Haematology

Chronic myeloid leukaemia

This is a myeloproliferative disorder, which can be characterised as a disease of the bone marrow in which excess cells are produced. Definition of CML: a malignant clonal proliferation of pluripotent haemopoietic stem cells (blood stem cells), with the ability to affect more than one cell line.

Prevalence 

CML affects around 1-2 people out of a population of 100,000, thus around 0.00001%, and has a slightly higher rate in males. It can occur at any age range, however it is more likely in the middle-aged to elderly age range. CML makes up around 14% of the leukaemias which  occur in adults. There are around 700 diagnosed cases per year within the United Kingdom. 

Cause

The cause of CML isn't very well researched, thus little is known about it. One theory is the exposure to ionising radiation, such as that present within Hiroshima after World War 2. CML does however have a link to a clear genetic abnormality, in fact it was the first malignancy to be related to this: 

• The BCR gene located on chromosome 22 can become fused with the ABL gene located on chromosome 9, and creates a BCR-ABL fusion gene. When translated into a protein, this forms tyrosine kinase. This fusion gene is found in roughly 90% of all CML cases. The translocation of the genes results in the elongation of one of the chromosome 9 chromosomes, and the shortening of one of the chromosome 22 chromosomes. Chromosome 22 is referred to as the Philadelphia chromosome.

The fusion gene is also located in adults and children with ALL (acute lymphatic leukaemia) (25-30% and 2-10% respectively). 

Fusion gene: T(9;22)(q34:q11).

Symptoms

A patient with CML can be asymptomatic, however the symptoms when they do occur come in the form of:

• Splenomegaly (enlargement of the spleen)
• Hepatomegaly (enlargement of the liver)
• Malaise
• Bruising
• Bleeding
• Anaemia
• Reduced platelet count

Diagnosis

A patient may be diagnosed with CML via the use of a full blood count, to detect a heightened white blood cell count, the predominance of myelocytes and metamyelocytes in the peripheral blood, the examination of the bone marrow, and also by cytogenetic techniques such as FISH and PCR. 

CML can be categorised into 3 stages:

1. Chronic phase: May last for a prolonged amount of time, typical CML, 85% of patients are usually in this phase. Usually asymptomatic, or mild symptoms such as fatigue, abdominal fullness etc. How long this will last will depend on how early the diagnosis was made, without curative treatment, it will progress.
2. Accelerated phase: In this phase there is approximately 10-19% of myeloblasts in the blood/marrow, and over 20% of basophils located in the blood or marrow. Platelet count can be less than 100,000 or over 1,000,000. Other genetic abnormalities occur alongside to the Ph chromosome. White blood cell count with increase and splenomegaly will increase. May be unresponsive to therapy.
3. Blast crisis: Terminal phase, behaves clinically like acute leukaemia, over 20% of myeloblasts are found within the blood/marrow. There are also large clusters of blasts in the marrow. A chloroma will form, this is a solid focus of leukaemia outside of the marrow. This is also found in AML, and can transform into megakaryocytic (the cell in which platelets originate from) leukaemia.

Treatment

The treatment of CML can involve the use of tyrosine kinase inhibitors, such as iminitab mesylate, alpha interferon and a whole bone marrow transplant, which involves the entire destruction of the patients bone marrow, and the injection of stem cells from a healthy suitable donor.

Iminitab was one of the first drugs designed around the molecular biology of CML, it is an inhibitor of the protein formed from the Ph (BCR-ABL) gene, it also inhibits proliferation and induces the action of apoptosis in cells which are positive for the BCR/ABL gene, and also Ph+ leukaemic clones. The iminitab molecule fits into the active sid e of the ABL protein, disallowing  the binding of ATP, meaning the ABL protein cannot phosphorylate its substrates. Around 90% of patients treated with this drug showed slowed or no further disease progression. 

Hodgkins Lymphoma

Lymphomas are the name given to the diseases associated with the malignancy of lymphocytes. There are two major sub-divisions:

1. Hodgkins lymphoma
2. Non-hodgkins lymphoma

Hodgkins lymphoma (HL) in the present day is now considered as a disease which we are able to treat and can be described as a "curable" disease. 

The name "Hodgkins" was derived from the man who first described the abnormalities of the lymphatic system in 1832. The abnormal cells which characterise HL were identified in 1898 by Reed and Sternberg, hence their name Reed-Sternberg cells, these are malignant cells.

