TI Flashcards
What is the definition of anaemia?
Anaemia is a condition in which the number of red blood cells (and consequently their oxygen-carrying capacity) is insufficient to meet the body’s physiologic needs
Insufficient oxygen carrying capacity is due to reduced haemoglobin concentration as seen with insufficient RBC
About haemoglobin…
Haemoglobin contains iron and transports oxygen. It is a metalloprotien within RBCs.
Reduction in haemoglobin = anaemia (reduction in oxygen carrying capacity)
About the production of blood cells and where they are…
Blood cells are made within bone marrow. As a child this is mostly within most of the long bones and it is being produced all the time. When we are older it is more predominantly in our pelvis, femur, sternum – the bones have become a bit dried out. We are continually producing RBCs and platelets etc
How are haemoglobin levels used to diagnose anaemia?
Every lab has its own normal range, and the normal range will vary throughout life and also between males and females.
We usually use g/L however some use g/dL and this is why sometimes values may seen to be out by a factor of 10 (eg 7 actually means 70).
From 6 months to 5 years there is a lower normal levels because the child is transitioning from breast milk to using other nutrients, but by adolescence it reaches around the normal range.
Women that aren’t pregnant have slightly lower levels than men, this is because they are menstruating. When women are pregnant it will drop even further – this is because their physiological volume increases so there is a decrease in concentration.
Men however, should stay at around 130 g/L.
Depending on how low you drop tells you how severe the anaemia is
What is normal erythropoiesis?
Maturation of RBCs required Vitain B12 and folic acid for DNA synthesis and iron for haemoglobin synthesis.
- Vitamins
- Cytokines (erythropoietin)
- Healthy bone marrow environment
What are the mechanisms of action of anaemia?
- Failure of production: hypoproliferation, reticulocytopenic
- Ineffective erythropoiesis
- Decreased survival: blood loss, haemolysis, reticulocytosis
For example, when you might have been stabbed you lose a lot of blood which could make you anaemic despite having healthy bone marrow
What deficiencies can cause anaemia?
- Iron deficiency
- Vitamin B12 deficiency
- Folate deficiency
Reduced concentration of Hb means that we can’t get the building blocks from food sources
About iron…
- Essential for O2 transport
- Most abundant trace element in body
- Daily requirement for iron for erythropoiesis varied depending on gender and physiological needs
- Daily requirement depends on TBC production amount, gender, age and physiological needs
What are the daily iron requirements?
Recommended intake assumes 75% of iron is from heme iron sources (meats, seafood). Non-heme iron absorption is lower for those consuming vegetarian diets, for whom iron requirement is approximately 2-fold greater.
Daily dietary iron requirements differ at various stages of development, between men and women, and between pregnant and nonpregnant women. The data reported in this table assume an average dietary iron absorption of 10%.
What food are rich in iron?
- Meats: Liver, Liverwurst, Beef, Lamb, Ham, Turkey, Chicken, Veal, Pork, Dried beef
- Seafood: Shrimp, Dried cod, Mackerel, Sardines, Oysters, Haddock, Clams, Scallops, Tuna
- Vegetables: Spinach, Beet greens, Dandelion greens, Sweet potatoes, Peas, Broccoli, String beans,Collards, Kale, Chard
- Breads & Cereals: White bread (enriched), Whole wheat bread, Enriched macaroni, Wheat products, Bran cereals, Corn meal, Oat cereal, Cream of Wheat, Rye bread, Enriched rice
- Fruits: Prunes, Watermelon, Dried apricots, Dried peaches, Strawberries, Prune juice, Raisins, Dates, Figs
- Other Foods: Eggs, Dried peas, Dried beans, Instant breakfast, Corn syrup, Maple Syrup, Lentils, Molasses
What is the distribution of iron in adults?
• Iron is an essential component of cytochromes, oxygen-binding molecules (ie haemoglobin and myoglobin), and many enzymes
• Dietary iron is absorbed predominantly in the duodenum
• Fe+++ ions circulate bound to plasma transferrin and accumulate within cells in the form of ferritin. Stored iron can be mobilized for reuse.
• Adult men normally have 35 to 45 mg of iron per kilogram of body weight. Premenopausal women have lower iron stores as a result of their recurrent blood loss through menstruation.
• More than two thirds of the body’s iron content is incorporated into haemoglobin in developing erythroid precursors and mature red cells.
• Most of the remaining body iron is found in hepatocytes and reticuloendothelial macrophages, which serve as storage deposits.
• Reticuloendothelial macrophages ingest senescent red cells, catabolise haemoglobin to scavenge iron, and load the iron onto transferrin for reuse.
• Iron metabolism is unusual in that it is controlled by absorption rather than excretion. Iron is only lost through blood loss or loss of cells as they slough.
• Men and nonmenstruating women lose about 1 mg of iron per day. Menstruating women lose from 0.6 to 2.5 percent more per day.
An average 60-kg woman might lose an extra 10 mg of iron per menstruation cycle, but the loss could be more than 42 mg per cycle depending on how heavily she menstruates
How is iron metabolised?
> 1 stable form of iron: Ferric states (3+) and ferrous states (2+).
Most iron is in the body as circulating Hb. Hb: 4 haem groups, 4 globin chains able to bind 4 O2.
The remainder is as storage and transport proteins: ferritin and haemosiderin. These are found in the cells of the liver, spleen and bone marrow.
Iron is absorbed form the duodenum via enterocytes into the plasma and binds to transferring and then transported to bone marrow to make RBCs. Excess absorption of iron is stored as ferritin
How is iron absorbed?
Iron absorption is regulated by GI mucosal cells mechanism: max absorption in the duodenum and proximal jejunum via ferroportin recetpors.
The amount absorbed depends on the type ingested: heme, ferrous (red meat, used to contain haemoglobin) > than non heme, ferric forms which is bound to other substances. Heme iron makes up 10-20% of dietary iron.
Other foods, GI acidity, state of iron storage levels and bone marrow activity affect absorption. Vitamin C helps absorption while milk/dairy decreases absorption, especially in children
How is iron regulated by Hepcidin?
“The iron regulatory hormone hepcidin and its receptor and iron channel ferroportin control the dietary absorption, storage and tissue distribution of iron…
Hepcidin cause ferroportin internalisation and degradation, thereby decreasing iron transfer into blood plasma from the duodenum, from macrophages involved in recycling senescent erythrocytes, and form iron-storing hepatocytes.
Hepcidin is feedback regulated by iron concentrations in plasma and the liver and by erythropoietic demand for iron.”
You can’t excrete it but there is a regulatory mechanism
How is iron transported and stored?
- Iron transported from enterocytes and then either into plasma or stored as ferritin
- Once attached to transferrin binds to transferrin receptors on RBC precursors
- A state of iron deficiency will see reduced ferritin stores and then increased transferrin
Transferrin will go up as you are trying to get whatever iron there is into RBCs as quickly as possible
What various iron studies are there?
- Serum Fe: hugely variable during the day
- Ferritin: primary storage protein and providing reserve, water soluble
- Transferring saturation: ratio of serum iron and total iron binding capacity – revealing % of transferring binding sites that have been occupied by iron
- Transferrin/Transferrin receptors: made by liver, production inversely proportional to Fe stores. Vital for Fe transport. Uptake of Fe from protein needs transferrin to be attached to the cell via the transferrin receptor
Some are useful, and some are not. We can test for the serum iron which gives us a number of the amount of iron in the serum. This doesn’t tell us how much is in the RBCs or how much it goes up however, and it is very different throughout the day and in response to meals – so it is not very useful.
Ferritin is relatively easy to do. It is very reliable for iron deficiency. If it is low we now that we don’t have enough stored iron.
Transferrin saturation can be measured. We can see how much there is and if it goes up. We can also test for saturation
What is total iron binding capacity?
This is a measurement of the capacity of transferring to bind iron. It is an indirect measurement of transferrin – a transport protein that carried iron. TIBC is technically easier to measure in the laboratory than transferrin levels directly.
In IDA, TIBC is high…
There is more transferrin produced, aiming to transport more iron to tissues in need
What are the causes of iron deficiency?
¬ NOT ENOUGH IN: poor diet, malabsorption, increased physiological needs
¬ LOSING TOO MUCH: blood loss, menstruation, GI tract loss, parasites
What iron deficiency investigations might be done?
- FBC: Hb, MCB, MCH, reticulocyte count
- Iron studies: ferritin, transferrin saturation
- Blood film
- BMAT (bone marrow aspiration trephine) and iron stores – but this is rarely done now
What are the stages in the development of IDA?
• Before anaemia develops, iron deficiency occurs in several stages.
• Serum ferritin is the most sensitive laboratory indicators of mild iron deficiency. Stainable iron in tissue stores is equally sensitive, but is not performed in clinical practice.
• The percentage saturation of transferrin with iron and free erythrocyte protoporphyrin values do not become abnormal until tissue stores are depleted of iron.
• A decrease in the haemoglobin concentration occurs when iron is unavailable for haem synthesis.
MCV and MCH do not become abnormal for several months after tissue stores are depleted of iron
What might the laboratory results of IDA be?
It shows red cells that are in general much smaller than a neutrophil, with marked anisocytosis (variation of the red cell size) and hypochromia (area of central pallor of red cells that is larger than normal, indicating a low MCHC).
The RBCs stain blue as the Hb is stained
What is the prevalence of iron deficiency anaemia?
World’s most common nutritional deficiency
o 2% in adult men (70 years old
o 10% in caucasion, non-Hispanic women
o 19% in African-American women
o Common cause of referral
o Excessive menstrual losses 1st cause in premenopausal women
Blood loss from the GI tract is the most common cause of IDA in adult men and postmenopausal women.
Iron deficiency is the most common nutritional deficiency as well as the most common cause of anaemia throughout the world and a common cause of referral.
Premenopausal women with excessive menstrual losses are particularly at risk of developing iron deficiency anaemia (IDA)
What are the signs and symptoms of iron deficiency anaemia?
Symptoms:
- Fatigue
- Lethargy
- Dizziness
Signs:
- Pallor of mucous membranes
- Bounding pulse
- Systolic flow murmurs
- Smooth tongue
- Koilonychias
What is microcytic anaemia?
low Hb and high MCV with normal MCHC
What are the two types of microcytic anaemia?
Magaloblastic: Low reticulocyte count:
- Vitamin B12/folic acid deficiency
- Drug-related
- (Interference with B12/FA metabolism)
Nonmegaloblastic:
- alcoholism ++
- hypothyroidism
- liver disease
- myelodysplastic syndromes
- reticulocytosis (haemolysis)
Vitamin B12 = cobalamin and folic acid are both are important for the final maturation of RBC and for the synthesis of DNA. We also need both for thymidine triphosphate synthesis.
