Blood grouping (1955)

blood grouping has become an important everyday procedure in hospital Laboratories it is a vital preliminary to blood transfusion and an essential investigation in the correct anti-natal treatment of the expectant mother this film is intended to show students and house officers some of the principal methods used in the laboratory the determination of a blood group depends upon a reaction causing red blood cells to Clump together when they are mixed with a serum containing an aguena antibody of the many blood group systems now known in hospital Laboratories it is usual to establish a person's blood group by two only the system and the Reus or RH system here are six specimens of clotted blood to be grouped first the serum is removed then normal sine is is introduced into each tube with gentle shaking cells from the clot are resuspended in Weak concentration it is important that the concentration of cells in Saline is not too thick thick suspensions mask weak utenation in practice it is sufficient to approximate to a 5% suspension of packed cells in normal saline these tubes show the sort of density required the one on the right being the weak suspension and on the left the ordinary blood in grouping cells are tested with anti-a and anti-b S which are supplied in distinctively labeled bottles it is convenient to have the tile ruled in squares two vertical columns are labeled antia on the left and anti B on the right and each pair of squares is marked 1 to six for the six samples of blood anti-a theum is added to the left hand column after rinsing the pipet first in distilled water then in Saline and finally drying off any remaining drops serum from the the antib bottle is taken up and added similarly to the right hand column after rinsing the pepet again the cell suspensions are added to each pair of squares the pipet used is of the dropping type calibrated to deliver 20 cubic mm it is essential to use small volumes as all anti-a and anti-b S are obtained from Human donors having added the cell suspensions the cells and serum are carefully mixed with a small round-ended glass rod between each mixing the rod is clean to avoid any carryover of cells and serum mixing in this way is very important without it the cells tend to stick to the surface of the tile resulting in Weak and delayed reactions the next step is to rock the tile this is done in such a way as to impart a rotatory motion to the drops of mixed serum and blood cell after 2 or 3 minutes the reactions are clearly seen number one has been agutin by anti-a serum only and is therefore group a number two has been agglutinated by antib serum and is therefore Group B number three shows no reaction at all these cells possess neither a nor B antigens they are group O number four has been agglutinated by anti-a and antib S having both antigens A and B it is therefore group a number five looks like group O but on closer examination a very fine utenation with anti-a serum can be seen this is in fact a subgroup of a known as A2 which is characterized by very weak utenation if this group had been incorrectly labeled group O and as the universal donor had been given to a group b person the result would probably have been fatal number six shows another A2 reaction in the anti-a square and a strong glutin in the antib Failure to observe the weak antia reaction would mean that this Blood would be wrongly labeled Group B and if given to a group b recipient a serious reaction would result blood number six is group A2B careful cross matching of blood with serum from the recipient avoids dangerous errors control tests are advisable in case the S used are defective for this purpose cells of groups a A2B and O are used here everything seems to be in order this closeup clearly shows the weak A2 reaction of the A2B cells in this method a block containing small tubes takes the place of the tile antib serum has just been added to the tubes in the back row and after rinsing the pipet antia serum is added to the front row the cell suspensions are then added to the pairs of tubes control tests are set up using group a A2B and O cells care should be taken to label the block clearly and accurately when the tests and controls are completed the tubes are covered with small glass caps to prevent evaporation as in this case the block will stand at room temperature for 2 hours this is the main difference between the tile and Tube methods after 2 hours the results are read with practice a concave mirror can be used to observe the negative and positive reactions the antib tube from the back row shows a group b of glutin and the anti-a tube from the front row shows no utenation results which appear to be negative must be checked microscopically a small quantity of the mixture is spread onto a slide and examined under a low power microscope objective it is a negative reaction all results are noted as they are determined now here is a weak A2 reaction from the anti-a tube in blood number six and positive utenation in the antib tube the A2 reaction should be checked microscopically here it is the tube method brings the A2 reactions up more strongly than the