HL may develop at any age, and is usually 1900 cases are diagnosed every year (1 in every 200 cancers). In men, it's most common between the ages 20-34 and 75-79 and 1 in 440 will develop HL. In women, it's most common between the ages of 20-24 and 70-74 and 1 in 500 will develop HL.

At the moment there is no one definitive cause for HL, however there are many factors associated with its occurrence:

• A possible cause of HL is the Epstein Barr virus, almost half of all HL cases have been related to a EBV infection.
• Previous NHL occurrence
• Hepatitis C
• Little research has been done of this, however the exposure to pesticides may play a role 
• Immuno-deficient patients, such as those with HIV, post transplant etc
• Lack of exposure to common infections, such as those from a higher socioeconomic background.
• Family history, via inheritance or a particular lifestyle trait.
• There is a supposed lowered risk of contracting HL in those who drink alcohol, however this does not affect those who also smoke.
• An increased risk can come from obesity, however this was only seen in males. 

Most people however do not show any signs of specific identifiable risk factors.

These malignant cells can accumulate in a single lymph node, and eventually may spread to other nodes, and also can pass into the blood stream and invade other organs of the body.

Reed-Sternberg cells and Hodgkin cells possess mechanisms which inhibit apoptosis.

In the pathogenesis of the disease, it is not fully understood as of yet. RS cells and H cells are all apart of the clones.

Clinical features

The clinical features of the disease include:

• Enlarged painless lymphadenopathy
• Lymph nodes can fluctuate in size
• The ingestions of alcohol can cause pain
• Itching
• Breathlessness and coughing
• Enlargement of the spleen and liver 
• Fever, weightloss, night sweats
• When the lymphoma leaves the lymph nodes and travels to other parts of the body, this is referred to as extra nodal disease, including the skin, lung, CNS, marrow etc
• Increase in the number of infections due to defective humoral and cell mediated immunity.

HL may arise in any part of the lymphatic system, however it is more common in some areas over others, such as the cervical nodes (located in the head/neck) where 60-70% of HL cases arise. The axillary nodes (located in the arm pit area) cause around 10-15% of cases, and the inguinal nodes (located in the stomach/hip/groin area) cause around 6-12% of cases. These areas will swell up, but will also be painless. Some people can be diagnosed as having extra nodal disease at the time of diagnosis. The most likely areas to have extra nodal disease are the liver and the bone marrow. The bone marrow can be associated with specific symptoms.

Diagnosis

A biopsy of the lymph nodes is carried out under the use of a local or general anaesthetic.

The WHO has varying histological classifications for HL.

Subtypes include classical:

• Nodular sclerosis: Most common type of HL in the UK, 60% of patients are diagnosed with this, most common in young adults. Found in the early stages where the lymph nodes in the neck have become swelled.
• Mixed cellularity: Around 15% of cases are of this type, it usually affects a number of lymph nodes, and these will each contain different types of lymphocytes and other cells.
• Lymphocyte depleted: 10% of HL cases are of this type. Lymphocytes appear to be very small, in a biopsy of the lymph nodes there are a lot of lymphocytes but very few Reed-Sternberg cells.
• Lymphocyte rich: This is quite a rare type of HL, the lymph nodes may either contain a lot of fibrous tissue, with very few Reed-Sternberg cells, or they may contain a high amount of a specific lymphocyte type referred to as the reticular lymphocyte, and many Reed-Sternberg cells.

Another subtype is the nodular lymphocyte predominant type (non classical type): Only 5% of HL cases are of this type, it is more common in the elderly however it can occur at most ages. The main difference between this specific type and the classical types, is that this type contains a very small amount of Reed-Sternberg cells (they're almost considered as absent), and they also contain a group of cells which are referred to as popcorn cells, these are RSC subtypes, they're small cells with a highly lobulated nucleus, and small nucleoli. This HL type is only ever diagnosed in localised HL. The treatment for this type differs from the classical types, and this type also has a slower growth rate.  

RSCs tend to have an owl like appearance which are multi-nucleated.