What is folate needed for?
folate is necessary for DNA synthesis: adenosine, guanine and thymidine synthesis
What are causes of folate deficiency?
Folate comes from most foods with 60-90% lost in cooking. It is absorbed in the jejunum and the body has enough stores usually for 3-5 months
INCREASED DEMAND: pregnancy/breastfeeding, infancy and growth spurt, haemolysis and rapid cell turnover e.g. SCD, disseminated cancer, urinary losses e.g. heart failure
DECREASED INTAKE: poor diet elderly, chronic alcohol intake
DECREASED ABSORPTION: medication, coeliac, jejunal resection, tropical sprue
About vitamin B12…
This is an essential co-factor for methylation in DNA and cell metabolism. There is intracellular conversion to 2 active coenzymes necessary for the homeostasis of methylmalonic acid (MMA) and homocysteine.
• Foods containing vitamin B12: animal sources – fish, meat, dairy
• UK intake recommendations are 1.5mcg/day
• EU: 1mcg/day and USA: 2.4mcg/day
• The average western intake is 5-30mcg/day
• Body (liver) storage: 1-5mg so many years for deficiency.
• Absorption of vitamin B12 in the terminal ileum requires the presence of intrinsic factor.
IF is made in parietal cells in the stomach. Transcobalamin II and transcobalamin I transport vitamin B12 to tissues
Why might there be an increased requirement for vitamin B12?
- haemolysis
- HIV
- pregnancy
- growth spurts
What are the clinical consequences of folate and vitamin B12 deficiency?
Brain: cognition, depression, psychosis
Neurology: myelopathy, sensory changes, ataxia, spasticity (SACDC)
Iron deficiency is the most common nutritional deficiency as well as the most common cause of anaemia throughout the world and a common cause of referral.
Premenopausal women with excessive menstrual losses are particularly at risk of developing iron deficiency anaemia (IDA).
Infertility
Cardiac cardiomyopathy
Tongue: glossitis, taste impairment
Blood: pancytopenia
What is pernicious anaemia?
- Autoimmune disorder
- Lack of IF
- Lack of B12 absorption
- Gastric parietal cell antibodies
- IF antibodies
What are the treatments for pernicious anaemia?
- Treat the underlying cuase
- Iron: diet, oral, parenteral iron supplementation, stopping the bleeding
- Folic acid: oral supplements
- B12: Oral vs intramuscular treatment
About normal haumatopoeisis…
- Blood cell production
- Bone marrow
- Long bones
- Maturation occurs in bone marrow
- Mature cells within peripheral blood
The purple tubes have EDTA in to stop the blood from clotting. As it doesn’t clot, if the blood is left for long enough it will segment out into its components
What is a full blood count?
Red blood cell results • Hb: concentration of Haemoglobin • Hct: percentage of blood volume as RBC • MCV: average size of RBC • MCH (mean cell Hb): Average haemoglobin content of RBC • RDW (red cell derivation width): range of deviation around RBC size • Reticulocyte count • Blood film
Hct: haematocrit (% of blood volume that is RBC) – usually around 50-52%
Not just RBC results…
• WHITE BLOOD CELL RESULTS: total WBC and differential neutrophils, lymphocytes, monocytes, basophils, eosinophils
• PLATELET RESULTS: platelet count and size
•
OTHERS: warning flags
What does a blood film show?
Confirming numbers
Morphology – are the cells ‘normal’
Are there cells present that shouldn’t be?
o PURPLE: clotting o PINK: EDTA cross matching in blood transfusions o BLACK: ESR o RUST: clots biochemistry o GREEN: lithium pagrin
Tube colour does matter!!
In a lab, around 1600 FBC samples are processed every day!
Peroxide stain picks up myeloperoxidase in neutrophils
What are tear drop poikilocytosis?
- These are seen in myelofibrosis or bone marrow infiltration with malignant disease
- This suggests there is something else sitting inside the bone marrow
- This wouldn’t be picked up in a numbers count
What is haemostasis?
This is a protective process evolved in order to maintain a stable physiology.
It forms a scaffold like structure so that the body can go on to heal
Haemostasis does more than just preventing blood loss. It also works in the context of someone who has an infection
Life preserving process designed to maintain blood flow:
- Respond to tissue injury
- Curtail blood loss
- Restore vascular integrity and promote healing
- Limit infection
What is disseminated intravascular coagulation (DIC)?
Coagulation is being activated by infection and blocking off the bacteria from getting to more vital places in the body. For example, the young child’s hand has become infarcted in trying to prevent the infection from getting to the liver or heart for example, and losing the hand as a result of this may not be as bad as the otherwise possible consequences.
Our biggest killer now is us (war, violence), rather than infection
What are the four key components of haemostats?
- endothelium
- platelets
- coagulation
- fibrinolysis
What makes a blood clot?
- Fibrin mesh
- Platelets
- Red blood cells
Platelets are the main features of a clot and they are very complex. Fibrin makes the mesh which is the proteins that come out of solution and is the final end point to strengthen the clot. Without this, people would bleed
What is the overview of haemostasis?
When you cut yourself, the first thing that happens is that you lose blood. Vasoconstriction occurs which reduces the size of the cut and therefore the amount of blood lost. There is lots of smooth muscle around the blood vessels to allow for this.
Next we get a platelet plug. The platelets recognise the damage to the endothelium and stick there to limit blood loss. This isn’t a very strong plug however, and it would only work for a little while. Fibrin comes out to strengthen this and maintain the clot by forming a mesh
What are the three phases of haemostasis?
Primary Haemostasis:
- Vasoconstriction (immediate)
- Platelet adhesion (within seconds)
- Platelet aggregation and contraction (within minutes)
Secondary Haemostasis:
- Activation of coagulation factors (within seconds)
- Formation of fibrin (within minutes)
Fibrinolysis:
- Activation of fibrinolysis (within minutes)
- Lysis of the plug (within hours)
There are several different aspects to haemostasis. There is a fibrinolytic mechanism that occurs almost straight away in order to modify the clot and make sure it only stays at the site of injury
What is VWF?
In the lumen there are lots of components that are very important for haemostasis; platelets, VWF etc.
VWF is very important and people without it bleed. It is the anchor for the platelets to adhere to at the site of injury.
In the subendothelium there are the initiators for when you injur yourself; collagen and tissue factor
What is primary haemostasis?
In a damaged vessel there are two crucial molecules exposed, forget tissue factor.
Primary haemostasis involves collage. It interacts with VWF in the blood and then VWF unravels and acts as the anchor for platelets to adhere to that damaged area. The platelets adhering transform from small and round into star shaped cells, releasing their components that they are carrying, which makes more platelets and VWF coming along. This provides a nice phospholipid surface for the fibrin
What happens to VWF activity under shear stress?
VWF in the endothelium us released when you damage the surrounding area. It is vital for platelets to adhere. VWF unravels when it gets released, allowing the platelets to identify there is a problem and stick to it. You then form a platelet clot
What does platelet aggregation do?
Platelet aggregation prevents excessive blood loss at the site of injury
What is the role of platelets in haemostasis?
The things inside are exposed to the blood. Tissue factor and collagen are the important ones. Collagen causes the platelets to adhere and release substances to cause more platelets to migrate to the site. A platelet plug then forms and we get primary haemostasis
What is the process of haemostasis?
¥ When a vessel wall is damaged, various signalling molecules are expressed / exposed, including tissue factor and collagen
¥ The TF leads to the production of a small local amount of thrombin, which is the initiation step of the coagulation process
¥ The exposed signalling molecules attract circulating platelets, which attach themselves to the exposed sub-endothelial tissue (mechanism to be discussed later): this is adhesion
¥ These platelets become activated – principally through the presence of the thrombin – and release further attractant chemicals, which attract more platelets: this is secretion
¥ These new platelets bind to the adhered platelets (mechanism to be discussed later) and themselves become activated: this is aggregation
¥ Through the conformational changes inherent in activation, the loose platelet plug contracts to form a dense, adherent plug: this is contraction
¥ Together these steps comprise primary haemostasis, which may well be sufficient to achieve haemostasis if the injury is relatively minor
¥ The activated platelets also present a substantial area of negatively-charged phospholipid membrane at the site of the injury, upon which the subsequent processes of coagulation (secondary haemostasis) can occur, if needed
What is an overview of haemostatic plug formation?
The fibrin mesh binds and stabilises the platelet plug and other cells.
In coagulation fibrin forms, there are lots of clotting factors that circulate in the plasma. Most of them are produced in the liver and are complex. In the last 20-30 years we have learnt the crystalline structures of compositions of them
What are coagulation factors?
There are 13 coagulation factors and we don’t really need to learn them all. A haematologist will refer to them as their numbers. We need so many because the more you have, the more easily you can achieve regulation – ie we don’t need to turn everything off or everything on. This about having lots of light switches, you would be able to alter the light more specifically
What does it mean that blood has an intrinsic ability to clot?
Blood has an intrinsic ability to clot. It can clot without any obvious provoking factors. We call this contact activation – surface contact. The easiest way to think of it is if you cut yourself and go and clean it and wrap it up in the process made a mess of blood on bench and the blood on the bench has clotted and this is the intrinsic ability – there was no tissue factor and it clots because of a foreign surface. Factor 12 and some others can recognise a foreign, non physiological surface. These in turn can cause a clot to occur. Factor 12 activates 11 activates 9 joins up with common area does 10 then thrombin and then fibrin
What does FVII deficiency cause?
FVII deficiency causes bleeding: without factors we get bleeding. For example, without factor 7 we have a bleeding disorder and cant drive forward coagulation.
What does FXII deficiency do?
FXII deficiency is not associated with bleeding: without 12 there is no bleeding problem. This shows that the role it plays physiologically in forming clots isn’t quite the same
What is the revised waterfall hypothesis?
Each reaction requires:
- Ca2+
- Phospholipid
- ± specific co-factors
In reality, these two limbs of the cascade, intrinsic and extrinsic, have a much more complex interaction
What is initiation of the cascade of events in haemolysis?
- TF is outside the lumen
- Formation of TF-FVIIa complex
- Recruitment of FX and formation of thrombin
When there is bleeding from the blood vessel the tissue factor is not usually exposed to the blood, but damage leads to 7a coming into contact with tissue factor. This leads to production of prothrombin into thrombin, and then into fibrin
What is the cell based model of coagulation?
