EFFECTS OF STORAGE TIMES AND TEMPERATURES ON T3, T4, LH, PROLACTIN, INSULIN, CORTISOL AND PROGESTERONE CONCENTRATIONS IN BLOOD SAMPLES FROM COWS l

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1 EFFECTS OF STORAGE TMES AND TEMPERATURES ON T3, T4, LH, PROLACTN, NSULN, CORTSOL AND PROGESTERONE CONCENTRATONS N BLOOD SAMPLES FROM COWS l T. J. Remers 2, J. P. McCann and R. G. Cowan Cornell Unversty 3, thaca, NY Summary Lttle s known about stablty of hormones n blood samples stored under varous condtons. Ths study was conducted to examne stablty of trodothyronne (T3), thyroxne (T4), lutenzng hormone (LH), prolactn, nsuln, cortsol and progesterone n blood and serum samples. Experment 1 was desgned to determne f concentratons of these hormones were affected by exposure to cellular elements of antcoagulated and coagulated blood when stored at 4 C and room temperature (22 to 26 C). Jugular venous blood was collected from sx destrous Holsten cows nto evacuated bottles contanng sodum ethylenedamnetetraacetc acd (EDTA), heparn or no antcoagulant. Subsamples of EDTA-treated and heparnzed blood were stored.25,.5, 1, 2, 4, 8, 24 and 72 h at 4 C or room temperature. Subsamples of blood wthout antcoagulant were stored n polypropylene tubes (clot tubes) or serum separator tubes for 1, 2, 4, 8, 24 and 72 h. Mean concentratons of T3, T4, LH, prolactn and cortsol dd not change n plasma or serum from ether of the four types of samples stored at 4 C or room temperature for 72 h. The mean nsuln concentraton decreased 18% by 72 h n serum from serum separator tubes stored at room temperature. At 4 C, mean progesterone concentratons decreased 55% by 24 h and 73% by 72 h n plasma from EDTA-treated blood; 41% by 72 h n serum from clot tubes, and 26% by 24 h and 36% by 72 h n serum from serum ' Supported by a grant from the Alumn Assoc. of the New York State College of Veternary Medcne. 2 Address all correspondence to ths author. 3 Dagnostc Endocrnol. Lab., New York State College of Vet. Med. Receved November 8, Accepted February 11, separator tubes. At room temperature, ptogesterone concentratons decreased 35% by 4 h, 61% by 8 h, 92% by 24 h and 93% by 72 h n plasma from EDTA-treated blood; 49% by 4 h, 69% by 8 h, 91% by 24 h and 94% by 72 h n plasma from heparnzed blood; 56% by 24 h and 83% by 72 h n serum from clot tubes, and 43% by 8 h, 60% by 24 h and 74% by 72 h n serum from serum separator tubes. n Exp. 2, we examned effects of storage tmes and temperatures on concentratons of the seven hormones n serum. Jugular venous blood from each of sx destrous Holsten cows was collected and allowed to clot for 3 h at 4 C. Subsamples of serum were stored 1 h (0 d) and 2, 4, 6 and 8 d at -20 C, 4 C or room temperature. Concentratons of none of the hormones changed when serum was stored for 8 d at -20 or 4 C. Storage of serum at room temperature for 8 d dd not affect concentratons of T3, T4, prolactn or LH. However, the mean progesterone concentraton was reduced 18% by 8 d and the mean nsuln concentraton was reduced 62% by 4 d, but dd not decrease further. Cortsol was reduced 12% by 2 d and remaned at ths level through 8 d. These data ndcate that mmunologcal actvtes of several hormones commonly measured n reproductve and metabolc endocrnology are very stable, whereas, others are qute unstable n blood samples. Stablty of hormones and other substances n bologcal samples must be consdered when estmatng ther concentratons by radommunoassay. (Key Words: Hormones, Stablty, Blood Samples, Radommunoassay.) ntroducton The use of radommunoassays to quantfy hormones n bologcal samples has greatly expanded our knowledge of endocrnology n JOURNAL OF ANMAL SCENCE, Vol. 57, No. 3, 1983

