Prostate

Prostate

Function

The vagina has an environment which is slightly acidic, with approximately a pH of 4, this is to protect against pathogens. Sperm however, are acid-sensitive, thus their environment, semen, is alkaline. Due to this, any ejaculate abolishes the vaginas protective acidity for many hours after it's entry, this is to ensure survival of the sperm. Seminal vesicle secretions are rich in fructose, as this is a primary energy source for sperm, and also rich in prostaglandins and proteins which promote semen clotting in the vagina. These secretions are also rich in Ca2+, zinc, citric acid, acid phosphatase, albumin and prostatic specific Ag (PSA). PSA has the ability to reverse the clotting enzyme affect from seminal vesicle fluid. he enzyme has the ability to make ejaculate gel and "glue" in place in the female cervix. Sperm will remain within this gel, until the PSA enzyme dissolves the clot and allows sperm to swim into the uterus.  

The exocrine gland is the size of a walnut, and weights approximately 20 grams. Within the adult prostatic parenchyma may be divided into 4 biologically and anatomically distinct zones or regions:

1. Peripheral
2. Central
3. Transitional zones
4. Region of the anterior fibromuscular stroma  

These are important as different types of proliferative lesions may arise in each region. For example, most hyperplasias arise in the transitional zone, and most carcinomas originate in the peripheral zone. 

This is important to mention in the exam. Try and draw it in the exams if needed (not a rude picture though!).

Figure 1. Picture provided by Roy Stewarts "Prostate" lecture presentation.

Histology

This is very important to include:

It is very important that the normal histology is known, as this is the main way to diagnose benign vs malignant tumours. This is also used in the grading of prostate cancer. Essentially, the prostate is a gland composed of tubular glands which have 2 layers of cells:

• Basal cuboidal layer: rests of a basement membrane
• Inner columnar luminal layer

The glands can be rounded or have papillary type (nipple like) projections. This can be useful diagnostically. The rest of the gland is background stromal cells consisting of fibro-muscular tissue, which has an influence of the glandular tissue itself, and not just inert background material. 

Normal histology and immunohistochemistry

The basal layer is positive for high molecular weight cytokeratins and the mouse antibody cocktail anti-cytokeratin 34bE12 reacts with cytokeratins 1,5,10 and 14. P63 is also positive, and it has been shown that without P63, mice do not develop a prostate, thus a cocktail of 34bE12 and P63 is a good marker, in order to pick up all of the basal cells. Remembering the names of this will give you more marks. Corpora amylacea - normally found in normal prostate. 

Benign prostatic hyperplasia (BHP)

Major theories attempting to explain the aetiology of pathological phase BPH include:

1. Dihydrotestosterone (DIT) hypothesis: this is a shift in prostatic androgen metabolism that occurs due to age, leading to abnormal accumulations of DHT, producing an enlarged prostate. 
2. Embryonic reawakening theory: a reawakening of the embryonic induction potential of prostatic stroma - a change in the prostatic stromal-epithelial interaction that occurs alongside ageing, leading to an a promoting effect on prostatic growth.
3. Stem cell theory: featuring an increase in the total prostatic stem cell numbers and/or clonal expansion of stem cells into amplifying and moving cells which occurs with ageing. 
4. Inflammatory theory: Prostatic inflammation may contribute to the growth of the prostate, due to the inducing of cellular growth because of the presence of inflammatory markers and agents which have growth stimulating abilities. 

BHP involves the enlargement of glandular and stromal tissue, stromal being mainly fibro muscular. Each can have varying amounts, providing a wide range of histological BPH presentations. The main activating component is DHT, that is converted within stromal cells from testosterone via enzyme 5alpha reductase, which is also produced by stromal cells (see fig 1). DHT has a function as an autocine/paracrine agent, causing direct cell stimulation, or up regulating mitogenic signals which act upon the proliferation of neighbouring cells. Testosterone as similar attributes to DHT, but it is nowhere near as potent. A reduction in the activity of DHT will cause the gland to shrink, however if the gland is very large, it may have to be dealt with surgically, e.g by use of trans urethral resection prostate (TURP). This can produce prostatic chippings, these must all be processed as there may be a background micro foci of cancer or PIN. 

Figure 1. Picture provided by Roy Stewarts "Prostate" lecture presentation.

DHT binds to nuclear androgen receptors in both epithelial and stromal prostate cells, DHT has a higher affinity for AR than T, forming a more stable complex. DHT binding promotes the transcription of androgen-dependent genes. DHT itself doesn't promote mitosis, however the DHT-mediated transcription of genes results in the production of many growth factors, such as FGF (fibroblast growth factor), with FGF-7 (keratinocyte growth factor) being important. Others produced are FGF 1 and 2, and TGF beta, that promotes the proliferation of fibroblasts. The overall cause of BPH isn't known, however DHT related growth factors ac by increasing stromal cell proliferation, and lead to a decrease in epithelial cells apoptosis.

The main feature of BPH is the presence of nodules. Their composition range from purely stromal and fibromuscular, to fibroepithelial with a glandular predominance. Glandular proliferation takes the form of aggregations of small to large cystically dilated glands, lined by 2 layers, inner columnar and an outer cuboidal or flattened epithelium.

BPH diagnosis cannot be made using a needle biopsy, as the histology of glandular or mixed glandular-stromal nodules cannot be appreciated in limited samples. They do not necessarily sample the transition zone where BPH occurs as this is in the middle of the prostate.    

5alpha reductase inhibition

Finasteride is used as an inhibitor for 5alpha reductase, it can inhibit its action by around 80-90%. As a result, the amount of DHT is reduced by 70% in serum, 90% in prostate and 34% in skin. The amount of testosterone increases by around 10%, and PSA is reduced by an average of 50%, as a result of this, the total prostate volume will decrease by 15-25%.  

BPH treatment

For cases which are moderate to severe that are resistant to medical therapy, there are a range of invasive procedures which can be used instead. TURP has been good in terms of reducing symptoms, improving flow rates and decreasing post-voiding residual urine. It is now used as the first line of therapy for those under certain circumstances, such as those with reoccurring urinary retention. However, as a result of its morbidity and cost, other procedures have been developed. Such as:

• High-intensity focused ultrasound
• Laser therapy
• Hyperthermia
• Transurethral electrovaporisation
• Transurethral needle ablation using radiofrequency
• Prostate artery embolisation as recently been trailed as a therapy also

Nodular hyperplasia is not considered to be a premalignant lesion. 

