Transfusion Flashcards
Reporting of transfusion reactions
Blood component storage
Which patients require CMV negative units as per the ANZBT?
Pregnant patients
IUT
- leucodepleted products are considered equivalent to CMV negative serology in all other cases ie. HSCT, immunosuppressed/chemotherapy patients
Key patients that require irradiated PRBC
IUT
Extreme prematurity and exchange transfusions
Allogenic patients for a minimum of 12 months
Autograft recipients for a minimum, of 6 months
CART - 7 days prior to the CART infusion and then at least a minimum of 6 months post.
Complications from massive haemorrhage
Targets during an MTP
Current emergency guidelines
Transfusion reactions
Types: Fever, chills/rigors, skin changes, pain, circulatory, pulmonary, coagulation/bleeding, psychological (doom)
Fever, chills and urticaria are the most common symptoms of adverse reactions.
Potentially significant and life-threatening reactions include acute/delayed haemolytic transfusion reactions, transfusion-transmitted bacterial infection (TTBI), anaphylaxis and transfusion-related acute lung injury (TRALI).
GENERAL RESPONSE TO RBC T/FUSION REACTION
Patient safety- ie. Stop transfusion, check observations, provide immediate care, keep IV open, MET call/Code blue etc.
Recheck blood product labelling + patient ID
Return of all blood products transfused and lines/giving sets + post t/fusion sample
transfusion reaction report by clinical team
Transfusion reaction registered in laboratory system- flags that no further blood products be issued until investigation complete/approval from haematologist
If severe- Notify ARCBS so associated products can be quarantined/donor investigated (esp if TRALI)
Our lab has a transfusion reaction worksheet, the scientists assess:
Clerical check on pretransfusion sample
Comparison of details on blood component, patient record and pre +/- post samples
Visual inspection of product for clots, discolouration etc.
If post reaction sample available also inspected
Depending on type of reaction suspected further Ix may be performed, eg. if acute haemolytic transfusion reaction complete serological workup including blood group and screen, XM, donor group check
Further investigations guided by haematologist based on assessment
Haematologist contacts treating team and assesses clinical history, assessment documented in laboratory system including likely type of reaction, severity and further recommendations eg. culture of products, no further ix required, special requirements for future transfusions
All transfusion reaction assessments are stored in laboratory records
Acute haemolytic reactions
ACUTE IMMUNOLOGICAL <24 hours
May be fatal - ABO incompatible t/fusion are due to human error, clerical error or improper ID of pre-transfusion blood samples
Clinical
> fever
>chills, rigors, dyspnoea, chest pain, flank pain, discomfort at infusion site, sense of dread, or abnormal bleeding, and may progress rapidly to shock/DIC
> oliguria, haemoglobinuria and haemoglobinaemia -> oliguria is a very bad sign within 24 hours of a transfusion
If anaesthetised patients – first signs may be hypotension and evidence of DIC
Causes
> Immune-mediated or non-immune-mediated
Immune-mediated:
>ABO/RhD mismatch - as little as 10ml
> Red cell alloantibodies (non-ABO) – immunisation from previous pregnancy or transfusion
>Rare cases when Group O donor platelets with high titres of anti-A or anti-B are transfused to a non-Group O recipient.
Non-immune-mediated:
Physical or chemical destruction of transfused red cells such as incorrect storage of red cells, transfusion through the wrong gauge of needle, incorrect use of a blood warmer, interaction with fluids transfused at the same time and bacterially contaminated red cell units.
Incidence: 1:76,000 (fatal 1:1.8 million)
Investigations:
- Check clerical records, such as ABO group of the patient and transfused unit.
- Blood group, antibody screen and DAT on both pre- and post-transfusion samples.
- Repeat crossmatch
- haemolysis screen/UEC/urine
- Septic screen
- Coagulation Screen ? DIC
- IgA levels/Anti-IgA Ab
Management
- Stop transfusion immediately + notify blood bank
- Maintain renal output. - fluids + inotropes
- Induce diuresis with intravenous fluids and diuretics.
