a control serum known to contain a weak anti-D antibody is included in each batch of tests ; only if this gives a macroscopic positive with the D positive and a clear negative with the D negative cells should the rest of the test be read . comment . this is a very sensitive and useful technique which is unlikely to fail to detect any Rh antibodies . it is recommended that it should always be used as a routine antibody detection method . occasionally sera are encountered which give pan-agglutination with trypsinised red cells . the antibody responsible for the pan-agglutination can usually be quite easily removed by incubating the serum with an equal volume of the patient &apos;s own trypsinised red cells . the absorbed serum can then be re-examined for the presence of specific antibodies with the standard trypsinised cells . no 16 . Lo&quot;w &apos;s papain technique for antibody detection . equal volumes of the serum to be tested , papain and a 2 per cent suspension of red cells are placed in a precipitin tube , taking care to adhere strictly to the order : ( a ) serum , ( b ) papain , ( c ) red cells . it is also important that the serum &amp;sol; papain mixture should not be allowed to stand on the bench for more than about 5 minutes before the red cells are added . it has been noted that the best results are obtained if the red cells are allowed to sink through the fluid during the incubation period . therefore the contents of the tubes should not be mixed up at the initial stage . the test is read after precisely one hour &apos;s incubation , controls of known D positive and D negative cells with a weak incomplete anti-D being included with each batch of tests . comment . this is a good and efficient technique and is excellent for the detection of Rh antibodies . in fact anti-D antibodies may be detectable when they are not apparent by any other technique , even the indirect Coombs . it does , however , give positives when certain other antibodies are present so that care must be taken in the establishment of the specificity of any antibody detected by this method . titration of Rh antibodies . technique no 17 . saline . serial dilutions of the serum are made in saline as in technique no 6 ( or if desired techniques Nos 7 or 8 ) and incubated at 37 &amp;deg; C for 2 hours with standard D-positive red cells ( 2 per cent suspension ) in saline . the tests are read taking the usual precautions against breaking down the agglutinates . the results are recorded as for ABO titres . ( see plates 6 and 7 . ) technique no 18 . albumin . the serial dilutions of the serum are made in AB serum and the standard cells are suspended in 30 per cent bovine albumin . in all other respects the method is identical with technique no 17 . technique no 19 . albumin addition . serial dilutions are made in AB serum . after 1 1/2 hours &apos; incubation an equal volume of bovine albumin is added without disturbing the cells . after a further 30 minutes &apos; incubation the tests are read in the usual manner . technique no 20 . indirect Coombs technique . serial dilutions of the serum are made in saline using double unit volumes ( 0.06 ml ) in the cell-suspension tubes . four volumes of a 5 per cent suspension packed washed D-positive red cells are added to each tube . from this point the procedure is exactly as in technique no 14 ( a ) . technique no 21 . trypsin . the serial dilutions of serum are made in AB serum and warmed to 37 &amp;deg; C before the addition to each tube of an equal volume of trypsinised D-positive red cells . the tests are incubated for 1 hour and read by tapping the tubes and examining the contents macroscopically and if necessary microscopically for agglutination . technique no 22 . papain . the serial dilutions are made as for technique no 18 , after which one volume of Lo&quot;w &apos;s papain is added to each tube . this is followed immediately by an equal volume of a 2 per cent suspension in saline of D positive red cells . the tubes are incubated for exactly one hour and then read , first tapping the tube twice gently before examining the contents macroscopically and if negative , microscopically . interpretation of results . sera are usually tested by at least two techniques . in the testing of Rh negative women antenatally , for instance , it is recommended that the saline ( technique 12 ) albumin ( technique 13 ) and trypsin ( technique 15 ) or papain ( technique 16 ) techniques are used in parallel . any reaction obtained , however weak , indicates that further tests are necessary to confirm the presence of an antibody and to establish its identity . if the serum of a D negative individual agglutinates the D positive but not the D negative control cells , there is a high probability that the serum contains anti-D , but the specificity should be confirmed by testing against several more examples of D-positive and D-negative red cells . if a pattern of reaction is obtained other than that expected for anti-D , the serum requires a more detailed investigation ( table 14 ) ; this is usually undertaken by a specialist serological laboratory . moreover it must be realised that a serum behaving like anti-D in the above tests may in fact be a mixture of Rh antibodies . rather less than half the Rh antibodies found in Rh negative persons are mixtures of anti-D and anti-C , a much smaller number are anti-D plus anti-E and a very few are mixtures of all three antibodies . a knowledge of whether or not a particular anti-D is mixed with anti-C or anti-E is usually unimportant clinically , but if the serum is required for Rh typing purposes its exact content must be known . it is dangerous to use for typing purposes a serum containing anti-C or anti-E in addition to the anti-D , for by this means certain individuals who are in fact D negative may be falsely classed as D positive . R&amp;prime;r(Cde.cde) cells which contain C without D will show the presence of anti-C in an anti-C plus anti-D mixture while R&amp;prime;&amp;prime;r(cdE.cde) which contain E without D cells will detect anti-E in an anti-E plus anti-D mixture . therefore , while the testing of a suspected anti-D against about 3 D positive and 2 D negative red cell samples followed by titration is adequate for normal purposes , a far more detailed investigation of the serum must be made ( probably by a specialist laboratory ) if it is required for Rh typing . it is , of course , possible as in example 4 ( table 14 ) that the antibody belongs to one of the other blood group systems such as Kell , Duffy , Kidd , Lutheran , etc . for a description of these another textbook , such as an introduction to blood group serology , must be consulted . antibodies related to these systems can only be identified by a laboratory possessing a panel of red cells extensively genotyped to cover them . chapter 8 . the choice of blood for transfusion and direct matching methods . blood transfusion has developed so rapidly in the last twenty years that it comes as something of a shock to realise that its history goes back into the remote past . in ancient thinking the words blood and life were almost interchangeable and many endeavours were made to transfer the healthy life blood of a young man to the aged and infirm . in most cases this was done by the recipient drinking the blood ; the results were of course , rather disappointing ! as early as the sixteenth century it was realised that the transference should be from blood vessel to blood vessel , but it is not known whether such an exchange was in fact performed . Harvey &apos;s discovery of the circulation of the blood in the early seventeenth century gave a new impetus to the interest in transfusion and Lower actually kept alive dogs , which had been exsanguinated , with blood from other dogs , transferred by connecting the carotid artery of the one to the jugular vein of the other by means of quills . the success of this venture led to attempts to transfuse man . animals ( sheep and lambs ) were used as donors , but the experiments were discontinued when the fourth recipient died . it is interesting to note that this patient had three transfusions in all , the first symptomless , the second showing typical symptoms of a haemolytic transfusion reaction and the third resulting in the patient &apos;s death . during the latter half of the nineteenth century experiments started again , sometimes using animal blood , sometimes human , but the results were so often serious or even fatal that transfusion was abandoned . then in 1901 Landsteiner discovered the ABO blood group system and realised immediately the importance of his discovery . it was not until some fifteen years later , however , that it was universally accepted that blood grouping and direct compatibility tests were a necessary prelude to transfusion . it was then realised that if the recipient had agglutinins active at 37 &amp;deg; C in his serum and the transfused blood had the corresponding agglutinogen , the blood would be destroyed in vivo and a haemolytic transfusion reaction would result . the possibility of the destruction of the recipient &apos;s red cells by transfused antibody was not considered to be a real danger because of the dilution factor . for this reason , up to about 1940 , group O blood was considered safe for transfusion to all groups and was called universal donor blood . nowadays it is realised that transfusion with homologous blood , i.e blood of the same type as the recipient , is to be preferred , not only because the transfusion of antibodies may be dangerous , but also because the number of potential donors is doubled ; an important point when the demand for blood is steadily increasing . the titre of anti-A and anti-B antibodies in most donor blood is not dangerous so that in emergency , one pint of group O can be given with little risk , but in massive transfusions of group O blood to patients of other groups the quantity of antibody transfused becomes considerable and may even result in the destruction of almost all the recipient &apos;s own red cells . in particular , it has been shown that exchange transfusion of infants suffering from haemolytic disease should be performed with blood of the infant &apos;s own ABO group . the discovery of the ABO blood groups was , however , merely the beginning . today many blood group systems are known , by means of which some hundreds of types of blood can be differentiated . should they all be taken into consideration in choosing blood for transfusion ? it is obvious that they can not be and except in special cases only two systems are in fact considered , ABO and Rh . when blood is transfused there are many dangers present , of which two are directly concerned with the antigen content of the transfused blood , the first being that of sensitisation , the second that of incompatibility . in the first case the recipient does not possess the antigen found in the transfused blood nor the corresponding antibody , but the transfusion acts as a sensitising dose so that antibodies are produced in response to the transfusion or to a subsequent stimulus by the same antigen . blood for transfusion can not be chosen so as to exclude every possibility of sensitisation but fortunately most of the blood group systems are not strongly antigenic in man and can usually be disregarded . the main exception is the Rh system , and here the problems of sensitisation must be faced . in the ABO system ( where antibodies occur naturally ) and in other systems whenever atypical antibodies active at 37 &amp;deg; C have been formed the problem is not that of sensitisation but of incompatibility . a consideration of the two systems , ABO and Rh , gives an idea of the factors involved and how best to arrive at the objective , the safe transfusion of blood . the ABO blood group system is still the most dangerous . this is because the antibodies are naturally occurring and over 95 per cent of all recipients will have anti-A and/or anti-B in their serum . on the other hand ABO blood grouping is a straightforward procedure and the simplest of direct matching techniques will detect any incompatibility . most of the mistakes which occur are clerical rather than technical . 