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Table Average Within Run Precision for ASAT Uric Acid and


									                                           Marvin   C. Feil                    pared with      the same tests run with                                           5-30 U/L                  for each). Linear                         least squares
                                        Alan D. Cormier                        washed rotors. The tests included               as-                               regression                 analysis yielded                         the following
                                        Kenneth   D. Legg                      partate    aminotransferase         (ASAT)     (1),                               equations:
                                                                               uric acid by an end-point         technique    (2),
Instrumentation               Laboratory,               Inc.                                                                                                       y       =    0.4 + 0.99x;                  r           0.999 for ASAT
                                                                               and the absorbance        of various reference
113 Hartwell             Ave.
                                                                               solutions.   The precision      of results from                                     y       =    1.0 + 1.OOx; r                            0.998          for ALAT
Lexington,             MA 02173
                                                                               four new rotors was compared              with re-
                                                                               sults obtained     with use of a single rotor                                     These regression         fits indicate     no major
                                                                               washed one to four times on successive                                            proportional     or systematic         bias.
                                                                               days.        For   ASAT         and      uric        acid,      each                  We also tested rotors for the removal
                                                                               rotor        was filled with 10 replicates      at a                              of specific  substances.        To the cuvettes
A Washer-Dryer                    System for                                   low concentration          and 10 replicates    at a                              in each of four rotors, various amounts
Cuvette          Rotors         Used in the Cobas-                             high concentration.          For the absorbance                                   of protein    were added from a serum
Blo Centrifugal                 Analyzer                                       measurements,         10 replicates were run on                                   pool. The rotors            were then washed,
                                                                               0.2 mmol/L        solutions      of potassium    di-                              dried, and analyzed for residual protein
To the      Editor:                                                            chromate,       at 350 nm; 0.04 mmol/L            4-                              by use                 of     a    sensitive                      turbidimetric
   The     Cobas-Bio                Centrifugal        Analyzer                nitrophenol,      at 401 nm; and 74 mmol/L                                        method                 (5).       In     no case                   did         residual
(Cobas;       Roche         Analytical            Instruments,                 cobaltous     ainmonium        sulfate,  at 512 nm.                               protein              exceed        0.03%                of that          added            mi-
Nutley,       NJ       07110)        is currently           used   for         As shown            in Table          1, no significance                          tially.
various     determinations,          both by enzy-                             difference         in average         precision    (F-test)                          Various    amounts    of the substrate       for
matic      and end-point            techniques.        A                       was observed           between new and washed                                     alkaline     phosphatase       (4-nitrophenyl
major     advantage         of the Cobas is its                                rotors at the 95% confidence                    interval.                         phosphate)      were put into a rotor, after
high-capacity        (30 cuvettes)        disposable                                These    same       data were used to deter-                                 which it was washed and dried in the
rotor.    Because       the rotor is discarded                                 mine if multiple             washings         led to pro-                         usual manner.       Then a specimen           with
after    each     run,      cross-contamination                                gressive deterioration                of rotor quality.                           high alkaline     phosphatase      activity    was
between runs-which               is a problem      with                        As shown in Table 1, there was no sta-                                            analyzed    for residual activity      in the ab-
centrifugal      analyzers      that have perma-                               tistically    significant        loss of precision for                            sence of additional       substrate.      The re-
nent      rotors-is             eliminated.            However,                any testing in which rotors washed two,                                           sults showed                  that       <0.1%                of the substrate
this increases    the expendables  cost per                                    three, or four times were used as corn-                                           remained.
test. This extra expense would be mini-                                        pared      with the average                precision       for                        Specimens         with      very high ASAT,
mized    if one could wash, dry, and reuse                                     these tests when new rotors were used.                                            ALAT,        creatine       kinase,     and alkaline
these rotors.                                                                       In a more extensive             study, we ran the                            phosphatase         activities     were selected, to
       We have        constructed           an apparatus           for         alkaline      phosphatase            procedure        (3) on                      study     their carryover          effects in subse-
washing          and      drying         Cobas     and
                                                     rotors                    a single rotor, used once and washed                                              quent     runs. Less than 1% of the residual
