| 1 - Laboratory services |
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ABO and Rh-D blood group
• ABO
By means of a red blood cell test, setting the red blood cells to be studied against anti-A, anti-B and anti-AB reagents, and a serum test, setting the serum to be studied against A1 and B red blood cells.
• Rh(D)
Using two monoclonal anti-D reagents to detect the most frequent variable, DVI.
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4.2.- DETECTION OF BLOOD GROUPS (COMMON X ANTIGEN)
Use of monoclonal reagents for the Rh (C, c, D, E, e), Kell (K) and Kidd (Jk a and b) antigens.
More specific and sensitive reagents are used to classify the rest of the common antigens.
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4.3.- DETECTION OF BLOOD GROUPS (PUBLIC/PRIVATE X ANTIGEN)
Use of specific commercial or own reagents obtained from sensitised individuals studied in the immunohaematology laboratory of the Banc de Sang i Teixits, or by means of collaboration agreements with other recognised centres.
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4.4.- MOLECULAR CLASSIFICATION FOR RhD/CE, DUFFY AND OTHER BLOOD GROUP SYSTEMS
Using DNA obtained from blood or amniotic fluid, using PCR techniques.
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4.5.-RESOLUTION OF COMPLEX PROBLEMS IN DETERMINING ABO GROUP, RH FACTOR AND OTHERS
The serological diagnosis of weak subgroups of ABH antigens, variants or modifications of the expression of other erythrocytosis antigens or double non-transfusional populations requires the use of one or several of the following strategies:
• absorption -elution of antibodies and study of eluate.
• research into the presence of antigens studied in other organic components different from red blood cells: saliva, plasma, etc.
• use of monoclonal antibodies for the separation of erythrocyte populations.
• use of polyclonal or monoclonal antibodies to detect and identify variants of the different antigens: B acquired, D, Kell, Duffy, etc.
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4.6.- DIRECT ANTIGLOBULIN TEST (PAD)
Two different polyvalent antiglobulins are used IgG+C3d.
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4.7.- STUDY OF A SAMPLE WITH LOW-COMPLEXITY POSITIVE PAD
Including the following studies:
• PAD carried out with monospecific reagents.
• Titration of the PAD with the monospecific reagent/s.
• ABO group, Rh factor and classification of the Kell, Kidd a and b antigens and monoclonal reagents
• In the case of a recent transfusion, separation of the rich fraction into neocytes to carry out the classification of the aforementioned antigens.
• Study of the eluate obtained from the patient's red blood cells in the conditions necessary to detect antibodies for the type found in the PAD.
• Study of the serum using the panel of red blood cells in an antiglobulin technique.
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4.8.- STUDY OF A SAMPLE WITH POSITIVE PAD AND FREE ANTIBODIES IN THE SERUM
Includes the determinations mentioned in the previous section in addition to studying the serum, with the aim of ruling out the presence of clinically significant alloantibodies, by carrying out:
• Self-absorption of the red blood cells treated with ZZAP or EDTA-Glycine and /or differential absorption with one or several samples of known red blood cells, depending on whether the determination of the patient's Rh factor and Kell and Kidd groups was possible.
• Study of the serum absorbed with the panel of red blood cells using an antiglobulin technique.
If C3d is detected in the patient's red blood cells: research on cold agglutination and IgM incomplete -type antibodies. If it is a PAD of the IgG + C3d type: Additional research into the IgG sub-class.
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4.9.- STUDY OF A SAMPLE WITH POSITIVE PAD PRESENTING COMPLEX SEROLOGICAL DIFFICULTIES
Includes the determinations mentioned in the two previous sections. The additional complexity is conditioned by some of the following situations:
• autoantibodies that resemble the alloantibodies
• identification of the alloantibodies in the eluate
• low affinity antibodies
• presence of antibodies directed against public antigens
• presence of antibodies related to medicines
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4.10.- TITRATION OF AGGLUTINATES OF THE ABO GROUP
By means of the sedimentation technique at 22ºC with double dilution of the serum.
The determination of the IgG component is carried out with the antiglobulin technique, with serum treated with 2ME or DTT.