Specific factors can be looked for in the diagnosis of HL:

• Non specific peripheral blood manifestations
• Normocytic and normochromic anaemia
• Eosinophilia (increase in RBCs)
• Neutrophilia (increase in WBCs, only in 1/3 of patients)
• In the advanced disease, low lymphocyte count
• Platelet count is either normal or increased in the early stages of HL, however in the later stages the count becomes low.
• The involvement of the bone marrow can be detected.
• Raised erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) are useful prognostic markers and good for monitoring response.
• Serum lactate dehydrogenase (LDH) can sometimes be raised.
• HL can cause irregular liver function test results (LFTs), and urea and electrolyte test (U&E) results 
• Increase in the levels of urate

There are a number of tests which can be carried out in order to establish the stage at which the lymphoma is at in a patient.

Within the laboratory:

• Full blood count (FBC) and bone marrow check, ESR, CRP, LFTs, LDH

Radiology techniques:

• Chest X-ray; detects if lymph nodes within the chest are enlarged. 
• PET; Positron emission tomography, using a low dose of radioactive glucose to measure the activity of the cells in differing parts of the body. This is helpful in the detection of minimal residue disease.
• MRI; Magnetic resonance imaging, uses the use of magnetism in order to compose an image of the body within, this is especially helpful in showing soft tissue and the CNS.
• CT scans; Computerised tomography, uses a small amount of radiation to build up a computerised image of the inside of the body.

It is important to be able to diagnose the stage of HL, as this will affect the treatment and prognosis of each individual patient. 

Stages

HL can be broken down into 4 stages:

1. Indication of node involvement within one area (I)
2. Indication of disease involving 2 or more lymph node areas, however these are contained to one side of the diaphragm. (II)
3. Splenic disease is involved at this stage (III)
4. Involvement of HL outside of the lymph nodes, and also refers to diffuse disease within the bone marrow, liver and other extra-nodal sites. (VI)

Each stage can then be sub-typed with the use of different letters, each acting as an indication towards other factors involved at each stage, such as stage 3, as this involves spleen enlargement, it can be called IIIs.

Treatment

The treatment of HL depends mainly on the stage of diagnosis. It is possible to just use radiotherapy on patients whose HL is localised (NLPHL). However, a combination of varying therapies may be used, such as chemotherapy and radio therapy, this is called combined modality therapy. Due to secondary complications resulting from radiotherapy, this combined method is now usually preferred, except in the presence of NLPHL. Radiotherapy can be useful in the elimination of bulky disease which can remain after chemotherapy, and also in the elimination of painful skeletal, nodal, or soft tissue deposits which can form as a result of previous chemotherapy.  

Chemotherapy is used for those patients suffering from stage 3 and 4 of HL, but also those who are suffering with stage 1 and 2 alongside bulky disease (widening of the mediastinum (undelinated group of structures in the thorax, containing the heart, lymph nodes etc) of more than a third, or the presence of measuring more than 10cm in any dimension), and also those who have relapsed after previous radiotherapy. ABVD is most commonly used, other variants used can be ChlVPP and BEACOPP, the latter mainly for those with a poor prognosis. 

Autologus stem cell transplantation involves:

1. Either bone marrow aspirated from iliac crest OR leucopheresis of stem cells from peripheral blood after chemotherapy and G-CSF injections
2. Attempts to remove residual tumour cells e.g via use of monoclonal antibodies
3. Stem cells infused intravenously (after storage)
4. Intensive support therapy e.g with RBC, platelets and antibiotics

However, a patient who responds well to chemotherapy will straight to 4 after treatment.

The response to a treatment is assessed by clinical examination using imaging, to identify any residual masses present. If a patient does carry out relapse, they can be treated alternatively with combination chemotherapy, or radiotherapy for bulky disease. If they undergo relapse again, if the patient is under 65 years of age, autologous transplant is considered. 

Prognosis

The prognosis for those in stage 1 or 2 of the disease at the time of diagnosis that 91-94% of suffers will live for at least another 5 years. For those in stage 3 or 4 of the disease, 50-90% of suffers will live for at least another 5 years. The type of HL and the age of the patient will also contribute to the prognosis. Cure rates do decrease if the patient relapses, however the disease will be able to be kept under control for a period of time. 