Prostate cancer

Prostate cancer is the most common cancer within men. Statistics show approximately every hour one man dies from prostate cancer, meaning over 10,000 every year. Over 40,000 men are diagnosed with prostate cancer every year, more than 100 a day. Estimated that in 2030, prostate will be the most common cancer type. 1 in 8 men will get this. There are many risk factors which aid in the formation of prostate cancer:

• Age
• Family history: this is one of the strongest risk factors, 5-10% of PC cases and 30-40% of early onset cases (those who develop it before the age of 55) are caused by inherited susceptibility genes. There is a 2-3x increase in risk for those who have a first degree relative diagnosed with PC, if the relative was under the age of 60, or the person has more than one relative with PC, the risk exceeds the average by 4x. If the person has more than one relative when an early onset of PC, the risk increases 7x. If there is a history of breast cancer, especially under the age of 60, this will also increase the risk, due to an association with BRCA1 and 2. Studies have identified a number of risk-associated loci, including one at 8q24 which appears to selectively increase the risk among African men. 
• Ethnicity: the incidences of which PC occur differ in varying locations in the world, suggesting ethnicity is a factor. In the UK, black caribbean and african men have 2-3 x the risk of developing PC than white men, and asian men have an even lower risk, this trend is also seen in the US.
• Life style: migration studies have shown that men moving from low risk to high risk areas have showed an increase in cases, suggesting that life style factors of the area also play a role, e.g a South Asian man living in the UK has a higher chance at developing PC than those living in South Asia. 
• Diet
• Alcohol and smoking
• Body weight and physical activity
• Medications, medical procedures and infections
• Endogenous hormones
• Diabetes mellitus

Cancer and gene arrangements

There is a common somatic mutation in prostate cancer which gives rise to chromosomal rearrangements (translocations) that juxtapose the coding sequence of an ETS family transcription factor gene (most commonly ERG and ETV1) next to the androgen-regulated TMPRSS2 promotor, testosterone increases. ETS over expression makes normal prostate epithelial cells more invasive, maybe by the up regulation of MMPs. Tumours with rearranged ETS genes do not necessarily have certain distinctive morphologic features, and a rarely have a differing gene expression signature than those who do not have different ETS arrangements, thus suggesting that ETS gene rearrangements define a specific molecular sub-class of prostate cancer. They may also have implications for prostate cancer during screening and early diagnosis, it's possible to detect ETS fusion genes in urine, through the use of PCR. Most aggressive fast growing PCs have these translocations. 

CAG repeat

Endogenous hormones, including androgens and oestrogens, have a likely influence of the carcinogenesis of the prostate. Men who have been castrated, such as eunuchs, whom have levels of testosterone equal to that of before puberty, will not develop prostate cancer. Thus, it is possible that a change in androgen biosynthesis and metabolism e.g the role of the androgen receptor (AR) CAG repeat in exon 1, could influence the risk of PC. It has been reported that AR CAG repeat length greater than, or equal to 20 repeats, conferred a protective effect for PC in men. Long repeats make the cell less likely to divide.

Androgen resistant cancer

The term "castration-resistant prostate cancer" or CRPC arose from what used to be referred to as androgen-independant prostate cancer. The AR is considered the principle driver towards prostate cancer progression. This concept showed that castration temporarily slowed the progression of PC, however subsequent castration-resistant growth of PC has been attributed to a variety of mechanisms, including: 

• activation by receptor tyrosine kinases from growth factors, 
• loss of cell cycle regulators and someones 
• genomic mutations in the AR allowing response to nonspecific AR ligands. 
• It has been showed that increased AR expression was the most common event associated with CRPC growth. 
• It has also been shown that metabolic adaptation is involved also, involving the production of androgen within the tumour cells, self stimulatory. 
• Many studies have revealed there are many alternative splice forms of AR, these variants have differing structures, however each variant lacks portions of the ligand-binding domain (LBD), a feature predicted to produce a constitutively active receptor. 
• The variants increased expansion was associated with a more rapid disease occurrence, following radical prostatectomy for localised disease. 

Aggressive cancer "Pussycat vs Tiger"

5 single nucleotide polymorphisms (variations within a gene) were located in, or associated with 5 genes which may affect PC progression:

1. LEPR: This is the strongest marker with PC mortality, it aids in tissue growth control, inflammation, development of blood vessels and the density of bones. These make this gene an interesting candidate for understanding disease progression, as the primary metastatic site for PC is bone, and these metastases are predictive of fatal disease.
2. RNASEL: This gene is associated with hereditary PC, and associated with apoptosis, inflammation and the proliferation and adherence of cells (cancer growth hallmarks).
3. IL4: Interleukin 4, associated with growth of tumours, blood vessel development and cancer cell migration.
4. CRY1: Cytochrome 1, gene that impacts the circadian rhythm this possibly affecting androgen levels. 
5. ARVCF: member of the catenin protein family, aids in the communication of inside and outside cells. Increased expression has been shown to disrupt adhesion of cells, possibly facilitating cancer progression.

Patients whom carried 4 or 5 of these markers had a 50% increased risk of dying of PC than  those with 2 or less. There is a correlation with death rate and the number of SNPs present.

BRCA2

BRCA2 is now not only associated with early development of PC, but also with the aggressive form. It is found in 1:100 of PCs. If there is a family history of breast or prostate cancer, then this gene should be tested for, however this isn't currently available within the UK. 

Prostate specific antigen (PSA)

PSA is often used as a tool for screening PC, however elevated levels can also be present in benign tumours, and some malignancies may have low levels, however the cut off is given at 4.0 ng/ml. A better way to measure this gene is via its velocity, or rate of change over time, the value 0.75 ng/mL per year best distinguishes between men with and without PC. In order to be considered efficient, there must be a minimum of 3 tests over a period of 1.5-2 years. Best used as a marker of treatment.

PSA vs volume: including the ratio of serum PSA value and volume of prostate gland (PSA density). Men with enlarged hyperplastic prostate glands have a higher overall serum PSA than men with smaller glands. Measuring serum PSA density factors out benign prostatic tissue contribution to serum PSA levels. This is calculated by diving the total serum PSA level by the estimated gland volume (determined by transrectal ultrasound measurements) in order to estimate the PSA produced per gram of prostate tissue. 

As men age, their prostates tend to become enlarged with BPH. Overall older men would have a higher serum PSA level than younger men. Thus there are reference ranges for upper age-specific PSA:

• 2.5 ng/ML - 40-49 years of age
• 3.5 ng/mL - 50-59 years of age
• 4.5 ng/mL - 60-69 years of age
• 6.5 ng/mL - 70-79 years of age

Free PSA: Immunoreactive PSA (the form which is detected by antibody tests) exists in 2 forms, a major fraction bound to alpha1-antichymotrypsin, and a minor free fraction. The % of free PSA (free PSA/total PSA x 100) is lower in men with PC than those with BPH. Free PCA higher than 25% indicates a lower risk of cancer, as compared to free PSA values of less than 10%, which can cause concern for cancer. 