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4.11.- SURVEY OF IRREGULAR ANTIBODIES
Study of the serum with a panel of two or three different red blood cells, widely classified and complementary, using the antiglobulin technique and an enzymatic technique if necessary. |
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4.12.- IDENTIFICATION OF MONOSPECIFIC IRREGULAR ANTIBODIES
Study of the serum with a panel of identification and "auto" control, isogroup and umbilical cord red blood cells, using the antiglobulin technique in a low ionic force medium (with red blood cells in a saline medium and treated with papein) and that of papein in two sessions.
Determination of the system's antigens against which the detected antibody is directed.
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4.13.- IDENTIFICATION OF IRREGULAR ANTIBODIES
Includes the determinations mentioned in the previous sections and others in relation to the obtained results:
Sometimes, the identification of simple specifications requires the carrying out of panels at different temperatures and/or the study of the serum with 2-ME or DTT.
The titration of an antibody is also considered a basic complementary study.
The prevision of new transfusion requirements could make the determination of the blood groups Rh complete, Ss, Kell, Duffy and Kidd necessary
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4.14.- COMPLETE IDENTIFICATION OF MIXTURES OF COMMON ANTIBODIES
With the aim of ruling out and/or confirming the presence of different antibodies, a combination of the following strategies is used:
• complete classification of the red blood cells obtained from the total blood or the part rich in neocytes.
• use of different panels composed of samples collected according to the specifications involved.
• carrying out differential absorptions.
• neutralisation of the serum with Lewis, P or plasma isogroup soluble substances.
• treatment of the serum with 2ME or DTT.
• study of the serum with special antiglobulin reagents, e.g., monoclonal antiglobulin without anti-IgG4 component.
• other techniques: antiglobulin test in an albuminous medium or with Polyethilenglycol (PEG), antiglobulin test with red blood cells of the panel treated with chloroquine.
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4.15.- RESOLUTION OF COMPLEX SEROLOGICAL PROBLEMS IN SAMPLES PRESENTING A POSITIVE ALLOANTIBODY IDENTIFICATION
The following types of complexity are included in this section:
• identification of antibodies directed against low- or high-frequency antigens.
• identification of mixtures of common antibodies with others directed against low- and/or high-frequency antigens.
• identification of antibodies related to the HLA system.
• identification of antibodies that resemble alloantibodies.
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4.16.- CROSSED TEST
The crossed test is used to set the receptor's serum against the red blood cells of the unit of red blood cells to be administered by an antiglobulin technique in a low ionic force medium.
In special situations, other methods are used: absorbed serum, antiglobulin without anti-IgG4 component, blood classified by common antigens, etc.
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4.17.- PREVENTION OF THE HAEMOLYTIC DISORDER IN A NEWBORN BABY: STUDY OF THE MOTHER
The following studies are included:
• Determination of the antigen Rh(D) with reagents enabling the detection of the DVI variant.
• Analysis of the irregular antibodies, using a technique with antiglobulin.
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4.18.- PREVENTION OF THE HAEMOLYTIC DISORDER IN A NEWBORN BABY: STUDY OF THE NEWBORN BABY
The following studies are included:
• Determination of the group ABO and Rh(D).
• Direct antiglobulin test with polyvalent reagents.
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4.19.- DETERMINATION / QUANTIFICATION OF FOETAL RED BLOOD CELLS IN THE MOTHER'S BLOOD
This determination can be carried out on mothers Rh(D) negative with a child Rh(D) positive to adapt the administration of the IgG anti-D to the amount of foetal red blood cells present in the mother's blood.
It can also be carried out in cases of double populations found in the determination of the blood groups during gestation or at the moment of childbirth.
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4.20.- QUANTITATIVE DETERMINATION OF THE ANTI-D (IgG)
Includes the titration of the anti-D detected in sensitised pregnant women. If the titration is greater than 1:16 in the antiglobulin with red blood cells CcDee technique in saline medium, an EIA quantitative technique will be carried out, as long as the functional study cannot be carried out. All the samples are studied in duplicate and in parallel with the previous sample. The calibration curve is determined with the anti-D international standard.