¥ Initiation of coagulation occurs when sub-endothelial tissue is exposed to the circulation at a site of injury. These tissues express tissue factor at their surface, which binds to endogenous activated FVII
¥ This complex binds small amounts of FX and FV to the exposed endothelial surface, which produce small quantities of thrombin
¥ The thrombin activates platelets that are attracted to the site by the process, as well as other plasma-borne clotting factors
¥ The activated factors (among them FVIII and FIX) enable the binding of activated FX and FV to the surface of platelets whose activation has produce conformational changes in their surface membranes to expose the ‘reaction sites’ necessary for continuation of the process
¥ This leads to the ‘thrombin burst’ that is necessary for the large-scale production of fibrin and so the development of an effective clot
¥ These three stages are called the initiation, amplification and propagation phases of coagulation
¥ Secondary haemostasis this time
¥ Tissue factor exposed to circulating blood
¥ Forms complies with factor seven
¥ Make a little bit of thrombin
¥ Very imprint regulator
¥ Need to make a lot
¥ Seven a cant do it alone
¥ Got to activate lots of other factors to do this
¥ Then make a huge amount of thrombin – thrombin burst and then get fibrin
¥ Thee is an initiator, form a small amount and then need to make more, amplification, the fed elopement of more and more sactivzted substances to PROPAGATE and produce a huge amount
Whats the main function of fibrinolysis?
The process of clot dissolution:
Main function
- Clot limiting mechanism
- Repair and healing mechanism
Series of tightly regulated enzymatic steps
- Feedback potentiation and inhibition
What are the main key players of fibrinolysis?
- Plasminogen
- Tissue plasminogen activator (t-PA) and urokinase (u-PA)
- Plasminogen activator inhibitor -1 and -2
- Alpha2-plasmin inhibitor
How does fibrinolysis occur?
First component of haemostasis and the process of removing the clot. If a clot formed and never went away you couldn’t replace it with normal tissue. Fibrinolysis is also important for limiting where the clot forms. It involves a lot of different steps. The main players are plasminogen, TPA which activated plasminogen, and things that turn it off.
The precursor wants to chew it up and is activated by TPA. It becomes active and will remove the clot. We can measure D-dimers in the lab to see if someone is going through a lot of the process. If they are then they may have a clot. This proves that it starts the moment that you are forming a clot.
If someone gets a clot it would be good if we could make this process a bit stronger. We can do this using TPA and similar substances to form plasminogen to be activated into plasminogen so it can chew up the clot
What happens in chronic venous insufficiency?
- Atrophic changes
- Hyperpigmentation
- Ulceration
- Infection
This can happen if you don’t get a DVT. There is damage to the blood vessels and they can become secondarily infected
Why might we get bleeding?
Normal haemostasis: a state of equilibrium
All the processes work in a fine balance. Blood is in a state of equilibrium between the processes
The imbalance can go the other way and we get bleeding. This would happen if you don’t have enough platelets or clotting factors.
What is ecchymosis?
Easy bruising: ecchymosis
This occurs in virtually all bleeding disorders and often in normal. If mild it may just result in easy bruising and heavy periods, or could be much more significant bleeding
What are methods of laboratory evaluation of bleeding?
There are different aspects for evaluating someone’s haemostasis. A FBC is interested in platelet number. It is not very specific but can get some degree of suspicion.
Fundamental tests are the coagulation tests. These aim to activate and produce fibrin. We can also do very specialised tests. We can look at if they have VWF which is the most common bleeding disorder. Global tests aren’t in routine diagnostic use but more at research level need standardising
What are the principles of clotting tests?
Incubate plasma with reagents necessary for coagulation
- Phospholipid, co-factors
- Trigger or activator
- Calcium
Measure time taken to form fibrin clot.
We need to take blood from the patient and not allow it to clot until we get to the lab. We want to isolate the coagulation factors from the sample by administering triggering factors
What is prothrombin time (PT)?
rothrombin time is one of the coagulation screen tests. It is trying to look at extrinsic path and see how long it takes to occur. 7 tf to fibrin. This is called the prothrombin time or PT.
PT takes from 9-13 seconds and each lab has a normal range. If someone is lacking one of the factors it will take longer so might have an abnormal PT
What is APTT?
Activated partial thromboplastin time.
APTT is measuring the intrinsic path. We have to add something to activate factor 12. It is a foreign substance and we used to add things like sand and then glass beads. Now we add agents that we know have the right ionic charge. APTT is the slower way to make a clot, taking 32-45 seconds. If prolonged then we must wonder what is missing. We know know what though, because this is only a screening test
What is Thrombin Time (TT)?
The third assay is thrombin time. This is trying to mimic the last reaction. We wan to make a clot so we add thrombin and make sure that the end point is right
How is haemostasis testing done on a blood sample?
- Accuracy of haemostasis laboratory tests depends on the quality of the specimen submitted.
- Blood is anticoagulated with 3.2% (0.109M) sodium citrate
- Most tubes contain 0.3mL anticoagulant and require 2.7mLs of blood
- Under filling the tube yields grossly inaccurate results
We use citrates in the blood. Blood is collected in a tube containing citrate. The proportions have to be right, not too little or too much compared to the volume of blood. If the tube is only half filled for example, it cant be measured – it has to be completely full to have the right proportions
What are possible pre-analytical errors?
Problems with blue-top tube
- Partial fill tubes
- Vacuum leak and citrate evaporation
Problems with phlebotomy
- Heparin contamination
- Wrong label
- Slow fill
- Underfill
- Vigorous shaking
- Difficult venipuncture
Biological effects
- Hct >55 or
What is manual coagulation?
We have go the plasma we can do a manual clotting test/manual APTT. When the things are added and it is kept at 37*C we just see how long it takes to clot whilst tilting 3 times every 5 seconds
What is automated coagulation?
This machine has reduced human error in seeing the end point and it is much easier to measure
What are mixing studies for factor deficiency?
- Clotting factor NR 50-150 IU/dL
- APTT normal (24-34s) if factor level within range
- 50:50 mix 32s/28s
If we have an abnormal clotting factor test and it takes a lot longer than it should it is usually due to a factor deficiency. We can mix their plasma with control plasma from healthy volunteers and this should give you a normal time. If its normal then we expect that their clotting factors are abnormal. If we mix them together, in theory we are replacing all the clotting factors from the patient with something right. If they are deficient and we mix it with normal it should replace the deficiency. We measure that, and whilst we might only get 50% of one of the clotting factors, it should be enough to correct the time. If it does correct it, then the patient was deficient in something.
Correction of a clotting test by mixing will investigate whether they are deficient in something
What are mixing studies for inhibitors?
- Antibody in excess
- Inhibits added factor
- 50:50 mix 75s/30s
This wont always correct it. When mixed there may still be a long APTT. This would show the patient to have antibodies so while the clotting factors have been replaced, there are still antibodies which will now interact with the normal plasma that you have added, and so we will still get a low factor as the antibodies may inhibit normal function. Something is interfering with the coagulation
How are mixing studies done?
- Mix patient and normal plasma in equal volumes (50:50 mix)
- Repeat abnormal coagulation test
- Test normalizes – factor deficiencies
- Test remains abnormal – inhibitor (usually antibody)
What is D-dimer testing?
D-dimer testing
• A measure of the D-dimer, a fibrin degradation product
• Found elevated in the situation of enhanced fibrinolysis (thrombosis, DIC)
• Not specific for thrombosis also elevated as an acute phase reactant
• A negative result is useful if clinical suspicion of VTE is low
The D-dimer is a breakdown product of fibrin. It can be broken down into D-dimer form when it undergoes fibrinolysis. Someone who clots a lot will have a lot of D-dimer and this can be useful if we suspect DVT. If there is no raised D-dimer then it is likely that we are heading towards the wrong diagnosis
What is a haemocytometer neubauer chamber?
This is low technology which was used until the 1940s. It took a long time to count and was cumbersome, taking easily more than five minutes to do a single blood count.
It use, you put a drop into the counting chamber with a fine grid allowing counting under the microscope once the drop has been allowed to spread
What is Advia 2120?
This is how the blood count is done now – the blood goes in, and a result comes out. This new technology has a high throughput and the blood goes through a capillary tube. The counting chamber applied an electrical field which is changed as the cells move through producing characteristic changes in current. This is then interpreted by a machine. We can also differentiate with different kinds of white cells
What is a normal WBC differential?
he most common white cells are neutrophils. This is reflected in the WBC count. We will see subtle differences in different labs, especially in neutrophils because there are ethnic variations. These variations do not indicate illness, they are just the normal ranges for different ethnicities. This may reflect on the normal range of the lab depending on where it is, even within a city.
Lymphocytes are the second most common, and have less variation between ethnicities – usually accepted as a global normal.
These cells do different things and are from different lineages within the development
About peripheral blood film…
If you have doubt about something and the results don’t quite fit, you can make a blood film. To do this you take a drop of blood and then take a slide and put a 40* angle onto it, pull back a bit so it spreads and then slide forward so you are spreading the blood thin enough to look at individual cells.
After this is done it needs to be fixed and stained to be looked at and investigated
What is Romanowsky Stain?
There are 2 main components:
- Azure B or Methylene Blue – basic dyes
- Eosin Y – acidic dye
Buffer: pH 6.8
To separate the white cells we will need two different stains. Basophillic stain (blue) and azurophilic stain (red). Some will stain blue and some will stain red allowing differentiation
What are normal neutrophils like on staining?
What you end up with is the staining of everything. Neutrophils are the most common cells. We can recognise that these are neutrophils because they have a segmented nucleus (polysegmented). Up to five segments is normal and this can be easily recognised. They become this way as they mature
What are lymphocytes like on staining?
They are different as they have a single round nucleus with a variable amount of cytoplasm. Some have more cytoplasm than others. When they become activated or reactive through infecition, they may change their morphology between
What is the role of lymphocytes?
The major role of the lymphocytes is adaptive immune response, the differentiation between self and non-self.
Eg viral attack, lymphocytes produce antibodies to destroy that virus, memory cells are produced
What are the divisions of the immune system?
The two basic divisions of the immune system are the adaptive and innate system. The innate system kills when and where it needs to. The two parts are integrated into one another and they don’t function independently but work as one
What are the two major types of lymphocytes?
There are three major types of lymphocytes:
- T cells
- B cells
- Natural killer (NK) cells
These work together to initiate an immune response when you need it. They are naïve when you are born. The first virus you come across will activate a certain number of clones of the pre existing lymphocyte repertoire and want to kill it.
T cells are triggered directly and detect the virus/infected cells and kill it. B cells make antibodies which bind to organisms and coat cells that are affected by viruses. Natural killer cells mostly directly kill the infected cells and cancer cells – abnormal cells.
T cells are involved in cell-mediated immunity
- CD4+ T-Helper cells
- CD8+ Cytotoxic T-cells
B cells in humoural immunity ie antibody production
NK cells are part of the innate immune system attacking virally infected cells and tumour cells
When do the lymphocyte cells become abnormal?