European Organisation for Research and Treatment of Cancer (EORTC) stage 1 and 2 prognosis features:

Favourable:

• Clinical stage 1 and 2 with a max. of 3 nodal areas involved
• Age less than 50 
• ESR less than 50mm/hr without B symptoms, or ESR less than 30mm/hr with B symptoms
• Mediastinal/thoracic ratio below 0.35

Unfavourable

• Clinical stage 2 with the involvement of 4 or more nodal areas
• Age over 50 
• ESR over 50mm/hr is asymptomatic or ESR over 30mm/hr with B symptoms
• Mediastinal thoracic ratio over 0.35

HL can also have later effects, which can include secondary malignancies, for example lung and breast cancer are related to the use of radiotherapy, MDS (myelodysplastic syndrome) and AML (acute myeloid leukaemia) are related to the use of chemotherapy. Other effects include cardiac disease, dysfunction of the endocrine system, intestinal problems, damage to the lungs, psychological trauma, lower general health levels, and can cause problems in getting loans. Attention must always be paid to these later effects. 




Malaria
Malaria is a tropical parasitic disease, it is caused by the transmission of a protozoan parasite which has the ability to infect RBCs. The most common route of transmission vessel for this disease is via the pregnant female anopheles mosquito.

It is the cause for a high morbidity and mortality rate in certain areas of the world, mostly distributed in tropical and subtropical areas. Malaria has been known to cause illness within the UK, this is generally due to importation. 

According to the WHO 2013 world malaria report. 97 countries had on-going malaria transmission, and an estimated 3.4 billion people were at risk, 1.2 billion being at high risk. In 2012, there were an approximate of 207 million cases with 670,000 deaths. 90% of deaths from malaria occur in sub-saharan Africa, with 77% being children under the age of 5 years old. All this information can be found on the WHO website. 

Malaria is a bigger problem in some areas more so than others, this can be for obvious reasons, such as the normal habitat for mosquitos, and varying climate types. Other reasons can also be due to lack of funding, and the cooperation of governments and charities. Education and preventative measures should be addressed also, such as teaching people in such areas to cover bare skin, and use insecticide sprays, buy nets etc, however access to these preventative measures is another factor as to the diseases abundance. Access to good doctors to provide a diagnosis, and varying types of treatments also affect where the disease can be most prominent, this is because the parasite can become resistant to the overuse of the same drug types. 

Malaria in the UK 
The ratio between cases of malaria from UK residents visiting friends and relatives, in comparison to cases of malaria acquired from holiday travellers is around 10:1. It should always be considered whether access to medical guidance is available before travelling, whether the guidance has been adhered to, full awareness of the risk and familiarity with the destination. Targeting these groups of peoples should be a priority. Other people within the UK who also tend to contract malaria, are those new to the country, foreign students studying in the UK, British citizens who have been working abroad in malaria strife countries, armed services and those who travel on business. 

An individuals risk can be determined by their awareness of areas which are at high risk areas, the time of year when they travel, the type of malarial parasite in the area, preventative measures taken and their immunity levels towards the disease.  

Transmission

• Mosquito bites
• Through the placenta (mother to child)
• Blood transfusions
• Transplantations
• Contaminated equipment
• Routes associated with air travel

Types

There are 5 main species of malarial parasites:

1. Plasmodium falciparum
2. Plasmodium vivax
3. Plasmodium ovale
4. Plasmodium malariae
5. Plasmodium knowlesi

Each one can be characterised through their differing features and symptoms. 

Plasmodium falciparum is the most common, and most severe form of malaria. The parasite has a cycle of development within the blood lasting around 36-48 hours and lasts within the liver for 8-10 days. The liver schizont contains around 40,000 merozoites, and a mature erythrocytic schizont contains 16-30 merozoites, although this is rarely seen unless the patient is extremely ill. Around 40% of all RBCs of varying maturity may be infected.

Plasmodium vivax has parasitaemia which is much lower than that of P. falciparum, its cycle of development within the blood lasts for around 48 hours, and all stages of the parasite are seen in the peripheral blood. Less merozoites are seen in the liver and erythrocyte stages. This parasite prefers to invade reticulocytes (immature RBCs making up around 1% of the overall RBCs in the body). This parasite has the ability to form dormant hypnozoites.

Plasmodium ovale is very similar to P. vivax, in that is also have a 48 hour erythrocytic stage, it also like prefers to invade reticulocytes and hypnozoites also can form. However it is more restricted in it's distribution, and there are around 8-12 merozoites seen in erythrocytic schizonts. 

Plasmodium malariae has an erythrocytic cycle of around 72 hours, it has low prevalence, and the liver and erythrocytic schizonts of this species produce fewer merozoites in comparison to other species, thus parasitaemia is low. Tends to infect older RBCs. 