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4.21.- DETERMINATION OF THE BIOLOGICAL ACTIVITY OF AN ANTIBODY
Chemiluminescence is used with mononucleated cells obtained from donors.
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4.22.- SEROLOGICAL DIAGNOSIS OF NEONATES WHO ARE DAT-POSITIVE FOR MATERNAL-FOETAL SENSITISATION
This section includes the following tests:
• Confirmation of nature of DAT with monospecific antiglobulin reagents.
• ABO and Rh (D) typing.
• Study of eluate obtained from umbilical cord blood with panel adapted to maternal-foetal incompatibility type.
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4.23.- 50% ANTIBODY SCREENING panel (3 ML)
This uses a panel comprising 2-3 red blood cell samples in preservation fluid at a 50% concentration.
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4.24.- RED CELLS SENSITISED WITH 3% IgG (X ML)
A suspension of red blood cells sensitised with IgG, used as the antiglobulin technique control.
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4.25.- ANTIGLOBULIN QUALITY CONTROL
The anti-IgG, anti-C3b, anti-C3d and anti-C4 content (progressive double-dilutions) of a specific antiglobulin is determined. The absence of other contaminating antibodies is checked. |
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4.26.- HLA-B27 GENOTYPE
Determined by PCR-SSP.
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4.27.- HLA-AB PHENOTYPE
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4.28.- LOW-RESOLUTION GENOTYPE HLA-A AND/OR B
PCR-SSP testing of samples with serological typing problems due to poor cell viability, complex specificities, or other reasons.
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4.29.- HLA SPECIFICITIES GENOTYPE
PCR-SSP testing for particular specificities.
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4.30.- LOW-RESOLUTION HLA-DR GENOTYPE
PCR-SSO testing in the reverse dot-blot variant combined or not combined with PCR-SSP. |
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4.31.- HIGH-RESOLUTION HLA-DR GENOTYPE
PCR-SSO testing in the reverse dot-blot variant combined or not combined with PCR-SSP.
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4.32.- HIGH-RESOLUTION HLA-DQ GENOTYPE
PCR-SSO testing in the reverse dot-blot variant combined or not combined with PCR-SSP.
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4.33.- IDENTIFICATION OF LYMPHOCYTOTOXIC ANTIBODIES
Conducted in situations where the following are required: organ transplant, neonatal cytopenia, identification of erythrocyte antibodies, febrile non-haemolytic transfusion reaction, ineffective platelet transfusion and other situations. A cytotoxicity method is used.
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4.34.- DIRECT ANTIGLOBULIN TEST (DAT) ON PLATELETS
The immunofluorescence technique is used with platelets treated with paraformaldehyde and polyvalent and specific anti-IgG, Ig-M and Ig-A antiglobulins.
A negative control is always conducted with platelets from healthy people.
Before the DAT, a platelet count is conducted on a sample of blood in EDTA and citrate.
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4.35.- ANTIPLATELET AUTOANTIBODY STUDY
This includes the following tests:
• platelet count in a sample in EDTA and citrate.
• DAT study.
• investigation of antibodies in eluate obtained from the platelets under study and in serum with polyvalent and monospecific anti-IgG, anti-IgM and anti-IgA antiglobulins.
• Evaluation of affinity with the antibody detected.
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4.37.- STUDY OF A SAMPLE WITH POSITIVE PAD PRESENTING COMPLEX SEROLOGICAL DIFFICULTIES
Includes the tests in the section above and those indicated below:
• identification of HLA antibodies and/or antibody mixtures in serum.
• study of antibodies directed against cryptic antigens: EDTA- and/or PFA-dependent.
• study of medication-related antibodies:
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4.38. - ANTIPLATELET ANTIBODY SCREENING
This study is conducted for febrile non-haemolytic transfusion reactions and neonatal cytopenias using the immunofluorescence technique with platelets treated with and without chloroquine.
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4.39.- IDENTIFICATION OF MONOSPECIFIC ANTIPLATELET ANTIBODIES
Using a panel of platelets classified by the HPA-1,-2, -3, and -5 systems in the solid-phase and/or immunofluorescence techniques.