Biopsies
Talk about this more (will be updated soon). Braccotherapy (implant therapy) - insert a core of radioactive material.

Histological diagnosis of PC

Most lesions are adenocarcinomas which produce well-defined, readily demonstrable gland patterns. These glands are typically smaller than benign glands, and are lined by a single uniform layer of cuboidal or low columnar epithelium. In contrast to benign glands, which have two layers, the outer basal cell layer found in benign glands is absent in adenocarcinomas. These inner cells cannot be stained, whereas the outer basal cells can. PC glands are crowded, and lack branching and papillary infolding. The cytoplasm of the tumour cells range from pale-clear, as noted in benign glands, to a distinctive amphophilic appearance. The nuclei in PC are large, and sometimes a cell can contain more than one. There is also variation is nuclear size and shape, however blatant pleomorphic features are not present. Mitotic figures are uncommon. 

Diagnosis

Difficulty in establishing an appropriate diagnosis with PC stem, not only due to the small amount of tissue available for examination from the needle biopsy, but also that a biopsy often only samples a new malignant glands among many benign glands. Morphologically, PC malignancy clues are very subtle, again making it hard to efficiently diagnose. There are many benign mimickers of cancer which can lead a pathologist to a misdiagnosis. There are very few histologic findings on a biopsy which are specific PC, however a perineural invasion is one. The general diagnosis is made based on a combination of morphological and ancillary findings. 

Diagnostic aids: A distinguishing feature between benign glands and malignant glands is the presence of basal cells, these are only present in benign glands. This can be exploited by the immunohistological use of markers which are specific to basal cells, such as high molecular weight cytokeratins 34bE12, 1, 5, 10, 14 and also P63. Another marker which is very selective for PC cells and not normal cells is AMACR (alpha-methylacyl-coenzyme A-racemase), this is up regulated in PC and can be observed by the use of immunohistochemistry. The majority of PCs are positive for AMACR, with sensitivities varying from 82%-100%. AMACR is a positive stain for malignancy, must be used with H&E.

AMACR

P504S is a 382 aa cytoplasmic protein which has been recently identified by microarray screening of prostatic carcinomas, it has been identified with AMACR, which is an enzyme that catalyses the racemization of alpha-methylacyl branches carbolic coenzyme A thioesters. It is located within the mitochondria and peroxisomes, where it aids in the oxidation of branched chain fatty acids. It is also responsible for the racemization of an intermediate in the synthesise of bile acids. In neoplasia, AMACR is most often expressed in tumours associated with a high fat diet, such as colonic and prostatic carcinoma. AMACR stains red, and CK and P63 stain brown. 

Prostatic intraepithelial neoplasia (PIN)

In around 80% of cases, prostatic tissue removed from a carcinoma will also contain presuming precursor lesions, referred to as PIN. These consist of architecturally benign prostatic acini (sac like cavity) lined by cytologically atypical cells with prominent nucleoli. PIN and carcinomas may be identical cytologically, however architecturally PIN involves larger branching glands with papillary infolding, compared to invasive cancer that is typically characterised by small crowded glands with straight luminal borders. PIN glands are surrounded by a layer of basal cells, alongside an intact basement membrane - there are many types of evidence relating PIN to invasive cancer. For example, both typically predominate in the peripheral zone, and are uncommon on other zones. 

Comparing a non cancerous prostate to one with cancer, those with have a higher frequency, and a greater extent of PIN. PIN is often observed near cancer, sometimes with cancer budding off from it. A high number of molecular changes observed in invasive cancers can also be witnessed in PIN, providing evidence that PIN is a type of intermediate between normal and invasive cancer. But how PIN behaves and develops into cancer is not known. thus as used in cervical cancer, the term "carcinoma in situ" cannot be applied to PIN. However, PIN is important in identifying BPH chippings. 

Benign glands have a continuous basal layer, PIN glands have a fragmented basal layer, and malignant glands have no basal layer. 

Grading

The Gleason system is used to grade prostate cancer. PCs are stratified into 5 grades, on the basis of their glandular patterns of differentiation. It's a subjective assessment. 

• Grade 1: represents the most well-differentiated tumours, in which the neoplastic glands are uniform and round in appearance and are packed into well-circumscribed nodules.
• Grade 5: tumours show no glandular differentiation, and the tumour cells infiltrate the stroma in the form of cords, sheets and nests. 

All other grades fall in between these. 

Most tumours contain more than one pattern, so a grade is given to the most dominant pattern, and another grade to the most frequent pattern, the two grades are then added together to give a combined Gleason score, thus the number can be between 2 and 10. The scores are combined into groups with similar biological behaviour;

• 2-4: represent well differentiated cancer, typically found in small tumours within the transition zone. In surgical specimens, low-grade cancer is typically an incidental finding on TURP, which is performed for symptoms of BPH.This is why all chippings are sampled. 
• 5-6: intermediate grade tumour: the majority of potentially treatable cancers detected on a needly biopsy as a result of screening have a score of  5-7. 
• 7: moderate to poorly differentiated tumour
• 8-10: high-grade tumour, these tend to be advanced cancers that are unlikely to be cured. 

Although PCs can become more aggressive with time, the score usually remains stable over several years. Grading is very important in PC as the grade and stage are the best prognostic predictors. 

The state of DNA ploidy can affect the Gleason score, with the score usually being higher when DNA ploidy is abnormal.

Staging and risk

Low risk; clinical stage T1c, or T2a, PSA level less than 10 ng/ML, GS of 2-6.

• Very likely the disease is within the prostate (confined)
• Minimal risk of spread to distant sites such as bones
• Prostate-only treatment (implant alone) is appropriate
• Observation is an appropriate option in selected individuals
• Extremely high cure rate with all therapies

Intermediate risk; clinical stage T2b, PSA 10-20 ng/mL, GS of 7.

• Possible disease is beyond the prostate (not necessarily confined)
• Low but significant risk of metastasis
• Implant alone is appropriate for some patients, however combination therapy is an excellent option
• Observation is rarely appropriate
• High cure rate with implant based therapies

High risk; clinical stage T2c, PSA over 20 mg/mL, GS of 8-10

• Very likely disease has progressed beyond the prostate
• Probable risk of metastasis
• Combination therapy is needed
• Observation is never appropriate
• Curable with intensive treatments - body wide treatments such as hormone therapy are often combined with radiation treatments.

