Compatibility Testing for Blood Transfusion

8/6/2019 Compatibility Testing for Blood Transfusion

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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


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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 fataltransfusion 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 shouldnot cause harm to recipient and will have acceptable survival.


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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 groupO donors.


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Can be divided into 3 categories:Can be divided into 3 categories:

yPreanalytical procedures

ySerological testing

yPost analytical procedures


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y Patient identification

y Specimen collection

y Review of patient history


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Must confirm recipients ID

from bracelet ON the

patient

Full patient name andhospital number.

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


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y The sample should alsohave the full patient name,CR number.

y Date and time ofcollection.

y All of this should be on therequest form and the

sample.


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Collected in tube with EDTA and NOADDITIVES.

If the venipuncture causes hemolysis, the sample may berejected.

True hemolysis in the patient is the result of complementactivation.

Samples are labeled at the bedside (pre-labeling is notrecommended)

A record of individuals who collect (or test) thespecimens should be documented in order to backtrackin case of an error


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

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


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y ABO/Rh Grouping

y Antibody detection/identification

y Cross match


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o In theABO typing, the forward and reverse MUST

matcho In the Rh typing, the control must be negative

o Both of these will indicate what type of blood

should be given


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The antibody screen will detect the presence of

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


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yy The procedure used to determine compatibility of donorThe procedure used to determine compatibility of donorand recipients blood by mixing their cells and serum orand recipients blood by mixing their cells and serum orvice versa to see if the mixturevice versa to see if the mixture agglutinates or formationagglutinates or formationof clumps.of clumps.


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y Purpose:

Prevent transfusion reactions

Increase in vivo survival of red cells

Double checks forABO errors

Another method of detecting antibodies


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1. Major cross-match2. Minor cross-match


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DonorRed Cells + Recipients serumIf there is any agglutination it should never be

transfused.

Recipients Red Cells + Donors serumIf there is agglutination, it may not be transfused butin case of emergency it can be transfused which maylead to Post Transfusion Reaction.


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Why is the minor cross-

match is less important?

yDonated units are tested

for antibodiesyMost blood is transfused

as packed cells, having

little antibodies


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Donor RBCs

(washed)

Patient serum

No agglutination ~ compatible

Agglutination ~ incompatible


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y Donor cells are taken from

segmentssegments that are attached

to the unit itself

y Segments are a sampling ofthe blood and eliminate

having to open the actual

unit.


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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 degreeCelsius.


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PRINCIPLE : the donorsserum and patients red

cells are incubated at 37

degree Celsius to becomecoated with a serum anti-

bodies . 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 .


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

the ionization of carboxyl group of sialic acid present at thecell surface as a result of Proteolytic action , some enzymes

are able to liberate sialic acid residues from the cellmembrane , thus decreasing negative charge & allowing thecells to approach one another closely , thus allowing IgMantibodies to bring agglutination of cells .Example-bromalin (pineapple) , papain (papaya) , ficin (fig)

, trypsin (pancreas)They are used in 2 ways:-

a) One stage technique

b) Two stage technique


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In this , the serum ,enzymes and cells arelayered in the tube and cells become enzyme treated as they

fall through enzymes.

Add one volume of patient

serum.

One volume of donors

Washed red cells

One vol. of enzyme

incubate the mix. at 37oC for one

hour

Examine


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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.


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

1. Unable to detect antibody inM,N,S and Duffy blood

group.

2. False results are obtained if red cells are over treatedwith enzymes causing fragmentation of

immunoglobulin molecule.


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y Can be performed in emergency cases as it decreases the incubation time andmaintaining a high degree of sensitivity in the IAT.

y In Normal saline Na+ and Cl- ions cluster around cells and partially neutralizethe charges on antigens and ab.Molecule .This shielding effect can bereduced by lowering the ionic strength of Rx medium, this LISS increases the

rate and degree of Ab uptake by the cells.y Composition :-

y NaCl-1.8g

y Na2HPO4-0.2g

y NaH2PO4-0.18g

y Glycine

-18g

y D/W-1 liter

y Osmolarity-270-285 mmol/l

y PH-6.55-6.85

y Conductivity-3.5-3.8 mmol/em at 23 degree Celsius


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Perform direct & reverse grouping on

the patient & donors blood.Make it 1% by adding 10 L. of

donor cells.