Plasmodium knowlesi has a similar morphology to P. malariae, however it has a much higher parasitaemia, and can be fatal. It is found in Southeast Asia.  

Immunity 

Immunity to malaria is most commonly associated with the Plasmodium falciparum parasite, in an area where the transmission of this parasite is high, if a child survives to 5-6 years of age then their immunity is seen as high, however this immunity will begin to deplete if there is no regular exposure to the parasite. Immunity will be lowered in pregnant females, thus the pregnant, and children will be most at risk. 

Symptoms

Fever, chills, headache, flu-like symptoms, muscle ache, fatigue, anaemia, diarrhoea, vomiting and coughing

Diagnosis

Diagnosis within the laboratory can be carried out using a variety of techniques:

• Full blood count 
• Rapid diagnostic tests
• Quantitative buffy coat
• Polymerase chain reaction (PCR), this is mainly used in reference laboratories
• Thin and thick blood films

If malaria is detected via the use of a full blood count, the haematological changes observed will include normocytic and normochromic anaemia, this will be due to the removal of  damaged RBCs by the reticuloendothelial system, and suppression of normal RBC formation. Thrombocytopenia will also be witnessed, this is decrease in the number of platelets. WBC count usually stays normal, however in a severe state of the disease, the number can elevate. Further tests after this will have to be carried out to properly confirm the presence of malaria. 

A kit is available called the NOW malaria kit (Binax), this is a rapid immunodiagnostic assay, it is composed of 2 antibodies which have been immobilised on a test strip, one is specific for the histidine rich protein II associated with Plasmodium falciparum, and the other antibody is specific for an antigen found on all malaria types which can infect humans. A colour will form if the test is positive. The test only takes around 15 minutes. The problem with this is, it can state whether malaria is present however it doesn't show how much in abundance the parasite is. 

Another test used for diagnosis is the optimal-IT test, this test uses monoclonal antibodies for the enzyme parasitic lactate dehydrogenase (PLDH), the test has 2 antibodies, one for specifically Plasmodium falciparum and the other as a pan-specific. When a parasite is detected, PLDH will react with both of the antibodies. 

The positives of using these kits are that they are quick, cheap, easy to carry out, and can be used anywhere.

The negatives of using these kits is that they can provide false positives and also false negatives. 

The use of a quantitative buffy coat is also a means of diagnosis. Blood from the capillary of a patient is placed into a glass haematocrit tube, which also contains a stain which colours parasite DNA, acridine orange and an anticoagulant to stop blood clotting, potassium oxalate. A cylindrical float is then placed into the tube, and the tube will be centrifuged in order to separate the cells into their different densities. This forms bands, which are also made larger by the presence of the float. The tube is then placed into a holder, and examined under a light microscope with a UV adapter, thanks to the stain, any parasitic organisms will fluoresce. The negatives to this technique is that it is impossible to distinguish between different species, and to know how much in abundance the parasite is within the blood. Also, it is possible to gain false negatives from this technique. 

The polymerase chain reaction technique involves DNA amplification in vitro, it is highly sensitive and highly specific.

The advantages to this method is that is can detect and differentiate between different malarial parasite types, the technique is 10 fold more sensitive than microscopy, and it's also more reliable at identifying the species of malaria present. 

The disadvantages of this technique is that it is time consuming, expensive, and expertise is needed. 

Thin and thick blood films have been describe as being the 'gold standard' in malaria diagnosis.The advantages is that they're cheap, and can be used for species identification and also their quantification (checking the parasites abundance within the blood). Their disadvantages it that the parasites can be overlooked, especially if the parasitic levels are low, this technique can be time consuming and it also requires a level of expertise. 

Once a malaria case has been detected, the results are entered into a hospital computer system so medical staff can view them, a patient report form is then filled out and sent to the School of Tropical Medicine, and also to the Health Protection Agency/Public Health England. 






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

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.




Transfusion hazards

All blood within the UK is supplied by unpaid volunteers, before taking their blood, all are questioning thoroughly about their lifestyles and medical health. Each of the donations are then screened, however mainly the UK system relies on the donors honesty. However, adverse events may occur between donation to transfusion, and some of these are unavoidable. 

Bacterial contamination
In order to minimise problems associated with bacterial blood contamination, the blood donation must be carried out in a way which is most aseptic as possible. The skin at the site of venepuncture must be cleaned with an antiseptic prior to donation. Bacteria which is airborne must also be taken into consideration. Patients may be suffering from an illness asymptomatically. 

