Clinical evaluation for lymphoproliferative disease prompted by finding of IgM warm autoanti-IT in two cases.

Anti-IT is an unusual specificity originally described as a naturally occurring cold agglutinin. The antibody reacts strongly with cord RBCs, weakly with adult I RBCs, and most weakly with the rare adult i RBCs. IgG anti-IT in patients with hemolytic anemia has been associated with Hodgkin's lymphoma. Difficulties in blood grouping tests and the presence of a warm reactive agglutinin in samples from two patients with hemolytic anemia led to further serologic studies and the identification of anti-IT. In both cases, the anti-IT was a rarely encountered IgM warm reactive agglutinin; in one case, the IgG component was also anti-IT, whereas in the second case the IgG antibody was broadly reactive. The unusual serologic finding of anti-IT prompted further clinical evaluation for lymphoproliferative disease in these two patients.

In vitro studies of the impact of transfusion on the detection of alloantibodies after autoadsorption.

BACKGROUND: In a patient with warm autoantibodies who has recently received a transfusion, it is not recommended to perform adsorptions using autologous RBCs to detect alloantibodies. Although not scientifically documented, this position is based on the theory that transfused RBCs in the patient's circulation would be capable of adsorbing alloantibodies that may be present. This in vitro study was designed to determine what percentage of transfused RBCs might completely remove alloantibodies in vivo. STUDY DESIGN AND METHODS: Selected D, E, K, Fy(a), and Jk(a) antibodies were adsorbed with mixtures of antigen-positive and antigen-negative RBCs to determine the lowest concentration of antigen-positive RBCs capable of removing all alloantibody reactivity. The percentage of antigen-positive RBCs in each mixture was determined by flow cytometry. RESULTS: Small amounts of antigen-positive RBCs (2-6%, as determined by flow cytometry) completely removed anti-D, -E, and -Fy(a) reactivity. Reactivity of two examples of anti-K was removed by 11 percent and 17 percent of K+ RBCs, respectively. Anti-Jk(a) reactivity was completely removed by 4 to 5 percent Jk(a+) RBCs using a PEG adsorption; the endpoint (>11%) was estimated, but complete adsorption with ZZAP-treated RBCs was not performed. CONCLUSION: Small amounts of antigen-positive RBCs are generally capable of removing all alloantibody reactivity. Thus, waiting for 3 months after transfusion before performing autologous adsorptions is a prudent policy.

Serology of antibodies to second- and third-generation cephalosporins associated with immune hemolytic anemia and/or positive direct antiglobulin tests.

BACKGROUND: First-generation cephalosporins rarely caused immune hemolytic anemia (IHA). Second- and third-generation cephalosporins, especially cefotetan and ceftriaxone, are increasingly associated with severe, sometimes fatal IHA. STUDY DESIGN AND METHODS: Samples from 53 patients with drug-induced IHA and/or positive direct antiglobulin test (DAT) were tested. Patients' sera were tested against drug-treated red cells (RBCs) and untreated or enzyme-treated RBCs, with and without the addition of drug solution. Eluates from patients' RBCs were tested against drug-treated and untreated RBCs. RESULTS: Forty-three patients had antibodies to cefotetan, 8 to ceftriaxone, 1 to cefoxitin, and 1 to cefotaxime. All patients had a positive DAT; only anticefoxitin and anti-cefotetan were demonstrable in RBC eluates. Sera containing anti-cefoxitin, anti-cefotaxime, and anti-cefotetan reacted with drug-treated RBCs (100%) and untreated or enzyme-treated RBCs in the presence of drug (98% or 100%, respectively). All of the ceftriaxone antibodies reacted with untreated or enzyme-treated RBCs in the presence of drug, but those tested did not react with ceftriaxone-treated RBCs. In addition to cefotetan-dependent antibodies, 19 (44%) and 14 (33%) of 43 sera contained drug-independent antibodies when tested with and without the presence of a polyethylene glycol potentiator, respectively. CONCLUSION: Cefotetan is by far the most common cause of drug-induced IHA. All cefotetan antibodies and the single examples of cefoxitin and cefotaxime antibodies reacted with drug-coated RBCs, and most, in contrast to the reactions of antibodies to first-generation cephalosporins (e.g., cephalothin), also reacted with RBCs (not treated with drug) in the presence of the drug. Ceftriaxone antibodies reacted only by the latter mechanism. Drug-independent antibodies (i.e., those reacting without any drug being present) were detected in 33 to 44 percent of patients' sera containing cefotetan antibodies, depending on the sensitivity of the method used.