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Then add 50 l of cell suspension (at 45o) in the gel cards which already centrifuged at

1

000 rpm for1

0 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.


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y Amount & strength of Ag present on RBCs surface.

y Ag/ab. Ratio present in the incubation.

y Time & temp. of incubation.

y Freshness of RBC(on storage, antigens RBC surfaces losses its sensitivity)

y Whether cells are pretreated with enzyme or not.

y 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

required in gel tech. whereas in case of tube method it is must.

y It gives more accurate results.

y More sensitive.

y Here the Ag : Ab ratio is different form that used in tube method i.e., (1:2=AB :Ag)

y LISS increases the rate of antibody binding.y (a)non specific agglutination may occur when NaCl ionic is < 2g/dl are used.

y (b)complement components are bound to the red cells at low ionic strength.


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(1) NoNo.. ofof washingwashing ofof RBCRBC ::--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)ImproperImproper incubationincubation ::--(time(time && temp)temp)yy

LesLess time gives false negative results, because most blood groups antibodies showreactivity over a restricted temp. range.

y E.g. IgM-4o- 27oC

y IgG-30o-37oC

(3)StrengthStrength ofof RBC(antigen)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 .T

hus, always use fresh sample.(4)Ag/AbAg/Ab ratioratio ::--EExcess of antibodies can gives false ve results.

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


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(1)Rouleax formation :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 enhancedwhich gives false + ve Rx.

(2)Certain type of cold antibodies likeTi -type can remain active at 22-33oC, which cangive false + ve results. It gives no reaction in body at 37oC, but gives Rx in-vitro test atRT.

(3)More time incubation may give false + ve.

(4)Polyagglutination :Polyagglutination :--Due to bacterial or viral infection or by storage, some receptor sites get activated whichagglutinate with anti-T substances which are present in serum of all normal adults.

(5)Multiple transfusion:Multiple transfusion:--Due to this, some other antibodies are produced by his/her body which gives false

+ ve Rx.


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1)

y It can occur in sera obtained form severally ill patients having raised serum globulin/orfibrinogen and dextran (given to patient)

y Rouleax formation and weak agglutination can be differentrated by adding 1-2 volumesof Normal saline to the cell suspension on the slide which causes Rouleax to break up toa greater or lesser extent and aids in differentation.

2)

y This will cause autoagglutination.if this suspected , the compatibility tests should berepeated at 37 oC at which the auto agglutination control must be ve.

3) gglutinins other than anti-a, anti-b andanti-d which give rise to agglutination at 37 oC are not commonly met with.

yE.g.

- O

ther 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)


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(1)

y Or when the patient undergoing other surgical procedures requiring massivetransfusion, 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 oneagainst the other, of the possibility that one are more blood samples may containimmune antibodies capable of reacting against the cells of some of the other or of therecipients.

y The compatibility test should be done in the usual way .if the patients is group A or ABserum 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 fromHDN.

y So , the simplest way is to match the donors cells with the mothers serum, unless thechild has some different, blood group than mother e.g. child-A Group,Mother-Ogroup.

y Thus, an antibody screen should always be carried out on the mothers serum.


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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.


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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 aninterval 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 subsequentsamples of blood, as antibodies that have formed may beadsorbed to incompatible cells and not present in the serum.


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WillVerify donor cell ABO compatibility

Detect most antibodies against donor cells

WillNot

Guarantee normal survival of RBCs

Prevent patient from developing an antibodyDetect all antibodies

Prevent delayed transfusion reactions

Detect ABO

/Rh errors


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Compatibility Testing for Blood Transfusion

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