These cells become abnormal when they are infected by viruses. Part of the virus is expressed on the surface of cells and they start looking different; altered self. This is how the immune system recognises and destroys cells. If you couldn’t do this, you would die of autoimmune disease and you wouldn’t be able to differentiate between your own cells. You also wouldn’t be able to absorb food etc as it would all see foreign. The immune system is programmed to tolerate, and it may switch and make an immune response when you need it to.
The nucleus of resting lymphocytes is roughly the same size as a red blood cell. The blue cytoplasm comes from antibodies staining blue
What is a large granular lymphocyte or natural killer (NK) cell?
A proportion of lymphocytes may look like this. They are large granular lymphocytes. It can be natural to see when fighting infection, for example in leukaemia there is a clinical expansion of these. Activated T cells into natural killer cells look like this
What are monocytes?
Monocytesa re the next most commonly seen WBC. They are part of the innate immune system and they are phagocytosing cells. These don’t have visible granules like the lymphocytes but the nucleus can be funny, irregular shapes. They are recognised by this characteristic as well as by their colour which is grey blue and no visible in cytoplasm and there are often vacuoles within the cytoplasm
What are eosinophils?
Eosinophils are also recognised quite easily. Eosinophils are recognised by the polynucleotide and granules. They also have granules but are more red than the neutrophils. There are very few basophilic granules. They are eosinophilic. These cells fight a specific corner that provides protection against parasites, especially in the gut. They also have involvement in allergic reactions. The gut reactions and allergies can show degranulation of eosinophils – antihistamines.
They are present in eczema, asthma, worms and GI parasites
What are basophils?
These are the rarest of the WBC in the blood. Sometimes none will be seen in a blood film. If there are too many it can be associated with certain malignancies. Blood is arguably the least important place for them to be – they go out into tissues to survey tissues and do their job and then they die. The typical life span of a basophil is less than one day – it goes where it is needed, fights infection and then dies
How do cells develop in the bone marrow?
Because they don’t live long we need good production of them in the bone marrow. We need millions produced every second to keep up with what you need. They start dividing in the bone marrow and are becoming more committed progenitors. The stem cell can be anything.
The reason we don’t run out of stem cells is because of asymmetric division. They go through a number of precursor stages in the bone marrow, and the whole process takes 7-10 days
What is the physiological response to most infections?
The physiological response to most infections is to increase neutrophils and push them out. Certain drugs can cause neutropenia, such as chemotherapy
What is toxic granulation?
when they become activated they change how they look
What are the steps of myloid maturation?
Myeloblast promyelocyte myelocyte metamyelocyte band form segmented neutrophil
What is a hzypersegmened neutrophil?
six or more segments make the neutrophil hyperhsegmented.
this can be caused by a deficiency in vitamin B12, for example.
What is a Dohle Body?
These are rather non-specific. Some innate problems can be seen. They are a glomerulus of large blue granules, forming one large blue longitudinal shaped thing. They can be seen in conditions of infection or inflammation and are a good thing to know.
They usually go alongside toxic granulation as well. There are three tongs in this neutrophil. Body, granulation and not fully mature segmented neutrophil is a slightly earlier precursor which sometimes pushes out into the blood, sometimes in response to infection/inglammation
What are possible causes for neutrophilia?
Normal physiological reaction:
- Post-operative
- Pregnancy
Other causes:
- Bacterial infection
- Inflammation eg vasculitis, myocardial infarction
- Carcinoma
- Steroid treatment
- Myeloproliferative disorders
- Treatment with myeloid growth factors
This can be in response to stress, autoimmune disorders, steroids, pregnancy. Neutrophils try to go to the side of the blood vessels because they leave the blood vessels. In steroids they become more in the middle which is why there is an increase when blood is drawn.
Is this a normal response to stress of the system? If it is without any real reason then it is called myeloproliferative disorder
What are investigations of neutrophilia?
- FBC and differential white cell count
- Blood film examination
- Bacterial culture screen for infection
- Bone marrow examination and chromosome analysis for chronic myeloid leukaemia – Philadelphia chromosome: translocation between chromosomes 9 and 22, molecular analysis for BCR-ABL oncogene
The white cell count makes you want to do a blood film. We want to look at the chromosomes in the cells of blood vessels. We may see characteristic translocation between chromosomes driving this growth and we now have treatments for these things. We can recognise, detect and monitor the treatment
What is neutropenia?
- Viral infection
- Drug induced eg sulphonamides
- Radiotherapy and chemotherapy
- Part of a pancytopenia in bone marrow failure (aplastic anaemia) or infiltration eg leukaemia
- Racial: ‘benign ethnic neutropenia’
What is eosinophilia?
They are bi-lobed with big red granules. This could also be a malignant process. Part of the stem cell is destined to be an eosinophil. Are there lost of basophils for example?
- Allergic diseases eg asthma, hayfever
- Parasitic infections
- Drug sensitivity
- Myeloproliferative diseases eg chronic myeloid leukaemia
- Hidgkin’s lymphoma
Allergic diseases will bring up eosinophils and the differential is very wide. It is important therefore not to jump to conclusions, but to report what you see and seek further information
What are investigations of eosinophilia?
- FBC and differential white cell count
- Blood film examination
- Stool examination for ova and parasites
- CLARIFICATION!
What is monocytosis?
These are recognised as bigger cells with irregular shaped nuclei. They have pale grey blue cytoplasm and there is no prominent granulations but some have vacuoles.
- TB
- Acute and chronic monocytic and myelomonocytic leukaemia
- Malaria (chronic, not so much acute)
What are the investigations of monocytosis?
- FBC and differential white cell count
- Blood film examination: for abnormal white blood cells and for malarial parasites
- Bone marrow examination – leukaemia
- TB cultures
What is lymphocytosis?
Some are clear cut, big square monocytes. The morphology alone may not get you to know what something is, so you could have to use certain stains etc and send them away to find out with phenotyping etc. There could be something malignant in this picture…
What are the possible causes of lymphocytosis?
Normal physiological status:
• Lymphocytosis of childhood (1-6 years) normal: 5.5-8.5x10^9/L
Other causes:
• Bacterial infection
• Viral infections eg hepatitis, mumps, rubella, pertussis, glandular fever, (infectious mononucleosis)
• Leukaemias and lymphomas
Higher in early life and drops down and reaches normal range in teenage life
What are the investigations of lymphocytosis?
- FBC and differential white cell count
* Blood film examination
What are atypical mononuclear cells?
This may be seen in glandular fever etc. All the glands produce these. They are not malignant, they are blasts – reactive T cells often look like this. They envelope the red cells. Peripheral basophilia is present which means there is a blue ring around the red cells. This may be how blood cells look after glandular fever for example, or anything that drives a strong T cell immune response
What is infectious mononucleosis?
Glandular Fever
Heterophile antibodies are antibodies which react against an antigen which is completed unrelated to the antigen which originally stimulated it eg human antibodies reacting against sheep or horse or bovine cells
What is clearview infectious mononucleosis?
This is the modern way of testing. There are antibodies that will detect antibodies. If they are in the serum moving upwards and we put on a secondary antibody that will recognise them, we will get a control line.
When we see the second line occurring this may show mononucleosis. This may not work in the first few days of infection because they take a while to develop – so a repeat should be suggested
What investigations might be done into lymphocytosis?
Mature lymphocytes: chronic lymphocytic leukaemia? Lymphoma?
Immunophenotyping to determine if lymphocytes are:
- B cells: demonstrate clonality by light chain restriction
- T cells: demonstrate clonality by T-cell receptor gene rearrangement studies
What are blood group antigens?
Antigens are part of the surface of cells
• All blood cells have antigens
Reactions to blood usually occurs when the antibody in the plasma reacts with an antigen on the cells.
- There are 26 known blood group systems
- ABO and Rh are clinically most important
- Antigens in transfused blood can stimulate a patient to produce an antibody but only if the patient lacks the antigen themselves
- The frequency of antibody production is very low but increases the more transfusions that are given
What are blood group antibodies?
Antibodies are protein molecules – immunoglobulins (Ig)
• Usually of the immunoglobulin classes: IgG and IgM
• Found in the plasma
• Produced by the immune system following exposure to a foreign antigen
How is antibody production stimulated?
- Blood transfusion: ie blood carrying antigens foreign to the patient
- Pregnancy: fetal antigen entering maternal circulation during pregnancy or at birth
- Environmental factors: ie naturally acquired eg anti-A and anti-B
What are example of in vivo and in vitro antibody-antigen reactions?
In vivo (in the body) – leads to destruction of the cell either: Directly when the cell breaks up in the blood stream (intravascular) Indirectly when liver and spleen remove antibody coated cells (extravascular)
In vitro (in the laboratory) Reactions are normally seen as agglutination tests
What is agglutination?
- Agglutination is the clumping together of red cells into visible agglutinates by antigen-antibody reactions
- Agglutination results from antibody cross-linking with the antigens
What can agglutination identify?
As the antigen-antibody reaction is specific, agglutination can identify:
• The presence of a red cell antigen: ie blood grouping
• The presence of an antibody in the plasma: ie antibody screening/identification
What is the clinical significance of the ABO grouping system?
- A and B antigens very common (55% UK)
- Anti-A, anti-B or anti-A,B antibodies very common (97% UK)
- High risk of A or B cells being transfused into someone with the antibody in a random situation
- ABO antibodies can activate complement causing intravascular haemolysis
(Almost) all serious/fatal transfusion reactions caused by technical/clerical error are due to ABO incompatibility
What is blood grouping?
The patients red cells and plasma are both tested.
Test patient’s red cells with anti-A, anti-B and anti-D
• Agglutination shows that a particular antigen is on the red cells
• No agglutination shows the antigen is absent
Test patient’s plasma with A cells and B cells
• Agglutination shows that a particular antibody is in the plasma or serum
• No agglutination shows the antibody is absent
What is the Rh grouping system?
50+ antigens:
• Most important antigen is called D
• People with D antigen are RhD positive (85% of UK)
• People who do not produce any D antigen are RhD negative (15%)
• The other 4 main Rh antigens are known as C, c, E and e
What is Rh (D) typing?
- Most important after ABO
- Must be tested in duplicate (or tested each time and compared to historical result)
- Patient/donor classified as RhD pos or RhD neg
What is the clinical significance of Rh?
Rh antibodies are clinically significant. Second only to ABO.
Transfusion:
• D antigen is very immunogenic and anti-D is easily stimulated – PREVENTION!
• All Rh antibodies are capable of causing severe transfusion reaction – ANTIBODY DETECTION
Pregnancy:
• Rh antibodies are usually IgG and can cause haemolytic disease of the newborn
• Anti-D is still most common cause of severe HDN
What is laboratory testing of HDN?
