Technical Notes Vox Sang. 30: 144-148 (1976)

Capillary Tube Testing and Enhancement with 30%Albumin MARYN. CRAWFORD, FAITH E. GOTTMAN and LANA C. ROGERS Pearson C. Cummin Memorial Laboratory, Villanova, and Department of Medicine, Temple University, Philadelphia, Pa.

Ahtract. Capillary tube methods are reviewed and modifications with albumin are described with emphasis on detection of antigens in the major red cell systems and in HL-A and related systems using red cell suspensions.

Rh typing tests in capillary tubes were first described by CHOWN [l] in 1944 and were subsequently developed for additional purposes [2, 41. The technique has had widespread though rarely publicized use. This report covers 15 years of experience using capillary methods to screen blood samples for both antigens and antibodies using small amounts of serum. Some recent results of testing for HL-A-related antigens are also included.

Materials and Methods Successful capillary tube testing is dependent upon the following rules: (1) Use glass capillary tubes measuring 0.4 (ID) x 90 mm (Diagnostic Technology Inc., Long Island City, N.Y.). (2) Use serum; plasma is unsatisfactory. (3) Remove particulate matter from serum. A Boerner filter (Arthur H. Thomas Co.) clarifies small amounts of serum quickly and easily. (4) Wash the red cells threc times to remove autologous plasma or serum. (5) Suspend red cells in saline, usually as a 50% suspension, and agitate just before testing to avoid cell aggregates and to obtain uniform concentrations. (6) Avoid air bubbles, during filling of the capillary tubes, to insure mixing of reagents. (7) Wipe the end of the capillary tube before entrance of cells to avoid contamination of cell suspensions. (8) Insert the serum end of the capillary tube into clay (Seal-Ease, Clay-Adams Inc.).

Received: January 20, 1975; accepted: March 26, 1975.

Variations in technique are described below. Estimates of reagent amounts in centimeters refer to the level in a 90-rnm capillary tube. For most tests washed 50% cell suspensions and room temperature incubation are used. Incubation time varies with different sera, and the incubation angle depends upon the viscosity of the reagents. Positive reactions produce beading or ribbons of cells. Negative tests give smooth unbroken cell columns. The results are viewed with a 6 x o r 7 x hand lens held against the hrow. while the capillary tube< are brought up within the focusing range. This enables one to look at several tubes simultaneously. When the cells have become packed at the bottom of the column, thc capillary tubes may be inverted for additional cell trips through the serum. Capillary tube tests may be preserved overnight in the refrigerator and inverted again the following day after reaching room temperature. They should not be stored upside down because the cells deteriorate at the open end of the capillary tubes. One-luyer olbirniin f e u . ( 1 ) Mix equal volumes of serum and 30% bovine albumin. (2) Insert the capillary tubc i n t o thc serum-albumin mixture and allow it to run-in for a distance of 1.5-2cm. (3) Wipe the end of the capillary tube and insert it into a 50% red cell suspension, allowing the cells to run through the length of the serum column. (4) Insert into clay at an angle of approximately 5 5 O . Two-layer cilbuniin /es/. ( 1 ) Admit 1.5-2 cin of serum into the capillary tube followed by an equal aniount of 30% bovine albumin. (2) After wiping the end insert it into a SO%-ccll suspension and allow the cells to run halfway through t h e serum-albumin column. (3) Insert i n t o clay at an angle of approximately 60".

Results

Over the years several rare red cell phenotypes have been found by screening donor populations with these techniques. A recent comparative study using capillary and microplate [?] methods was performed by a previously untrained college student, testing 264 donors for 18 antigens. Among the approximately 4,500 te5ts performed by each technique, there were 12 discrepancies, nine of which occurred early in the study. The majority were capillary test errors. Our best results in detection of Bga (HL-A7), Bgb (W17) and Chido antigens on red cells have been achieved with capillary tube methods (fig. 1). Consistent red cell reactions have been observed with some HL-A sera from the NIH tissue-typing collection. These tests have required the use of albumin. With many antibodies the two-layer method is remarkably better than the one-layer albumin test, as illustrated by figure 2. In the few studies we have carried out, comparing different commercial 30% bovine albumin reagents, the product made by Pentex Inc. (distributed by Miles Laboratories Inc.) has given the best results.

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Fig. I. Anti-Chido. Cells in tube No. 4 are Chido-negative. The other five tubes contain Chido-positive red cells. The anti-Chido serum was mixed with three parts of neutral serum (from a Chido-negative donor) and four parts of 30% albumin for use as a one-layer test.

SIMMONS et al. [ S ] have reported y-globulin contamination of Pentex albumin, a finding which suggests the importance of this or other contaminants in capillary tube testing. Capillary tube methods have several virtues. They are simple and rapid, using little equipment or space. The amounts of serum required are small: 1 ml of reagent is sufficient for 300-400 tests. Furthermore, many sera requiring antiglobulin testing by conventional tube methods do

Capillary Tube Testing and Enhancement with 30% Albumin

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Fig. 2. Anti-M. The red cells in the six tubes on the left were tested by the one-layer albumin method. The tubes o n the right contained the same serum and cells tested by the two-layer albumin method. Cells No. I and 5 are M-negative.

not need this additional procedure when used in capillary tubes. T h e method is valuable for narrowing antibody possibilities when a n ‘unknown’ serum is in short supply. An entire red cell panel requires only one d r o p of serum. I f cells receive preliminary washing in a laboratory, tests may be easily performed in the field. T h e method should receive consideration in disaster planning. However, capillary tube testing should not be used by unsupervised inexperienced personnel because different sera give marked individual variations in reaction patterns.

Acknondedgements For gifts of reagents relating to Chido and HL-A systems we a r e indebted t o Mrs. M A R I E C. CROOKSTON, Mr. WALTERE. HAESLER, jr., Mrs. ELEANOR D. HOWELL, Dr. SYLVIAD. LAWLER, Mrs. JANE L. SWANSON, Dr. PATRICIA A. TIPPETT,Miss MINNIEWEINBERC and the Tissue Typing Bank of the National Institutes of Health.

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CRAWFORDIGOTTM ANIROGERS

References 1 CHOWN, B. : A rapid, simple and economical method for Rh agglutination. Am. J. clin.

Path. 14: 114 (1944). 2 CHOWN, B. and LEWIS,M.: The slanted capillary method of rhesus blood grouping. J. din. Path. 4: 464 (1951). 3 CRAWFORD, M. N.; GOTTMAN, F. E., and GOTTMAN, C. A.: Microplate system for routine use in blood bank laboratories. Transfusion, Philad. 10: 258 (1970). 4 Lrwrs, M.; KAITA,H., and CHOWN,B.: Kell typing in the capillary tube. J. Lab. d i n . Med. 52: 163 (1958). 5 SIMMONS, A.; JONES, J., and HENDRIX, D. : Gamma-globulin contamination ofcommercial bovine albumin. Transfusion, Philad. 13: 142 (1973).

Dr. MARYN. CRAWFORD, Pearson C. Cummin Memorial Laboratory, 125 Ashwood Road, Villanova, P A 19085 (USA)

Capillary tube testing and enhancement with 30% Albumin.

Capillary tube methods are reviewed and modifications with albumin are described with emphasis on detection of antigens in the major red cell systems ...
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