Storing the sample at a temperature of 4-6 degrees C is best in order to inhibit most bacterial growth, however some organisms such as Pseudomonas fluorescens are able to live at this temperatures, and if the bags are allowed to warm, the risk of bacterial proliferation increases, thus removing the bags from fridges in order to test them can cause issues.Bacterial proliferation which occurs in platelets is more of an issue than within RBCs, this is due to storage temperatures.

There will always be a risk of contamination from the donor, however in recent years this has decreased dramatically thanks to screening. 

Human error
People may be at fault when it comes to transfusion contamination, such as placing the wrong blood in a tube, labelling a sample wrong, incorrect techniques in the lab, misidentification of an antibody on a contaminant, wrong patient transfused etc. Stress and tiredness can cause these mistakes to occur, however they can be life threatening in some cases. This is why doctors are required to have adequate training to carry out such a procedure.

A scientist has a large responsibility from beginning to end, they test, store and issue blood, and also investigate any adverse reactions.

Blood safety regulations
Any serious adverse blood reactions and events (SABRE) from a transfusion must be reported to the Medicines and Healthcare Products Regulatory Agency (MHRA), this agency is associated with the Department of Health, and is responsible for ensuring medications and medical devices work and are safe. The agencies role is to enforce standards laid down by the EU Blood Directive. Hospital blood banks must demonstrate they're complying with standards.

SHOT
Serious hazards of transfusion: was created due to concern over absence of data available in relation to safety of transfusion and adverse events. It is a voluntary, anonymous confidential reporting scheme. It collects and reports data for adverse events in transfusions, it has a wider scope than the MHRA as the reports come from clinical and professional areas of practice as well as transfusions carried out in a hospital. They provide an annual report giving their findings and recommendations. SHOT is beneficial as it provides an aid in guideline production, improving standards and minimising adverse events via education. 

SHOT abbreviations for adverse reaction reasons:

• IBCT: incorrect blood component transfused
• I&U: inappropriate, unnecessary and under/delayed transfusions
• HSE: handling storage errors
• ATR: acute transfusion reactions
• HTR haemolytic transfusion reactions
• TRALI: transfusion-related acute lung injury
• TACO: transfusion-associated circulatory overload
• TAD: transfusion-associated dyspnoea
• PTP: post-transfusion purpura
• Ta-GvHD: transfusion associated graft versus host disease
• TTI: transfusion transmitted infection

Acute intravascular haemolysis
This is the adverse reactions caused from the transfusion of red blood cells, this usually occurs due to the incompatibility of the donors blood type with the patient, such as ABO. The RBCs will be destroyed rapidly by antibodies against that blood type, which will release free haemoglobin into the blood, this can lead to disseminated intravascular coagulation (DIC), shock, acute renal failure and maybe death. 

Shock phase: symptoms include vomiting, headache, shortness of breath, fall in blood pressure and a raise in body temperature (pyrexia), this can occur at any time from the blood being transfused, even if it's only a small amount. This will be accompanied by increase RBC destruction and DIC.  

Oliguric phase: acute renal failure and acute tubular necrosis may occur.

Diuretic phase: fluid and electrolyte imbalance may occur during recovery.

AIH can be tested for in the laboratory, tests can be conducted in order to determine, within the blood, the amount of:

• Haptoglobins (free haemoglobin transporter, for recycling, produced by the liver)
• Bilirubin (a product of haemolysis)
• Haemoglobin
• RBC count
• Reticulocytes
• Nucleated RBCs
• Polychromasia (abnormal amount of RBCs due to being released from bone marrow prematurely)
• Fragmented RBCs
• Antiglobulin

Compatible blood types

Patient A - can receive donor A and O

Patient B - can receive donor B and O

Patient O - can receive donor O

Patient AB - can receive donor A and B and O and AB

Other factors do play a role such as antigens for other proteins that the patient or donor may possess. 