- Blood group and antibody screen at antenatal booking to identify pregnancies at risk of HDN – RhD negative women who may need anti-D prophylaxis
- Blood group and antibody screen at 28 weeks
- Atypical antibodies are quantified periodically to assess their potential effect on the fetus
What is RAADP?
- An injection of anti-D will bind to and remove any fetal RhD positive red cells in the circulation
- 1500 iu of anti-D is given routinely at 28 weeks and a smaller dose (usually 500 iu) after delivery if baby RhD+
- in some hospitals 2 smaller (500 iu) doses are given at 28 and 34 weeks instead of the 1 larger dose
- anti-D is also given after any event that may cause a feta-maternal haemorrhage (bleed between mum and fetus) such as:
- abdominal trauma
- intrauterine death
- spontaneous or therapeutic abortion
What is antibody screening?
- there are other clinically significant antibodies that can cause a haemolytic transfusion reaction
- it is important that we screen for these antibodies so that if detected, antigen negative blood can be provided to avoid causing an immune reaction
- patients serum is mixed with 3 selected screening cells, incubated for 15 minutes at 30^C and then centrifuged for 5 minutes
- any clinically significant antibodies reacting at body temperature should be detected and then identified using panel of known phenotyped red cells
- specific antigen negative blood can then be provided for these patients to avoid stimulating an immune response
What must be do if an antibody is detected in an antibody screen?
- identify the antibody
* assess its clinical significance: for transfusion, in pregnancy
How do we detect an antibody in an antibody screen?
- compare pattern of reactions with each reagent cell of ID panel with the pattern fo antigens on the reagent cells
- matching pattern will identify the antibody
What is the zeta potential?
the potential difference existing between the surface of a solid particle immersed in a conducting liquid (e.g. water) and the bulk of the liquid.
IgM antibodies can span the gap between RBCs. IgG can not, because it is too small to overcome the ZETA potential (positive chage). LISS (low ionic strength saline) is negatively charged, so neutralises positive ZETA potential. Therefore, IgG can now span the gap
What is the indirect anti-globulin test (IAT)?
• used to detect IgG antibodies • LISS counteracts Zeta potential • Results in agglutination • Used for: • Screening for antibodies • Identifying antibodies Cross matching donor blood with recipient plasma when there are known antibodies or a previous history of antibodies
What is an intermediate spin cross-match (ISX)?
- Antibody screen is negative
- Checking donor red cells against patients plasma: ABO check, incubate for 2-5 minutes (room temp), spin and read
ISX – basically just checking the ABO group. Therefore IgM antibodies (therefore no problem with ZETA potential, therefore no need to IAT).
What is a full indirect anti globulin test (IAT) cross match?
- Antibody screen positive or patient has known antibody history
- Select antigen negative donor red cells and incubate with patient serum for 15 minutes at 37^C
How is donor blood tested?
- Blood establishment: MHRA licensed manufacturer of blood and products
- Donor selection: questionnaire; lifestyle, health, not previously transfused
- Collection procedure arm cleansing/diversion pouch
- Comprehensive testing of all products: Viral; HIV 1+2, hepatitis B, hepatitis C, syphilis, HTLV
- Platelets: bacteria
What are the relative risks of transfusion?
- 1 in 910,000 for hepatitis B
- 1 in 70 million for hepatitis C
- 1 in 5 million for HIV infection
- 1 in 23 million for HTLV infection
There are believed to have been 4 cases of infected with vCJD from blood transfusion
About fresh frozen plasma…
• FFP contains all clotting factors
- Given for coagulopathy with associated bleeding
- Requires clotting screens to monitor
• Only has 24 hour life after thawing
About platelets (donor)…
- Adult pool of platelets fro 4 donors (suspended in plasma from 1 donor)
- 85% of doses are apheresis units
- platelets required to create clots to reduce bleeding
- some drugs given to reduce efficacy of platelets (anti-platelet agents) so patient history is important
What is cryoprecipitate?
Contains factor VIII, VWF and fibrinogen
- 2 units usually given at one time
- monitor fibrinogen levels by clotting screens
How is donor blood regulated?
- EU Blood Safety Directive
- Blood Safety Quality Regulations
- Better Blood Transfusion 3
- MHRA inspections
- CPA inspections
What are methods of haemovigilance?
Serious hazards of transfusion (SHOT):
- Voluntary reporting
- Report all serious adverse events (SAE) and serious adverse reactions (SAR)
Serious adverse blood reactions and events (SABRE):
- Mandatory reporting
- Report all SAR and SAE where the root cause error was the quality system
What sources maintain blood glucose levels?
Blood glucose levels are maintained from several sources:
• Dietary carbohydrate
• Glycogenolysis
• Gluconeogenesis
Livers role:
• After meals: stores glucose as glycogen
• During fasting: makes glucose available through glycogenolysis and gluconeogenesis
Why should glucose levels be regulated?
- Brain and erythrocytes require continuous supply – avoid deficiency
- High glucose and metabolites cause pathological changes to tissues; eg, micro/macro vascular diseases, neuropathy – avoid excess
What is insulin?
- Peptide hormone (51 amino acids)
- Synthesised in -cells of pancreas as proinsulin; cleaved to insulin and C-peptide
- Secretion is stimulated by rise in blood glucose
What are glucose counter-regulatory hormones?
Glucagon
• Secreted by alpha-cells of pancreas in response to hypoglycaemia
• Stimulates glycogenolysis and gluconeogenesis
Adrenaline
• Increased glycogenolysis and lipolysis
Growth Hormone
• Increased glycogenolysis and lipolysis
Cortisol
• Increased gluconeogenesis, glycogen synthesis
What is diabetes mellitus?
Metabolic disorder characterised by chronic hyperglycaemia, glycosuria and associated abnormalities of lipid and protein metabolism.
• hyperglycaemia result of increased hepatic glucose production and decreased cellular glucose uptake
• blood glucose > ~10mmol/L exceeds renal threshold – glycosuria
• long term complication – micro-macrovascular disease
What is the prevalence of diabetes?
- globally 415 million people currently have diabetes; estimated to double by 2030
- estimated prevalence of diabetes in UK 2013 was 3.2 million (ages 17+)
- number of people in UK with diabetes has more than doubled since 1996 (1.4 million)
What are the reasons for an increase in the prevalence of diabetes?
- increased awareness
- more complete recording
- poor diet
- low physical activities
How is diabetes diagnosed?
In the presence of symptoms: (polyuria, polydipsia weight loss)
• random plasma glucose > 11.1 mmol/l (200 mg/dl) OR
• fasting plasma glucose > 7.0mmol/l (126 mg/dl) fasting is defined as no caloric intake for at least 8 hours OR
• 2-h plasma glucose > 11.1 mmol/l (200 mg/dl); 2hrs after 75g oral glucose tolerance test (OGTT)
In the absence of symptoms:
• test blood samples on 2 separate days
What is IGT (pre-diabetes) and IFG?
Impaired glucose tolerance (IGT)
• fasting plasma glucose 6.1-6.9 mmol/L**
• OTTG value of
What is the oral glucose tolerance test (OGTT)?
• OGTT should be carried out:
o In patients with IFG
o In unexplained glycosuria
o In clinical features of diabetes with normal plasma glucose values
o For the diagnosis of acromegaly
• 75g oral glucose and test after 2 hours
• blood samples collected at 0 and 120 mins after glucose
• subjects tested fasting after 3 days of normal diet containing at least 250g carbohydrate
What are the classifications of diabetes?
TYPE 1:
insulin secretion is deficient due to autoimmune destruction of beta-cells in pancreas by T-cells
TYPE 2:
insulin secretion is retained but there is target organ resistance to its actions
GESTATIONAL:
occurs for first time in pregnancy
SECONDARY:
chronic pancreatitis, pancreatic surgery, secretion of antagonists
About type 1 diabetes mellitus…
- predominantly in children and young adults’ but ages also
- sudden onset (days/weeks)
- appearance of symptoms may be preceded by ‘prediabetic’ period of several months
- commonest cause is autoimmune destruction of B-cells
- interaction between genetic factors and environment
- strong link with HLA genes within the MHC region on chromosome 6
What is the pathogenesis of type 1 DM?
• HLA class II cell surface present as foreign as self antigens to T-lymphocytes to initiate autoimmune response
• Circulating autoantibodies to various-cell antigens:
o Glutamic acid decarboxylase
o Tyrosine-phosphatase-like molecule
o Islet auto-antigen
• The most commonly detected antibody associated with type 1 DM is the islet cell antibody
• More than 90% of newly diagnosed persons with type 1 DM have one or another of these antibodies
• Destruction of pancreatic beta-cell causes hyperglycaemia due to absolute deficiency of both insulin and amylin
• Amylin, a glucoregulatory peptide hormone co-secreted with insulin
lowers blood glucose by slowing gastric emptying, and suppressing glucagon output from pancreatic cells
What is the presentation of type 2 DM?
- Slow onset (months/years)
- Patients middle aged/elderly – prevalence increases with age
- Strong familiar incidence
- Pathogenesis uncertain – insulin resistance; beta cell dysfunction may be due to lifestyle factors – obesity, lack of exercise
- Emergency presentation as hyper-osmolar, non-ketotic state (HONK)
What are metabolic complications of type 2 DM?
Hyper-osmolar non-ketotic coma (HONK) • Development of severe hyperglycaemia • Extreme dehydration • Increased plasma osmolality • No ketosis, minimal acidosis • Impaired consciousness • Death is untreated
What are ways of biochemical monitoring DM?
- Immediate: blood glucose
- Few hours: urinary dipstick
- 3-4 months: blood HbA1c (glycated Hb, covalent linkage of glucose to residue in Hb)
- Others: orinary albumin (index of risk of progression to nephropathy), abnormalities in serum lipids common in T1 and T2 DM – increased risk of MI and stroke
What are the aims of monitoring DM?
- Prevent complication through tight control
- Avoidance of hypoglycaemia
- Self-monitoring: capillary blood measurement, urine analysis: glucose in urine gives indication of blood glucose concentration above renal threshold
- Glycated Hb (HbA1c) – aim at
What are possible long term complications of DM?
- Occur in both type 1 and type 2 DM
- Micro-vascular disease: retinopathy, nephropathy, neuropathy
- Macro-vascular disease: related to atherosclerosis heart attack/stroke
- Exact mechanisms of complications are unclear
How might we reduce the CV risk in DM?
- Attain normal weight and waist circumference
- Eat food low in fat and salt
- Exercise
- Stop smoking
- HbA1c
What is hypoglycaemia?