2 684 REMERS ET AL. recent years. However, even though radommunoassays have been used for 20 yr, relatvely lttle s known about stablty of hormones n blood samples that are collected, handled and stored under varous condtons. Ths artcle descrbes two experments desgned to evaluate stablty of seven bovne hormones n coagulated and antcoagulated blood and n serum when samples were stored for varous lengths of tme. The seven hormones ncluded three protens, lutenzng hormone (LH), prolactn and nsuln; two amno acds, trodothyronne (T3) and thyroxne (T4), and two sterods, cortsol and progesterone. These hormones were chosen for these studes because of ther mportance n anmal scence and because of ther dversty n molecular sze and chemcal structure. Materals and Methods Exp.. n ths experment, we determned f concentratons of T3, T4, LH, prolactn, nsuln, eortsol and progesterone n serum and plasma were affected by exposure to cellular elements of blood when stored at two temperatures for 72 h (.e., precentrfugaton stablty). Decreases n concentratons of some of the hormones due to effects of storage tmes and temperatures mght have approached the lmts of senstvty of our radommunoassays. Therefore, sx destrous Holsten cows were selected for ths experment to assure hgh concentratons of progesterone n blood and were njected wth varous stmulatng hormones to assure hgh concentratons of the other sx hormones. Each cow receved 20 U of thyrotropn (TSH) 4 v 4 h before collecton of blood to ncrease concentratons of T3 and T4, 200 U of adrenocortcotropn (ACTH) s m 2 h before collecton to ncrease concentratons of cortsol, 40 /ag of gonadotropn-releasng hormone (GnRH) 6 v 30 mn before collecton to ncrease concentratons of LH, 100 ~g of thyrotropn-releasng hormone (TRH) 6, and 1.5 mg of glucagon ~ v 15 mn before collecton to ncrease concentratons of prolactn and nsuln, respectvely, n blood. Treatment wth TRH rased concentratons of prolactn n blood of two of the cows to levels beyond the range of *Jensen-Salsbery Laboratores, Syracuse, NY. s Armour Pharmaceutcal Co., Phoenx, AZ. 6 Sgma Chemcal Co., St. Lous, MO. El Llly and Co., ndanapols, N. s Comng Glass Works, Cornng, Nu the standard curve for the prolactn assay. Rather than dlutng the serum or plasma, two addtonal cows were bled wthout pretreatment wth TRH and blood from them was used nstead. Two-hundred mlllters of jugular venous blood were collected from each cow nto four evacuated bottles. One bottle contaned 300 mg of sodum ethylenedamnetetraacetc acd (EDTA), the second contaned 3,000 unts of lqud heparn and the remanng two bottles contaned no antcoagulant. The order n whch the bottles were used for collectng blood from each cow was randomzed. Subsamples of EDTA-treated or heparnzed blood were dspensed mmedately after collecton nto 16 polypropylene tubes; subsamples of blood from one of the bottles wthout antcoagulant were dspensed nto 12 polypropylene tubes, hereafter referred to as "clot tubes," and subsamples from the second bottle wthout antcoagulant were dspensed nto 12 glass serum separator tubes s contanng a chemcally nert serum separator and clot actvator. One-half of the subsamples of each type of blood was stored at room temperature (22 to 26 C) and one-half was stored at 4 C. One subsample of EDTA-treated blood and one subsample of heparnzed blood were centrfuged (15 mn at 600 x g).25,.5, 1, 2, 4, 8, 24 and 72 h after collecton. Subsamples of blood n clot and serum separator tubes were centrfuged 1, 2, 4, 8, 24 and 72 h after collecton. Plasma or serum was recovered from each subsample and stored at -20 C untl assayed for the seven hormones. All 56 subsamples of plasma and serum from each cow were analyzed for a partcular hormone wthn a sngle radommunoassay to avod potental effects of nterassay varaton. Effects of storage tme at each temperature on concentratons of hormones were examned by randomzed complete block analyss, wth cows consdered as blocks (Danel, 1978). When the F-test for treatment (storage tme) was sgnfcant, Dunnett's test (Steel and Torre, 1960) was used to compare mean concentratons n plasma stored.25 h and serum stored 1 h wth concentratons for each subsequent storage tme. Exp. 2. Effects of storage tmes and temperatures on concentratons of the seven hormones n serum were nvestgated n ths experment (.e., postcentrfugaton stablty). Sx destrous Holsten cows were treated wth

3 STABLTY OF HORMONES N BLOOD SAMPLES 685 TABLE 1. RELATVE ACTVTES OF CROSS-REACTNG HORMONES N THE PROGESTERONE, LH AND PROLACTN RADOMMUNOASSAYS Radommunoassay Compound Relatve actvty a Progesterone Progesterone la-hydroxyp rogesterone a-pregnane-3,20-done l#-hydroxyprogesterone.2210 Deoxyeortcosterone.0450 Cortcosterone a-hydroxyprogesterone.0078 Pregnenolone a-hydroxy-& 4-pregnen-3-one #-hydroxy-A 4-pregnen-3-one.0007 Testosterone.0007 Cortsone #-estradol <.0001 Hydrocortsone <.0001 Cholesterol < a-hydroxypregnenolone < #-pregnene-3 ~,20~-dol <.0001 LH NAMMD-bLH-4 (bovne LH) NAMMD-bTSH-9 (bovne TSH).1614 NH-GH-B 18 (bovne growth hormone).0044 NH-P-B6 (bovne prolactn).0006 LER (bovne FSH).0005 Prolaetn NH-P-B6 (bovne prolactn) NH-GH-B18 (bovne growth hormone).0084 NAMMD-bLH-4 (bovne LH).0004 LER (bovne FSH) <.0003 NAMMD-bTSH-9 (bovne TSH) <.0003 arato of the concentraton of the specfc hormone to the concentraton of the potentally nterferng hormone needed to nhbt bndng of tracer to antbody by 50%. ACTH and GnRH to ncrease concentratons of cortsol and LH n blood, respectvely, as descrbed above. n addton, each receved 60 ml of a 50% glucose soluton v 5 mn before blood collecton to ncrease nsuln levels. One-hundred ffty mlllters of blood wthout antcoagulant were collected by jugular venpuncture from each cow and allowed to clot at 4 C for 3 h. Serum was collected after centrfugaton and subsamples were dspensed nto 15 polypropylene tubes. Fve subsamples were stored at -20 C, fve at 4 C and fve at room temperature (22 to 26 C). One subsample from each storage temperature was transferred to -70 C after 1 h (0 d) and 2, 4, 6 and 8 d of storage. All 90 subsamples were analyzed for each hormone wthn a sngle radommunoassay. 