Delayed transfusion reactions

Adverse reactions due to red blood cell transfusions, involving extravascular haemolysis. Involves antibodies that are not able to activate complement, e.g Abs to the E or Kidd Ags. This is usually a result of a patients response who has previously been sensitised against a particular Ag by transfusion or pregnancy. It is less severe than intravascular haemolytic reactions. Abs are present at low levels, thus not always detected in the pre-transfusion samples. Cells acquired from the donor will become coated with IgG, and are then removed by the reticuloendothelial system. The symptoms of this can be progressive anaemia, possibly with the presence of jaundice. The patient must have their blood pressure and renal perfusion maintained, if failure of these occur, this may be managed with dialysis until the patient is recovered. The patient will require more transfusions with the appropriate blood type 

Iron overload

Those patients who undergo many transfusions may result in acquiring an iron overload. Examples of this include patients who are dependent on transfusions, such as those suffering from thalassaemia, myelodysplasia, or myeloproliferative disease. The consequences of this will cause adverse effects on the liver, heart and endocrine glands, all with clinical consequences. The treatment for this is iron chelation therapy. 

TA-GVHD

Transfusion-associated graft-verses-host disease, occurs when live lymphocytes are transfused into immunocompromised patients, causing clonal expansion of viable lymphocytes. Examples include premature babies, and bone marrow transplant patients. This is quite rare, however when it does occur it can be fatal; thus its prevention is a high priority. 

TRALI

Transfusion related acute lung injury, this is caused by HLA Abs from donor plasma, which has been transferred to the patient during a transfusion. These Abs cause complement-mediated cellular damage to the endothelial and epithelium of the lungs. Symptoms for this include fever, chills, coughing and increased distress on the respiratory system.

TACO

Transfusion-related circulatory overload. Can be caused via inappropriate transfusions from sampling errors, or inappropriate blood requesting. It can also be caused by a laboratory error, and also transfusing blood into patients too quickly. This can result in cardiac failure and cardiac arrest. Some patients will be more prone to circulatory overload than others, such as those with renal disease and previous cases of heart failure.   

PTP

Post-transfusion purpura, this is another reference for acute thrombocytopenia, which is basically the presence of anti-platelet antibodies already within the recipient patients body, which could have developed from a previous transfusion or during pregnancy. This can occur 5-12 days after a transfusion of RBCs or platelets has taken place, this can lead to severe thrombocytopenia and subcutaneous bleeding. Both the transfused and original platelets are destroyed by immune complexes. 

FNHTR

Febrile non-haemolytic transfusion reaction, caused by antibodies present in the recipients blood against donor leukocytes, and also HLA antigens. The use of cytokines is used to mediate the reaction, and leukoreduction of any future transfusions can be performed. The symptoms of this can be fever.

Allergic reaction

Most common reaction from a transfusion, occurs in all types of product however less so with RBCs. Most reactions are mild. 

Anaphylactic reactions 

These tend to be associated with IgA deficiency, of haptoglobin deficiency. Very common in Asian patients. 

Ways to reduce the need for transfusion

Correction of anaemia prior to operation, attempting to salvage cells, lower the trigger levels for RBC transfusion. Avoiding aspirin also helps, also trying biological alternatives, such as recombinant clotting factors and erythropoietin. 

Transfusion monitoring and adverse incident reporting

Patients whom are receiving a transfusion of any type of blood product or component must be monitored closely for the first 15 minutes of after a transfusion, this is so that any early signs of incompatibilities or bacterial contamination can be detected. A patient is monitored via the monitoring of their temperature, pulse, blood pressure and respiration rate. Monitoring will be undertaken regularly afterwards incase of any delayed reactions. All transfusion bags should be kept for a minimum of 24 hours. If an adverse reaction occurs, the hospital transfusion lab would be informed, as well as the haematology medics. If there are any suspected bacterial or viral infections caused by transfusions, this should be reported as quickly as possible to the local blood service centre. 

If a reaction has occurred:

Stop the transfusion, re-test the samples from the patient to check their blood type and cross match with the donor type, post and pre transfusion to ensure original test was correct. Identify any Ab present. Carry out an anti-globulin test, check the colour of a patients plasma and urine for any signs of haemolysis. Check for clerical errors, and check appearance of blood, plus ABO group and Rh group of donor units. Always send for microbiological testing: samples will be taken from the donor unit, side tube and giving set, this is to check where the contamination or error arose from.  

Overall, the majority of transfusions are successful. This is due to the current quality procedures in place. It has been estimated that the number of reports in regards to transfusions reactions are less than what may occur, so it is suggested that the real number of cases is unknown. However, SABRE and SHOT reports still give a good insight and important information about the adverse events that do occur.