Crossmatching Transfusion – cross matching Quality assurance § Clearly defined / documented quality management system
§ Audit § Full audit trail of laboratory steps § Regular internal audits
§ Risk management § Critical points in pretransfusion testing should be identified and security should be built in to these points § SHOT / regular reviews of untoward incidents / near misses
§ Quality control § External quality assurance schemes (NEQUAS) § Equipment and processes need to validated against the requirements of the laboratory § Reagents § In accordance with manufacturers instructions § Batch numbers and expiry dates need to be documented § Need to validate all changes in the testing process in accordance with BCSH guidelines § Regular maintainance § Automated equiptment § Needs to be validated with clinical samples after any servicing with standardised algorithms § Validated manual backup system with defined procedure to determine when it should be used
§ IT systems § Computerised algorithms should be used compare current group with historical controls § System needs to be validated by using clinical scenarios prior to introduction of electronic issue
§ Training of staff § Documented programme which includes action limits for retraining § Retraining after software upgrades
§ Hospital transfusion committee § Define procedures for sampling, collecting and handling blood products and ensure that staff are trained to comply with this § Define procedure for massive transfusion § Formulating and reviewing maximum surgical blood orders § Commissioning audits to assure compliance with the defined procedures § Investigate adverse events and report to SHOT § Review operational performance of the laboratory (response times for emergency requests)
Sample identification § Minimum (on form and sample) § Surname § Name § DOB § Hospital number § SOP for dealing with inadequately labelled samples § In trauma / unconscious A&E patients single identifier and sex will suffice providing sample is signed § If poorly labelled should issue O and RhD- if female and under age of 60 § Telephone requests should be documented as per SOP § Identity of requester and person in the blood bank receiving the request § Minimum information should be taken and confirmed (name, hospital number, location etc.)
Repeat antibody screening § Every 72h
Antibody screening § Should be performed in advance to warn the clinician that there may be a delay in crossmatching § More sensitive than crossmatching against donor red cells for detecting antibodies § Should be done against C, c, D, E, e, K, k, Fya, Fyb, Jka, Jkb, S, s, M, N, and Lea (underlined in bold need homozygous cells eg Fya+b- (Fya+b+ are insufficient)) § If an antibody is found it should be typed § If there is any doubt about the specificity it should be sent to the reference centre
Transfusion dependent patients § Extended RBC phenotype prior to transfusion for C, c, E, e, MNSs, P1, Lu a/b, K, k, Kpa/b, Le a/b, Fy a/b, Jk a/b. U (if S/s-), Jsb if afrocaribean § Antigen match for D, C, c, E, e and K § Select large units (>300mL) and < 14d if transfusion dependend to minimise donor exposure and maximise lengthe of survival (<7d for exchange transfusions)
Types of crossmatch Electronic issue § Sample grouped twice if full automation and data transfer (otherwise need two samples) § No antibodies dectected § Advantages § Reduces crossmatching workload and response time § Reduces out dating § Doesn’t prevent the wrong patient being bled
Immediate spin § Quick check that ABO and RhD compatible
Saline § Detects antibodies capable of direct agglutination (principally IgM) § Some IgG antibodies will also cause positive reactions
IAT (Serological gold standard) § Usually done at room temperature and will usually detect IgG antibodies (but specific reagents for IgG or IgM are available) § Strict 37oC will prevent clinically insignificant reactions (particularly M/P1) § Low ionic strength (LISS) considered to be most suitable for detecting clinically significant antibodies § LISS IAT at 1/3 dilution can be used (at reference centre to dilute out autoantibodies to look for underlying alloantibodies in patients with AIHA) § Technique § Patient plasma mixed with red cells and incubated § Read for agglutination – IgM antibody § Centrifuge and wash § Add Anti-human immunolobulin
Enzyme = LIP (Papain) § Use in a standard panel is strongly recommended § Strengthens reaction with Rh and Kidd § Reduces MNS (not s) and Fya and Fyb Summary table
