red compensator (py = positive)



(the only other crystal arthropathy)
* Occurs in patients with osteoarthritis
* Pyrophosphate crystals
* Self-limiting 1-3 weeks
* Affects different joints (osteo joints)
*Positively birefringence, blue in line with axis of red compensator


Diagnostic requirement for coeliac disease

endomysial antibodies and tissue transglutaminase antibodies


Duodenal biopsy of pt with coeliac disease will show

On gluten rich diet showing villous atrophy
Off gluten showing normal villi


MALToma associated with Coeliac found in the _______ and is B/T lymphocyte origin

MALToma associated with Coeliac found in the duodenal and is of T lymphocyte origin (Enteropathy Associated T-cell lymphoma)


Treatment for essential thrombocytopenai

* Aspirin - prevent thrombosis
* Anagrelide - inhibition of platelet function (SE palpitations and flushing)
* Hydroxycarbamide - antimetabolite, suppress other cells, mildly leukaemogenic


What should you think of when you see leuco-erythroblastic blood film?

Bone marrow
Means there is infiltration


What causes leuco-erythroblasti blood film?

* Root issue: bone marrow infiltration by
* Cancer
* Haemopoietic: leukaemia/lymphoma/myeloma
* Non-Haemopoieotic: breat/bronchus/prostate
* Severe infection
* Military TB
* Severe fungal infection
* Myelofibrosis
* Masive splenomegaly
* Dry tap on BM aspirate


Morphological features of leuco-erythroblastic blood film

* Teardrop RBC
* Nucleated RBC
* Immature myeloid cells


Fe deficiency is always due to ____ unless proven otherwise

from GI cancers or urinary tract cancers


Common distinguishing features of haemolysis of RBC

* Anaemia (though may be compensated)
* Reticulocytosis
* Raised bilirubin (unconjugated)
* Raised LDH (intracellular enzyme released into plasma when RBC lyse)
* Reduced haptoglobin (because haptoglobin binds to free haemoglobin in the blood released from lysed cell)


Causes of inherited haemolytic anaemia

Think of the structure of RBC

* hereditary spherocytsis
* hereditary elliptocytosis

* G6PD def
* pyruvate kinase def
* Pyrimidine-5'-nucleotidase def

* Thalassaemia (number)
* Sickle cell disease (functional)
* unstable Hb variant


Features that suggest this haemolytic anaemia is inherited

Are the common lab features of all Haemolytic anaemia present?
Ethnic background (why are these anaemias so common worldwide?)
Is there a family history/lifelong history with first presentations in childhood/recurrent episodes?
Pigment gallstones?


What is one test to distinguish between immune and non-immune causes of haemolytic anaemia

Direct Antiglobulin (DAT or Coombs test)


Autoimmune and non-immune causes of acquired haemolytic anaemia

Auto-immune causes of acquired haemolytic anaemia
* Spherocytes, DAT+ve
* Cancer of immune system: lymphoma
* Disease of immune system: SLE
* Infection affecting the immune system: EBV, HIV

Non-immune causes of acquired haemolytic anaemia
* Infection (malaria)
* Red cell fragments
* Low platelets
* Bleeding/DIC
* Underlying adenocarcinoma


Haemolytic aneamis associated with systemic disorders

Associated systemic disorder
Cancer of immune system: lymphoma
Disease of the immune system: SLE
Infection (disturbing the immune system)


Explain how adenocarcinomas or DIC causes acquired, non-immune haemolytic anaemia

1. Consume platelets (in DIC)
2. Coagulation activation (cancer)
3. Formation of fibrin strands - cheese strings
4. Red cells pushed through this mesh at high pressure due to BP
5. SHEEEEEAAAARRRR - red cell fragmentation (microangopathy)
6. Haemolysis


List the primary and secondary causes of TRUE polycythaemia

primary (low EPO)
* Polycythaemia vera - clonal proliferation of red cells from acquired mutations in JAK2

Secondary (high EPO)
* Appropriate causes - high altitude, hypoxia, cyanotic heart disease, high affinity haemoglobin
* Inappropriate - liver cancer, renal disease (EPO tumours), lung cancer


Female aged 39 treated breast cancer 4 years previously
- recent onset jaundice and hepatomegaly
-GP bloods
- Hb 87g/l
- Reticulocyte 15x10^9/L (20-92)
- Bilirubin 50 micromol/l conjugated

DAT negative

Blood film
- leuco-erythroblastic anaemia

- leuco-erythroblastic anaemia think bone marrow

Infiltrates from cancer or infections

In this lady probably her breast cancer mets to liver and bone marrow


Investigation of raised white cell from a blood test

What do you want to do/investigate?

Ask for blood film - are the cells mature or immature? And there features

What lineage - specific e.g. granulocytes or all (granulocytes + lymphocytes)

How high?


Causes of neutrophilia

- infection (reactive)
- cancer (myeloproliferative disorders, leukaemia disorder)
- tissue inflammation (e.g. pancreatitis, colitis)
- underlying neoplasia
- corticosteroids


What infections classically do not produce neutrophilia

brucella, typhoid, many viral infections.