have      evaluated           the  reliability of the                          once each working               day for three weeks.                              protein       remained.        The washer         effec-
washed       rotors       for use in some laboratory                           No deterioration              in precision         was ob-                        tively removes             these      enzymes,        and
tests.                                                                         served.     However,        after repeated washing                                carryover       is negligible.
     The     washer,       constructed          mostly        of               and handling,          the rotors       tended to break                               Thus we have shown that washed ro-
 polyvinyl         chloride        and operated              by                apart. Even though                 the pieces        can be                       tors can be reused for successive runs of
 pressurized        water, measures 71/2 X 71/2 X                              reassembled          into the Cobas and reused,                                   the same test: ASAT,                ALAT,      alkaline
4 in. A rotor is placed on a platform                      and                 we discard them once they break. Thus                                             phosphatase,                      creatine                    kinase           (6),    uric
spins at about one revolution                  per minute                      it appears that reuse of rotors may be                                            acid, calcium           (7), phosphorus         (8), and
 under the impact              of the water, during                            limited      by physical         strength       more than                         cholesterol        (9).
 which      the individual           cuvettes        are re-                   by chemical         contaminations.                                                   To study            the effects       of reagent
 peatedly       rinsed as they pass each water                                      Results     for ASAT           and alanine          ami-                     carryover       from one test to another,              we
jet, flushing         away the mixture            of serum                     notransferase            (ALAT)          (4) were com-                            evaluated      the results of running a single
and reagents.            A 5-mm timer is set to                                pared for 120 different                patients’      speci-                      test in a washed              rotor that had been
 minimize         water use.                                                   mens for which we used new and washed                                             previously       used for another test. ASAT,
     The dryer measures 9Y4 X 161/4 X 94                                       rotors. Activities           for ASAT and ALAT                                    ALAT,       alkaline        phosphatase,       and cre-
 in. and is operated               by pressurized           air                were all <440 U/L (reference                       interval                       atine    kinase       showed       no inter-test      in-
warmed to 40 #{176}C,        which is sent into the
 individual        cuvettes       of the rotor. Up to
 five rotors can be dried simultaneously                                              Table 1. Average Within-Run Precision for ASAT, Uric Acid, and
 in 20 mm. Detailed                      plans       of the                      Spectrophotometric    Absorbance of Reference Solutions with New Rotors
 washer/dryer            are available           upon       re-                                     and Rotors Washed One to Four Times
quest.                                                                                                                              Av.rag.       rotor                                                   IndIvidual
     Several clinical tests were used in the                                                             Nominal               pr.cislon,        cv,         a                        waahsd        rotor             pr.ciston,          CV,      %
 evaluation         of the cleaning         efficiency        of               Test                       value                Now               Wash.d                        Day     1            Day           2                Day    3            Day     4
 the washer and dryer system,                      in which
                                                                               ASAT,                        25                   1.5                   1.8                     2#{149}9b            0.9                             1.2                2.3
 we compared           washed and new rotors for
 within-run         precision,      day-to-day         preci-                         U/L                   75                  1.1                    1.0                     0.9                      1.1                         1.2                0.9
 sion, and agreement              between test results                         Uric acid,                   47                  7                      8                       8                    7                              7                   9
 for patients’        specimens       run on both new
                                                                                   mg/L                     82                  4                      6                       6                    6                              5                   6
 and washed rotors. The effectiveness                         of
 the washer was also evaluated                        for re-                  Absorbance,           A at:
 moving        selected      substances          from the                             350 nm            0.620                    1.1                   1.1                     0.50                     1.1                         1.2                 1.7
 rotors,     including       serum       proteins,        sub-                        401 nm              0.720                 0.7                    0.7                     0.4’                 0.6                             0.8                 1.0
strates,        and specific            enzymes          from                         512nm              0.332                  1.7                    1.4                     0.6c                 0.8c                            2.9                 1.3
specimens          with high activities,          as well as
 reagent carryover             from other tests.                                    Average of fotx trials of 10 replIcates                 each. b Washed-rotor                    results sigeificantly                (p         0.05) less precise
                                                                               than average precision     for new rotors.              C    Washed-rotor  results                  significantly    (p                 0.05)       more precise than
     Within-run         precision      for several tests
                                                                               average precision   for new rotors.
 was evaluated           with new rotors as corn-