Febrile non haemolytic transfusion reaction
ACUTE <24h IMMUNOLOGICAL
Occurs during or within four hours of a transfusion
Diagnosis of exclusion
Clinical:
- temp rise >38C or >1C above baseline during or shortly after transfusion.
- May also include chills, rigors, increased respiratory rate, change in blood pressure, anxiety and a headache.
Incidence: 0.1% to 1% of transfusions (with universal leucocyte depletion).
Usual causes :
- Most common – cytokines accumulate during storage of cellular components (esp. plt) –> Reduced by pre-storage leucodepletion as cytokines are released by WBC
- Recipient leucocyte antibodies (formed as a result of previous transfusions or pregnancies) –>Abs react with HLA/other antigens found on donor lymphocytes, granulocytes, or platelets.
Investigation:
- Exclude an acute haemolytic reaction
+/- DAT, full blood count and repeat ABO grouping
+/- investigations for transfusion-associated sepsis (blood cultures from unit and patient)
In patients with repeated FNHTRs – investigation for HLA antibodies may be useful
Management:
- Stop the transfusion immediately
- Antipyretic
- Rule out an acute haemolytic reaction, transfusion-associated sepsis, TRALI
- If other causes of a fever have been excluded, can restart transfusion at slow rate
Allergic reaction to transfusion
ACUTE <24h IMMUNOLOGICAL
MINOR: hives or urticaria
Causes: Hypersensitivity to allergens or plasma proteins in the transfused unit
1-3% of transfusions
Management: stop, antihistamine, recommence at slower rate.
Premeds for future transfusions
SEVERE: Anaphylaxis
Causes: IgA deficient patients who have anti-IgA Abs. Antibodies to plasma proteins
Or transfusing allergen to sensitized patient (eg peanuts)
1;20,00-50,000 rare
Ix: check for IgA deficiency and presence of anti-IgA abs
Management: Stop t/fusion + notify blood bank, adrenaline, antihistamine, vasopressors .
Wash red cells/plts for future transfusions. Pre med with antihistamine
TRALI - Transfusion related lung injury
ACUTE <24h IMMUNOLOGICAL
Sx: hypoxia, acute resp distress within 6 hours of transfusion. New bilateral infilitrates on CXR.
Most common cause of transfusion assoc fatalities.
Transfusions indicated: Red cells, plts and plasma
Causes: passive transfer of HLA/HNA abs in donor plasma directed against recipient leucocyte antigens - neutrophils activation in lung microcirculation - blood vessel damage - leakage
2 event model - Usually occurs in patients in which have had a primed by illness/insult that activates the endothelium,.
eg liver surgery, etoh, smoker, fluid overloaded, mechanically ventilated.
Ix: If clinically suspected, test the donor and recipient serum for HLA/HNA antibodies and perform a HLA type I/II on the recipient. If these antibodies are present, it supports diagnosis of TRALI .
Occur at reference lab - red cross.
Mgt: Supportive care w oxygen. Diuresis can worsen TRALI and no evidence for steroids
Needs to quarantine related components from donor
Two significant Lifeblood changes to reduce TRALI risk:
- Male-only plasma for FFP and cryoprecioitate.
- No female apheresis
- Reduced plasma volume for plt components - platelets are suspended in Platelet Additive Solution (PAS) with reduced volume of plasma
Massive transfusion complications
NON-IMMUNOLOGICAL ACUTE (<24 HOURS)
Defined as replacement of more than one blood volume in 24 hours or >50% of blood volume in four hours.
–> metabolic and haemostatic abnormalities.
Some of the complications of massive transfusion include:
1. Hypothermia
2. Dilutional coagulopathy
3. Hypocalcaemia, hypomagnesaemia, citrate toxicity (Citrate binds calcium, works as anticoagulant in blood products -> leads to metabolic alkalosis)
4. Metabolic acidosis
5. Hyperkalaemia and hypokalaemia
6. Immune haemolysis
7. Air embolism
Management
- temp > 36°C.