Severe immune hemolytic anemia associated with prophylactic use of cefotetan in obstetric and gynecologic procedures.

Second- and third-generation cephalosporins, especially cefotetan, are increasingly associated with severe, sometimes fatal immune hemolytic anemia. We noticed that 10 of our 35 cases of cefotetan-induced hemolytic anemias were in patients who had received cefotetan prophylactically for obstetric and gynecologic procedures. Eight of these cases of severe immune hemolytic anemia are described.

Evaluation of methods for detecting alloantibodies underlying warm autoantibodies.

BACKGROUND: In pretransfusion testing of patients whose sera contain autoantibodies reacting optimally at 37 degrees C, it must be determined whether alloantibodies are also present. Two approaches, testing a 1-in-5 dilution of patients' sera and the adsorption of sera in the presence of polyethylene glycol (PEG), have been proposed as alternatives to the time-consuming approach of adsorbing sera with ficin- or ZZAP-treated red cells (RBCs). The three approaches were compared. STUDY DESIGN AND METHODS: Patients' sera containing warm autoantibodies, with and without alloantibodies, were retested 1) after dilution (1-in-5) and 2) after adsorption with allogeneic RBCs in the presence of PEG. Results were compared to those after adsorption with ZZAP-treated allogeneic RBCs. RESULTS: Dilution (1-in-5): Twenty-seven of 119 sera (7/26 [27%] with and 20/93 [22%] without alloantibodies) did not react; one example each of alloanti-D, -E, -e, -Fy(a), and -Jk(a), and two examples of anti-Jk(b) were not detected at a dilution of 1 in 5. Alloantibodies were identified in 5 (19%) of 26 1-in-5 diluted sera containing alloantibodies; 87 (73%) of 119 sera still reacted with all cells and would have required further workup. PEG adsorption: Thirty-nine sera were tested after parallel PEG and ZZAP adsorptions. The PEG adsorptions required a total of 55 aliquots of adsorbing cells and 13.75 hours, whereas ZZAP adsorptions required 61 aliquots and 30.5 hours. All alloantibodies (anti-D [3], -C [2], -c [1], -E [4], -K [2], -Fy(a) [1], -Jk(a) [2], -Jk(b) [1]) reacted in the adsorbed serum-PEG mixtures at a strength equal to or greater than that in the ZZAP-adsorbed sera. CONCLUSION: Although the 1-in-5 dilution approach is convenient, only 22 percent of warm autoantibodies without alloantibodies were nonreactive, and 27 percent of alloantibodies of potential clinical significance were not detected. PEG adsorption appears to give similar results to those of ZZAP adsorption, but it has the advantages of eliminating the cost and time of prior treatment of the allogeneic adsorbing cells and of a reduction of at least a 50 percent in adsorption time.

False-positive eluate reactivity due to the low-ionic wash solution used with commercial acid-elution kits.

BACKGROUND: During the use of commercial red cell (RBC) acid-elution kits for adsorption and elution (adsorption/elution) studies with anti-D, unexpected reactive eluates (anti-D) were obtained from D- RBCs. Such results were not obtained with a parallel xylene method or, historically, with heat and ether methods. STUDY DESIGN AND METHODS: Single-donor and commercial polyclonal anti-D samples were incubated with D+ and D- RBCs. Acid eluates were prepared by the manufacturers' directions. Variations in the wash step of the eluate preparation included the use of commercial kit wash solution versus phosphate-buffered saline versus solutions of various ionic strengths. RESULTS: Anti-D was eluted from 20 of 22 samples of D- RBCs after incubation with commercial polyclonal anti-D (titer 512) and from 2 of 3 samples of D- RBCs incubated with single-donor anti-D (titer 256). With a low-titer (16) single-donor anti-D, 0 of 4 eluates from D- RBCs reacted. When phosphate-buffered saline was substituted for the commercial wash solution, 0 of 11 D- RBC eluates reacted, as compared with 9 of 11 D- RBCs that yielded positive 1+(-)2+ eluates with the commercial wash solution. If the recommended initial phosphate-buffered saline wash was omitted before the use of the commercial wash solution, the eluate reactivity was stronger (2+(-)3+). When low-ionic-strength (< 0.03 M) saline was substituted, anti-D was eluted from D- RBCs. All last washes were nonreactive. Antiglobulin tests on all adsorbing D- were negative. CONCLUSION: Commercial wash solutions used for acid elution are at low ionic strength and commonly yield superior eluates, but in the presence of high-titer antibodies, false-positive eluates can result. It is our belief that the low-ionic-strength wash solution caused aggregation of IgG and nonspecific attachment of IgG on RBCs. Aggregates will contain IgG serum antibodies in proportion to the titer of the antibody. It is this nonspecifically bound antibody that is eluted from antigen-negative RBCs.

