Technology Section

Microplate System for Routine Use in Blood Bank Laboratories

M. N. CRAWFORD, F. E. GO-ITMAN, AND C. A. GOTIUAN

From the Philadelphia Blood Center and Temple University School of Medicine, Philadelphia, Pennsylvania

Modifications of the microplate system are de- scribed which permit this system to be used for all laboratory phases of blood bauking with no- table economy of time and reagents and without loss of accuracy. Antibody screening, identification] and typing studies are summarized.

THE microplate system, as described by Sever,3 has long been useful to microbiol- ogists. In recent years, adaptations to the blood-grouping field have been made by Wegmann and Smithies, and McCloskey and Zmijewski.zu5-7 In this reference labor- atory the system has been modified for antibody screening and identification] titra- tion, and cell typing. This has resulted in marked economy of equipment, reagents, and time without loss of sensitivity in com- parison with standard technics carried out in tubes.

Materials and Methods

Rigid transparent styrene plates (128 rnm long, 86 mm wide, 14 mm high) containing 96 “U” shaped wells were used for all tests nor- mally performed in glass tubes. The wells are permanently numbered from 1 to 12 across the top of the plate and lettered from A to H along the side. Two holes drilled in opposing sides of the plates (Fig. 1) permit the use of wire slings (Fig. 2) so that the plates can be centrifuged in heads of at least 17 inches diam-

eter. Two styles of slings were designed, one for the International size-2 centrifuge which holds four plates, and another type for the Sorvall GLC-1 which accepts two plates.

New plates are unsatisfactory because the cells adhere to them. This problem is avoided by pretreatment of each well with approxi- mately 0.1 ml of albumin or plasma for a few minutes. The plates are then washed and can be reused routinely after water washings which are accomplished by ten rinses under fast- running tap water, followed by one rinse with distilled water. As much water as possible is knocked out by a sharp rap on an absorbent towel and the plates are then air dried upside down.

Several types of dispensers are used for reagents: plastic pipettes+ with tips delivering 25 @liter drops (Fig. l), small glass capillary tubes (70 mm long, 1.2 mm ID), glass pipettes with capillary stems of 1 mm OD and plastic sticks+* (Fig. 1) with a loop at one end for delivering serum or plasma, and a notch at the other end for picking up small amounts of cells or whole blood.

Washed saline suspensions of cells were used for most of the work. Ficin-treated cells for screening and identification were prepared by exposing washed four per cent cell suspensions to a ficin solution (1 g ficin powder+++ in 100 ml of Hendry’s buffer)4 for 15 minutes at 37 C, followed by packing, rewashing, and resuspen- sion of the cells to four per cent. The volume of ficin solution used was approximately equal to the packed cell volume.

Received for publication April 22, 1970; accepted June 8, 1970.

Presented in part before the American Associa- tion of Blood Banks, November 16-20, 1969, Hous- ton, Texas.

+ Cooke Engineering Company, Alexandria, Va. ++ Plastic sticks, Al-Mar Precision Company,

+++ Nutritional Biochemicals Corp., Cleveland, Philadelphia, Pa.

Ohio.

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loop end of plastic stick /*

notched end of.plostic stick '\.\ 25microliter plastic pipette

plastic tubing -----

two of these for each sling

FIG. 1. Basic equipnient and method for making slings from piano wire, plastic-covered bell wire and plastic tubing.

The dilutions of .2HO- ant1 Rh-typing sera varied from 1:3 to 1:6. Aiitiglol)ulin serum was diluted routinely with equal volunies of saline solution. All these sera met N I H standards prior to dilution.

A hand-held 2-ml automatic Cornwall pi- pettet with a 14-gauge cannula was used for cell washing prior to addition of antiglobulin serum. The syringe and tubing were rinsed and filled with distilled water for overnight storage and, prior to use, were rinsed with 40 to 50 ml of 0.9 per cent NaCI.

'I'he plastic buckets and aluminum sieve used for these washings, and the magnifying mirrors for viewing the final sediment were obtained from department stores. Each mirror was placed at an angle at the bottom of an ope~ l card- board box which was covered with a clear plastic sheet to support the plates. Cutting out part of the front of the box permitted easier viewing of the plates through the mirror.

t Arthur H. Thomas Company, Philadclphia. Pa.

A11 incubation was at room temperature except for tests specifically set up for cold antibodies.

Centrifugation varied from 45 seconds for light packing of final sedimented cells to three or four minutes for firm packing of the cells during washings. The speed was kept at 50 x g for all centrifuge operations.