- Monitor K+, ion Ca and acid base status.
- Monitor dilutional coagulopathy by measuring platelets, INR/APTT and fibrinogen often.
- cardiac monitoring
- consider thromboelastography
- blood warmers where appropriate for all blood components and IV fluid.
- calcium replacement and tranexamic acid infusions.
Non-immune mediated haemolysis
NON-IMMUNOLOGICAL ACUTE (<24 HOURS)
Physical or chemical destruction of blood from non immune causes such as:
- extended storage of red cells,
- improper shipping or storage temperatures (stored directly on ice)
- improper use of blood warmers and mechanical damage by administration though a narrow-gauge needle or use of a rapid infuser.
RARE
Most are benign.
Clinical : symptoms of haemolysis - fever and tachycardia +/- haemoglobinuria and haemoglobinaemia.
Ix: Exclude immune haemolysis by performing a direct antiglobulin test (DAT) and repeating the patient’s ABO group.
Test the transfused unit for haemolysis.
Transfusion-transmitted bacterial infection
NON-IMMUNOLOGICAL ACUTE (<24 HOURS)
Can occur early particularly in bacteria that have pre-formed toxin
Causes:
- bacteria from the donor’s skin during the collection procedure (usual source)
- unrecognised bacteraemia in the donor
- environmental contamination or contamination through product processing
Serious morbidity and mortality most frequently with GNB.
More common with:
- Platelets (as these are stored at room temperature)
- Previously frozen components thawed by immersion in a water bath
Sx: Rigors, high fever, severe chills, hypotension, tachycardia, nausea and vomiting, dyspnoea –> sepsis or DIC
Incidence: 1:250,000 platelet transfusions, 1:2.5 million red cell transfusions (Australia)
Investigations :
- Request for blood cultures from the patient, and perform culture and Gram Stain on the remainder of the blood component.
- key is identifying same organism from the patient and component.
Management : Stop the transfusion immediately, bloods/cultures, broad spec antis,
Notify lifeblood to ensure quarantine and testing of related components from the same donation/donor.
TACO - Transfusion associated circulatory overload
NON IMMUNOLOGICAL < 24h
Pulmonary oedema due to excess volume or circulatory overload results in the patient experiencing acute respiratory distress
Leading cause of transfusion-related deaths and major morbidity
Patients over 60 years of age, infants and the severely anaemic are particularly susceptible.
Usual causes –>
Typically if receive a large volume of transfused products rapidly or those with underlying cardiovascular or renal disease.
Risk generally increases with the number of blood products that are transfused.
Incidence: 1:100 transfused patients (1%)
Ix: BNP/CXR
Differential = TRALI
- Hypertension is a constant feature in TACO whereas it is infrequent and transient in TRALI.
Management : Stop, position pt upright, O2, diuresis, resp support as needed.
Prevention:
- at risk pts have transfusion delivered slowly and consider preemptive diuresis + fluid assessments.
Delayed hemolytic transfusion reactions (DHTR)
IMMUNOLOGICAL DELAYED (>24 HOURS)
Characterized by fever and anemia days or weeks following a red cell transfusion.
24 hours to 28 days after a red cell transfusion.
Sx: Fever, inadequate post-transfusion rise in Hb, spherocytic haemolysis, positive antibody screen, positive DAT.
> mainly extravascular (IgG mediated) , so although haemoglobinuria may occur in rare cases, acute renal failure and DIC are not normally seen.
Pathogenesis :
If exposed patient may become alloimmunised after previous transfusion/pregnancy –> level of Ab may diminish below level of detection on pretransfusion screening.
IF patient then transfused with red cells with those antigens –> an anamnestic (or secondary immune) response may occur which causes the antibody level to rapidly rise leading to a DHTR.
