Abstract:
Red cells are exposed to an unsaturated aldehyde such as acrolein (propenal) under conditions sufficient to increase the stability of the cells without impairing the ability of a lysing reagent to lyse the cells. After treatment, the treated cells are washed and are suspended in a stabilizing suspension medium.

Description:
The present invention relates to hematology controls and calibrators, and especially to the lysable red cell component of such controls or calibrators. 
     Controls or calibrators for hematology instruments are suspensions in isotonic medium of a cellular component that mimics a patient&#39;s red cells, a cellular (or synthetic) component that mimics a patient&#39;s white cells and, frequently, a cellular component that mimics a patient&#39;s platelets. Unlike the white cell component, the red cell components should be lysable by the lysing reagent (typically one or more quaternary ammonium salts). In the red cell channel of the instrument, the red cell component should act like normal red cells and yield reproducible values of such parameters as mean cell volume (MCV), total cell count, hemoglobin, RDW and hematocrit. Once the red cell component is lysed (and converted by cyanide to cyanohemoglobin), a reproducible hemoglobin value should be obtained (from which the instrument can calculate hematocrit and other derived parameters. 
     The limited shelf life of hematology controls and calibrators is normally due to the red cell component changing over time in one of the measured parameters: either hemolyse in isotonic medium to change the cell count or swelling or shrinking in the isotonic medium to change MCV, hematocrit and RDW values. 
     Various attempts have been made to stabilize the red cell component either by pretreatments prior to suspending in the isotonic medium or by incorporating additives in the isotonic medium (osmotically balanced). For example, U.S. Pat. No. 4,358,394 to Crews, et al (1982) discloses red cells treated in a multicomponent preconditioning diluent (containing, e.g., lactose and a non-ionic surfactant) and then separating the red cells and adding them to an isotonic medium containing lactose, bactericides and fungicides, albumen and a bile salt, cholic acid derivative or phenothiazine. See also, U.S. Pat. No. 4,299,726 to Crews, et al (1981). U.S. Pat. No. 3,873,467 to Hunt (1975) discloses washing, swelling and fixing the cells with a multicomponent fluid (which may include a small amount of aldehyde to toughen the cell membranes, col. 2, lines 47-49) and then suspending the cells in a suspension fluid. The treated cells are still lysable when later exposed to lysing reagent. The washing fluid indicated as typical contained 0-0.00008 molar glutaraldehyde. Other uses of aldehydes are disclosed, e.g., in U.S. Pat. No. 4,390,632 to Carter (1983), and in copending, commonly-assigned U.S. Ser. No. 889,748 of Wong, et al, filed July 28, 1986, now U.S. Pat. No. 4,698,312. 
     U.S. Pat. No. 4,579,024 to Louderback, et al discloses red cells treated with aldehyde (e.g., 40 ml of 37% formaldehyde and 500 ml of 0.9% saline) for two hours at 18°-28° C. whereby they retain their plasticity for up to about 5 days. Then, before the cells have become rigid, they are retained in Alsevier&#39;s Solution with about 0.01% to 0.21% NaCl to provide an osmotic pressure to yield a desired cell size (MCV). Formaldehyde and glutaraldehyde on the aldehydes disclosed. Sucrose is disclosed for use in the modified Alsevier&#39;s Solution for certain levels. 
     U.S. Pat. No. 4,489,162 to Hawkins, et al (1984) discloses hematology controls and calibrators wherein the suspending medium contains 5-50 grams per liter of a disaccharide such as lactose or sucrose. 
     In a field unrelated to hematology controls and calibrators, it is known that aldehydes such as formaldehyde, acetaldehyde and glutaraldehyde are commonly used in fixing tissue for microscopic examination. The unsaturated aldehyde acrolein (or propenal) has also been used for fixing tissue, especially for electron microscopic examination. For such tissue fixation, acrolein is reported to rapidly stabilize cells or tissue with minimal morphological change. 
     BRIEF DESCRIPTION OF THE INVENTION 