Recent studies have indicated the presence of the gammaretrovirus XMRV being present in a significant amount of PCs, this may be of relevance?

Haemolysis

Haemolysis

This can be diagnosed by the use of a full blood count, and determining whether there is a presence of:

• Low haemoglobin count
• Low RBC count
• Low MCV (average volume of a single red blood cell)
• Increased reticulocyte count 

Within a haemolytic full blood count, the following traits can be observed:

• Polychromasia: this is the presence of an abnormally high number of red blood cells, whom have been released prematurely from the bone marrow. This is a sign of stress being applied to the bone marrow. These RBCs will have a slight gray/bluish hue to them. 
• Spherocytes: there are RBCs whom are sphere in shape, they have a smaller surface area for oxygen/carbon dioxide transport however they still have enough in order to maintain a healthy transport. The main different is their osmotic potential, they are more likely to carry out haemolysis in water than normal, biconcave disc shaped RBCs.
• Red cell fragments: these would be present in haemolytic anaemia as a result of burst RBCs. 

A direct antiglobulin test can carried out, however it is not 100% specific or sensitive to haemolytic anaemia. 

Urinary haemosiderin

Haemosiderin is an iron storage complex, however unlike ferritin, it is always found within the cells as apposed to circulating in the blood. It's molecular structure is poorly defined, however it appears to be a complex of denatured ferritin and other material. The iron within haemosiderin is poorly accessible when it is required. 

We look for urinary haemosiderin as this indicates the presence of haemoglobin in the glomerular filtrate. This is useful in proposing chronic intravascular haemolysis as haemosiderin may be present in the urine, even if no haemoglobin is detectable. IT will be detectable for many weeks after the incidence of haemolysis, but it will not be present at the initial onset of haemolysis, as the iron in the haemoglobin has to first be metabolised. 

Stains in order to detect urinary haemosiderin at applied parallel with a bone marrow slide, acting as a positive control, from a patient who has previously been found to have adequate iron stores. The Prussian blue stain can be used to visually identify haemosiderin, staining it a dark blue colour. 

If the staining causes an ambiguous result to form, this should always be consulted with a senior haematologist.  

If a sample is thought to be contaminated, it should not be used in diagnosis.

Biochemical tests

Testing for bilirubin is useful as this indicates the break down of haemoglobin as it is a product of such an event. 

Testing for haptoglobin is useful as this tends to bind to free haemoglobin in the blood, thus there will be less free haptoglobin in the blood. 

Testing for lactate dehydrogenase (LDH) in the blood is useful as this is increased when haemolytic anaemia is present, this is because LDH can be found within RBC, and is consequently released as a result of their haemolysis. 

Blood transfusions

Blood transfusions

A blood product can be clotting factor products, albumin, or immunoglobulins. These can be obtained via the use of chemical processes. A blood component such as RBCs, platelets, cryoprecipitate, plasma and WBCs, are separated and extracted from the blood without the use of chemical processing. 

Blood donation
Blood is donated voluntarily from people who adhere to a specific criteria, this is to make sure there is safe transfusion of blood products and components to recipients who require them. The criteria involves the following factors:

• Age, weight, donation frequency
• Health status 
• Sexual orientation and activity
• Body piercings, acupuncture, tattoos
• Infectious agents such as HIV, hepatitis 
• Illegal drug use, some legal drugs, and vaccinations
• Travel

Donors attend a donation centre. They sign consent forms and read leaflets. Haemoglobin levels are also checked. 

A cuff is tied around the arm, arm is sterilised and a sterile needle is used. Samples as well as the donation are taken. The bag the blood collects in also contains an anti-coagulant and a preservative. The process takes around 10 minutes. The donors can then rest and are given refreshments. Blood is then delivered and screened before being supplied to patients. 

Leucodepletion

This is a process that is carried out in order to filter the entire blood sample and remove WBCs. This was brought about in 1999. Leucodepleted blood is considered as having <5x10^6/L WBCs present. Carrying this out reduces vCJD infection, as leucocytes can act as a reservoir for prions. This also lowers the risk of CMV infection, and reduces incidence of febrile transfusion reactions to platelet and RBC transfusions. It also lowers the bacterial rate of contamination of RBC concentrates.

Routine donated blood tests

Checks for infectious agents such as:

• Hep B
• Hep C
• HIV 1 and 2
• HTLV (human t cell lymphotropic virus) 1 and 2
• Syhpilis
• Cytomegalovirus (patient depending)
• Bacteria

Blood groups and antibodies, each is tested to determine the ABO and RhD group of the RBC from the donor. Group O donors are tested to detect high levels of anti-A or anti-B Abs.

SAG-M
This is the main additive in use within the UK. It is composed of:

• S: Saline (providing volume expansion)
• A: Adenine (as an energy source)
• G: Glucose (as an energy source)
• M: Mannitol (prevents red cell lysis by WBC enzyme action)

Blood component preparation

Different blood components can be obtained dependent on what the recipient needs:

• Whole blood: Fresh plasma, buffy coat, red cells
• Fresh plasma: FFP for clinical use, cyroprecipitate
• Buffy coat: Platelets
• Red cells: Red cell concentrate

These are prepared from the donor blood by the use of differential centrifugation, where the densest component (RBCs) separate out first, followed by leucocytes, then platelets, then with plasma at the top. Cyroprecipitate is prepared from rapidly frozen, then thawed plasma. It is then centrifuged to sediment the cyroprecipitate.

Donated blood component containers contain the following:

• Unique donation number
• Blood group
• Expiry date
• Cautionary notes
• Special requirements

Blood component transportation

It is transported by a secure system within transit containers, along with packing materials and specific procedures, such as temperatures, security and hygiene control. Monitoring of routine transport temperatures is carried out periodically. The containers must be appropriately labelled, and secure in order to protect the components and samples from damage. In order to permit their identification, documentation must accompany the components. A record of the date time and person transporting the container and the person receiving it must be kept. Once attained, appropriate checks must be made, guidelines must be followed at all times, for example each red cell unit must be transfused over a max period of 4 hours after it has been removed from a controlled storage environment. Failure to carry out appropriate ID checks can results in the wrong patient being transfused. If any signs of a transfusion reaction show, the transfusion must be stopped immediately. 

Blood grouping and compatibility testing for RBCs
Surface Ags present on RBCs are determined. In the absence of RBC Ags, individuals may make alloantibodies, through a process called alloimmunisation, if exposed during transfusion/placental transfer. ABO antibodies are naturally occurring, this are called isohemagglutinins. Antibodies to other red cell Ags occur after sensitisation, in the form of IgG. There are 2 main systems: ABO and Rhesus (Rh) system. 
Alloantibodies may give rise to:

• Intravascular (ABO incompatibility) or extravascular (Rh incompatibility) haemolysis of donor RBCs in the recipient.
• Haemolytic disease of the newborn due to placental passage. 