Clinical severity of a DHTR depends on the immunogenicity or dose of the antigen and how quickly the new antibody is produced.
Red cell antibodies associated with DHTRs:
Kidd, Duffy, Kell and MNS systems antigens.
Incidence: 1:2,500 of transfusions
Investigation- DAT, antibody screen, LFT, LDH and markers of haemolysis (e.g. serum haptoglobin, bilirubin). Blood film- spherocytes.
Management :
- Most DHTR have benign course and require no treatment, however life-threatening haemolysis with severe anaemia and renal failure may occur.
- antigen-negative blood if further transfusion is needed.
Patients who frequently receive transfusions, such as those with sickle cell disease or thalassaemia, should have extended red cell phenotyping and receive red cells matched for at least ABO, Rh (D,C,c,E,e) and Kell antigens.
IMMUNOLOGICAL > 24 h
Rare delayed transfusion reaction where a patient develops dramatic, sudden and self-limiting thrombocytopenia (platelet counts <10 x 109/L in 80% of cases), typically 5-7 to 10 days after a blood transfusion.
Clinical : rapid onset marked thrombocytopenia, pupura, bleeding/bruising
Mortality is rare but may be due to intracranial haemorrhage
Thrombocytopenia can be expected to persist for 7-28 days if not treated (or longer)
F >M
Pathogenesis: caused by antibodies to platelet-specific antigens, mostly HPA-1a.
Affects HPA-1a negative patients who have been alloimmunised by pregnancy/transfusion.
The immune specificity is against a platelet-specific antigen yet BOTH autologous and allogeneic platelets are destroyed (unclear mechanism)
FNAIT mothers are at an increased risk of this -> platelets should be HPA negative
Can occur with red cell transfusions as well - HPA antigens can be floating free and cause a reaction
Investigation:
- Demonstrate anti platelet antibody in the patient’s plasma (anti-HPA Ab)
Management :
- IVIG
- Steroids and plasma exchange may be tried in refractory cases.
- Plts can be given with poor increments
Antigen negative RBC and platelets may be indicated if subsequent transfusion is required but this is controversial (no evidence that Ag neg plts more effective than pooled donor)
Consider washed RBC or HPA compatible in future
Transfusion-associated graft vs host disease (TA-GVHD)
IMMUNOLOGICAL > 24h
Extremely rare
Sx: fever, rash and diarrhoea commencing 1-2 weeks post-transfusion.
Lab: pancytopenia and LFT derangments.
TA-GVHD leads to profound marrow aplasia with a mortality rate >90%.
Death typically occurs within 1 -3 weeks of first symptoms, most commonly due to overwhelming infections.
CAUSES: Occurs in immunodeficient recipients whose immune system is unable to recognise the transfused T lymphocytes as foreign –> these T lymphocytes engraft in the recipient and react against the host.
The three primary risk factors for developing TA-GVHD are:
- Degree of immunodeficiency of the recipient
- Number of viable T lymphocytes transfused
- Genetic diversity between donor and recipient. Greatest risks are donations from blood relatives and with HLA-matched blood products.
Investigation- Diagnosis is normally made on skin biopsy, occasionally on liver or bone marrow biopsies. Lymphocyte chimerism
Management :
Treatment is supportive and often, corticosteroids and cytotoxic agents are used, but are largely ineffective which means prevention is crucial.
For patients at risk (ANZBT guidelines), it’s critical to provide irradiated cellular blood components
Leucocyte depletion is not sufficient to prevent TA-GVHD in at risk patients, but irradiation does.