Excretion of phenytoin into semen of rabbits and man. Comparison with plasma levels.

The concentration of phenytoin (diphenylhydantoin, DPH) was measured in plasma and semen of rabbits and man. In the rabbit, a single iv injection of DPH (4.64 mg) resulted in a concentration-time curve for DPH in semen parallel to the concentration-time curve for DPH in plasma (t1/2beta = 171 +/- 29 min). A semen/plasma drug concentration ratio of 0.20 was maintained for at least 8 hr, demonstrating that DPH concentrations in semen are directly proportional to DPH concentrations in plasma. In epileptic subjects maintained on oral DPH the mean drug concentration in semen was 2.31 microgram/ml while that in plasma was 13.8 microgram/ml. The mean semen-plasma DPH concentration ratio in man was 0.17; this closely approximates the observed ratio in rabbits.

The metabolism of d-, l- and dl-methadone in the isolated perfused rat liver.

Five-milligram doses of d-, l- and dl-methadone were added to the perfusate of the isolated perfused rat liver and the disposition of each compound was monitored over a period of 120 minutes. There was an initial rapid decline in concentration of each drug in the perfusate, with approximately 70% disappearing in the first 10 minutes of the perfusion. Thereafter, the rate of decline of each compound slowed and the apparent half-life was about 100 minutes. In experiments with d- l- and dl-methadone the mean hepatic extraction from the perfusate at 10 minutes was 72, 66 and 69%, respectively. A comparison of the rate of disappearance of the d- and the l-isomers from the perfusate showed that the concentration of the d-isomer at each sampling time was lower than the concentration of the l-isomer and this concentration ratio ranged between 68 and 79%. A study of the disposition of dl-methadone in the isolated perfused liver indicated that the initial rapid decline of the drug in the perfusate in the first 10 minutes was caused primarily by the uptake of the unchanged drug into the liver. Thereafter, the concentration of methadone in the perfusate and liver declined slowly with time and was paralleled by an increase in the concentration of the primary mono-N-demethylated metabolite, which appeared in the liver, perfusate and bile. Smaller quantities of more polar metabolites, including hydroxylated compounds and conjugates, were also present in the liver and bile. Addition of SKF 525-A retarded the rate of decline of methadone from the perfusate and pretreatment of the rat with phenobarbital increased the rate of biotransformation of methadone in the isolated perfused liver.

Clinical effects and pharmacokinetics of racemic methadone and its optical isomers.

The respiratory and pupillary effects of oral l-, d-, and d,l-methadone were studied in healthy male volunteers 21 to 35 yr of age. The mean half-life of drug in blood was 22 hr for racemic methadone, 24 hr for l-methadone, and 25 hr for d-methadone. The effects of d-methadone were not significantly different from the placebo response at a 7.5 mg dose, whereas a 50 and 100 mg dose slightly depressed respiration in one subject each. Both 7.5 mg of l-methadone and 15 mg of d,l-methadone induced intense and sustained respiratory depression and miosis. The changes induced by l-methadone were of longer duration than those of d,l-methadone, lasting more than 72 hr in some subjects. Whole blood drug concentration correlated well with respiratory depression and miosis for l- and d,,l-methadone. The potency ratio of l-methadone to d,l-methdone, calculated from blood drug concentration data, was found to be 3.0 for respiratory depression and 2.7 for miosis. The antiduretic effect of 15 mg of d,l-methadone was investigated in three subjects and was found to persist for as long as measurements were taken, namely 11 and 12 hr in two subjects. d,l-Methadone administered frequently for pain may have cumulative effects on respiratory control and ability to excrete a water load.

New gas chromatogrphic assay for the quantification of methadone. Application in human and animal studies.