§ 4 types of peripheral core structures (largely Band 3 and glycosphingolipids) § Type 1 found in secretions § Type 2 common on red cells § Type 3 and 4 lower prevalence on red cells only A1 can add to these residues (creating the antigenic epitope for anti A1 antibodies – hence the lack of A2 antigens)
§ H antigen § Manufactured by H-transferase - the product of the FUT1 gene (ch19) – null null gives Bombay phenotype – adds fucose to core structures § H Ag O>A2>A2B>B>A1>A1B § A1B and A1 individuals may make anti H antibodies which can result in the haemolysis of O cells § FUT2 – encodes gene for secretion of ABO antigens – 80% of UK population are secretors
§ Alpha1,2 fucosyltransferase catalyses the addition of the peripheral core structure – the H antigen § A-transferase = GalNAc-transferase Adds GalNAc to Fuc § B-transferase = Gal-tranferase Adds Gal to Fuc § A1 glycosyltranferase differs from A2 in that A2 has a single nucleotide deletion resulting in a frameshift which deletes the stop codon resulting in a slightly longer protein which has a reduced specificity for Type 3 and 4 peripheral core structures § B differs from A1 in having 4 changes in the AA sequence in the catalytic domain of the protein § Several different types of O mutations exist – the most common being a mutations that result in premature stop codons § cisAB also exists where the single enzyme can transfer both A and B antigens onto core structures
§ Most people have ABO antibodies by the age of 6 months § Largely IgM – reacting with Saline best at <37 § IgG(2) found in some people – largely group O - capable of activating complement but only rarely cause HDN (usually in O mothers) § Cause rapid intravascular transfusion reactions
§ Anti A1 (2% of A2 and 25% of A2B patients) § Agglutinates A1 but not A2 cf anti-A that agglutinates both § Not usually clinically significant § Dolychos biflorus (lectin) will specifically agglutinate A1 rather than A2 cells § Rarely can result in destruction of red cells in vivo – then need to use blood typed for A2 § Clinical importance of the distinction is that A2 only reacts weakly with anti-A and may be missed. In addition if an A1 cell is only used for the reverse group anti-A1 in the patients serum may react and the patient will wrongly be identified as group B.
ABO grouping § Anti A monoclonal § Anti B monoclonal § Should not react with acquired anti B § Reverse group using A1 and B reagent cells § Except when there is a historical control § Neonatal samples § Confounders of reverse group § Absent responses (immunosuppressed, young, old, agammaglobulinaemia) § Mixed field (post transfusion, post BMT, MDS) § Other antibody present in the sample reacting on the cell
Acquired B
§ 1939 Levine and Stetson (discovered anti-D) § Anti-D – IgG (thus crosses placenta)
Genetics § RHD and RHCE genes both on chromosome 1 transcribed in opposite directions separated by 30kb § Minor changes in amino acid sequency cause conformational changes that create new epitopes
D-negativity § All Europeans have complete deletion of D gene § Africans also have RHDy (66%) a nonsense mutation well as RHD-CE-Ds (15%) a hybrid of CE and RhD
D variants § Weak D has all D epitopes and therefore can’t make anti-D § Should be considered as D positive § Defect inside the membrane § Receive D+ blood in the UK § Blood donors should be types as D pos, § Partial D Lacks some D epitopes and can make anti-D § Defect outside the membrane § Anti-D reagents should not detect DVI, so typed as D negative in patients, but in donors should be typed as D positive § Receive D- blood § Anti-D in D variant women can cause severe HDN and Anti-D Ig should be given to women following pregnancy
D Grouping § Each sample should be tested in duplicate § IgM anti D which does not detect DVI § If clear cut results can’t be obtained should classify patient as D negative until results from reference laboratory available § Partial D (eg VI) should be typed as D negative § Weak D should be typed as D positive
Controls § Positive and negative controls should be used on a regular basis (at least twice daily)
Anomalies § Retesting should be performed using washed cells taken from the original sample § Autocontrol should be used § If the reverse group cannot be reliably obtained (eg due to the age of the patient / immunodeficiency) then the original group should be repeated § May need to obtain a fresh sample and send to the reference laboratory
Frequency of Rh genotypes R1 (41%) r (38%) R2 (14%) R0 (8%) r’ and r’’ (1%) Rz and ry (<1%) R0R0 Common in afrocaribean population
Cw § This is an additional epitope on the C antigen. § Would usually use C –ve blood if an anti-C were present unless both anti-Cw and anti-c were present in which case would use Cw –ve blood