PRODUCTS:
Use gamma or x-ray irradiation - 25 Gray required dose
Red cells >21 days old are now considered ‘irradiated equivalent’
Alloimmunisation - transfusion reaction
IMMUNOLOGICAL > 24H
Alloimmunisation occurs when a patient is exposed to foreign antigens as a result of blood transfusion, pregnancy or tissue transplant and they amount and immune response with the stimulation of alloantibodies to red cell antigens, HLA or HPA
Clinical :
HLA and HPA antibody formation may lead to platelet refractoriness where less than an expected rise in platelet count is seen after a platelet transfusion. This may occur in about 25-70% of patients receiving multiple platelet transfusions.
PTP can also occur as a consequence of development of HPA and HLA antibodies.
Alloimmunisation by red cells results in an acute or delayed haemolytic transfusion reaction or haemolytic disease of the newborn
Alloimmunisation against HLA antigen is also implicated in transplant rejection, FNHTR and TRALI.
Incidence : ~ 1% red cell transfusions are associated with alloantibody formation but may be much higher (up to 30%) in patients who are frequently transfused, such as those with sickle cell disease and thalassaemia.
- HLA antigens 10%
Investigation- Perform an antibody screen on the patient’s plasma to detect clinically significant red cell, HLA or HPA antibodies (red cell reference lab).
Management: Treatment depends on the type and severity of the transfusion reaction with most reactions being mild.
Alloimmunisation can’t be completely prevented
In Australia, all cellular blood components are leucodepleted –> reducing the risk of HLA sensitisation.
TRhD immunoglobulin is also given to pregnant women who are RhD negative to prevent haemolytic disease of the newborn.
To reduce the risk of TRALI, plasma for FFP and cryoprecipitate is only accepted from males, and apheresis platelets collected from males and nulliparous women only
If a red cell alloantibody is identified, antigen-negative blood should be selected if further transfusion is needed.
With patients requiring long-term transfusion support, e.g. those with thalassaemia, giving phenotyped-matched red cells early in their treatment course may reduce the risk of further antibody development
NON IMMUNOLOGICAL DELAYED >24h
transfusion reactions
Iron Overload
Transfusion-transmitted infections
VIRAL (< 1 in a million) vs BACTERIAL (more common in plt than RBC transfusions)
> reduced by donor questionnaire (travel history, tattoo history, high risk behaviours)
Mandatory testing includes screening of donations for presence of Treponema pallidum
Mandatory testing includes: HIV 1 and 2, Hepatitis B and C, HTLV I and II
Some products also undergo screening for CMV in order to provide a CMV seronegative inventory of cellular components
Other viruses are not routinely tested for including dengue, West Nile virus, Chikungunya virus. Some agents cannot be routinely tested for eg. Chagas disease, variant Creutzfeldt-Jakob Disease
Forward and Reverse ABO grouping
Forward (cell) group
- Identifies ABO antigens on RCs cells by testing with antisera
- Anti-A1, Anti-B and Anti-AB
Reverse (plasma) group
- Identifies ABO antibdoies in plasma by testing against RCs with known ABO groups
- A1, (A2), B, (O)
Forward and Reverse grouping serves as check of each other –> MUST AGREE
Exceptions: neonates, ABO subgroups, eldelry & hypogamm
Group Discrepancies
Clerical
Technical
Unexpected cell group reactions
- Weak or absent cell reactions
- Novel antigens
Unexpected reverse group reactions
- Weak or absent reverse group reactions
- Other Abs – allo or AutoAb
Mixed field reactions
-Transplantation or massive transfusion
-IUT or exchange transfusion
-Chimera
-Polyagglutination
-subgroup
Age
Contamination
Methods to potentiate agglutination
- LISS (low ionic strength saline): decreases repulsive charges between RBCs; tends to enhance cold antibodies and autoantibodies
2. Polyethylene glycol (PEG) excludes H2O, tends to enhance warm antibodies and autoantibodies.
- Enzymatic treatment: decreases zeta forces, destroys some antigens, and exposes/enhances some antigens.