• Defined as plasma glucose
What are the causes of hypoglycaemia?
- Insulinoma
- Drugs eg sulphonylureas, insulin, alcohol abuse
- Inherited metabolic disorders eg glycogen storage diseases, galactosaemia, hereditary fructose intolerance
- Endocrines disease; eg cortisol disorder
- Others: severe liver disease, non-pancreatic tumours, postgastrectomy
What are the adaptations to falling glucose levels in fasting?
• Insulin levels fall, limiting glucose entry into non-cerebral tissues
• Hepatic gluconeogenesis stimulated
• Glycogen breakdown activated
• Fatty acid oxidation activated
• Release of counter regulatory hormone raising glucose
- Glucagon – (
What are the symptoms of hypoglycaemia?
Neurogenic (autonomic):
- Triggered by falling glucose levels
- Activated by ANS and mediated by sympathoadrenal release of catecholamines and Ach
- Sign and symptoms include shakiness, anxiety, nervousness, palpitations, sweating, dry mouth, pallor and pupil dilation
Neuroglycopaenia:
- Due to neuronal glucose deprivation
- Sign and symptoms include confusion, difficulty speaking, ataxia, paresthesia, seizure, coma and death
Insulinoma:
- Tumour in the insulin secreting beta-cells of pancreas
Symptoms include:
- Fasting hypoglycaemia, patients may present with behavioural changes
- Inappropriate high insulin concentration at time when plasma glucose is low (
What are examples of inherited metabolic diseases?
- Glycogen storage disease type I (von Gierke’s disease); deficiency of G-6-Phosphatase: impaired glucose release from glycogen
- Galactosaemia: deficiency of galactose-1-phosphate uridyl transferase: liver damage
- Hereditary fructose intolerance: deficiency of fructose-1-phosphate adolase B: accumulation of fructose-1-phosphate in liver
What is glycogen storage disease type Ia?
- Autosomal recessive disorder (described in 1929)
- Glucose synthesis from glycogen or by gluconeogenesis is blocked
- Presents in early infancy; severe fasting hypoglycaemia as only source of glucose is dietary carbohydrate
- Accumulation of glycogen causes hepatomegly; inability to produce glucose from lactose causes acidosis
- Tx: uncooked cornstarch; a slow releasing glucose prep
What is galactosaemia?
- Defects in 3 enzymes can cause galactosaemia; most common is galactose-1-phosphate uridyl transferase deficiency
- Autosomal recessive disorder (1st described in 1935)
- Deficiency of G-1-PUT impairs conversion of galactose-1-phosphate to glucose-1-P gal-1-phosphate accumulates in liver – toxicity
- Hypoglycaemia and vomiting/diarrhoea after starting milk feeds
- Galactose excreted in urine. Tx – exclude galactose from diet
What is hereditary fructose intolerance?
- Autosomal recessive disorder (1st described in 1956)
- Deficiency of fructose 1-phosphate aldolase B when ingested fructose accumulates – inhibits glycogenolysis at phosphorylase step
- Severe hypoglycaemia and vomiting after ingesting fruit, sweetened foods
- Fructose detected in urine
- Avoid ingestion of fructose, sucrose
- Benign fructose intolerance: this is due to absence of fructokinase
What are the hypothalamic/pituitary connections?
- These are neural connections between the hypothalamus and posterior (but not anterior) pituitary
- Blood form the hypothalamus supplies the anterior pituitary
- The secretion of anterior pituitary hormones is regulated by hypothalamic releasing factors
What is the regulatory factor to growth hormone?
GR-RH (stimulatory) and somatostatin (inhibitory)
What is the regulatory factor to prolactin?
PIF (dopamine)
What is the regulatory factor to TSH?
TRH
What is the regulatory factor to ACTH?
corticotrophin (CRF)
What is the regulatory factor to LH?
GnRH
What is the regulatory factor to FSH?
GnRH
About thyroid hormones…
¥ Essential for normal growth and development
¥ Increase basal metabolic rate (BMR) and affect many metabolic processes
¥ Synthesized in thyroid via series of enzyme catalyzed reactions, beginning with uptake of iodine into gland
¥ Synthesis and release controlled by TSH
¥ T4 main hormone secreted by thyroid, T3 is more biologically active – mostly formed by peripheral conversion from T4
¥ Effects are mediated via activation of nuclear receptor
About thyroid hormones in blood…
- Both T4 and T3 are extensively protein bound, principally to TBG
- Free (non-protein bound) fraction physiologically active
- Changes in protein binding affect total hormone concentrations
- Measurement of free hormones is not affected by variation in TBG and so discriminates more reliably between normal and abnormal thyroid function
What are possible causes of hyperthyroidism?
- Graves disease: most common (TSH-R antibodies)
- Toxic multinodular goiter
- Toxic adenoma thyroiditis
- Excessive TSH production (rare)
What is hyperthyroidism?
excessive production of thyroid hormones
What are the clinical features of hyperthyroidism?
- weight loss
- heat intolerance
- palpitations
- goitre
- eye changes
What investigations might be done into hyperthyroidism?
- Serum TSH concentraitons suppressed
- fT4 raised
What is hypothyroidism?
deficient production of thyroid hormones
What are possible causes of hypothyroidism?
- primary: autoimmune thyroiditis, iodine deficiency, post-surgery/antithyroid drugs, congenital
- secondary: pituitary or hypothalamic diorder
What are the clinical features of hypothyroidism?
- weight gain
- cold intolerance
- lack of energy
- goitre
- congenital - development abnormalities
What investigations might be done into hypothyroidism?
- serum TSH concentrations raised
- fT4 reduced (primary)
- reduction in both TSH+fT4 consistent with hypopituitarism
What are treatment and monitoring options for hyperthyroidism?
- anti-thyroid drugs (g carbimazole)
- partial thyroidectomy
- radioactive iodine
- fT4 levels fall rapidly with successful treatment
- TSH may remain suppressed for several months
What are treatment and monitoring options for hypothyroidism?
- Thyroxine replacement
- fT4 may remain slightly raised
- best monitored by TSH (elevated values indicate under/and suppressed values over replacement)
What are the zones of the adrenal cortex?
- Aldosterone is the main mineralocorticoid synthesized in (outer) zona glomerulosa
- Cortisol is the principal glucocorticoid produced by the zona fasciculate
- Glucocorticoids and adrenal androgens are synthesized in zona fasciculate & zona reticularis
What are the biological effects of adrenal steroids?
Cortisol
- antagonizes effects of insulin by enhancing gluconeogenesis - promotes the central deposition of fat - increases catabolism of proteins - weak mineralocorticoid effects - anti-inflammatory
Aldosterone
- increases sodium reabsorption in renal DCT
Androgens
- virilizing effects when secreted in excess
How is adrenal steroid secretion controlled?
- Synthesis of cortisol regulated by hypothalamic - pituitary - adrenal axis
- Aldosterone synthesis is controlled by the renin-angiotensin system
- Adrenal androgen synthesis is regulated by ACTH, not gonadotrophins
What are examples of hyper functioning of the adrenal cortex?
Conn’s syndrome:
excess secretion of aldosterone due to tumour or hyperplasia of adrenal - sodium retention
Cushing’s syndrome: excess cortisol secretion - causes: - pituitary tumour (Cushing’s disease) - ectopic ACTH production - adrenal tumour - exogenous administration of steroids
What is cushings syndrome and what are the clinical features?
Due to excessive cortisol production by adrenal.
Clinical features include: • muscle weakness • menstrual disturbances • psychiatric disturbances • moon face, truncal obesity • striae, bruising • hirsutism, acne • hypertension • glucose intolerance • osteoporosis
What are the diagnostic steps to cushion’s syndrome?
- Demonstration of excessive cortisol production
Screening Tests:
- Loss of cortisol circadian rhythm
- Elevated 12am serum cortisol
- Increased 24hr urine cortisol
- Inadequate suppression of serum cortisol levels following low dose (1mg dexamethasone) - Elucidation of cause
- High dose dexamethasone suppression test
- ACTH assay
What are dynamic tests of endocrine function?
Dynamic tests are important tools in the investigation of endocrine function.
There are two types:
- Stimulatory test may be used to investigate endocrine gland hypofunction
- Suppression tests are used to investigate suspected hyperfunction
What is the dexamethasone suppression test?
Dexamethasone
- synthetic glucocorticoid
- binds to GC receptors in pituitary to suppress
- ACTH release (and cortisol secretion from adrenal)
Low dose dexamethasone suppression test
- 1mg dexamethasone given at night. Serum cortisol is measured at 9am next morning
- in normal subjects, cortisol
What are the causes of andrenocortical insufficiency?
Causes:
Primary adrenocortical failure –
Addison’s disease due to :
autoimmune disease
tuberculosis
less common; metastases, haemochromatosis,
haemorrhage
Secondary to impaired ACTH release eg –
tumour, head trauma, surgery,
rapid withdrawal of steroids
What are the clinical features of adrenocortical insufficiency?
- fatigue
- weakness
- hypoglycaemia
- hypotension
What is the short syncathen test?
Short synacthen test
- to assess the ability of the adrenal to produce cortisol in
response to ACTH
Serum cortisol is measured after giving synacthen (synthetic ACTH)
(normal response – cortisol increases by >200nmol/L over basal
level with peak of >550nmol/L).
What is the long syncathen test?
Long synacthen test
- 3 day stimulation with 1mg depot synacthen i.m. daily
In secondary adrenal hypofunction, serum cortisol levels increase
to at least 200nmol/L over baseline values
What is congenital adrenal hyperplasia?
¥ Group of inherited metabolic disorders of adrenal steroid hormone biosynthesis
¥ Clinical features depend on the position of enzyme defect in metabolic pathway
¥ 21-hydroxylase deficiency > 90% cases, prevalence 1:5000-12,000
Leads to impaired production of cortisol and aldosterone. Raised ACTH stimulates production of adrenal androgens
What are neonatal manifestations of CAH?
¥ Virilization of female infants
¥ Salt wasting and dehydration occurs during first 4 weeks of life, serum Na 2 days after birth
¥ Requires glucocorticoid and mineralocorticoid replacement
¥ Treatment monitored by measurement of either plasma 17a-hydroxyprogesterone or androstenedione
What is polycystic ovarian syndrome?
¥ Condition of excess androgen secretion and chronic anovulation
¥ Ovaries are the source of excess androgens and there are multiple ovarian cysts
¥ Associated with menstrual disturbances, hirsutism and infertility
¥ Many patients with PCOS are overweight and a link has been made with insulin resistance
¥ Increased serum LH:FSH ratio
¥ Decreased SHBG
What are possible causes of hypopituitarism?
