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Presented to:

Respected S.K.Sharma Sir Respected A.P.Chauhan Sir Deptt. Of Haematology

Presented By: Munish Dogra B.Sc MLT- II year

 As, transfusion is used as a part of treatment for patients it

and thus it has become mandatory to perform compatibility test so as to ensure safer blood transfusion. y Otherwise, mismatched blood transfusion can cause fatal transfusion reactions. y Compatibility tests are also called as pre- transfusion test. y Compatibility testing of blood is also based upon the fact that either the whole blood or blood components therapy should not cause harm to recipient and will have acceptable survival.

1907 - Hektoen suggested that the safety of transfusion might be improved by cross-matching blood between donors and patients to exclude incompatible mixtures. Reuben Ottenberg performed the first Blood transfusion using Blood typing and cross-matching. Ottenberg also observed the of Blood groups and recognized the universal utility of group O donors.

 Can be divided into 3 categories:


y Preanalytical procedures y Serological testing y Post analytical procedures

y Patient identification y Specimen collection y Review of patient history

Must confirm recipients ID from bracelet ON the patient Full patient name and hospital number.

http://www.usatoday.com/tech/news/techinnovations/2006-07-17-chips-everywhere_x.htm

y The sample should also

have the full patient name, CR number. y Date and time of collection. y All of this should be on the request form and the sample.

 Collected in tube with EDTA and NO ADDITIVES.  If the venipuncture causes hemolysis, the sample may be

rejected.  True hemolysis in the patient is the result of complement activation.  Samples are labeled at the bedside (pre-labeling is not recommended)  A record of individuals who collect (or test) the specimens should be documented in order to backtrack in case of an error

y Testing should be performed on samples less than 72 hrs.

or else complement dependent antibodies may be missed (complement can become unstable).

y ABO/Rh Grouping y Antibody detection/identification y Cross match

o In the ABO typing, the forward and reverse MUST

match o In the Rh typing, the control must be negative o Both of these will indicate what type of blood should be given

The antibody screen will detect the presence of

any unexpected antibodies in patient as well as donor serum. Proceed to the Cross-match

y The procedure used to determine compatibility of donor

and recipients blood by mixing their cells and serum or vice versa to see if the mixture agglutinates or formation of clumps.

y Purpose:
Prevent transfusion reactions Increase in vivo survival of red cells Double checks for ABO errors Another method of detecting antibodies

1. Major cross-match 2. Minor cross-match

Donor Red Cells + Recipients serum If there is any agglutination it should never be transfused.

Recipients Red Cells + Donors serum If there is agglutination, it may not be transfused but in case of emergency it can be transfused which may lead to Post Transfusion Reaction.

Why is the minor cross-

match is less important? y Donated units are tested for antibodies y Most blood is transfused as packed cells, having little antibodies

No agglutination ~ compatible

Agglutination ~ incompatible Donor RBCs (washed) Patient serum

y Donor cells are taken from

segments that are attached to the unit itself y Segments are a sampling of the blood and eliminate having to open the actual unit.

 Immediate Spin Tube Technique  As no single test is capable of disclosing

all types of incompatibility satisfactory , 4 tests are recommended:1) Saline test at Room Temperature. 2) Saline test at 37 degree Celsius. 3) An albumin test carried out at 37 degree

Celsius. 4) I A T , sensitizing the cells at 37 degree Celsius.

PRINCIPLE : the donors serum and patients red cells are incubated at 37 degree Celsius to become coated with a serum antibodies . After sensitization , the coated cells are washed and AHG serum is added and mixed , incubated at RT and centrifuge at1000 rpm for 1min. And look for the presence of agglutination .


 The net negative charge which the red cells carries is due to

the ionization of carboxyl group of sialic acid present at the cell surface as a result of Proteolytic action , some enzymes are able to liberate sialic acid residues from the cell membrane , thus decreasing negative charge & allowing the cells to approach one another closely , thus allowing IgM antibodies to bring agglutination of cells . Example- bromalin (pineapple) , papain (papaya) , ficin (fig) , trypsin (pancreas)
They are used in 2 ways:-

One stage technique b) Two stage technique


a)

In this , the serum ,enzymes and cells are layered in the tube and cells become enzyme treated as they fall through enzymes.
Add one volume of patient serum. One vol. of enzyme One volume of donors Washed red cells incubate the mix. at 37oC for one hour

Examine

y In this the cells are pretreated. y Prep of papain : 1 volume of activated papain sol.  9 volume of Sorensons buffer.

Procedure :1)Equal volume of papain + washed packed cells of donor Incubate at 37oC for 30 minutes, agitating frequently. Washing( 3 times in normal saline)and make 5% cells suspension. 2)Then, 2 vol. of patients serum. 1 vol. of papain treated cells Mix and incubate at 37oC for 1 hour Read agglutination.

1. Enables both IgM and IgG antibodies to be detected.

1. Unable to detect antibody in M,N,S and Duffy blood group. 2. False results are obtained if red cells are over treated with enzymes causing fragmentation of immunoglobulin molecule.

y Can be performed in emergency cases as it decreases the incubation time and y

y y y y y y y y y

maintaining a high degree of sensitivity in the IAT. In Normal saline Na+ and Cl- ions cluster around cells and partially neutralize the charges on antigens and ab. Molecule . This shielding effect can be reduced by lowering the ionic strength of Rx medium, this LISS increases the rate and degree of Ab uptake by the cells. Composition :NaCl-1.8g Na2HPO4-0.2g NaH2PO4-0.18g Glycine-18g D/W-1 liter Osmolarity-270-285 mmol/l PH-6.55-6.85 Conductivity-3.5-3.8 mmol/em at 23 degree Celsius

Perform direct & reverse grouping on the patient & donors blood.