Why are blood groups important?

If an individual is exposed to a blood group Ag which is not recognised as a self cell, the immune system may produce Abs that will specifically bind to that Ag, thus an immunological memory against that can be formed, this is referred to as sensitisation. These Abs can binds to Ags on transfused cells, often leading to destruction of the cells by recruitment of other immune system components. Maternal Abs may also cause haemolytic disease of the new born, (HDNB), if the foetus has a differing blood group to the mother. 

Cross matching

This is the compatibility testing of donor's to recipients blood to ensure that no adverse reactions occur when the blood transfusion is carried out. This is done by:

• Suspending red cells from donor with recipients serum
• Incubate at 37 degrees
• Examine mix for any agglutination occurrence (when RBCs clump together)

From the patient: the ABO and Rh group is determined; the serum is screened for Abs

From the donor: Selection of appropriate ABO and Rh unit

At the crossmatch: Patient serum + donor RBCs = check for agglutination.  

Transfusion laboratory work

Within the laboratory, identification of the patient and the sample must be made, along with the ABO and Rh group of both. Antibody screenings must be done (and identification if necessary). Selection of bloof og appropriate Rh and ABO type, and cross matching. Issuing of compatible blood with reports. Check the blood bag for any defects. Detect autoantibodies. Check any antenatal work. 

RBC transfusion

This is carried out in order to increase the oxygen delivering capacity of the blood, perhaps in the case of acute or chronic anaemia contributes to inadequate oxygen delivery to tissues. RBCs are then stored and transported at 2-6 degrees C, and generally have a 35 day shelf life. This transfusion type is only requires when a massive bleed occurs, such as gastrointestinal bleeds, road traffic accidents, wounds, operations, pre/post-op top ups, chemotherapy patients, haematological malignancies, haemorrhages, severe anaemia etc. A patient is not transfused if haemoglobin concentration is over 10 g/dL. A safe level of haemoglobin is 8-0 g/dL. If less than 7 g/dL, this is a strong indication that a transfusion should be carried out. It is essential to carry out a transfusion if haemoglobin concentration reaches 5 g/dL. The cells transfused must be ABO and Rh compatible. 

RBC transfusion procedure

1. Identity of patient must be confirmed
2. Blood compatibility label must be checked to ensure blood is the correct type
3. Expiry date should be checked
4. Bag should be inspected for damage
5. Blood left out of fridge for 30+ minutes should be transfused within 4 hours or be discarded.
6. Details of the unit of blood transfused should be recorded on the anaesthetic chart or as an entry in the clinical notes
7. Volume of blood transfused should be recorded once administered
8. 100% traceability of all allogenic blood transfused is a legal requirement from the European Blood Directive. 

Fresh frozen plasma (FFP)

Plasma is stored and transported at -25 degrees C. It will have a shelf life of 2 years. When needed for transfusion, it can be thawed rapidly at 37 degrees C. FFP contains high levels of coagulation factors. FFP is indicated for massive bleeds. Can also be used for the reversal of the effect of warfarin (an anticoagulant) in the event that the correct coagulation factors are unavailable. It is also transfused into patients with liver disease and disseminated intravascular coagulation (DIC). Giving the adequate dose is important, 12-15ml/kg is a good starting point.    

Platelets

These can be stored at room temperature 22 degree C +/- 2 degree C, for up to 5 days. A constant gentle agitation is required. It can be produced from several whole blood donations or by apheresis of a single donor. Transfusion is patients whom are suffering from, or are at a significant risk of haemorrhage due to platelet dysfunction and/or thrombocytopenia. Platelets express HLA class 1 antigens, care must be taken if the patient has HLA Abs, as this can pose of risk of alloimmunisation due to the RBC fragments. Care must also be taken in order to avoid bacterial contamination. Clinical uses include prophylactic use in patients with bone marrow failure. A platelet count should always be maintained above a value of 1 x 10^10/L, as this reduces the risk of haemorrhage, when haemorrhaging does occur in a patient, the count should be kept above 7.5 x 10^10/L. Platelets are also indicated in those with DIC with thrombocytopenia and bleeding. They may also be required in surgical procedure where thrombocytopenia is present, the platelet count trigger points for transfusion vary depending on the type of surgery taking place. 

Cryoprecipitate

This must be stored and transported at less than -25 degree C, and can last for up to 2 years. It contains factor 8, the von Willebrand factor and is a rich source of fibrinogen. It can be used in cases of DIC, hepatic failure, in severe bleeding or immediately prior to an invasive procedure in patients with significant hypofibrinogenemia. It is thawed at 37 degrees C when needed for transfusion. It is usually provided as one therapeutic dose, (2 packs) and the patients fibrinogen level is checked again in order to ensure it is above 1 g/L. 

Other products used therapeutically

• Immunoglobulins (Igs): These are a source of Abs for patients with immune disorders
• Specific Igs: Obtained from donors with high titres of Igs (e.g anti-D antibodies)
• Freeze dried factor 8 concentrates: used to treat factor 8 deficiency of von Willebrand disease.
• Protein C concentrate: used for DIC patients to reduce thrombosis
• Other factor concentrates: various ones are available, for example containing factors 2, 7 and 10. These are used for treating factor deficiencies. Prothrombin concentrates and recombinant 7a can be used in life threatening bleeds. 

Human albumin solution

Albumin solution, 4.5%: undergoes heat pasteurisation to inactivate viruses. It is also tested for serological viral markers of infection. It is used as a fluid replacement and a plasma volume expander, in order to reach the desired osmotic effect prior to blood administration.

Salt poor albumin, 20%: used in hypoalbuminaemia when minimal electrolyte content is required.

Donor blood alternatives

In an early assessment, early FBC and a good diet is suggested in order to ensure haemoglobin is as high as possible at the time of the operation. Planned approach such as stop taking medication such as warfarin (anti-coagulant). Start medication such as B12/folate if needed. 

In a predeposit/pre-operative autologous blood donation (PAD), this should be taken weeks before surgery, and only used for patients with a rare phenotype. More than one unit may be required. 

In cell salvaging, blood is collected and then re-infused.

Pharmacological agents may also be used, such as  erythropoietin an intravenous iron. 