A new gas chromatographic assay utilizing 2-dimethylamino-4,4-diphenyl-5-nonanone as the internal standard was developed for the quantification of methadone. The method involved extraction of methadone with 1-chlorobutane from tissue at pH 9.8, re-extraction of an aliquot of the organic solvent with 0.5 M sulphuric acid, alkalinization and final extraction into chloroform. The assay was used to determine the concentration of methadone (i) in whole blood samples from a normal volunteer following a single 9.4-mg oral dose of d-methadone hydrochloride, (ii) in whole blood, saliva and gastric juice from a methadone addict maintained on 90 mg of dimethadone hydrochloride per day, (iii) in mouse liver microsomes incubated with methadone, and (iv) in the perfusate of the isolated perfused rat liver.

Identification of glucuronide metabolites of benzomorphan narcotic analgesic drugs in bile from the isolated perfused rat liver by gas chromatography and mass spectrometry.

The metabolism of four benzomorphan compounds was studied in the isolated perfused rat liver, and glucuronide metabolites were identified by combined gas chromatography-mass spectrometry (GC/MS). Cyclazocine, ketocyclazocine, volazocine, and pantazocine were each added to the perfusate of the isolated perfused rat liver and the bile collected for 3 hours. The residue from evaporation of the bile was derivatized with the dimethylsulfoxide anion and methyl iodide, and the permethylated glucuronide metabolites were identified by GC/MS. The four compounds were hydroxylated by the liver and excreted in the bile as phenolic glucuronides. For example, permethylated hydroxycyclazocine glucuronide had a mass spectrum with a molecular ion at m/e 533 and fragment ions at m/e 301 (aglycone), m/e 260 (loss of cyclopropyl group) and prominent ions at m/e 232, 201, 169, 141, and 101 caused by fragmentation of the permethylated glucuronic acid moiety. Perdeuteriomethylation demonstrated that pentazocine, volazocine, and cyclazocine were further metabolized by methylation of one hydroxy substituent and glucuronidation on the other. Pentazocine, cyclazocine, and ketocyclazocine were also metabolized to phenolic glucuronides of the parent drugs. N-deakylated metabolites of pentazocine, volazocine, and cyclazocine were identified both as permethylated glucuronic acid conjugates and as the trimethylsilyl derivatives of the aglycones, obtained by enzymatic hydrolysis on the conjugates in bile.

Identification of glucuronide metabolites of d- and 1-methadone in bile from the isolated perfused rat liver.

Three hydroxylated metabolites of methadone, 2-ethyl-3-(dihydroxyphenyl)-5-methyl-3-phenylpyrroline (di-HO-EMDP), 2-ethyl-3-(hydroxy-methoxy-phenyl)-5-methyl-3-phenylpyrroline (HO-MeO-EMDP) and 2-ethyl-3-(hydroxy-phenyl)-5-methyl-3-phenylpyrroline (HO-EMDP) were excreted as O-glucuronide conjugates in bile from the isolated perfused rat liver following the addition of either d- or 1-methadone to the perfusate. These glucuronide metabolites were identified as intact molecules by combined gas chromatographic-mass spectrometric (GC-MS) analysis of their permethylated derivatives.

The secretion of methadone and its major metabolite in the gastric juice of humans: comparison with blood and salivary concentrations.

Four healthy subjects and four addicts on high daily maintenance doses of methadone each received a parenteral dose of methadone hydrochloride following an overnight fast. The concentration of methadone in blood was compared with that in the gastric juice obtained over 8 hr by continuous low-pressure suction via a nasogastric tube. The concentration in the gastric juice was 25-200 times that measured at the same time in the blood. Thus, 8 hr after the injection mean blood concentrations of 28 and 210 ng of methadone per ml were recorded in the normal subjects and the addicts, respectively. The corresponding concentrations in gastric juice were 2,200 ng/ml and 18,000 ng/ml, respectively. In the normal subjects about 2% of the administered dose was recovered in the gastric juice in 8 hr, whereas in addicts about 7% was recovered. The greater recovery of methadone from the addicts appears to be the result of the larger volume of gastric juice recovered from the latter subjects. Methadone was also excreted in the saliva of both groups of subjects. In addicts, salivary concentrations were often 10 times those recorded in the blood. The N-monodemethylated metabolite of methadone was identified in the gastric juice of addicts by gas chromatography and mass spectrometry.