Reliability of polymerase chain reaction in the detection of human immunodeficiency virus infection in children.

During a 3-year period we followed 128 human immunodeficiency virus (HIV) antibody-positive children ages 6 days to 11 years clinically and with an HIV diagnostic panel consisting of antibody (by enzyme-linked immunosorbent assay and confirmed by indirect fluorescence assay or Western blot), p24 antigen detection, HIV isolation from peripheral blood culture and molecular detection of HIV nucleic acids by polymerase chain reaction (PCR). The PCR procedure included 30 cycles of amplification using two separate gag primer pairs (SK38/39 and SK101/145), followed by detection with probes to areas amplified (SK19 and SK102). Results of PCR were available within 48 hours and were sensitive (97%) and specific (100%). PCR should be obtained on all children exposed perinatally, and aggressive management should be undertaken for those found to be positive.

A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-1.

The sensitivity and specificity of the polymerase chain reaction (PCR) for the detection of HIV-1 proviral DNA was determined in five laboratories with extensive experience in PCR testing. Five panels consisting of 105 HIV-1-seronegative specimens from regularly repeating blood donors with no risk factors for HIV infection and 99 HIV-1-seropositive and culture-positive specimens from a cohort of homosexual/bisexual men were sent under code to each laboratory. Amplification procedures and testing algorithms by which specimens were judged positive, negative, or indeterminate varied between laboratories. The average sensitivity for the five laboratories was 99.0%, with two laboratories achieving 100%. The average specificity was 94.7%, varying between 90.5 and 100%. The overall false-positive rate was 1.8%, the false-negative rate was 0.8%, and the indeterminate rate was 1.9%. Of 1,005 determinations made by the five laboratories, 32 (3.2%) were misclassifications. Most of the classification errors occurred in specimens from uninfected individuals and were distributed among the laboratories in such a way as to indicate laboratory error rather than the inherent reactivity of some samples. This emphasizes the need for standardization of PCR testing and caution in interpreting positive PCR reactions in HIV-1-seronegative persons.

Detection of human immunodeficiency virus DNA using the polymerase chain reaction in a well-characterized group of homosexual and bisexual men.

The polymerase chain reaction (PCR) for human immunodeficiency virus type 1 (HIV-1) DNA was performed on specimens from 197 homosexual and bisexual men enrolled in studies of HIV-1 infection. Thirty cycles of amplification were conducted, followed by detection with probes corresponding to two gag primer pairs (SK 38/39 and SK 101/145). Of 107 men who were HIV-1 antibody-negative, 105 (98%) were PCR-negative. Two who were initially PCR-positive antibody-negative were PCR- and antibody-negative on repeat testing of both the same specimen and specimens drawn 8-10 months later; this suggests that the first PCR results were false-positive. Of 90 men who were antibody-positive, PCR was positive in 87 (97%), including all 13 with AIDS, all 22 with AIDS-related conditions, all 11 with generalized lymphadenopathy only, and 41 (93%) of 44 without signs or symptoms of HIV-1 infection. On repeat testing, all 3 PCR-negative, antibody-positive men were PCR-positive. In this population and with this technique, PCR had excellent agreement with the HIV-1 antibody test.

Preparation of white cell-depleted red cells for 42-day storage using an integral in-line filter.

A new blood pack system for the preparation of white cell-depleted red cells was studied. The system is a modified additive-solution quadruple-unit blood pack that incorporates a cellulose-acetate fiber depth filter in-line between the AS-3 additive bag and the CP2D collection bag. Mean +/- SD white cell removal from 156 units processed under standard production conditions was 97.7 +/- 2.7 percent; residual white cells were 1.1 +/- 1.0 x 10(8) per unit. Red cell loss was 10.0 +/- 1.0 percent (n = 43). Mean platelet removal was 80.9 percent from units from which platelet concentrates were not prepared (n = 47). Microaggregates did not form during storage, and hemolysis of filtered red cells was lower than that of unfiltered controls. Filtered AS-3 red cells stored for 42 days had a 51Cr survival of 80.1 +/- 5.7 percent (mean +/- SD) as compared with 78.9 +/- 6.2 percent for unfiltered controls (n = 17). This in-line filter system provides white cell-depleted, microaggregate-free red cells that can be stored for 42 days.

Extended survival of neocytes produced by a new system.