tile method and permits them to be checked microscopically the final results on these six samples of blood are now complete and the tube results agree with those obtained by the tile method in resar grouping a Serum is used to test for the presence or absence of the antigen Big D this being the most important of the recess antigens the serum agglutinates positive cells suspended in Saline one drop of the serum is added to each tube a very fine droper pepet is essential as all satisfactory Reus antisera are extremely scarce and come from Human donors after rinsing the pipet one drop of each cell suspension is added to each tube it is important of course to set up control tests again using known recess positive and recess negative cells after capping and shaking the tubes are incubated at 37° Centigrade for 2 hours capping is essential at this temperature to avoid complete drying up of the small volumes used in Reading Reus results care must be taken to avoid agitating the agutin cells Reus clumps are particularly easily disintegrated after much experience it is possible to ascertain a blood group with the aid of a concave mirror but it is far safer to examine all reactions microscopically here is a very strong positive reaction visible to the naked eye it is the positive control and here is the negative control there is no naked eye glutin in this case apparently nevertheless it is being checked microscopically air bubbles and undue agitation in manipulating the cell suspensions should be avoided positive control negative control tube number one in the concave mirror has a nice hard Edge indicating strong positive utenation the result is noted tube number two appears to be negative however it's being checked microscopically it is not negative but a definite though weak positive reaction clearly demonstrating the importance of checking all recess results microscopically here are the completed results for the six specimens of blood for both and RH systems unfortunately saline anti-d is scarce and albumin anti-d serum which is more readily available is generally used as a substitute this serum only agglutinates RH positive cells when they are in the presence of abundant protein the preparation of albumin cell suspensions is now being shown for this purpose Bine albumin is used in a concentration of 20% and this solution is being made up from the 30% stock two parts of Albin are measured and one part of saline is added and mixed the cell suspensions having been centrifuged are transferred to a rack the supernatant saline is removed and replaced by an equal volume of the 20% Al solution when all the cell suspensions have been prepared in this manner one drop of each suspension is added to one drop of the incomplete anti-d serum from now on the procedure is identical with that of the saline anti-d method the tests being incubated for for 2 hours at 37° centigrade and checked in the usual way notice the characteristic wine color imparted to cells suspended in Bine albumin another albumin method of particular value when there are large numbers of specimens to test is the dilution of alamin anti-d serum in 30% bovine alamin using cells suspended in Saline the dilution must not be overdone and should be kept well within the aguena Teeter of the serum the procedure is now exactly as used in the saline anti-d method one drop of anti-d serum in albumin is added to each tube followed by one drop of saline cell suspension after the usual 2 hours incubation at 37° the results are read microscopically There is a disadvantage with all albumin methods albumin encourages the formation of Rulo this is a low power picture of Rulo formation showing some resemblance to positive utenation on higher power examination the cells appear to be arranged in Columns of pennies and not in clumps this difficulty can be overcome with practice and experience which are essential elements in all methods of blood grouping in conclusion here is a sample of clotted blood which is going to be rapidly grouped prior to transfusion this will be completed in 30 minutes instead of the customary 2 hours the cells are suspended in sine to the required density then one drop is added to a tube containing one drop of albumin anti-d serum in albumin this together with suitable control tubes is incubated at 37° in a water bath so that the required temperature is reached as rapidly as possible while this is going on the group of the person concerned can be established by the tile method which gives quick results requiring no 2-hour incubation the controls must be set up as usual a A2B and O in this case the result is group O after incubating the Rh test for half an hour the tubes are centrifuged at low speed not more than 1,000 revolutions for about 1 minute high speed is particularly dangerous at this stage as it gives false positive utenation the tubes are removed from the centrifuge and read microscopically in the usual way however it is advisable to let the 2 hours test proceed normally to check up any possibility of error rapid RH groupings are often tricky and are no substitute at all for the routine 2 hours test here the result is recess positive