Quantitation of hemoglobin F. An interlaboratory study.

Samples of whole blood from four hematologically normal adults and from two individuals with increased fetal hemoglobin levels were shipped to laboratories participating in the 1976 and 1977 Center for Disease Control (CDC) hemoglobinopathy proficiency testing surveys. The data from these surveys were used to evaluate the interlaboratory variability of current methods used to quantitate hemoglobin F (Hb F). Results of Hb F quantitation obtained from more than 100 laboratories than voluntarily participated in the survey were compared with those obtained from 21 reference laboratories. Individual values for all samples varied greatly among laboratories and among methods. Results returned by most of the laboratories were outside two standard deviations of the reference laboratory mean and were not accurate enough to differentiate between a normal level and an increased, abnormal level.     

Quantitation of A2 hemoglobin by polyacrylamide gel disc electrophoresis: a method with individual specimen standardization

A polyacrylamide gel electrophoresis technic for the determination of hemoglobin A2 is presented. Rapid separation is an advantage. The use of a diluted hemolysate as a 4 percent standard avoids overestimation of the A2 fraction by densitometry and also provides a reference for visual comparison. Normal range of A2 hemoglobin by this method is 1.12 to 4.13 per dkg. Coefficient of variation was 5.84 percent.     

HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry. A study of interlaboratory agreement

Immunohistochemistry (IHC) is used commonly for evaluating HER-2/neu protein expression in breast cancer. Given the potential clinical importance of HER-2/neu status in patient management, interlaboratory variability in HER-2/neu IHC results in a matter of legitimate concern. We compared the results from 2 laboratories for HER-2/neu determined by IHC on paraffin sections of the same 100 consecutive invasive breast cancers. Both laboratories used the same primary antibody; however, different methods for heat-induced epitope retrieval (microwave or steam) and immunostaining (automated equipment from different manufacturers) and different scoring systems (positive-negative and 0-4+) were used. Slides were read in a blinded fashion and the results from the 2 laboratories were compared. Of the 93 cases evaluable in both laboratories, 24% were scored as HER-2/neu-positive at 1 laboratory, and 23% were scored as positive at the other. Complete concordance in categorization of HER-2/neu status between the 2 laboratories was achieved in 90 of 93 cases. Excellent interlaboratory agreement for HER-2/neu IHC was attained using the same primary antibody to HER-2/neu, even without standardization of assay method or scoring criteria. However, standardization of these parameters remains an important objective to optimize interlaboratory agreement.     

An interlaboratory study of a candidate reference method for platelet counting

A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 x 10(3)/microL (1-400 x 10(9)/L). Pooled analysis of the serial dilutions showed that RBC-platelet and RBC-RBC coincidence events became negligible at sufficiently high dilutions (i.e., > 1:1,000). All laboratories demonstrated excellent intra-assay and acceptable interlaboratory precision. Two antibodies (CD61 and CD41) were used for identifying platelets and individually gave acceptable results, but in a minority of samples, staining differences were observed. The optimum method thus uses a double-labeling procedure with a final dilution factor of 1:1,000. The study demonstrated that this method meets the criteria for a reference platelet count.     

An immunohistochemical study of hemoglobin A, hemoglobin F, muramidase, and transferrin in erythroid hyperplasia and neoplasia

The bone marrow biopsy specimens of 35 patients with benign and malignant erythroid hyperplasias were examined for the presence of hemoglobin A, hemoglobin F, muramidase (lysozyme), and transferrin, using an indirect immunoperoxidase method (PAP) on Zenker's-fixed paraffin-embedded bone marrow biopsy specimens and particles. Five cases of each of the following entities were studied: erythroleukemia and erythremic myelosis, acute granulocytic leukemia with maturation (FAB M2), polycythemia rubra vera, myeloproliferative syndrome in childhood, megaloblastic anemia (B12 and folate deficiency), erythroid hyperplasia (regenerating bone marrow and hemolytic anemia), and Ph' chromosome positive chronic granulocytic leukemia. Hemoglobin A was present in both the early and late erythroid precursors in all conditions. Hemoglobin F was the predominant hemoglobin in early erythroblasts of pernicious anemia and in both early and late erythroid elements in erythroleukemia and erythremic myelosis. Small quantities of hemoglobin F were present in a few isolated clusters in other conditions. Staining for hemoglobin F may be useful in identifying immature erythroid precursors and in distinguishing some cases of dysplastic erythroid hyperplasia from neoplasia. Additionally, these findings suggest that the maturational switch in hemoglobin synthesis operates with distinct pathways under different conditions.     

An improved method for the quantitation of A2 hemoglobin utilizing cellulose acetate electrophoresis and densitometry.

An improved method of A2 quantitation by cellulose acetate electrophoresis is describes. Densitometry is performed on uncleared membranes. The height of the peak obtained for the A2 band is compared with a standard curve derived from peaks obtained by serial dilutions of hemoglobin. Noral A2 hemoglobin as measured by this method is 2.55 percent plus or minus 0.60 (2 S.D.). Twenty patients samples were run in parallel with a column chromatographic method. Eight samples were found to contain elevated A2 levels by the column method. The same eight samples were correctly identified by the electrophoretic technic.     