Red cells (RBCs) prepared by a new system using centrifugation to produce neocyte enrichment were studied in two laboratories. The system used a blood bag with a geometric configuration such that younger, less dense cells could be separated from older, denser cells. Phthalate ester density gradient curves determined that neocyte enrichment was 81.3 percent in one laboratory and 82 percent in the other. RBC viability was studied by 51Cr autologous transfusion in normal volunteer donors. A randomized, paired design was used in which each donor was transfused once each with neocytes and with RBCs of all ages. The mean control half-life was 34.8 +/- 5.4 days in one laboratory and 34.0 +/- 3.6 days in the other. The mean half-life of the neocyte-enriched RBCs was 45.2 +/- 8.2 days in one laboratory and 45.1 +/- 4.4 days in the other. This represented a more than 30 percent increase in half-life for the neocyte-enriched RBCs, a significant difference. This new system, a two-bag set that costs +15, allows the simple, efficient separation of neocyte-enriched RBCs that would have a longer half-life and could reduce the transfusion requirement in patients receiving chronic transfusion therapy.

Five-week red cell storage with preservation of 2,3 DPG.

The 2,3 diphosphoglycerate (2,3 DPG) content of red cells stored in current anticoagulant-preservative products decreases rapidly after the first few days of storage, and by 3 weeks the red cells are essentially depleted of 2,3 DPG. Because ascorbic acid and ascorbate-2-phosphate (A-2-P) are effective in maintaining erythrocyte 2,3 DPG during liquid preservation, ascorbate was stabilized through autoclaving and subsequent storage by adding it as the trisodium salt of A-2-P to a phosphate-adenine-saline solution at a pH of 8.5 to 9.0. Red cell concentrates prepared from blood drawn into citrate-phosphate-double-dextrose were supplemented with the A-2-P additive solution (AS-4) and studied in vitro and in vivo. Mean 2,3 DPG values for 22 units were 147.6, 113.5, and 82.3 percent of initial value after storage for 3, 4, and 5 weeks, respectively. Maintenance of 2,3 DPG was at the expense of adenosine triphosphate (ATP), which fell to as low as 22.2 percent of initial value after 5 weeks. Despite the low ATP values, the 24 hour 51Cr-labeled red cell recoveries averaged 80.8 and 74.1 percent after 4 and 5 weeks of storage, respectively. The AS-4 system provides a red cell product with acceptable viability and improved oxygen off-loading function.

Proposed standardization of methods for determining the 24-hour survival of stored red cells.

Two methods of determining the survival of stored red cells are described: one using a double label and one using a single label. It is not suggested that one is superior, but rather both methods are presented in the hope that the personnel of laboratories engaged in determining the effectiveness of anticoagulant-preservation will follow one of the protocols presented. Were this to occur, many of the interlaboratory variations that have decreased the value of data from red cell survival studies would be decreased.

In vivo viability of red blood cells stored in CPDA-2.

The marginal viability of erythrocytes stored for 35 days as red blood cell concentrates in citrate-phosphate-dextrose-adenine-one (CPDA-1) was attributed to inadequate nutrient support with adenine and glucose. In an effort to improve the viability of red blood cells following extended storage, a new CPD-adenine and 1.4 times more glucose than CPDA-1. The efficacy of CPDA-2 was evaluated in vivo by measurement of 24-hour postinfusion recovery of 51Cr-labeled erythrocytes which had been stored as whole blood or red blood cell concentrates for 5 to 8 weeks. All red blood cell concentrates, and the whole blood units stored for 35 and 42 days, were held at room temperature for 8 hours prior to processing and/or refrigeration. CPDA-2 yielded significantly higher 51Cr survivals than CPDA-1 and exceeded the accepted criterion for anticoagulant preservative efficacy of 70 percent postinfusion survival of red blood cells after storage for a period of 42 days. Preliminary data supports possible usage to 49 days. Plasma glucose and red blood cell ATP concentration were maintained better in CPDA-2 than in CPDA-1. When compared to historical controls for CPD and CPDA-1 the data suggest that red blood cells stored in CPDA-02 will have superior viability throughout the entire storage period. CPDA-2 is a candidate to replace CPDA-1 as the anticoagulant-preservative solution of choice for red blood cell concentrates.

Effect of delayed refrigeration on plasma factors in whole blood collected in CPDA-2.