For antibody identification with cell panels the sera were distributed into the appropriate number of wells using plastic pipettes that delivered 25 pliter drops. One set of wells received the serum, an equal amount of SO per cent bovine albumin, and four per cent saline suspensions of the panel cells. A second set of wells received the serum and four per cent suspensions of ficin-treated cells. The four per cent cell suspensions were added with plastic or Pasteur pipettes. Small amounts of six to eight per cent suspensions were added with the glass capillary tubes touching one side of the well (Fig. 1). The contents were mixed by gentle agitation of the plate; the degree of motion

260 CRAWFORD. ET AL. Transfusion Scpt.-Oct. 19 70

must be small to prevent spilling. The plate was centrifuged for an immediate reading which required gentle agitation to release the cell buttons. The cells must be viewed from the bottom of the plate. T o prevent drying, plates were covered by spare plates, securely strapped with large elastic bands during incubation at room temperature. After incubation the plate was again centrifuged and inspected after gentle agitation. For the antiglobulin test the plates required

a preliminary spin for three or four minutes. The supernatant fluids were then dumped with a rapid flick of the wrist into a sink or plastic bucket covered by a sieve to prevent splashing.

FIG. 2. Left-hand sling for Sorvall GLC-1 centri- fuge which demonstrates plastic-covered copper wire in position for centrifug. ing. Right-hand sling for International size-2 cen- trifuge with small wires in raised position for re- moval of the sling.

The plates were then agitated very vigorously to disperse the cells. Saline solution, 0.22 ml, was jet-delivered into each well with a Corn- wall pipette, hand-held vertically with the can- nula tip a few millimeters above the well to prevent the fluid from splashing off the curved bottom. This technic is easily acquired and an entire plate may be filled in about a minute. Each of three washes was followed by three minutes of centrifugation. One 25-lliter drop of antiglobulin serum was added to each well and the plate centrifuged once again for 45 seconds. When these plates were held verti- cally, the cells of negative reactions ran down- ward while those of positive reactions remained

FIG. 3. Antibody screen- ing of 96 donor sera. The plate is held in a verti- cal position for interpre- tation of antiglobulin tests. Positive reactions occur in wells: B4. C11, D7, F2, F7.

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FIG. 4. The sane plate as shown in Fig. 3 after “settling” in horizontal position. Positive reac- tions: B4, C11, D7, F2, and a very weak one in F7.

fixed in their original position (Fig. 3). How- ever, some very strong reactions may slide intact in the tilted position. After gentle agitation the plate was covered with a transparent piece of glass or plastic and left, undisturbed, over a magnifying mirror. Reactions were observed again after cell settling. The cells of negative reactions formed smooth round buttons, whereas positively-reacting cells were more dispersed and irregular in their settling pattern (Fig. 4).

For antibody screening, sera were delivered into the microplate wells with pipettes, straws,

FIG. 5 . “Immediate spin” of 96 donors typed with anti-Rh. (D) after stick delivery of ells into diluted ficin solution. There are 22 presump- tively Rh-negative cells. Cell C9 is Du positive. The presumptively Rh- negative cells are next evaluated on the same plate by antiglobulin test.

or plastic loops (Fig. I).** The volume of reagents was limited by the capacity of the wells. If the wells were more than half full, it was difficult to mix the contents without spill- ing or splashing. If the sera were introduced by plastic loops, the loops were dipped into the albumin or saline solution in the wells to free the contents of the loop. Twenty-five pliters of 30 per cent albumin were put into each well of those plates which received normal screening cells, and 25 aliters of saline solution were added to each well of those plates which

262 CRAWFORD, ET AL. ‘Tram fusion Scpt.-Oct. 1970

TABLE 1. Antibodies Delected in Total Donor Donor Sera-5,204

Philadelphia Blood Center by microplate American Red Cross

by pooled tube method method

Antibodies detected R h 8 Rh 10 Kell 5 Kell 5 M.P,or Le 12 hl,P, orLe 19

Rh 2 M, PI or Le 13 M.P,orLc 6

Antibodies not detected

received ficin-treated cells. When the sera were being added, a small rubber capillary bulb was placed in the next well to mark the place (Fig. 1). Twenty-five-pliter drops of four per

cent saline suspension of the screening cells were added. With bulbs of 3 or 4 ml capacity on the plastic pipettes, one filling held enough for an entire plate. Incubation reading of antiglobulin tests and inspections were con- ducted as described.

Antibody titration may be done with Takatsy steel loops as described by Sever,J Feizi and Monger.1 and McCloskey and Zmijewski.7

Blood-typing of cells was done by introducing 25 pliters of the typing reagent into the wells followed by similar volumes of washed saline suspensions of the cells to be tested. More rapid ABO- and Kh-typing was done using whole blood delivered with the notched end of the plastic stick (Fig. 1). -10 avoid plasma clots which result from mixing whole blood with recalcified typing reagents, the ABO plates were prefilled with 25 pliters of 6.25 per cent trisodium citrate solution. The cells were then introduced with the notched stick into the citrate solution and the typing serum was added last. For rapid Kh-typing the plates were prefilled with 25 pliters of ficin solution prepared as described above but diluted 1:3 with 0.9 per cent NaCI. The cells were added with the notched stick and then the Rh-typing serum was added. Gentle agitation of the con- tents was followed by centrifugation for 45 seconds for the initial inspection. The plates were covered and inspected again after incu- bation and recentrifugation. Wells with pre- sumptively Rh-negative cells were subjected to the antiglobulin test. The diluted ficin solu- tion also may be useful for ABO-typing, a possi- bility now under study.