Make it 1% by adding 10 L. of donor cells.

Then add 50 l of cell suspension (at 45o) in the gel cards which already centrifuged at 1000 rpm for 10 min. Then add 25 l of patient serum at an angle of 90o. Incubate at 37 oC for 15 min. Centrifuge at 1000 rpm for 10min. See for agglutination.

 Gel cards are 1st centrifuged before use at 1000 rpm for 10mins to;1. Gel get onto a uniform layer which may disturbed while transportation. 2. An air column is formed.

y y y y y y

Amount & strength of Ag present on RBCs surface. Ag/ab. Ratio present in the incubation. Time & temp. of incubation. Freshness of RBC(on storage, antigens RBC surfaces losses its sensitivity) Whether cells are pretreated with enzyme or not. The method of reading results.

 Advantages:Advantages:y Gel acts as a trap for particles other than RBC that is why no washing of all is y y y y y y

required in gel tech. whereas in case of tube method it is must. It gives more accurate results. More sensitive. Here the Ag : Ab ratio is different form that used in tube method i.e., (1:2=AB :Ag) LISS increases the rate of antibody binding. (a)non specific agglutination may occur when NaCl ionic is < 2g/dl are used. (b)complement components are bound to the red cells at low ionic strength.

(1) No. of washing of RBC :No. y Red cells of infants Rh+ve (of mother Rh-ve who is sensitized) will be covered all over by antibodies , so they may give false- ve results and thus must be washed properly. y Also the cord blood cells covered by whartsons jelly which may give false-ve results. (2) Improper incubation :-(time & temp) y Les time gives false negative results, because most blood groups antibodies show Less reactivity over a restricted temp. range. y E.g. IgM-4o- 27oC y IgG-30o-37oC RBC(antigen): (3) Strength of RBC(antigen):y It may vary during storage ,as on storage the sensitivity of antigens is lost thus decreasing the reactivity of RBCs & leading to false ve result .Thus, always use fresh sample. (4) Ag/Ab ratio :-Excess of antibodies can gives false ve results.

Enzyme: (5) Enzyme:- Over treatment of RBC by enzyme may give false ve results.

(1) Rouleax formation ::Increased rouleax form give false +ve result . In detection of IgG, in myeloma cases, dilute the cells and serum with saline which removes rouleax. In many dehydrated cases like a patient is on dextran, rouleax formation is enhanced which gives false + ve Rx. (2) Certain type of cold antibodies like Ti -type can remain active at 22-33oC, which can give false + ve results. It gives no reaction in body at 37oC, but gives Rx in-vitro test at RT. (3) More time incubation may give false + ve. (4) Polyagglutination ::Due to bacterial or viral infection or by storage, some receptor sites get activated which agglutinate with anti-T substances which are present in serum of all normal adults. (5) Multiple transfusion:transfusion:Due to this, some other antibodies are produced by his/her body which gives false + ve Rx.

1)
y It can occur in sera obtained form severally ill patients having raised serum globulin/or

fibrinogen and dextran (given to patient) y Rouleax formation and weak agglutination can be differentrated by adding 1-2 volumes of Normal saline to the cell suspension on the slide which causes Rouleax to break up to a greater or lesser extent and aids in differentation. 2)
y This will cause autoagglutination.if this suspected , the compatibility tests should be

repeated at 37 oC at which the auto agglutination control must be ve.


oC

3)

gglutinins other than anti-a, anti-b and anti-d which give rise to agglutination at 37 are not commonly met with. y E.g.- Other Rh antibodies, anti-M, anti-s, anti-Lu and anti-k. y An attempt should be made to identity the agglutination by using a panel of cells of known genotype. 4)

(1) y Or when the patient undergoing other surgical procedures requiring massive transfusion, when the blood of many donors is mixed together . y The problem is whether the large no. of bottles of blood req. should be matched one against the other, of the possibility that one are more blood samples may contain immune antibodies capable of reacting against the cells of some of the other or of the recipients. y The compatibility test should be done in the usual way .if the patients is group A or AB serum should also be tested with pooled A, cells to exclude the presence of anti-A. y Thus, any atypical antibody should be demonstrated. (ii)
In them the only alloantibodies present in the serum are those derived from the mother,

the compatibility tests are worth while if we are sure of the infant, not suffering from HDN. y So , the simplest way is to match the donors cells with the mothers serum, unless the child has some different, blood group than mother e.g. child-A Group, Mother-O group. y Thus, an antibody screen should always be carried out on the mothers serum.

3.
y Blood for intrauterine transfusion should be tested for

compatibility with the mother serum . the blood should be of same group as of mother or group O and always Rh ve( except in Rh + mothers)the compatibility tests for subsequent transfusions must be tested every time using the fresh serum to a certain the possibility of escape of fetal cells into the maternal circulation , leading to the formation of antibodies of new specifity. y So, it is essential to test the mothers serum against a fully genotyped panel b/w each intrauterine transfusion.

4. alloantibodies may develop quickly following a transfusion early in a series, so perform the cross match before each transfusion from the fresh serum of the recipient, if they are separated by an interval of 2 days or longer, whereas in daily transfusions, only blood that is likely to the used in the 2 days. y Following the collection of the serum should be matched . It is advisable to do a DCT on the red cells of subsequent samples of blood, as antibodies that have formed may be adsorbed to incompatible cells and not present in the serum.

Will
Verify donor cell ABO compatibility Detect most antibodies against donor cells

Will Not
Guarantee normal survival of RBCs Prevent patient from developing an antibody Detect all antibodies Prevent delayed transfusion reactions Detect ABO/Rh errors