9 Mcromede Systems, Horsharn, PA. Randomzed complete block analyss was used to determne treatment (days of storage) effects for each storage temperature (Danel, 1978). Mean concentratons of hormones after 2, 4, 6 and 8 d were compared wth the ntal concentraton (0 d) by Dunnett's test (Steel and Torre, 1960) f the F-value for treatment was sgnfcant. Radommunoassays. The radommunoassays for T3, T4, nsuln, cortsol and progesterone used 8 50 mm antbody-treated polypropylene assay tubes and radoodnated tracers 9. Procedures and valdaton data for the T3, T4 and cortsol assays for other speces have been publshed (Remers et al., 1981). However, these assays were valdated also to quantfy these hormones n bovne samples. Seral dlutons of bovne sera contanng low, medum and hgh concentratons of T 3, T4 or cortsol nhbted bndng of tracer to the respectve antbody n a manner that was

4 686 REMERS ET AL. parallel to nhbton by standard preparatons. Relatve actvtes of potentally nterferng compounds n each assay are publshed (Remers et al., 1981). Procedures for and valdaton of the sold-phase radommunoassay for nsuln n bovne samples have been publshed (Remers et al., 1982) n the radommunoassay for progesterone, [l,2,6,7-3h]progesterone was added to each subsample of serum and plasma before extracton wth petroleum ether (Nswender et al., 1975) to montor extracton recovery. The extracted progesterone was dssolved n.05 M phosphate-buffered salne (ph 7.2) contanng.5% bovne serum albumn (PBS-BSA). One hundred mcrolters of extract or standard, 800/~ of PBS-BSA and 100/~ (about 20,000 cpm) of progesterone- l~-succnyl-1~ tyrosne methyl ester were added to polypropylene tubes coated wth rabbt antbody aganst progesterone-1 lasuecnyl-bsa 9. The tubes were ncubated for 3 h at 37 C and then rnsed twce wth deonzed water. Seral dlutons of extracts of bovne sera produced nhbton curves that were parallel wth the standard curve. Concen- tratons of progesterone n extracts wth and wthout pror purfcaton on columns of Sephadex LH-20 agreed very well (12.1 vs 11.3 ng/ml; 10.4 vs 9.9 ng/ml;.6 vs.5 ng/ml, respectvely). Recovery of progesterone (1.25, 2.5, 5, 10, 20 ng/ml) added to three samples of serum was quanttatve. The correlaton coeffcent was 1.0, the slope of the recovery curve was 1.02 and the y-ntercept was -.11 ng/ml. Relatve actvtes of other sterods n the progesterone assay are shown n table 1. The ntraassay coeffcents of varaton (CV) obtaned from 10 duplcate determnatons of progesterone n sera contanng low, medum and hgh concentratons of progesterone are shown n table 2. The nterassay CV for three pools analyzed n 10 consecutve assays are shown n table 2. The radommunoassay for LH used antbody #15, kndly suppled by Dr. G. D. Nswender and the assay was performed as descrbed by Nswender et al. (1969). The radommunoassay for prolactn was performed as descrbed by Davs et al. (1971) usng a prolactn antserum (DJB ) kndly suppled by Dr. D. J. Bolt. TABLE 2. PRECSON OF THE PROGESTERONE, LH AND PROLACTN RADOMMUNOASSAYS Rsdommunoumy Sample X, ng/ml CV, % Progesterone Low P4-1 Medum P4-1 Hgh P4-1 LH Low LH- Medum LH-1 Hgh LH-1 Prolactn Low Prl-1 Medum Prl-1 Hgh Prl- 1 Progesterone Low P4-2 Medum P4-2 Hgh P4-2 LH Low LH-2 Medum LH-2 Hgh LH-2 Prolactn Low Prl-2 Medum Prl-2 Hgh Prl-2 ntraauay precson a ,4 ~., lnterassay precson b , aeach sample was analyzed 10 tmes n duplcate wthn a sngle radommunoassay. beach sample was analyzed n duplcate n 10 separate radommunoassays.