Special requirements

• Irradiated blood: used for immunocompromised patients. It removes viable lymphocyte components that may give rise to problems such as transfusion related graft versus host disease (TR-GVHD). Gamma irradiation renders lymphocyte DNA incapable of replication with little or no effect on RBCs, platelets or granulocytes. 
• Components confirmed to be cytomegalovirus (CMV) negative: used for patients who are immunocompromised
• Red cells for neonates: derived from an adult unit split into up to 8 paediatric packs, CMV neg, neg for high anti-A/B titre.
• Platelets for neonates: derived from adult unit split into 4 paediatric packs
• Paediatric FFP: Similar volumes of methylene blue treated FFP is available for this group
• Components confirmed to be negative for specific RBC Ags: used for patients with significantly clinical Abs.

Blood transfusion hazards

Donor: fainting (2-5% of donors), infections of the venepuncture site

Recipient: Clerical errors and lab errors, congestive heart failure from circulatory overload, immunological reactions, infection transmission, iron overload, TA-GVHD.

Tuberculosis

Tuberculosis (TB)

Tuberculosis can take many forms. The most common found to infect humans is Myobacterium tuberculosis. Other forms include:

• Myco canetti - a rare variant
• Myco bovis - affecting mainly cattle and other mammals
• Myco microti - mainly found to affect small mammals, such as voles 
• Myco africanum - this type is considered to be between the human and bovine types, this type is mainly found within equatorial Africa.

Mycobacterium tuberculosis

This type is an acid alcohol fast bacilli, which is a term used to refer to the bacteria being rod shaped, and any gram stain cannot be washed away, even when treated with acid or alcohol. Stains used are Ziehl-Neelson stains (red, slightly curled appearance, beaded, almost looks as if they're branched) or Auramine Phenol stains, this is because these stains can infiltrate the mycolic acids (consisting of 60-90 waxy carbon compounds, these are a protective layer) located within the cell walls of the bacterium, whereas gram stain cannot.

MTB is an obligate aerobe, thus require the presence of oxygen in order to grow. They are non-motile are do not create spores. They are also not contained within capsules. They cordate in broth culture, this is where they form the shape of a heart? They grow slowly due to the presence of their complex mycolic acid wall. 

Can be grown on a Lowenstein-Jensen medium, which is egg/glycerol based. It can go within 2 weeks of a subculture, but it can take up to 8 weeks on a primary isolation. During this time the sample can be contaminated, if this occurs, in order to decontaminate sodium hydroxide is used, and then it is neutralised.

MGIT and BacT Alert systems can be used, these are a continuous monitoring liquid culture. 

For diagnosis, nucleic acid probes can be used with the use of PCR. This is rapid and reliable, and can be used to detect resistance. 

The T-SPOT test can be used to look for activated T cells. Useful for when TB is latent and also in outbreaks. The organism can come out of latency if the patient is given a high dose of steroids.

Interferon gamma test and tuberculin skin tests can also be used. 

Treatment

Antibiotics can be used to treat this bacterium, however many can struggle to penetrate the waxy layer. Ones that are generally used are Rifampicin, Ethambutol and Isoniazid, these are 1st line antibiotics. Multiple treatments may be used in order to prevent any resistance, as this can cause treatment failure. Multiple drug resistance tuberculosis - when resistant to 1st line antibiotics. 

Primary tuberculosis

This is usually contracting from inhalation of TB droplets in the air. The organism is then engulfed by the alveolar macrophages, where they then replicate in order to form the initial lesion. This is then carried by the phagocytes to the lymph nodes. This can then further spread to other organs and other parts of the lungs. In 90-95% of infected individuals, haematogenous spread occurs. The DNA of the bacterium can be detected in tissues by in situ PCR. In 5-10% of infected individuals TB will progress to cavitary TB, this is where the cavities open into the bronchi allowing spread of TB through coughing. 

Myco bovis can be contracted through the ingestion of milk. It affects the tonsils, lymph nodes and the intestines. It can be spread by macrophages to the local lymph nodes through the bloodstream. It can also reach other organs such as the spleen, kidneys, CNS and bone marrow. It can become dormant and resurface years later.

Clinical disease

5% of patients exposed to the active disease show it developing in 2 years. 5-10% later in life. Those who are HIV positive, 10% show the active disease within 1 year. It's more likely to spread to extrapulmonary sites, which can progress quickly to death.

Pulmonary TB is present in 80% of cases, and extrapulmonary in 20%.

Extrapulmonary cases are made of:

• Meningitis
• Lymphatics
• Pleural
• Genito-urinary
• Bone/joint
• Others

Symptoms of the disease are malaise, weight loss, coughing, night sweats and haemoptysis.   

The organisms can be protected within necrotic or caseous granulomas. These can become calcified. This is referred to as cavitating disease, this can cause tissue destruction. This infection can be lifelong. 

Central nervous system infection

TB infecting the CNS, this can cause meningitis. Meningitis has an insidious onset, can cause confusion and headaches, it is an acute rapid disease. It results in a raised lymphocyte count, and is very difficult to diagnose as there can be very low amounts. Will cause an altered mental state. 

Miliary TB

This is a disseminated primary disease, it is named due to the many "millet seed" sized lesions on chest X-ray, which look like bird seed. Patients can also have meningitis and a sepctic of typhoidal presentation.

In accordance to the WHO, it was confirmed a few years ago that the emergence of extensively drug-resistance tb (XDR-TB resistant to 2nd line drugs) poses a serious threat to public health, particularly when associated with HIV.  

NTU students 2014 Project Abstracts

http://www.joomag.com/magazine/ntu-undergraduates-research-april-2014-biosciences/0163496001398433142

Above is the link for the project abstracts in our year, (mine is on page 115!) enjoy! :)

Tumour Pathology

Tumour Pathology

Cancer is a general term for over 200 diseases, used to indicate the presence of malignant neoplasms (abnormal mass of tissue). These can be characterised by the rapid and uncontrollable division of cells forming malignant tumours, whom can invade and destroy healthy tissues, and whom also persist in the same manner even when the primary stimulus causing the abnormal growth is removed. These cells have the ability to spread via invasion or implantation.

• Invasion: direct growth into tissue adjacent to the tumour.
• Implantation: growth in distant sites caused via metastasis

A tumour can be defined as any unknown lump or tissue, and can be benign of malignant. 

• Benign: non-cancerous mass of localised cells which will not spread.
• Malignant: cancerous mass of cells which can spread invasively to nearby tissues (known as malignant) or to distant tissues (known as metastatic).
• Neoplasm: swelling or lump comprised of an abnormal proliferation of cells, whom are no longer under normal physiological control, of uncertain origin. The ratio of proliferation to apoptosis favours the first. Neo - new, plasma - that which is formed.

Tumours and neoplasms are not synonymous with cancer.

Carcinoma
Carcinoma is essentially another name for cancer, referring to a malignant tumour of epithelial origin. 