Results l h r e e comparative studies were made with

1. .4ntibody identification with a Hyland Laboratories collection of “self-check” hu- man sera; 2. Antibody screening in parallel with the Philadelphia branch of the .4meriran Red Cross; 3. ABO- and Rh-typing in parallel with our routine laboratory.

The plate method described for antibody identification was used to make a blind panel study of 42 Hyland testing sera which con- tained 20 different antibodies. Half these sam- ples contained mixtures of two antibodies. The Anti-M in one could no longer be detected by either the plate or conventional methods. Otherwise, all the results matched exactly those reported by Hyland Laboratories.

For antibody screening, the Ked Cross labora- tory pooled one “straw” drop of serum from each of three consecutive donors in single tubes. Each tube also received one drop of a four per cent suspension of commercial screening cells. ‘The tubes were centrifuged and inspected for agglutination both before and after adding two drops of 22 per cent bovine albumin. The tubes were then incubated at 37 C for one hour and again centrifuged and inspected. Finally, an antiglobulin test was performed. Each do- nor serum also was tested individually at room temperature, inspecting the test cells before and after a one-hour incubation. 1’ap;iin solu- tion was added, and after incubation at 32C for 30 minutes. the tubes were centrifuged and inspected.

In the microplate study the donors were screened individually using two sets of plates, one with saline solution for ficin-treated screen- ing cells and another with 30 per cent albumin for untreated cells. Each donor serum was de- livered with a plastic stick (Fig. 1) into one well on the ficin plate and one well on the albumin plate. A four per cent saline suspen- sion was made of a combination of two screen- ing cells. Twenty-five-pliter drops of the cell suspension were delivered with plastic pipettes. The plates were centrifuged, inspected, and then incubated at room temperature. Anti- globulin tests were done only on the albumin plates. Anti-H and anti-I have been omitted from the tabulation of the results in Table 1.

A previously untrained high school student, after one week of instruction, performed 3,203

the microplate system:

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donor typings during two summer months in parallel with the routine blood donor labora- tory. The cells were delivered into citrate solu- tion for ABO, rh’ (C), and rh” (E) typing, and into freshly diluted ficin solution for Rho(D) typing. This involved 20,145 determinations. There were two errors in cell-typing for ABO and 14 in serum tests for expected agglutinins. There were three errors in C and E typing. Most errors occurred during the first month of the study.

Discussion

The microplate system has not been a p plied to routine blood banking for several reasons. Commercially available microplate centrifuge carriers require large rotors which unnecessarily prolong time in the centrifuge. Untreated plastic plates cause red blood cells to adhere to the plastic. Lastly, washing for the antiglobulin test seems impossible to perform with such small volumes of saline solution.

T h e simple wire slings described for two popular laboratory centrifuges permit effi- cient and rapid centrifugation.

Initial treatment of microplates with al- bumin or plasma permits multiple use of these plates without cell adherence. T h e plastic surface combined with rapid d u m p ing of supernatant fluid results in an un- usually “dry” cell button, even if the wells are filled with serum.

T h e pipette used for washing requires some dexterity to prevent the contents of one well from splashing into another, but a multiple outlet manifold to fill an entire row with one stroke may become available commercially.

Because the amount of serum per well is not a limiting factor for antibody screen- ing, straws may be the most economical and convenient dispensers for distribution of large numbers of serum or plasma samples.

The efficiency and speed of the antiglob- d i n test on plates compares favorably with that of mechanical cell washers because one person can perform 500 to 600 cell washings per hour. The test cells are always in view and important information about the com- parative strength of reactions may be gained during the washing process. For reading reactions, agitation of the plate gives more uniform cell dispersal than is achieved by shaking individual tubes.

Acknowledgment

The authors thank Miss Mary F. Eichman and the Philadelphia Regional Red Cross Blood Center and the Hyland Laboratories for the sera used in the comparative studies, and for their laboratory results.

References

1. Feizi, T., and E. Monger: Microtitration of serum cold agglutinins. Transfusion 1 0 33, 1970.

2. McCloskey, R. V., and C. M. Zmijewski: A semi-microtechnic for the detection OE human blood group isohemagglutinins. Techn. Bull. A.S.C.P. 37: 184, 1967.

3. Sever, J. L.: Application of a microtechnique to viral serological investigations. J. Immun. 88:320, 1962.

4. Technical methods and procedures of the American Association of Blood Banks, 4th ed. Chicago, Twentieth Century Press, Inc., 1966, p. 99.

5. Wegmann, T. G., and 0. Smithies: A simple hemagglutination system requiring small amounts of red cells and antibodies. Trans- fusion 6: 67, 1966.

6. - , and 0. Smithies: Improvement of the microtiter hemagglutination method. Ihid. 8:47, 1968.

7. Zmijewski, C. M.: I!nnlunohematoloe;y. NCW York, Appleton-Century-Crofts, 1968, p. 2.10.

Mary N. Crawford, M.D.. Associate Director. The Philadelphia Blood Center, 2630 ”B” Street, Phila. delphia, Pa. 19125.