5 STABLTY OF HORMONES N BLOOD SAMPLES 687 Reference preparatons for the LH and prolactn radommunoassays were NAMMD-bLH-4 and NH-P-B6, respectvely. Relatve actvtes of other ptutary hormones n these assays as determned by us are shown n table 1. nterassay and ntraassay CV are presented n table 2. Results Exp.. Shown n fgures 1,through 7 arc mean concentratons (+ 95% confdence ntervals) of Ts, T4, LH, prolactn, nsuln, cortsol and progesterone n plasma from EDTA-treated or heparnzed blood and n serum from clot and serum separator tubes (SST) after storage for 72 h at 4 C or room temperature. Mean EDTA CLOT f,.25.s HEPARN SST TE hl Fgure 3. Concentratons (.~ 95% C) of LH n plasma from EDTA-rteated or heparnzed blood and n serum from clot and serum separator tubes (SST). Samples were stored at 4 C (a a) or room temperature (o o). 2,0 t! to 4,~ll 1.! 1,1 rdyta t~pa~ $ST s~ ~ ~ & ~72 SrORA~ TE N S, Fgure 1, Concentatons (X ~ 95% C) of T s n plasma from EDTA-treated or heparnzed blood and n serum from clot and serum separator tubes (SST). Samples were stored at 4 C (s. m) or room temperarare (o--o). : EDTA CLOT.S.$ 1 HEPARN SST $ * * 9 ~, 1 4 S B a ~4 72 STORAGE Tlde N Fgure 4, ConcenU'atons (~ 95% C) of prolactn n plasma from EDTA-treated or heparnzed blood and n serum from clot and serum separator ~bes (SST). Samples were stored at 4 C (a~a) or room temperature (o o). E~A HEPN~N EDTA * a * HEPARN C~T SST 4( 3( CLffr s,; ; ; ; ; ~,72 S,S 1 4 S 114 $1DR~! h) Fgure 2. Concentratons (X 95% C) of T 4 n plasma from EDTA-treated or heparnzed blood and n serum from clot and serum separator rubes (SST). Samples were stored at 4 C (s s) or room tempera- Cure (o--o). '.~,S S S ~ S Fgure S. Concentratons (X 9S% C) of nsuln n plasma from EDTA-treared or heparnzed blood and n serum from clot and serum separator tubes (SST). Samples were stored at 4 C (m m) or room temperature (o~o). **Dfferent (P<.01) from concentraton at h.

6 688 REMERS ET AL. concentratons of T3, T4, LH, prolactn and cortsol were not affected by storage tme or temperature. Whereas nsuln concentratons were not affected by storage at 4 C, the mean concentratons Of ths hormone n serum from clot and SST decreased 26% (P>.05) and 18% (P<.01), respectvely, after storage for 72 h at room temperature: Progesterone was the least stable of the seven hormones studed. After storage at room temperature, the mean progesterone concentraton n plasma from EDTA-treated blood decreased 35% (P<.05) by 4 h although an apparent, but nonsgnfcant, decrease occurred between 1 and 2 h. The mean concentraton of 8C 60 E'= 4O EDTA CLOT HEPARN SST ,4 72 ~5.S ?2 STORAGE TME lhl Fgure 6. Concentratons (X -+ 95% C) of cortsol n plasma from EDTA-treated or heparnzed blood and n serum from clot and serum separator tubes (SST). Samples were stored at 4 C (=--,) or room temperature (o o). progesterone was reduced (P<.01) 61% by 8 h, 92% by 24 h and 93% by 72 h at room temperature. Mean progesterone concentratons n plasma from heparnzed blood stored at room temperature decreased (P<.01) 49% by 4 h, 69% by 8 h, 91% by 24 h and 94% by 72 h. At room temperature, mean progesterone concentratons decreased (P<.01) 56% by 24 h and 83% by 72 h n serum from clot tubes and 43% by 8 h, 60% by 24 h and 74% by 72 h n serum from SST. Progesterone concentratons decreased even when the samples were stored at 4 C, albet at a slower rate. At 4 C, the mean progesterone concentraton decreased (P<.01) 55% by 24 h and 73% by 72 h n plasma from EDTA-treated blood. t decreased 41% by 72 h (P<.01) n serum from clot tubes and 26% by 24 h (P<.05) and 36% by 72 h (P<.01) n serum from SST. Exp. 2. Effects of postcentrfugaton storage condtons on concentratons of the seven hormones n serum are shown n fgures 8 through 13. Concentratons of none of the hormones changed n serum when stored for 8 d at -20 C or 4 C. Storage of serum at room temperature for 8 d dd not affect concentratons of T3, T4, LH or prolactn. The mean progesterone concentraton was reduced 18% by 8 d (P<.05), but not sooner. The mean concentraton of nsuln was reduced 62% by 4 d (P<.01) of storage at room temperature, but l: CLOT l 9 25.S 2 4 l l l l, 8 ~ ~ STORAGE ~ hj Fgure 7. Concentratons(X 95% C) of progesterone n plasma from EDTA-treated or heparnzed blood and n serum from clot and serum separator tubes (SST). Samples were stored at 4 C (a--a) or room temperature (o--o). *Dfferent (P<.05) from concentratons at.25 h (EDTA) or 1 h (SST); **dfferent (P<.01) from concentratons at.25 h (EDTA, HEPARN) or 1 h (CLOT, SST). 5 1"5 f ~ ~'~terone l F Fgure 8. Concentratons (.X 95% C) of nsuln, LH, progesterone and T 3 n serum stored at -20 C.