M/N § Glycophorin A § IgM – only found in saline § Occasionally IgG Abs seen on IAT § Destroyed by enzyme treatment § M rarely causes transfusion reactions but can transfuse if not reacting at 37 by IAT § N does not cause transfusion reactions
S/s/U/N-like § Glycophorin B § S destroyed by enzyme – IgM / some IgG § s/U not affected by enzyme § IgG antibodies and can cause severe transfusion reactions and HDN § Can only be U- if both S and s are negative
Duffy (Fy) § IgG – can cause both HTR and HDN § Degraded by enzyme treatment § Fy(a+b-) 20% / Fy(a-b+) 33% / Fy(a+b+) 47% § If Fya-/b- (0% whites / 68% blacks) will be Fy3 –ve as well § Confers resistance to vivax malaria
Lewis (FUT3) § IgM – Haemolytic transfusion reactions rare / HDN not seen § Primarily a system of soluble antigens that are adsorbed onto red cells § The Lewis phenotype is governed by the gene controlling H secretion (FUT2) § Lewis a antigen is converted to b if the patient is H secretor positive
§ In oriental patients there is competition between the Lewis and weak secretor tranferases which leads to Le(a+b+) phenotype § Babies are Le a-b- then initially type as a+b+ if they are destined to become a-b+ (up to age 6) § Antibodies predominantly IgM and not clinically significant § Some patients (particularly Le(a-b-) have antibodies – Lea is usually more potent than b § However, transfused cells lose their antigens in the patients circulation so only the first few cells are haemolysed § Therefore recommended that ABO identical cells are crossmatched at 37
Kell § Four sets of antithetical antigens on the Kell glycoprotein § All can induce IgG antibodies capable of causing haemolytic transfusion reactions and HDN § K/k § IgG both can cause severe transfusion reactions and HDN § 91% kk so most patients at risk of making anti-K antibodies but K-ve blood is easy to find § Anti selano very rare but extremely difficult to find compatible blood (same true for Kpb and Jsb)
§ Kell is bound to the Kx protein which is encoded by an X-linked gene. § Absence of Kx results in weak expression of Kell and the McLeod phenotype (neuroacanthocytosis, muscular, neurological and neuropsychiatric disorders. Occasionally the deletion also involves CYBB – and this leads to chronic granulomatous disease as well as the McLeod phenotype § If these patients are transfused they are likely to make anti-Kx antibodies, which can cause severe HTRs and compatible blood can be very difficult to find Anti-K § Important antibody in white populations – IgG and nearly always complement binding (causes both HTRs and HDN) § Around 10% of K- people develop the antibody after 1 unit of blood. § HDN caused by K differs from HDN caused by Rh in that the fetal anaemia is caused by suppression of erythropoiesis. Anti-k § Very rare antibody – reacts with 99% of random samples § Always immune and can cause some cases of HDN
Kidd § Urea transporter § Can be difficult to detect § Enhanced by enzyme § Anti Jka rare and anti Jkb very uncommon § IgG - can cause severe or even fatal haemolytic transfusion reactions § Jka-/Jkb- very rare - cells resistant to urea lysis – can develop antibodies = JK3
I / i § Cord blood usually I- i+ (i is the synthetic precursor of I) § Adult red cells usually I+ i- § Homozygotes for mutations in GCNT2 have been described who have adult persistence of i Auto anti I - CHAD § Agglutinates at 20 but not 30C - need to test at strict 37 § Anti-I IgM not reacting at 37 – so the DAT will be negative in CHAD § Occasionally thermal range and strength of antibody increases after mycoplasma infections resulting in a postitive DAT and haemolysis § Can treat CHAD with I- blood Anti-i § Reacts with cord blood but not adult cells § Can cause haemolytic anaemia in the context of EBV
P system
Lutheran system § Complex system generally with antigens of very high frequency § Anti Lu antibodies are uncommon § Anti Lua is considered insignificant and does not need to be typed for § Anti Lub may cause mild delayed HTRs
Rare, significant blood groups Fy (a-,b-) U- Bombay Jsa Colton (Coa) Dombrock V Vs Vel
Polyagglutinable red cells Sera from many different adults causes agglutination of the patients red cells (property of the red cells rather than the serum) § Can be inherited or acquired § Inherited divided into 4 types 1. Cad 2. CDA type II (or HEMPAS) 3. NOR 4. Hyde park
§ Acquired divided into microbial and non microbial § Microbial caused by either: 1. Microbial antigens on the surface of red cells 2. Alteration of red cell oligosaccharides (T, Tk, acquired B) by bacterial enzymes