What features of the neutrophil on the blood film and pt would suggest this is a reactive neutrophilia?

Presence bands, toxic granulation (vacuoles), and clinical signs of infection/inflammation (pyrexia, abscess, raised X? protein


What features on a blood film suggest malignant increase in WBC?

Neutrophilia, basophilia plus immature cells myelocytes, and splenomegaly. Suggest a myeloproliferative (CML)

Neutropenia plus Myeloblasts (AML)


Causes of reactive eosinophilia?

Parasitic infestation

allergic diseases e.g. asthma, rheumatoid, polyarteritis, pulmonary eosinophilia.

Underlying Neoplasms, esp. Hodgkin’s, T-cell NHL (reactive eosinophilia)
- release of IL5 from neoplasms causing einsophilia

Drugs (reaction erythema mutiforme)


Chronic Eosinophilic leukaemia

Eosinophils part of the “clone”
FIP1L1-PDGFRa Fusion gene


Causes of monocytosis

Monocytosis is generally rare but seen in certain chronic infections and primary haematological disorders

TB, brucella, typhoid
Viral; CMV, varicella zoster
chronic myelomonocytic leukaemia (MDS)


Interpreting a lymphocytosis

Determine if this primary (cancer) or secondary (benign)
- primary = monoclonal
- secondary = polyclonal, reactive to infection or inflammation

Symptoms suggestive of infection or lymphoma
Massive lymphadenopathy or splenomegaly

Degree of lymphocytosis
- Modest lymphocytosis likely reactive
- Massive lymphocytosis more likely primary
Light microscopy/ morphology
Mature cells (reactive or CLL/NHL) or primitive lymphoblasts (ALL)

Flow cytometry
Lineage; B or T cells
Stage of differentiation

Molecular genetics
Rearranged: T cell receptor or Immunoglobulin gene


Causes of reactive lymphocytosis

infection and inflammation

EBV, CMV, Toxoplasma
infectious hepatitis, rubella, herpes infections

autoimmune disorders


Blood film of CLL

Small lymphocytosis
Smear cells


CD19 marks a

B lymphocyte


CD3 and CD8 marks

ALL T cells


Tdt marks

A lymphoid stem cell


45 year old male
3 week history of sore throat
Recent episode of shingles
EBV IgG serology positive

Lymphocytes 12x109/L (raised)
Neutrophils 7x109/L (normal range)
Film: reactive lymphocytes no abnormal cells

Flow cytometry shows:
CD19 - 55++% CD20 - 69++%
B cell: 87++% kappa, 0% lambda
CD5/CD19 - 5%


Mature B cell CLL

Do to get tricked by the EBV IgG+ because IgG (suggest past infection not active infection)


Two principal mechanisms of anaemia

Decreased production
-BM infiltration, leucoerythroblastic film

Increased destruction
- Lab markers of haemolysis (retics, LDH, Bilirubin)
- Immune (spherocytes and DAT) lymphoid tumours
- Microangiopathic (red cell fragments) adenocarcinoma


In the context of PNH, what is the role of GPI in red cell function? Do PNH have more and less GPI?

* GPI in the context of RBC is important for complement regulation such that RBC are protected against complement destruction

* Hence def of GPI seen in PNH result in complement mediated red cell destruction


How do you classically test for hereditary spherocytes? What has that test now been superseded by?

Hallmark: Increased sensitivity to lysis in hypotonic saline (osmotic fragility test) - superseded by simpler diagnostic test of reduced binding of dye oesin-5-maleimide (flow cyto, reduced mean cell fluorescence)


What is the inheritance pattern for hereditary spherocytosis?

Majority AD
25% recessive and a little de novo


What is the link between hereditary ellipocytosis and hereditary pyropoikilocytosis?

Hereditary pyropoikilocytosis is the more severe form of hereditary ellipocytosis where the pt is homozygote

In hereditary elliptocytosis the pt is usually asymptomatic and do not have anaemia


G6PD def is a cause of inherited haemolytic anaemia. What does G6PD actually do?

* G6PD catalyses first step in pentose phosphate pathway - main function to generate NADPH to maintain intracellular glutathione (GSH)

GSH is impt for combating oxidative stress

* Hence def = lack of NADPH and vulnerable to oxi stress

Hence G6PD HA is triggered by oxidative stress, infections and fava beans


Characteristics of G6PD blood film - buzzwords

Nucleated RBC
Bite cells
Contracted cells


Which key clotting factor activates both factors V and VIII, and also activates protein C?



How do you tell the difference between petechiae, purpora and ecchymosis?

Purpura measure 0.3–1 cm (3–10 mm), whereas petechiae measure less than 3 mm, and ecchymoses greater than 1 cm.


What important study is conducted in all newly diagnosed AML pts? and why is this study done?

Cytogenetic studies is done in all AML pts. Molecular studies are done in selected pts.

Prognostic value + helps select treatment
Good prognostic e.g. Inv(16)= treat with medication and no bone marrow transplant
Bad prognostic e.g. del5/7, complex karyotype = achieve remission with meds and push for bone marrow transplant