H Antigen, A & B Antigen Type 1 vs Type 2
ABH antigens are built from on oligosaccharide chains, most common forms are type:
Type 1 soluble in plasma and secretions
- FUT2 gene – responsible for formation of H antigen in secretions (Se= dominant, se= inactive)
- therefore se/se = non secretor
Type 2 present on RBC membranes
- H gene (FUT1) - encodes for fucosyl transferases vs h which encodes for a non-functional enzyme = Bombay (Oh)
Proteins vs Carbohydrate blood groups
Carbohydrate (ABH, Le, MNS)
- Indirect gene product – gene codes for an enzyme –> enzyme creates the antigen by transferring a sugar molecular onto a protein or lipid
- Abs to these antigens are usually IgM
Protein (Rh, Kell, Fy)
- Direct gene product – gene codes for the protein
-Specifically determined by the inherited amino acid sequence +/- conformation of the protein
- Abs to these antigens usually IgG
Blood group LIKE antigens
Red cell antigens are expressed on other organisms
Can by implicated in AIHA:
ABH & Le - E.Coli
P1 - Echinoccocus
O - Trepnoema
i - EBV
I - Mycoplasma, Listeria
H/h and Se/se phenotypes
Bombay phenotype vs para Bombay
Ulex europaeus (lectin with anti-H specificity) –> used to identify Bombay (no reaction)
Bombay make naturally occurring anti-H antibodies and can only receive transfusions from other Bombay patients
Para-Bombay retain FUT2 activity, so express H in plasma and secretions
> May make clinically insignificant cold reacting anti-H and anti-HI antibodies; but still also can make strong anti-H antibodies
> Should be transfused with Bombay or Parabombay blood group if anti-H or anti-HI is clinically significant (i.e. reacting at 37 C)
When do ABO antibodies appear?
Appear at 4 months of age, and reach adult levels by age 10
They can fade in older age
A subgroups
A1 and A2 are the most common, others include (A3, Ax Aend, Am, Ay and Ael )
Two main subtypes occur:
A1 (80%) and A2 (20%)
A1 has 5 x more antigen than A2
Distinguished by using Dolichos bifloros lectin which has anti-A1 specificity (in forward group) –> agglutinates A1 but not A2
ABO/Rh Blood grouping discrepancies
Weakened or loss:
- Subgroups
-Neonatal/cord
- Disease states (eg haem malignancy)
Novel Antigens:
- Acquired B
- Polyagglutination (T, Tk, Tn etc)
Polyagglutination
RBC antigen exposure - due to modifcation from infection OR somatic mutation (eg T, Tk, Acq B and Tn)
- patient RBCs which agglutinate in almost all donor/test plasma BUT not cord serum
Cells may show variable degrees of polyagglutination
ABO/RhD grouping problems
Autocontrol and DAT positive
Approach:
- test with different polyclonal reagents
- test pts RBCs with lectin panel
- test pts RBC with fresh normal AB plasma (adult and cord)
–> pos with adults, neg with cord
- repeat ABO with enzyme treated cells
Unexplained reverse group reactions
Alloantibodies
- immune vs passive
Autoantibodies
- warm vs cold
Hypogam/agamma
Rouleux
Haemolysis (when using serum)
Weak D vs Partial D
Weak D:
- Decreased D antigen expression
- Not detectable on immediate spin
- require the IAT test to detect D antigen
Causes:
- Mutations in the RhD gene causing altered amino acids in the membrane/inner part of the RhD protein
Who is required to have Weak D testing?
- Blood donors (if testing RhD-, these patients require IAT testing)
- RhD- babies with RhD- mothers (if the baby expresses weak D, the mother may form a clinically significant anti-D allo-antibody)
The converse situation, Weak D mothers with RhD- babies, does not necessitate anti-D prophylaxis
Partial D:
- Lack portions (epitopes) of the D antigen
- Antibodies form against absent parts of RhD; the antibody appears to be anti-D
- Presents as: anti-D positivity in a D-positive person
- Most common variant is variant VI –> most likely to form Anti-D if exposed
- Do NOT want to detect this in a hospital lab –> reagents are specifically insensitive to DVI and pts treated and typed as RhD neg and recieved RhD neg products
- Blood banks frequently uses monoclonal/mor sensitive anti-D in tests as want to ensure screening donors as D positive.