2188        CLINICAL            CHEMISTRY.           Vol.    28,   No.   10,   1982
terferences                as judged by the run-to-run                              use of enzymes.                  Clin.          Chein.       19, 476-482                               BOOmmoL/L                    6rnmot/L
variance            expected            with        new      rotors.                (1973).
   In general, end-point          tests suffer more                                 11. Li, P. K., and Shull,                          B. C., Fixed-time
from     inter-test       contaminations           than                             kinetic          assay     of      plasma            ammonia       with
enzymatic        tests.   For example,       triglyc-                               NADPH             as cofactor,           with      a centrifugal              ana-
                                                                                    lyzer.         CUrt. Chem.         25, 611-613              (1979).
erides (10) cannot            be run after an am-
monia test (11 ) because of the difficulty                                                                                            AlanB. Wut H.                                 .4-

of removing         glycerol,    which is used in                                                                                                                                     LI

                                                                                                                              James     Ohneck2
high         concentrations                    in one            of the       re-                                                                                                   ‘4-
                                                                                                                                    Eric Klaus
agents in the ammonia     test. Thus we do
                                                                                                                       Robert     B. McComb3                                         C-
not recommend      that washed rotors that
originally have been used in one end-                                               Gun.     Chem.     Lab.
point    test be reused   with a different                                          Hartford       Hospital
end-point             test.                                                         Hartford,       CT 06115
       The         washer-dryer                     system          as     de-                                                                                                       x
scribed           in this      paper          can    be an effective
means of reducing       laboratory       costs with                                                                                                                                  LI