7 STABLTY OF HORMONES N BLOOD SAMPLES 689 dd not decrease any further through 8 d. Cortsol was reduced 12% by 2 d (P<.05), 13% by 4 d (P<.01), 15% by 6 d (P<.01) and 14% by 8 d (P<.01). Dscusson Researchers n the anmal scences frequently and justfably are very concerned about the performance of a partcular radommunoassay n terms of specfcty, accuracy, precson and senstvty when estmatng concentratons of a hormone or other substance n bologcal samples. However, an equally mportant factor determnng relablty of such estmates s the qualty of the samples n whch the hormone s measured. Our data clearly ndcate that the mmunologcal actvtes of several hormones commonly measured n reproductve and metabolc endocrnology are very stable, whereas others are qute unstable (e.g., progesterone). Short (1958), Vahdat et al. (1979, 1981), Holdsworth (1980), Owens et al. (1980) and Olmer and Edqvst (1982) have reported prevously that progesterone dsappeared from antcoagulated bovne blood, even when the samples were refrgerated. Our data confrm these reports and also ndcate that progesterone decreases n coagulated blood, but to a lesser extent than n antcoagulated blood. Short (1958) reported that progesterone n antcoagulated (oxalated) bovne blood was metabolzed to another sterod, tentatvely dentfed as 2013-hydroxypregn-4-en-3-one. Van der Molen and Groen (1968) reported that the product of ths metabolsm n ovne blood was 20c~-hydroxypregn-4-en-3-one and that the converson was favored n the presence of glucose. ncubaton of blood cells wthout glucose favored the oxdaton of the 20ametabolte to progesterone, possbly explanng the dsappearance and then reappearance of progesterone n plasma from whole blood observed by Vahdat et al. (1981) and Olmer and Edqvst (1982). Our experment dd not show ths reappearance as reported by these nvestgators. 3~ f 0-1: f 4'- ns~k~ ~r Progesterone 1.5[ T3 ~1.0 ~ ~ r Fgure 10. Concentratons (X 95% C) of nsuln, LH, progesterone and T 3 n serum stored at 4 C. 7O "Prolactn 5O ~4o[- Prolactn L ' l Fgure 9. Concentratons (X -+ 95% C) of prolactn, cortsol and T 4 n serum stored at -20 C. 40 L- "s f 35'- 0 T4 l Fgure 11. Concentratons (X -+ 95% C)of prolactn, cortsol and T, n serum stored at 4 C.