- Tumours eg pituitary adenoma
- Trauma/ head injury
- Hypothalamic disorders
- Vascular disease
- Infection – eg meningitis
- Iatrogenic – eg therapeutic skull irradiation
- Miscellaneous – eg sarcoidosis, haemochromatosis
What is a combined pituitary function test?
Destructive lesions of the pituitary tend to present with pituitary hypofunction, thus stimulatory tests are used toassess the ability of the gland to secrete hormones.
In assessing patients with suspected anterior pituitary dysfunction, it is often convenient to test the capacity of the gland to secrete GH, ACTH, TSH and the gonadotrophins in a single procedure – the combined pituitary function test.
This involves measurement of anterior pituitary hormones following adminstration of TRH, GnRH and insulin
What is isolated growth hormone deficiency?
- GH deficiency in children is a cause of growth retardation
- The diagnosis depends on the demonstration of subnormal growth velocity and subnormal serum growth hormone concentrations
- While a random growth hormone concentration >20mU/L excludes significant deficiency, a low level is not diagnostic
- GH status can be measured by a number of stimulatory tests
About pituitary tumours…
¥ Pituitary tumours may be functional, producing excess hormone secretion
¥ Prolactin, GH and ACTH secreting tumours are well recognised
¥ Gonadotrophin and TSH secreting tumours are rare
¥ Since the presence of the tumour may give rise to destructionof normal pituitary tissue, decreased production of other hormones should be investigated with the combined pituitary function test
What is acromegaly?
¥ Acromegaly is the result of excessive GH secretion by a pituitary tumour leading to increased growth of soft tissue/bone increase in size of hands, feet, jaw change in facial appearance
¥ GH concentration in a random serum sample usually raised
¥ Diagnosis confirmed biochemically by demonstrating failure of suppression of GH levels in response to oral glucose
¥ Treatment options – surgery, external irradiation, drugs
¥ Accompanying hypopituitarism requires appropriate treatment with cortisol, sex steroids and thyroxine
What are the major functions of the liver?
Carbohydrate metabolism
• Gluconeogenesis
• Glycogen synthesis and degredation
Lipid Metabolism • Fatty acid synthesis • Cholesterol synthesis and excretion • Lipoprotein • Ketogenesis • Vitamin D 25-OH • Bile acid synthesis and excretion
Protein metabolism
• Plasma protein
• Ammonia detoxification
Bilirubin metabolism
Hormones
• Conjugation of steroid hormones
• Degradation of peptide hormones
Xenobiotic metabolism
Storage • Vitamin A • Glycogen • B12 • Iron
About liver function tests…
¥ Liver has a considerable functional reserve, and therefore simple tests of liver function as bilirubin or albumin concentrations are insensitive indicators of disease
¥ Tests reflecting liver cell damage are more sensitive, e.g., hepatic enzymes in plasma
¥ True tests of liver function, e.g., bromsulphophthalein (BSP), indocyanin green, caffeine clearance and radio-labelled bile acid uptake and clearance tests are available for research purposes
Biochemical tests usually reflect the basic pathological processes common to many conditions, and rarely provide a precise diagnosis, However, they are cheap, non-invasive, and can direct other investigations, e.g., liver biopsy and imaging. They are useful in detecting the presence of a liver disease and in monitoring progress
What are the objectives of liver function tests?
- Aid in establishing liver disease by showing abnormal values
- Inform or help specific diagnosis by showing different patterns
- Establish the severity of liver damage and provide prognostic insight
- Monitor disease progression
- Assess response to therapy
About bilirubin and urobilinogen (bile pigments)…
o Derived from the break down of haem of senescent PBCs, and to a lesser extent from myoglobin, cytochromes and some enzymes
o Being water insoluble, it is transported in the plasma to the liver by albumin. At this stage, bilirubin is unconjugated and can not pass in urine even if present at high level
o Unconjugated bilirubin is taken up by liver cells to be conjugated with glucuronic acid by the enzyme UDP-glucuronyl transferase to produce bilirubin diglucuronide which is water soluble and s called conjugated bilirubin which is then secreted into bile and eventually reaches the intestine where more of it is oxidised by bacteria to urobilinogen and stercobilin
o A small amount of urobilinogen and bilirunin are reabsorbed through entero-hepatic circulation to be secreted in urine
Total bilirubin reference range (RR): 3-17 micromol/l
Plasma bilirubin rises when significant liver damage occurs and causes yellowish discolouration of skin and mucous membranes (Jaundice) which can be clinically detected at bilirubin level of 50 umol/l
What is monitoring bilirubin used for?
- Investigate liver diseases and assess subclinical hyperbilirubinaemia
- Determine the need for exchange transfusion in neonatal jaundice
- Assess surgical treatment of bile duct stricture
- Determining correct doses of drugs, e.g., cytotoxic therapy
What is alkaline phosphatase?
Alkaline phosphatase (ALP): (RR: 30-130 U/I)
o A group of enzymes that hydrolyse phosphate esters at alkaline pH and are present in liver, bone, intestine and placenta. Plasma ALP increases in the presence of obstruction to the biliary passages or cholestasis. The block could be intrahepatic as in PBC, or due to a stone in the bile duct or pressure by a tumour, e.g., head of pancreas or lymph nodes
o ALP increases in liver and bone disease, with pregnancy, growing children and to enhance the specificity, ALP isoenzymes can be measured using electrophoresis or utilising different thermal stabilities of iso-enzymes, as liver isoenzyme activity drops by 40% after 15 min incubation at 56oC while bone isoenzyme activity decreases only by 15% or gGT is used
About alanine aminotransferase (ALT) and aspartate aminotransferase (AST)…
Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST)
(RR: 0-40 U/l for both)
o Also called transaminases as they transfer an amino group from an amino acid to a keto acid. They increase in the plasma in presence of liver cell damage.
o They have wide tissue distribution, with AST present in liver, heart, skeletal muscle, kidney, brain, erythrocytes and lung. ALT has also a similar wide tissue distribution but activities are lower in extrahepatic tissues, and increased plasma ALT is more specific for liver disease (CK for muscle and troponins for heart)
o Aminotransferase activity above 10 times the upper limit of RR are most frequently due to hepatocellular damage, e.g., viral or drug induced hepatitis or autoimmune hepatitis
o Aminotransferase activity below 10 times the upper limit of RR, but higher than upper limit of RR are less specific but could occur in chronic liver diseases
o Aminotransferase activities within RR do not exclude liver disease that could be assessed by histological examination of a biopsy specimen
o The higher the AST/ALP ratio, the more likely the underlying condition to be a form of hepatitis. The lower the ratio, the more it is indicative of cholestatic disorder
o An AST/ALT ratio of more than 2 in a patient who clinically appears to have hepatitis strongly suggests that alcohol is involved
What is mitochondrial AST?
Accounts for 80% of total AST in liver cells and the ratio of mAST/total AST is a good marker for chronic alcohol consumption
What is gamma-glutamyl transferase?
o A microsomal enzyme that transfers glutamyl groups between peptides and aminoacids and is involved in the transport of peptides across plasma membranes.
o Plasma activity is mainly derived from liver, despite its wide distribution, and it has a poor specificity and high sensitivity for liver disease
o Its measurement helps in linking an elevated ALP to the liver and is elevated with chronic alcohol consumption and is more elevated in the presence of chronic liver disease with alcohol consumption
About albumin…
Albumin: (RR: 30-50 g/l)
o Synthesised exclusively by liver and its plasma half-life is 21 days. It maintains plasma oncotic pressure, binds calcium, fatty acids, bilirubin, some hormones and drugs.
o Its level tends to fall with progress of chronic liver disease and therefore plays a major prognostic role. It also falls with inadequate protein intake
What is prothrombin time (PT)?
Measures the rate at which prothrombin is converted to thrombin in the presence of thromboplastin, calcium, fibrinogen, and coagulation factors V, VII and X.
What is the international normalised ratio (INR)?
INR is the ratio of the PT of the patient to that obtained using a control preparation. Normal individuals have an INR
How can PT and INR be affected?
In the presence of severe acute or chronic liver damage, the synthesis of clotting proteins by the liver is impaired . Prothrombin half life is 6 hours and therefore, a prolonged INR which can only be improved by fresh plasma transfusion or providing the deficient clotting factors is a good indicator of severe liver damage, e.g., acute liver failure
Vitamin K is a fat soluble vitamin and requires bile salts from liver for absorption. It is also needed for the synthesis of clotting factors by the liver. In case of cholestasis, vitamin K absorption is defective and the synthesis of clotting factors is impaired causing an increased INR which can be corrected within 24 hours by an injection of vitamin K
What is the epidemiology of hepatocellular carcinoma?
Is relatively common in Southeast Asia, Sub-Saharan Africa, Japan, Pacific Islands and Mediterranean area. UK incidence ranges between 1-7/100 000. Incidence in some tribes in Mozambique is 107/100 000. Individuals who migrate from areas of high incidence to areas of low incidence of HCC have lower incidence compared with the population remaining in lands of origin. Therefore, environmental factors, rather than genetic or racial predisposition, play a key role in pathogenesis of HCC.
In recent years, the incidence of HCC seems to be rising in the West, as a result of improved treatment of alcoholic and other cirrhosis (e.g., after hepatitis B or C) and more prolonged survival. Incidence rates of about 10/100 000 have been reported
What is the clinical presentation of hepatocellular carcinoma?
In low-incidence populations, the tumour usually presents in a patient who is known to have had cirrhosis or haemochromatosis for some time. In high incidence areas, it appears in individuals who were apparently healthy. However, the majority of affected patients are found to have cirrhosis in non-tumour part of liver
What are the symptoms of hepatocellular carcinoma?
Usually runs a silent course until advanced where it presents with:
• Upper abdominal discomfort or pain, in the right hypochondrium or epigastrium, radiating to the right sub-scapular region or shoulder
• Weight loss and weakness
• Feeling of fullness in the upper abdomen after meals/ anorexia
• Generalised swelling of the abdomen (due to ascites)
• Nausea, vomiting, constipation
• Peritoneal haemorrhage (acute abdomen due to tumour rupture )
• Bone pain due to skeletal metastasis
What are the signs of hepatocellular carcinoma?
- Early: Mild to moderate hepatomegaly, but later: Massive enlargemen, tenderness
- Hepatic arterial bruit, ascites heard, splenomegaly, jaundice, wasting, fever
- Stigmata of chronic liver disease: gynaecomastia, caput medusae, feminisation
- Ectopic Hormonal Syndromes: erythrocytosis, hypercalcaemia, feminisation (dehydroepiandrosterone converted to oestradiol by the tumour)
- Hypoglycaemia, hypercholesterolaemia, porphyria
- Elevated a-fetoprotein, carcino-embryonic antigen (CEA), dysfibrinogenaemia
How is hepatocellular carcinoma diagnosed?