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4.40.- IDENTIFICATION OF ANTIBODY MIXTURES
Includes the tests in the above section and the MAIPA technique with different monoclonal antibodies directed against different membrane glycoproteins to rule out or confirm the presence of alloantibody mixtures.
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4.41. - RESOLUTION OF COMPLEX SEROLOGICAL PROBLEMS
This section includes the presence of:
• mixtures of autoantibodies and alloantibodies.
• antibodies against public antigens.
• antibodies directed against cryptic antigens.
• alloantibodies in eluate.
To solve these problems, a combination of the solid-phase, immunofluorescence, MAIPA and lymphocytotoxicity techniques is used. On occasion, it may be necessary to perform auto-adsorptions and/or differential adsorptions.
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4.42. - HPA SYSTEMS GENOTYPE
This uses DNA obtained from blood, amniotic fluid, hair, etc.
It is tested by the ASRA and/or SSP variants of the PCR-SSP.
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4.43.- MEMBRANE GLYCOPROTEINS STUDY
Monoclonal antibodies are used to evaluate possible abnormalities in platelet glycoprotein expression with the immunofluorescence or MAIPA techniques.
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4.44.- SCREENING panel
Comprises 2-3 samples of platelets classified by the HPA-1, -2, -3, and -5 systems.
For use with the immunofluorescence (frozen) or MAIPA (suspension) techniques.
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4.45.- POSITIVE SERUM CONTROL
1 ml of polyclonal and polyspecific HLA serum for use with the immunofluorescence technique.
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4.46.- COMPLETE NEUTROPENIA STUDY
The immunofluorescence technique is generally used with granulocytes treated with paraformaldehyde. Occasionally, the agglutination technique is used.
Polyvalent and specific anti-IgG, IgM and IgA antiglobulins are used.
Study of the presence of antibodies in serum and eluate.
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4.47.- SCREENING FOR GRANULOCYTE CYTOPLASMIC ANTIBODIES, ANCA (ETHANOL OR FORMALIN)
Using the immunofluorescence technique on neutrophils fixed by ethanol or formalin.
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4.48.- IDENTIFICATION AND QUANTIFICATION OF ANCA
Using the EIA technique, specific for anti-PR3 and anti-MPO antibodies.
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4.49.- ANTIGRANULOCYTE ANTIBODY SCREENING
This study is conducted in cases of febrile non-haemolytic transfusion reactions and neonatal cytopenias using the immunofluorescence technique and occasionally, the agglutination method.
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4.50.- IDENTIFICATION OF ANTIGRANULOCYTE ANTIBODIES
By studying the specificity of antibodies detected using a panel of granulocytes classified by the NA, NB, ND and 5a antigens.
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4.51.- NA GENOTYPE
With DNA obtained from blood, amniotic fluid, hair, etc., using the PCR-SSP technique.
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4.52.- NB, ND OR 5A TYPING
Using the immunofluorescence or agglutination techniques. The reagents used are obtained from sensitised persons analysed by the Immunohaematology Laboratory of the Banc de Sang i Teixits or through exchanges with other laboratories.
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4.53.- FcRIII TYPING
Using the immunofluorescence technique with a specific monoclonal antibody. |
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4.54.- FEBRILE TRANSFUSION REACTION STUDY
Includes screening of antiplatelet, antigranulocyte and antilymphocyte antibodies.
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4.55.- CROSS TESTING WITH PLATELETS AND LYMPHOCYTES (X1 A 3)
Tests the recipient's serum against the platelets and lymphocytes of the donor using immunofluorescence and cytotoxicity techniques.
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4.56.- POST-BMT CHIMERISM MONITORING
This study is conducted on pre-transplant samples from the recipient and donor and on post-transplant samples from the recipient.
It is based on analysis of the polymorphic microsatellite DNA markers (STRs).
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4.57.- SPECIAL MOLECULAR STUDIES BASED ON STUDY TYPE
Study of mutations associated with cystic fibrosis using the PCR-OLA technique.
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LIRAD