T activation § V. Cholerae, Clostridium perfringens, Strep pneumoniae, influenza virus § Produce sialidases which remove sialic acid and expose T antigen § Need to have high quantities of enzyme as inhibitors in the plasma § Most adult sera have naturally occurring IgM anti-T § May make ABO typing difficult – can be done at 37 § T polyagglutinin also associated with haemolytic anaemia, HUS and neonatal NEC
Tk activation § Bacteroides, clostridia, Candida albicans § Remove beta-galactose from ABH polysaccharide chains – reduces ABH expression § Tk cells specifically agglutinated by BSII lectin
Tn activation § Persistent abnormality caused by an abnormal clone of red cells § Associated with other haematological abnormalities such as haemolytic anaemia, leucopenia and thrombocytopenia § Results in the depression of M and N antigens § Just produces a mixed field in agglutination tests
High titre low affinity Antibodies (HTLA) § Weakly positive reactions that persist despite dilution § Clinically insignificant but will mask other alloantibodies § Usually have to be removed by elution
Epidemiology § 1 in 700 are IgA deficient, but only 30% have anti-IgA § 1996 – 2000, 3 reports to SHOT
Suspected reaction - Test IgA levels § If normal it is unlikely § If IgA <0.5, send sample to the NBS for estimation of IgA level and the presence of anti-IgA § However, there is a small group of patients who have IgA antibodies of limited specificity who have normal levels of IgA and can still have reactions to transfused IgA but these tend to be mild. Thus if convincing clinical history test for IgA Abs even if IgA levels are normal
Management IgA deficient without Abs § Transfusion dependent try and use IgA deficient products unless an emergency § Use standard products if not going to need regular transfusion and no previous reactions
IgA deficient with Abs § Try to use IgA deficient products but the great majority of patients are unlikely to have a severe reaction and the risk of a reaction needs to be balanced against the consequences of delaying the transfusion.
IgA deficient with previous history of severe anaphylactic reaction § Use IgA deficient products irrespective of the IgA antibody status
Blood products Red cells 1. Washed red cells (24 hour shelf life) 2. IgA deficient red cells can be obtained from Sheffield or Colindale Platelets 1. Platelets suspended in PSM 2. Platelets from apherisis IgA deficient donors (need prior arrangement with NBS – unlikely to be available on the shelf) FFP § IgA deficient donors – available from Colindale / Sheffield Cryo § IgA deficient cryo can be manufactured on request only.
Consider giving with hydrocortisone and piriton cover
Major mismatch (e.g. A donor; O patient)
Minor mismatch (e.g. O donor; A patient)
Bidirectional (e.g. A donor; B patient)
Red cells § Give O red cells until the DAT is negative and there is no mixed field on the group.
Platelets § Give A if either donor or recipient is A or AB § Give B if donor or recipient B to O or AB § Bidirectional – give recipient type
FFP § Give AB unless only A or B antigens present.
ABO and RhD grouping § Forward and reverse grouping should not be problematic in warm AIHA § Cold agglutinins may cause difficulties – need to wash the cells in warm saline (37oC) for the forward group. Need also to do the reverse group at strict 37oC
Elutions Purpose to separate antibody from cells Technique 1. Wash cells x6 and keep last wash as a negative control (this step removes antibodies not attached to red cells) 2. Change pH or temperature of the solution to dissociate the antibodies from the red cell antigens (can use ZZAP to destroy the antibodies if only want to use the cells for autoadsorption) 3. Centrifuge and separate cells and serum 4. Wash cells
Autoadsorptions § Use the cells which have had the autoantibody removed from them to deplete the serum of auto-antibody (repeat x3) § Can then use the treated serum to look for alloantibodies using a standard cell panel § This technique cannot be used if the patient has been transfused as the transfused cells may remove an alloantibody in the serum Alloadsorptions § Uses commercially available cells (OR1R1 OR2R2 and Orr) to remove autoantibodies from the serum § Will do one of three things!: 1. Adsorb autoantibody and leave alloantibodies (what you want to happen!) 2. Adsorb alloantibodies and leave autoantibodies or adsorb both
Emergency transfusion § ABO grouped § Attempt to phenotype the patient and give phenotype matched blood. |
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