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.