8 690 REMERS ET AL. No matter what the cause or eventual product, t s qute apparent that accurate estmates of progesterone concentratons n blood samples requre prompt separaton of plasma or serum from the cellular elements. Once separated, progesterone s qute stable. The concentraton n serum dd not change even after 6 d at room temperature. Data shown n fgure 4 suggest that storage ~ 16~ LH,, Fgure 12. Concentratons (~ ~ 95% C) of nsuln, LH, progesterone and T s n serum stored at room ternperanare. 'Dfferent (P<.05) from concentraton on d 0; "'dfferent (P<.01) from concentraton on d O Fgure 13. Concentratons (~ 95% C) of prolactn, cortsol and T~ n serum stored st room temperahare. "Dfferent (P<.0~-~o-m concer~cratnn on d O; "dfferent (P<.01) from concentraton on d O. T of blood n SST caused a reducton n prolacun concentratons relatve to other types of samples. The consderably lower concentratons of prolactn n SST, however, can be explaned as an artfact of the samplng procedure. As ndcated above, the order n whch types of blood samples were collected from each cow was randomzed. n one partcular cow, blood samples were collected whle prolactn concen; tratons were ncreasng due to njecton of TRH. The order of blood collecton from ths cow was as follows: sample for SST, sample for EDTA antcoagulant, sample for heparn antcoagulant, sample for clot tubes. The respectve concentratons of prolactn n serum and plasma from these samples were approxmately 17, 30, 80 and 95 ng/ml. Ths was much more varable than for other cows and ths one cow accounted for the varaton n prolactn concentratons among sample types. Nevertheless, t s stll apparent that storage dd not affect concentratons of prolactn once the samples were collected. L terstu re Cted Danel, W. W Bostatstcs: A Foundaton for Analyss n the Health Scences (2nd Ed.). John Wley and Sons, New York. Davs, S. L, L. E. Rechert, Jr. and G. D. Nswender Serum levels of proactn n sheep as measured by radommunoass&y. Bol. Reprod. 4:145. Holdsworth, R. J Measurement of progesterone n bovne plasma and preserved whole blood samples by a drect radommunousay. Brt. Vet. J. 136:135. Nswender, G. D., A. M. Akbar and T. M. Nett Use of specfc antbodes for quantfcaton of sterod hormones. n: B. W, O'Malley and J, G. Hardman (Ed.) Methods n Enzymology, VoL 36. Academc Press, New York. Nswender, G. D., L, E, Rechert, Jr., A, R. Mdgley, Jr. and A. V. Nalbtndov Radnmmunoassay for bovne and ovne lutenlzng hotmone. Endocrnology 84:1166. Olmer, R. and L. -E. Edqvst Changes n plasma progesterone levels durng storage of heparnlzed whole blood from cow, horse, dog and pg. Acta Vet, Scand. 23:1. Owens, R. E., D. T. Atklns, C. H. Rahe, J. L. Fleeger and P. G. Harms Tme-dependent loss of radommunoassayable levels of progesterone followng ambent temperature nc~tbaton of heparnzed bovne blood. Therogenology 13: 305. Reamers, T. J., R. G. Cowan, H. P. Davldson and E. D. Colby Valdaton of radommunoassays for trodothyronne, thyroxne, and hydrocortsone (consol) n canne, felne, and equne sere. Amer. ]. Vet. Res. 42:2016. Remers, T. J., R. G. Cowan, J. P. McCann and M. W.

9 STABLTY OF HORMONES N BLOOD SAMPLES 691 Ross Valdaton of a rapd sold-phase radommunoassay for canne, bovne, and equne nsuln. Amer. J. Vet. Res. 4-3:1274. Short, R. V Progesterone n blood. The chemcal determnaton of progesterone n perpheral blood. J. Endocrnol, 16:r Steel, R.G.D. and J. H. Torre Prncples and Procedures of Statstcs. McGraw-Hll Book Co., New York. Vahdat, F., J. P. Hurtgen, H. L. Whr.more, S. D. Johnston and C. L. Ketelsen Effect of tme and temperature on bovne serum and plasma progesterone concentraton. Therogenology 12:371. Vahdat, F., J. P. Hurtgen, H. L. Wh~nore, B. E. Segun and S. D. Johnston Declne n assayable progesterone n bovne plasma: Effect of tme, temperature, antcoagulant, and presence of blood cells. Amer. J. Vet. Res. 42:521. Van der Molen, H. J. and D. Groen nterconverson of progesterone and 20a-dhydroxyprogesterone and of androstenedone and testosterone n vtro by blood. Acta Endocrnol. 58:4-19.