• Determination of a-fetoprotein: markedly elevated
• Biochemistry: similar to cirrhosis: markedly elevated alkaline phosphatase with normal or slightly elevated bilirubin and transaminases, low albumin and elevated IgG and IgM
Radiologic Investigations: isotope liver scanning (figures), hepatic angiography, ultrasonography (solid vs cysts), CT scans
What is the treatment of hepatocellular carcinoma?
- Surgical Resection: partial hepatectomy, hemihepatectomy offer the only chance of cure of HCC. Tumour should be confined to one lobe and the rest of the liver free from cirrhosis or only have mild cirrhosis
- Liver transplantation
- Chemotherapy: 5-Flurouracil, cyclophosphamide, methotrexate, vincristine.
- External irradiation
What is helicobacter pylori infection?
H. Pylori infection is the main cause of duodenal or gastric ulcers and plays an important role in gastric cancer.
It can be diagnosed by microscopical examination of biopsy specimens. Alternatively, biopsies could be tested for the presence of urease, which is specific for H. Pylori, in the presence of an indicator that changes colour with the release of ammonia in case of positive biopsies. Antibodies for H. Pylori can also be checked but C13 or C14 urea breath test of PCR are preferred
What are the pancreatic functions?
Exocrine:
• Alkaline secretion
• Enzymes: amylase, lipase, trypsin, chymotrypsin, carboxypeptidases, elastase, esterases, phospholipases
• Bile salts and lipases are essential for fat digestion and absorption and absorption of fat soluble vitamins A, D, E, K
Endocrine:
• Insulin
• Glucagon
• Somatostatin
What is acute pancreatitis and its aetiology?
Pain: upper abdominal, radiates to the back
Enzymes marked elevation of amylase and lipase in blood and urine
Aetiology • Alcohol • Gallstones • Post-operative • Hyperlipidaemia • Traumatic • Carcinoma • Drugs: steroids, azathioprine, diuretics
What are the investigations of acute pancreatitis?
Biochemistry: • serum or urine amylase or lipase (high) • serum calcium (low) • falling arterial O2 tension • coagulopathy
Radiology and imaging (figures) • plain radiography • ultrasonography • computerized tomography (CT) • ERCP • PTC
What are gallstones?
affects 10-20% of world population
- Cholesterol stones: >70% cholesterone, mucoprotein ± calcium
• Supersaturation
• Nucleation
• GB motor dysfunction - Pigment stones: calcium bilirubinate, cholesterol, calcium soaps and mucoprotein matrix
• Bacterial beta-glucuronidase
What is the natural history of gall stones?
- Symptomless
- Biliary obstruction
- Cholangitis
- Cholecystitis
- Jaundice
- Intestinal obstruction
What investigations might be done into gall stones?
- Polymorphonuclear leukocytosis
- Increased alkaline phosphatase
- Elevated bilirubin
- Increased amylase
Abdominal radiograph: calcified gallstone
Ultrasonography: gallstones in GB
What is intrahepatic cholestasis?
Hepatocellular:
- Viral or alcoholic hepatitis
- Sex hormones and pregnancy
Biliary:
- Intrahepatic atresia
- Primary biliary cirrhosis
- Sclerosin cholangitis
- Malignancy
About carcinoma of the pancreas and what are the risk factors?
The incidence has doubled in UK over the past 50 years to 10/100 000/year. It is twice as high in men than in women, and over 80% of cases occur in the 60-80 y age group.
Risk Factors:
• Tobacco: cigarette smoking is associated with PC, with a relative risk of 2-3 over non-smokers.
• Diet: Increased animal fat, combined with increased alcohol consumption, are associated with increased incidence of PC. The fish oil fatty acids (eicosapentaenoic and docosahexaenoic acids) seem to antagonise the carcinogenic effect. Vegetables and fruit are protective
• Coffee: prolonged intake of higher amounts of coffee might be linked to PC
• Chronic Pancreatitis
• Diabetes mellitus
• Gastric resection
What are the signs and symptoms of carcinoma of the pancreas?
Symptoms:
- Pain: is the presenting feature in 50-80% of cases, usually dull aching or boring pain in the epigastrium and radiates to the back. It is aggravated by food intake and may be worst in recumbent position and eased by sitting.
- Jaundice: is the 1st symptom in 10-30% of patients and the presenting symptom in 30-65%. It is progressive until releived by surgery or stenting. Pruritus is common.
- Weight Loss: is the presenting feature in 90% of patients, usually rapid and progressive.
- Other Symptoms: Fatigue, weakness, epigastric bloating, diarrhoea, steatorrhoea, or constipation. Nausea and vomiting occur in 25% of patients
Signs:
- Jaundice
- Hepatomegaly
- a palpable gallbladder and epigastric tenderness
- ascities
- enlarged lymph nodes
What investigations might be done into carcinoma of the pancreas?
- Imaging Techniques: CT scanning, ERCP, PTCA, US, angiography (Figures)
- Blood Tests: ESR, CRP, Blood count, LFTs, enzymes: amylase, lipase, pancreatic elastase, tumour markers: DU-PAN 2, CEA, CA 19-9, CA 50
What are treatments for carcinoma of the pancreas?
- Pre-operative biliary drainage
- Palliative surgery
- Curative resection: prognosis and complications
- Chemotherapy: 5-flurouracil, cyclophosphamide, methotrexate, vincristine
- Radiotherapy
What biochemical tests are done in clinical medicine?
- Screening (subclinical conditions)
- Diagnosis (normal vs abnormal values)
- Monitoring (course of disease)
- Clinical management (treatment/response)
- Prognosis (risk stratification)
What are cardiac markers?
- Located in the myocardium
- Released in response to cardiac overload
- Released in response to cardiac injury
- Released in response to cardiac failure
- Can be measured in blood samples
What is the classification of laboratory tests in cardiac disease?
- Markers of risk factors for development of coronary artery disease
- Genetic analysis for candidate genes of risk factors
- Markers of cardiac tissue damage
- Markers of myocardial function/overload
What can biochemical makers of cardiac dysfunction/damage contribute to?
- Rule in/out an acute MI
- Confirm an old MI
- Help to define therapy
- Monitor success of therapy
- Diagnosis of heart failure
- Risk stratification of death
What are the characteristics of the ideal cardiac marker?
Analytical characteristics
- Measureable by cost-effective method
- Simple to perform
- Rapid turnaround time
- Sufficient precision and accuracy
- Reasonable cost
Clinical Characteristics
- Early detection of disease
- Sensitivity vs specificity
- Validated decision limits
- Selection of therapy
- Risk stratification
- Prognostic value
- Ability to improve patient outcome
Why is it important to define the type of IHD?
Stable angina vs acute myocardial infarction
- Treatment
- Prognosis
- Management
What are some possible causes of chest pain?
- Broken rib
- Collapsed lung
- Nerve infection (shingles)
- ‘pulled’ muscle
- infection
- heart burn (hernia)
- pericarditis
- blood clot in the lungs (PE)
- angina
- myocardial infarction
How might we assess IHD?
- medical history
- risk factors
- presenting signs and symptoms
- ECG
- Biomarkers
- Imaging/scans
How does ECG and biomarkers define the type of acute coronary syndrome?
In general, an ST elevation MI is caused by complete obstruction of a coronary artery, and causes damage that involves the full thickness of the heart muscle.
A non-ST elevation MI is caused by partial obstruction of a coronary artery, and causes damage that does not involve the full thickness of the heart wall
What is myocardial injury?
- Irreversible injury typically requires 30 minutes of ischaemia
- High risk that 80% of cardiac cells die within 3 hours and almost 100% by 6 hours
- Cellular content leak out through membrane dependent on size and solubility
- Concentration gradient from inside to outside important (high gradient improves detection of early damage)
What are markers of myocardial damage?
- 7-36h peak after MI
- heart muscle specific markers troponin-T and troponin-I
- creatinine kinase (increase by 90% MIs, but less specific as also released from skeletal muscle)
- heart specific isoforms of creatinine phosphokinase (CPK-MB)
- myoglobin raised early but less specific for heart damage
What are troponins?
The troponin complex is a component of the thin filaments in striated muscle complexed to actin.
There are three types of troponins:
o Troponin T (tropomyosin binding)
o Troponin I (inhibits actomyosin ATPase)
o Troponin C (calcium binding)
The troponins are three different proteins structurally unrelated with each other.
Cardiac troponin T and I differ significantly from troponin T and I found in skeletal muscle
What are the advantages of cardiac troponin?
Advantages of cardiac troponin:
- An index of cardiac damage
- Blood levels related to severity of cardiac damage
- Predicts major adverse cardiac events such as myocardial infarction
What are some major causes of heart failure?
- Coronary artery disease
- Chronic hypertension
- Cardiomyopathy
- Heart valve disease
- Arrhythmias – AF, VT
- Infective endocarditis
- Pulmonary hypertension – PE, COPD
- Alcohol and drugs (eg cocaine)
What are signs and symptoms of congestive heart failure?
- Shortness of breath
- Swelling of feet and legs
- Chronic lack of energy
- Difficulty sleeping at night due to breathing problems
- Swollen or tender abdomen with loss of appetite
- Cough with frothy sputum
- Increased urination at night
- Confusion and/or impaired memory
Sensitivity and specificity of signs and symptoms of heart failure is relatively poor
What is the clinical utilisation of cardiac biomarker testing in heart failure?
- Initial evaluation of heart failure
- Screening for cardiac dysfunction
- Guiding management of heart failure
- Assessment of prognosis and survival
Natiuretic peptides as markers of cardiac damage/overload. An A,B,C of natriuretic peptides
What are the advantages of N-terminal precursor forms over ANP or BNP?
Assays are now available for the active peptides and the N-terminal precursor forms of ANP and BNP.
Advantages of N-terminal precursor forms over ANP or BNP
- Longer half life
- Higher plasma concentrations
- Less sensitive to rapid fluctuations
When are plasma levels of ANP and BNP markedly raised?
- Congestive heart failure
- Aortic stenosis
- Dilated cardiomyopathy
- Hypertrophic cardiomyopathy
- Myocardial infarction
- Chronic renal failure
What are possible causes for raised plasma natriuretic peptides in heart disease?
- Haemodynamic
- Structural
- LVH
- Ventricular dilation
- Damage/remodelling after MI
- Hypoxia/necrosis
What are some conditions that are investigated for possible use of plasma ANP/BNP?
- Assessment of severity of congestive heart failure
- Screening for mild heart failure
- Monitor response to treatment in congestive heart failure
- Prognostic outcome/risk stratification