     Unsaturated aldehydes such as acrolein (propenal) have been found efficacious in the stabilization of erythrocytes (human, particularly) for use as the lysable red cell component of hematology controls and calibrators. Accordingly, the present invention provides a hematology control or calibrator comprising a suspension of treated erythrocytes in a stabilizing medium, characterized by the treated erythrocytes having been exposed to a aliphatic unsaturated aldehyde of 3-6 carbons under conditions sufficient to increase the stability of the treated erythrocytes in the medium without impairing the ability of a lysing reagent to lyse the treated erythrocytes in a hematology analyzer. 
     The present invention further provides a process for preparing a hematology control or calibrator comprising the steps: 
     (a) Exposing washed erythrocytes to an unsaturated aldehyde at a treatment concentration and temperature for a treatment time period, 
     (b) After the treatment time period, washing the treated erythrocytes, and 
     (c) Suspending the treated and washed erythrocytes in a stabilizing suspending medium, the treatment concentration, temperature and time being sufficient to increase the stability of the treated erythrocytes in suspending medium without impairing the ability of a lysing reagent to lyse the treated erythrocytes in a hematology analyzer. 
     DETAILED DESCRIPTION OF THE INVENTION 
     In the present invention, red cells are stabilized by treatment with an unsaturated aldehyde under controlled conditions. The conditions (especially aldehyde concentration, time and temperature) are not individually critical but are rather selected together, as described and illustrated below, to yield a result of improved stability (particularly of MCV values) without detracting from ability to lyse the cells in the white cell channel of a hematology instrument. 
     The red cells used can be human (fresh or expired) or from various mammalian sources (e.g., bovine). The red cells are normally washed to remove contaminants and endogenous white cells and platelets (the buffy coat) before treatment; however, final removal of remaining white cells (such as by filtration through a filter to which white cells, and especially granulocytes adhere), if employed, can either precede or follow treatment with aldehyde. 
     The aldehyde can be propenal (acrolein) or another aliphatic unsaturated aldehyde of 3-6 carbons such as 2-butenal, 3-butenal, 2-hexenal, 4-methyl-2-pentenal or other similar compounds. Such aldehyde can be of varying concentration in the liquid phase at the time of treatment; representative concentration is in the range of 0.01% to 1.0%. By comparison, the formaldehyde concentration in U.S. Pat. No. 4,579,824 is 37% diluted 40/540 or 2.7%. It should be apparent that propenal can be used herein at concentrations lower than proposed for formaldehyde in certain prior art. 
     The treatment temperature can be from about 0° C. to about 30° C., and even higher if treatment time is controlled very carefully. Room temperature conditions (e.g., 20°-25° C.) can be employed with careful control of temperature and time. Thus, if 150 minutes at 24° C. is chosen, essentially identical results will be obtained even if temperature varies one degree or time varies 15 minutes. Wider variations will yield controls or calibrators with suitable properties; those properties may, however, depart from the expected values one would obtain if the design conditions are maintained. Refrigeration conditions, such as 0°-5° C. (e.g., 4° C.) can be employed with more latitude on treatment time and temperature. This enables better control during large scale processing. Thus, properties are not expected to vary appreciably whether treatment at 4° C. proceeds for 15, 20 or 25 hours or whether refrigeration temperatures vary from 2° C. to 5° C. Freezing temperatures should be avoided. 
     At the conclusion of the treatment by unsaturated aldehyde, the red cells are separated from the aldehyde solution and washed in normal saline or osmotically-controlled medium. The treated cells can then be suspended in the desired final stabilizing media. The process of this invention can be employed with fresh blood cells (i.e., within 5 days), aged red blood cells and/or expired red blood cells for use in a whole blood controls/calibrators. Formulations of stabilizing media for conferring long-term stability on hematology controls and calibrators are given below: 
     