no loss of analtyical      precision         or accu-                                    1   Present       address:           Dept.          of Pathol.            and
racy if used within the limits described.                                           Lab.       Med.,       Univ.       of Texas,              Houston,             TX
A normal 5-mm wash requires about 32                                                77025.
L of water. Thus a substantial             savings is                                    2   Present       address:      Beckman               Instruments,
                                                                                    Inc., Brea, CA 92621.                                                                                                              20                         40
realized (the current price of new rotors
                                                                                       3 Address correspondence                              to this      author.                                  Fraction             number(4mL)
is $1.95 each). The use ofwashed                rotors
combines the advantages           of a disposable
                                                                                                                                                                               Fig.          1. Elution            profile           of     125l-labeled
rotor,  by maintaining        cleanliness,         and                                                                                                                         human placental     lactogen   (hPL) from                                        a
the advantages      of a permanent          rotor, by                                                                                                                          Phenyl-Sepharose-CL       4B column
keeping replacement         costs down.                                                                                                                                        Shaded          areas       represent        the extent of antibody.
                                                                                    Radloiodinated                     Hormones                   Purified                     binding of the          recovered   materials;             ionic Strength        of
                                                                                    by Hydrophobic                       Interaction                                           eluent        indicated     by arrows
References                                                                          Chromatography
1. Expert           Panel      on Enzymes:                 Committee           on
                                                                                                                                                                               ed, then     purified       by HIC on Phenyl-
Standards       of IFCC provisional    recommen-                                    To       the     Editor:                                                                   Sepharose-CL         4B (Pharmacia,          Uppsala,
dations      on IFCC methods for measurement
of catalytic     concentration  of enzymes.  Part                                       Recently,     Baxter      and Brown          (1 ) re-                                  Sweden). We passed this second portion
2. IFCC            method    for aspartate   aminotrans-                            ported    the advantageous            purification,                                        through     a 1 X 15 cm column                packed
ferase.           Clin.   Chim.    Acta’   70, F19-F42                              by hydrophobic            interaction        chroma-                                       with Sephadex          G-50 and equilibrated
(1976).                                                                             tography      (HIC),     of an iodinated          tracer                                   with    phosphate           buffer     (pH    7.5, 50
2. Haeckel,          R., The reliability     of a mecha-                            for use in a somatomedin            C radioim-                                             mmol/L)       containing           5 g of bovine
nized        procedure     for the enzymatic determi-                               munoassay.       We have compared             the                                          serum    albumin      per liter. We eluted with
nation        of uric acid according     to Kageyama.    J.                         performance      of HIC with that of the                                                   this buffer,      collecting       1-mL fractions.
Clin.        Chem.     Clin. Biochem.        14, 175-179                            classical gel-exclusion      chromatography                                                We then applied           fractions     correspond-
                                                                                    for purification    of radioiodinated      poly-                                           ing to the protein          peak to a 1 X 20 cm
3. Bowers, G. N., Jr., and McComb,             R. B.,                               peptide            hormones,               and give here our                               column      packed         with     Phenyl-Sepha-
Measurement       of total alkaline    phosphatase
                                                                                    results further              evidencing                  the usefulness                    rose-CL 4B, equilibrated              in Tris-NaC1
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                                                                                    of HIC.                                                                                    (25 mmol/L     Tris, 775 mmol/L    NaC1, pH
                                                                                         Pituitary  and placental       hormones                                               8.6). We eluted     80 mL at this same ionic
4. Expert           Panel on Enzymes:    Committee   on
                                                                                    from Radioassay       Systems (Carson, CA                                                  strength,  then a further      80 mL at an-
Standards            of IFCC provisional   recommen-
dations           on IFCC methods for measurement                                   90746) were iodinated     with                                          other          ionic        strength           (0.25        mmol         Tris,
of catalytic concentration                          of enzymes.           Part      1251, according to Thorell and Johansson                                                   7.75 mmol     NaCl,                     pH    8.6).        We collected
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(1980).                                                                             by exclusion      chromatography          (3) on                                           these fractions,    we promptly        diluted
5. Grant,   G. H., and Kachmar,           J. F., The                                Ultrogel    AcA 54 (LKB,      Bromma,       Swe-                                           them to 200 000 cpm/mL        with, accord-
proteins  of body fluids. In Fundamentals          of                               den). The second was promptly            desalt-                                           ing to their ionic strengths,     either dis-
Clinical Chemistry,        2nd ed., N. W. Tietz, Ed.,
Saunders,    Philadelplia,       PA, 1976, pp 371-
6.     Rosalki,       S., An improved                      procedure          for        Table     1. Decrease in a’ T Observed for Some 1251-Labeled Polypeptide
serum creatine      phosphokinase   determina-                                                 Hormones during Storage in Lyophilized Form after Purification by
tion. J. Lab. Clin. Med. 69, 696-705      (1967).                                              Conventional Gel Filtration (AcA 54), and Hydrophobic Interaction
7. Liedtke,  R. J., Kroon, G., and Batjer, J. D.,                                                                                               Chromatography                             (HIC)       a
Centrifugal               analysis        with        automated               Se-
                                                                                                                         125l.Iaboled                                          1251-labeiod                                    1251.labei.d
quential reagent addition:   Measurement     of
serum calcium.   Clin. Chem.   27, 2025-2028                                                                        chorlogonadotropin                                   __________________
                                                                                                                                                                             foliltropin _______________
                                                                                                                                                                                          placental Iactogsn
(1981).                                                                               Weeks           of         AcA     54                      HIC                     AcA   54                      HIC                   AcA54                     HIC
                                                                                         storage                                                                               % bInding
8.     Drews,  A., Direct colorimetric deter-
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a!., Enzymatic determination      of total serum
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(1974).                                                                                        4                     48.9                        58.1                      58.1                        72.0                    83.7                    98.6
10. Bucolo,               C., and David,       H., Quantitative                          a j        decrease     in 9’   T is       expressed          relative     to the initial 8/ T, considered              as 100 % binding.
determination                 of serum   triglycerides      by the

                                                                                                                                                                  CLINICAL CHEMISTRY,                         Vol. 28, No. 10, 1982                     2189

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