This would be a tumour located in the epithelial cell layers which cover body surfaces and line the organs. Carcinomas approximately make up 80% of all cancers. Common examples would be:

• Skin
• Gut
• Breasts
• Prostate

Cancer hallmarks
Cancer has six main hallmarks, these are:

• Avoidance of apoptosis
• Self-sufficiency of growth signals
• Insensitivity to anti-growth signals
• Limitless replication potential
• Tissue invasion and metastasis
• Sustained angiogenesis

These are a basic overview, as there are extremely intricate steps into the processes of cancerous cells. Others which can be associated with cancer are:

• Deregulation of cellular energetics
• Avoiding immune destruction
• Mutation and instability of the genome
• Tumour-promiting inflammation

There is a link with the increase in age and the increase in the instances of cancer. This is to be expected, as an increase in time and number of cellular divisions, along with an increase in the exposure to other factors, increase the chance of a genomic mutation occurring.

Another interesting trend is the difference in the types of cancers commonly found in children in comparison to adults in 2004. In adults, 54% of cancers were derived of breast, lung, bowel and prostate cancers, whereas in children, 56% of cancers were derived of leukaemias, lymphomas and CNS/brain cancers. These findings, in my opinion, show that life style and age have a severe determining factor with adult cancers. Lung cancer is a good example of this, making up 13% of adult cancers, this will be due to the presence of smoking within the population, and the abundance of asbestos which would have been inhaled in the past couple of decades.  

Cancer stages

• Transformation stage: Single normal cell mutates into a single tumour cell
• Progression: Tumour cell replicates, and cellular variants begin to emerge, after 30 "doublings" the mass is around 1 gram in weight and contains 10^9 amount of cells. This is also known as the smallest clinically detectable mass. These cells are now mutating and becoming genetically unstable, forming different cancer cell types. Small metastases may be found in other organs. After another 10 doublings, the tumour has increased in weight to 1 kilogram, consisting of 10^12 cells, this size is considered the maximum size compatible with life. At this stage the varying cancer cells with have caused heterogeneity of the tumour. Large metastases will be present in other organs. 

The varying cancer types can be:

• Non-antigenic
• Invasive
• Metastatic
• Some will require fewer growth factors to carry out mitosis.

Neoplasia
Growth of a cancer. Size is usually bearing on no biologic behaviour. It can have varying growth configurations, such as:

• Infiltration and projection onto the surface
• Infiltration and ulceration of the surface
• Infiltration forming tumour mass
• Papillary
• Cauliflower appearance
• Nodular
• Lobular
• Cystic

Varying amounts of proliferating neoplastic cells and supportive stroma give tumours their excessive features.

Diagnosis of the tumour will then allow for it's prognosis, allowing to predict the tumours future activity, such as:

• Mode of spread
• Therapy response
•  Genetic implications

Benign tumours

As a general rule, if -oma is a suffix then the tumour is benign, however there are exceptions, such as lymphoma, carcinoma.

The prefix coincides with the growth type, e.g:

• Adenoma (glandular)
• Papilloma (finger like)
• Cystadenoma (Cystic)
• Polp (Mucosal projection)
• Combined features such as papillary cystadenoma

A benign tumour tends to be well circumscribed, can be encapsulated, is slow growing, resembles the tissue of origin and will push other tissues aside, in comparison to a malignant tumour, where there is infiltrative growth, no capsule, and will cause destruction of other tissues.

Malignant tumours
Those derived from epithelial cells, from any of the 3 germ layers, are referred to as carcinomas. A carcinoma with a glandular growth pattern would be referred to as an adenocarcinoma. Carcinomas producing squamous cells are referred to as squamous cell carcinoma. It's also general practice to mention the organ of origin, e.g renal cell adenocarcinoma. Those composed of undifferentiated unknown tissue origin, are referred to as poorly differentiated or undifferentiated malignant tumour.

Many tumours are a result of chromosomal translocations, this is an abnormal rearrangement between non homologous chromosomes, where a part of one detaches, and reattaches to another, examples of these are lymphomas, such as follicular, mantle cell, Burkitt's, and MALT.

Soft tissue sarcomas
Typically rare, only an estimated 1,300 people diagnosed per year in the UK. Occur in muscle, fat, blood vessels or any organ that supports, protects and surrounds the organs. Soft tissues of the body are referred to as mesenchyma tissues, thus sarcomas being referred to mesenchymal tumours. Generally these occur in those over 30, however some do occur in young persons. Almost half occur in the limbs, mainly near the knee joint. The chest, abdomen and pelvis are common areas. Less common are the head and neck.

Blue small round cell tumours
Small blue round cells are typically found in children and young adults with cancer, the cancers are similar in their histological features, however their diagnosis is essential as their prognosis and treatment can differ greatly. The use of karyotyping (establishing the number of chromosomes and their appearance) will provide very specific information, however in order to do this, cells must be cultured. The use of FISH would be useful.

Karyotyping
Cells which have been grown within a culture are arrested during metaphase using colchicine, they'e then placed in hypotonic solution to make them swell, and are then fixed in methanol-acetic acid. Once fixed they're placed onto slides to create metaphase spreads, and their chromosomes can be stained to create the characteristic coloured bands. An example of such a stain is "G banding", this works by the use of trypsin partially digesting histone proteins, allowing the chromosome to relax and the dye to attach itself to the exposed DNA, in this case dense A&T regions. The size and location of these bands allow cytogenetics to compare and distinguish homologous chromosome pairs. This process is extremely specific but is not cost or time effective.

In Situ Hybridisation
This is a method of localising mRNA and DNA within a cell by the hybridisation of the sequence of interest via the use of a complementary nucleotide probe. This is a morphological technique and does not cause destruction of the tissue. It's detection level is 10-20 copies per cell. The sequence of interest can range in size, from 20bp long up to 1000 bp long. It's useful for untranslated or secreted proteins. It avoids the problems of anything with shared epitopes as it's very specific. There are 5 types of probes:

1. Double stranded DNA: can be produced within bacteria via the use of plasmids, or through PCR, it has to be denatured to allow hybridisation with DNA or mRNA. They are possible to be produced in bulk, however when done so there is a risk of them re-annealing to themselves. If they're large, they may have problems when attempting to access the cell.
2. Single stranded DNA: around 200-500bp in length. Can be produced via the process of reverse transcription of mRNA, or via asymmetric PCR using one primer. 
3. Riboprobes: these have an advantage as RNA-RNA hybrids are very strong, and are also resistant to digestion by RNases, thus post hybridisation washing with RNase can remove non-hybridised RNA and give a very clear background. These probes are hard to produce by in vitro transcription of a linearised plasmid, with an RNA polymerase from a promoter site which can produce either sense or antisense probes. 
4. Oligonucleotides: produced synthetically as in PCR primers, usually around 20-40bp in length. Their size is ideal for penetrating cells, however they do cover less of the target, so they're usually mixed into a cocktail in order to increase sensitivity.
5. PNA probes: mimics DNA, however it has a repeating backbone of N-(2 aminomethyl)-glycine unites linked by amide bonds, The bases are attached to the backbone via methylene carbonyl linkage. It cannot be used as a PCR primer. 