Partial RhD mothers require anti-D
- Distinguishing partial and weak D can be impossible without molecular typing; if in doubt, give anti-D
Lectins and red cell reactivity
Mixed field reactions
- BM transplant
- Chimera
- Polyagglutination
- Maternal contamination of cord sample
- Feto-maternal haemorrhage
- Twin to twin in utero transfusion
Most common partial D Variant?
Variant VI
Rh Antibodies
-RhD antigen most immunogenic (then c, E)
-Most Rh antibodies are due to previous transfusion and/or pregnancy
- Usually IgG, occasionally IgM
-Some naturally occurring IgM forms of anti-E and anti-Cw
-Rh antibodies do not fix complement
-Cause severe HDFN & haemolytic transfusion rx
High incidence Antigens
Antigens which occur in >90% of most populations.
Development of Abs is rare
Issues
- hard to find donros if antibodies present
Examples:
Kell system (Kpb, Jsb and Lub)
Low incidence Antigen
Antigens occurs <1% of most populations
Antibodies:
- Commonly encountered
-Clinical significance varies – generally not highly significant
- Cannot usually identify them
- Easy to find compatible blood as the Ag occurs at low frequency
Examples: Kpa, Di, Jsa, Cw and V
I antigen system
little i antigen is present on fetal and newborn red cells.
big I antigen develops gradually after birth, becoming the predominant form in adult red cells.
Change occurs due to enzymatic modification
i phenotype is autosomal recessive <1% of all individual
Antibodies:
- usually cold reacting IgM autoantibodies
- uaturally occurring
- Commonly have anti-H specificity as well (anti-IH)
Disease associations:
- Anti I
> CAD
> Mycoplasma
Anti (i)
> Infectious mononucleolsis
Anti-Pr
> CAD with anti-Pr specificity is uncommon
(Pr antigen found at the distal N terminus of glycophorins)
Anti-IH
> Rarely found or clinically significant
Screening/determining antibody specificity in Cold Agglutinins.
When is a TITRE done?
Ensured sample is collected warmed and maintained at 37 degrees
Separate out the plasma – then can be stored at room temperature until testing
Testing requires group I adult (OI), cord (Oi), papainised RBC (OI enhanced) + patient RBCs
Antibody specificity:
1:11 and 1:66 titres (other references say 1:2, 1:4, 1:8, 1:16, 1:32)
Incubate with OI and Oi cells @ 4 degrees (2 hours or overnight)
> If agglutination occurs then proceed to full titration
Full titration includes further dilutions 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048
> Uses OI, Oi, OI enhanced, auto RBC to plasma
> Incubation and test 4 temperatures (4 degrees, 20-24, 30, 37
Reaction pattern :
- OI enhanced improves reactions with anti-I anti-i, and anti-IH
- Anti-I has better reaction OI than Oi
- Anti-i has better reaction with Oi than OI
- Anti-Pr reacts with OI, Oi, auto equally. Not enhanced with OIE
- Can only tell anti-IH from anti-I if test using bombay blood group I
Titre:
- Performed at 4 degrees C
- Done once specificity identified
- Identify lowest dilution at which agglutination occurs
- Titre = reciprocal of lowest dilution
> Accepted threshold for diagnosis is 64 (titres often MUCH higher than this)
Thermal amplitude of cold agglutinins
Determines clinical significance of the antibody
Determines the highest temperature at which autoantibody binds RBC antigen
Positive at 30 and 37, suggests high thermal amplitude
Positive 4 degrees or room temperature only “limited thermal amplitude”
–> does titres with 11 tubes with doubling dilutions (1:1 to 1:1024) and done at 37C, 30C, 22C and 4C