Extension of the time within which whole blood may be separated into components offers logistic advantages for the operation of remote mobile drawing teams. We evaluated the effect of an 8-hour hold of whole blood at room temperature before preparation of components. Plasma coagulation activity and opsonic factor content were studied in 14 units drawn into the anticoagulant-preservative solution citrate-phosphate-dextrose-adenine (CPDA-2). At the time of collection, an additional 7-ml aliquot was drawn into 1 ml of CPDA-2, the plasma separated and frozen immediately. Components were prepared from whole blood units allowed to rest undisturbed at 22 +/- 1 degrees C for 8 hours. After 8 hours, a significant decrement of about 10 percent was found in the concentration of fibrinogen, plasminogen, fibronectin, and activity of Factor V. Factor VIII activities (VIIIAHF and VIIIAGN) were not significantly different after 8 hours. Our results indicate that room temperature storage for 8 hours before component processing has minimal effects on potentially labile plasma protein factors using CPDA-2 anticoagulant-preservative solution.

Platelet counts during rapid massive transfusion.

The effect of rapid massive transfusion upon platelet counts has been studied retrospectively in 24 patients treated for severe trauma. Multiple linear regression analysis showed that a platelet dilution model with an exponential form described the quantitative relationship between the pre- and posttransfusion platelet counts and the volumes of blood products administered. Intravenous salt solutions had very little effect upon the platelet count even when large volumes were infused. Red blood cell transfusions decreased the platelet count to an extent two and one-half times greater than that of colloid infusions. The differences in the magnitudes of the dilution effects are attributable to the different physiologic volumes of distribution of the various classes of infusates. If only red blood cells (RBC) and fresh frozen plasma (FFP) are transfused, two useful equations obtain. First, the current platelet count (Pt) can be predicted knowing the initial platelet count (Po) and the number (#) of units of RBC and FFP administered: Pt = Po (0.634) e-0.046 [#RBC + #FFP/8]. Second, the point during massive transfusion when platelet transfusions will be necessary can be predicted knowing the initial platelet count.

Red blood cell membrane abnormalities during storage: correlation with in vivo survival.

Experiments with citrate-phosphate-dextrose supplemented with adenine, 0.5 mM final concentration (CPD-A2), provided an opportunity for study of red blood cells (RBC) membrane storage lesions which could limit extension of storage. Impairment in drug-induced endocytosis in intact RBC occurred with storage, but the changes produced did not quantitatively predict or reflect the 24-hour in vivo 51Cr-labelled RBC survival. Abnormalities in RBC membrane protein did appear after storage, but these alterations neither paralleled nor predicted the in vivo 24-hour 51Cr-labelled RBC survival. The RBC membrance protein changes were not reminiscent of those produced by either acute adenosine triphosphate depletion, oxidative attack, or calcium accumulation. Therefore, while storage produces significant alterations in RBC membrane protein and function, the changes detected were not those that determined in vivo RBC survival.

Comparative effects of phosphoenolpyruvate on selected mammalian erythrocytes.

1. Incubation of human, rat, cow, sheep, dog, rabbit and monkey erythrocytes with phosphoenolpyruvate (PEP) resulted in increased intracellular 2,3-diphosphoglycerate (2,3-DPG). 2. Physiologic temperature (37 degrees C) and a pH less than 6.5 were required for transport and metabolism of PEP in rat and monkey erythrocytes. 3. Although erythrocytes from all species (except pig) exhibited PEP transport and metabolism, hemoglobin oxygen affinity (HOA) was affected only in species whose hemoglobins are sensitive to 2,3-DPG. 4. These results suggest that the effect of PEP incubation on HOA is mediated through 2,3-DPG.

Some properties of blood stored in anticoagulant CPDA-1 solution. A brief summary.

Approval of the new anticoagulant preservative, CPDA-1, was based in part on data from human clinical trials of CPDA-1 performed by six cooperating laboratories and two blood bag manufacturers. Biochemical and red blood cell survival properties of 123 units of blood stored for 28 to 35 days in CPDA-1 (as reported by cooperating laboratories) are briefly summarized.

The significance of 2,3-DPG in red blood cell transfusions.

This review will begin by giving the highlights of the history and explain development of the basic science knowledge of hemoglobin chemistry, function, and physiology. The necessary involvement of red cell metabolism, as it pertains to the maintenance of 2,3-diphosphoglycerate (2,3-DPG) levels, both normally and under the perturbed and experimental conditions of blood storage, will be given as part of the basic science data. The clinical science and transfusion data will comprise the main critical aspects of the paper. Analysis and comment of over 20 studies will be given on the effects of animal and human transfusions with altered 2,3-DPG levels. Decreased survival and organ function have been demonstrated with transfusion of low 2,3-DPG red cells, with or without anemia, in the conditions of exercise, shock, hypotension, ischemia, cardiac surgery, hypoxia, sepsis, and acidosis. By critical analysis of these studies, recommendations on general and specific patient needs for red cell transfusions with normal or high 2,3-DPG levels are given.