Normally, probes are labelled with radioisotopes, however recently non-radioactive probes have been developed and have an equal sensitivity, produce faster results and avoid all hazards associated with handling radioactive waste, making them more suitable for a routine laboratory.  

Originally probes were labelled with biotin, however it has recently been discovered that endogenous biotin exists within some tissues, thus a different label is now used, such as digoxigenin (Dig) or fluorescein. The Abs for these labels are made with reporter systems attached, such as alkaline phosphatase. The advent of fluorescent labels has allowed the visualisation of multiple probes simultaneously, and resulted in the development of the FISH technique. Once only applied to metaphase chromosome spreads, it can now be used on interphase cells (interphase cytogenetics).

Benign neoplasm features
Benign tumours have specific characteristics, they:

• Grow slowly
• Are encapsulated within a fibrous capsule
• Have a smooth surface
• Compress surrounding organs and tissues, pushes them to the side
• Small in size (usually)
• Do not cause death unless occurring in the central nervous system, or compressing vital organs
• Cells are very differentiated, thus resemble the tissue of origin
• Cells are uniform and almost identical
• Blood vessels around the tumour are well formed
• Doesn't have necrosis
• Doesn't metastasise
• DNA content is normal
• Karyotype is normal

Malignant neoplasm features
Malignant tumours have specific characteristics, they:

• Grow rapidly
• Do not have a capsule
• Have an irregular surface
• Tumour invades and destroys surrounding tissue
• Large in size (usually)
• Causes death if not treated
• Cells are not very well differentiated, especially in comparison to benign tumours
• Malignant neoplasms mostly do not resemble origin tissue
• Cellular nuclei can be enlarged and hyperchromatic (darker nucleus)
• Numerous prominent nucleoli 
• Cellular pleomorphism
• Increased mitotic activity with abnormal mitotic figure
• Blood vessels surrounding the neoplasm are numerous and poorly formed, some do not have any endothelial lining, and this can leads to tissue haemorrhaging
• Necrosis is present
• Metastasis does occur 
• DNA content of the cells tends to be increased, additional chromosomes can be present
• Karyotyping abnormalities such as aneuploidy and polyploidy occur.

An eponymous tumour type is a type which has been named after a person, e.g the founder of the type or the first diagnosed case. 

Pleomorphism: variation of size and shape of cells located within a neoplasm. Mitoses on their own are not a sign of malignancy, however the presence of abnormal mitoses are, and these alongside the presence of pleomorphism and hyperchromatism (development of excess chromatin) definitely favours malignancy. 

Ki 67 is a protein associated with cellular proliferation. It is found only in cells which are carrying out the cell cycle, and is brought to the surface during mitosis, however in resting cells it is not present.  

Carcinoma of unknown origin
Some diagnostic techniques for malignancies can fail to determine the primary origin site where the cancer developed, if the cancer has carried out metastasis. This is referred to as carcinoma of unknown primary origin (CUPO) or occult primary malignancy. In 2007, 32,100 people (roughly 50:50 gender ratio) were diagnosed with unspecified primary cancers. In post mortem examination, 15-25% still could not be identified. This is a problem as the primary site of the cancer in most cases decides the treatment and prognosis.

The majority of CUPO are adenocarcinomas or undifferentiated tumours,however it is possible they are squamous cell carcinomas, melanomas, sarcomas or neuroendocrine tumours. 

Cellular differentiation
Tumours can be "graded" on how closely they imitate parent tissues which they originate from:

• Well-differentiated: the cells are very similar in both appearance and conformation to the tissue of origin 
• Poorly-differentiated: the cells only show minimal resemblance's to the normal parent tissue
• Anaplastic: tumour shows no obvious similarities to the origin tissue, this type is associated with having aggressive behaviour. 

The grading is based upon the appearance of the cells under a light microscope along with H&E staining. A higher grades means a smaller degree of differentiation, thus the worse the biological behaviour. Most systems have 3 or 4 grades:

• Grade 1: Well differentiated
• Grade 2: Moderately differentiated
• Grade 3: Poorly differentiated
• Grade 4: Anaplastic

Gleason grading system involves 5 grades, and then the two most common cell patterns are added together, providing a number between 2-10, with 10 being the most violent type of malignancy. Doctors often find it difficult to determine whether to grade someone as 6 or 7, this is because once a patient is deemed as having a grade of 7, their treatment becomes much more aggressive. 1-4: 10 year probability of local progression 25%, 5-6 50% and 7-10 75%. 

Functional differentiation

Well differentiated tumours produce the normal product of the origin tissue, however poorly differentiated tumours are less likely to carry out the standard functions. This differentiation provides clues as to the clinical aggressiveness of the tumour. Tumours can lose their differentiation features over time as they become more malignant, and acquire more genetic mutations. The level of differentiation within the cells depict how they will respond to certain therapies and treatments. 

Spreading pathways

There are different pathways taken during metastasis of malignant cells:

• Lymphatic spread: usual route for carcinomas, involves the lymph nodes
• Haematogenous spread: usual route for late stage carcinomas
• Transcoelomic (body cavity) spread: seeding of cavities surfaces by tumour, typical in ovarian carcinomas

TNM
This is a system for determining the anatomical extent of a disease based on the assessment of 3 components:

1. T: the extent of the primary tumour
2. N: the presence or absence and extent of regional lymph node metastasis
3. M: the presence of absence of distant metastasis.

The addition of numbers to these 3 components indicates the extent of the malignant disease. The system is a shorthand notation for describing the extent of a particular malignant tumour:

TX - Primary tumout cannot be assessed

T0 - No evidence of primary tumout

Tis - Carcinoma in situ

T1,2,3,4 - Increasing size and/or local extent of primary tumour

NX - Regional lymph nodes cannot be assessed

N0 - No regional lymph node metastasis

N1 - Regional lymph node metastasis

MX - Distant metastasis cannot be assessed

M0 - No distant metastasis

M1 - Distant metastasis

Both grading and staging have prognostic value, but the staging is the most valuable as it indicates extent of the disease.