         ______________________________________Distilled Water     800         mlSodium Citrate, dihydrate               3.20-6.32   gmCitric Acid         0.22-0.43   gmSodium Chloride     1.68-3.32   gmAdenine Sulfate     0.08-0.16   gmSodium Salt of Nalidixic Acid               0.064-0.126 gmSodium Omadine (Sod. Pyrithione)               0.003-0.006 gmChloramphenicol     0.040-0.080 gmAmpicillin          0.064-0.126 gmAmikacin            0.064-0.126 gmGentamicin Sulfate  0.064-0.126 gmBovine Albumin (30%)               20.0-39.5   ml.q.s. to 1 liter with distilled water______________________________________ 
    
     For the best results, the above ingredients are added to distilled water in the order listed in the table, allowing each ingredient to dissolve completely before the following ingredient is added. The solution then filtered through 0.2μ filter and used for resuspending washed red blood cells and/or formulation. Different levels are achieved by initially selecting small, medium or large cells and by adding different concentrations of such cells to media of different specific formulation with the above ranges. 
     Upon suspension in such media, the cells will normally shrink or swell to an equilibration value of MCV. Thus, for example, cells with an MCV of 77.8 before treatment and 74.7 after treatment (with 0.1% acrolein) have been found to stabilize in the above suspending medium for Level I. Similar stability was achieved with red cells having an MCV before treatment of 82.1 and after treatment of 80.0 (used for Level II) and with red cells having an MCV before treatment of 90.0 and after treatment of 90.2 (used for Level III). Equilibrations took approximately 35, 25 and 10 days for Levels I, II and III, respectively. Once, however, the MCV values stopped changing, they remained constant (within tolerances of 1 MCV unit) for 90 days or longer. These equilibrations are of a different type than the five day or less equilibrations of U.S. Pat. No. 4,579,824 used to enable a single pool of cells to be employed for all three 
    
    
     EXAMPLE 1 
     Packed red blood cells were separated from a CPD-A whole blood by conventional blood bank techniques to remove platelet rich plasma and buffy coat (white cell concentrated layer), washed (each unit individually) with 30 mm citrate-saline, pH 6.0, and expressed buffy coat by conventional aseptic techniques. To the washed packed cells (in a plastic bag) were added equal volume of 0.2% acrolein in 6.5 mm phosphate-saline, pH 7.2 (PBS). The suspended cells were kept at 4° C. over-night (16-19 hours), then washed with PBS twice by conventional centrifugation method and, at the time, expressed to remove buffy coat material if needed. The washed/stablilized cells were filtered through leukofilter (polyester filter) and were ready to formulate with white cells and platelet components into appropriate levels of control or calibrator in suspending medium. The real-time, open vial and other stabilities of the control, or calibrator were analyzed on a Coulter S+IV hematology analyser. The major problem/drawback of a long-stability product is usually the real-time stability if the MCV of the red cell component in hematology control or calibrator. Therefore, the improved stability of MCV is the most significant index for improvement. The real-time/long term stability results of MCV from various levels formulation as shown in Table1. 
     
                       TABLE 1______________________________________(0.1% Acrolein)MCVDays    Low           Normal   High______________________________________ 1      77.8          82.1     90 2      77.3          82       89.7 8      76.5          80.5     89.611      76.3          80.5     89.615      76            80.8     90.221      75.8          80.7     90.525      75.1          80.1     90.139      75.2          80       90.549      74.8          79.6     90.256      74.8          79.7     90.266      74.6          79.5     89.878      73.8          78.8     89.487      74.4          79.4     89.998      74.5          79.7     90.3107     74.3          79.5     90119     74.1          79.4     89.6128     74.8          79.7     90.2137     74.2          79.2     89.5163     74            79.1     89.6176     74.2          79.2     89.6190     74.7          79.4     89.9______________________________________ 
    
     EXAMPLE 2 
     The buffy coat removed packed red blood cells were washed with the citrate-saline twice by conventional techniques as in Example 1.Buffy coat residue was processed in the bag. The washed cells were filtered through leuko-filter and collected in an appropriate sterile container and an equal volume of 0.1% acrolein in PBS added. The well suspended cells were kept at room temperature (ambient temperature) for 21/2 hours, and then 1/20 volume of 1 M glycerine in PBS was added to neutralize excess aldehyde for 10-20 minutes. The suspension was then centrifuged to remove supernatant, then washed with PBS buffer twice and then used for pilot formulation as in Example 1. The real-time stability of MCV was measured over time, with the results shown in Table 2. 
     
                       TABLE 2______________________________________(0.05% Acrolein)MCVDays    Low           Normal   High______________________________________ 0      94.4          94.2     94.6 5      92.9          93.1     93.8 15     90.8          92       94.5 26     90.1          92.2     95.9 40     89.3          91.9     97.1 57     86.5          89.8     95.5 77     87.8          91.2     98.2 96     87.2          90.9     97.9106     87.3          90.6     97.6130     87.5          90.7     98173     87.6          90.8     98.6188     86.8          90.7     98.4189     87            90.8     97.8190     87            90.6     97.2195     87.7          90.5     97.7209     87.5          90.5     97.6226     87.5          90.7     97______________________________________