An immunochemical investigation of SV40 T-antigens 2. Quantitation of antigens and antibody activities

The interaction of the two major forms of simian virus 40 (SV40) T-antigen, large-T and small-t, with antisera has been studied using immunoprecipitation followed by adsorption on to fixed Staphylococcus aureus Cowan 1. With sera derived from hamster bearing bearing tumors of SV40-transformed cells, the amounts of serum required for optimum precipitation of the two antigens were markedly different. Small-t required 15 to 30 times more serum than large-T. This effect can lead to underestimation of small-t or even failure to detect this species. The amount of small-t synthesized in SV40-infected CV-1 cells at late times after infection is substantial, more than equimolar with respect to large-T. Specific antiserum against the large-T polypeptide also precipitates small-t and the two antigens are immunoprecipitated coordinately, consistent with their sharing antigenic determinants. The only antiserum tested which failed to react with small-t was anti-U serum. Even in a sensitive radioimmunoassay, using the denatured polypeptide purified by gel electrophoresis as probe, anti-U serum had no detectable activity against small-t. Coordinate precipitation of large-T and a 53,000-dalton protein from extracts of SV40-transformed mouse cells did not appear to be due to their having shared antigenic determinants, but rather to the existence of a complex of large-T with the 53K protein. The latter protein appears to be a host protein, unrelated to large-T but complexed with it.     

A report on the interlaboratory quantitation of haemoglobin A2 and haemoglobin F

An interlaboratory trial of the quantitation of Hb A(2) and Hb F has been carried out on two samples of blood by 90 laboratories in the British Isles. The results have been compared with those obtained by four reference laboratories. Overall, the correlation was very poor and indicated that the methods used have to be standardized before interlaboratory values become meaningful. Moreover, in many laboratories the level of Hb A(2) in a patient with proven beta thalassaemia was not reported as being increased.     

An interlaboratory study of the use of PapNet in the quality control of cervico-vaginal cytology]

In this feasibility study of the utilization of the PapNet System (Neuromedical Systems, Suffern, NY) for computer-assisted cervical/vaginal cytology diagnosis, a random sample of 329 negative smears and a series of 68 positive smears reported as such by the Caltagirone laboratory, underwent PapNet review at the Imola laboratory. False-positive (FP) cases (smears originally classified as ASCUS, LGSIL, AGUS, e HGSIL and interpreted as negative on PapNet) and false-negative (FN) cases (reverse discrepancies) were further and conventionally re-evaluated by the staff of the originating laboratory. On PapNet review, there were 16/68 FP cases (23.5%) e 20/329 FN cases (6.1%) with a FP:FN rate ratio of 3.8 (95% confidence interval, 2.2-6.3). At final re-examination of these diagnostic errors, most FP cases (14/16) were confirmed as such whereas the FN cases significantly decreased from 20 to 9, with a final rate of 2.7%. As a consequence, the ratio of the FP rate (14/68) to the FN rate (9/329) rose to 7.5 (4.1-12.6). The study suggests one potential approach to the preliminary utilization of PapNet by those laboratories that are interested in this technology. The results are in accordance with those of the previous PapNet review studies which have generally shown a greater frequency of FP cases compared with FN cases.     

Reliable estimation of hemoglobin A2 concentration by electrophoresis with densitometry.

Elevated hemoglobin A2 (Hb A2) levels can be identified conveniently by densitometry after electrophoresis on cellulose acetate strips. Because a recent report questioned the accuracy of this technic, the method was re-evaluated by paired comparison with microcolumn chromatography. Analysis of 100 patient specimens showed high correlation (r = +0.84), but an average Hb A2 concentration 0.7% higher by densitometry than by chromatography (P less than 0.001). With upper limits set at 4.5%, and 3.8%, respectively, results were divided into "normal" and "high" for each method. Concordant results were obtained in 97 of the 100 cases (82 normal, 15 high). Another densitometer of improved design was used for paired analysis of 50 additional specimens, 25 normal and 25 with beta-thalassemia trait. The two groups were well separated by both procedures, and Hb A2 levels were similar (r = +0.92, P greater than 0.6). This study demonstrates that it is possible, with carefully controlled technics and properly calibrated instruments, to use electrophoresis with densitometry as a reliable means of identifying abnormal Hb A2 levels.     

Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin

In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia from sickle-beta-thalassemia. The purpose of this study is to assess the Hb A2 levels in samples containing sickle hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC-Variant beta-thalassemia Short Program). The blood samples analyzed were from individuals of African descent living in the state of Tennessee who had either sickle cell trait (Hb AS), sickle cell disease (Hb SS), or sickle cell-hemoglobin C disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in samples containing Hb S. The Hb A2 mean in Hb AS samples (n=146) is 4.09% (SD +/- 0.42, range 2.20 to 5.20%); in Hb SS samples (n=33) it is 3.90% (SD +/- 1.08, range 0.60 to 5.90%); and in Hb SC samples (n=27) it is 4.46% (SD +/- 0.70, range 2.30 to 5.91%). The Hb A2 mean by HPLC in normal individuals (Hb AA, n=70) is 2.57% (SD +/- 0.25, range 2.1 to 3.0%), and the Hb A2 range in beta-thalassemia carriers is 4 to 9%. Our results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from beta-thalassemia carriers. The hemoglobinopathy laboratory should be aware of this apparent elevation in Hb A2 levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle cell anemia is made.     

Human interleukin 2. Quantitation by a sensitive radioimmunoassay

Using polyclonal and monoclonal antibodies to human recombinant IL-2 (rIL-2), we developed a sensitive radioimmunoassay (RIA) for quantitation of human IL-2. In this assay, microtitration plates pre-coated with an anti-rIL-2 monoclonal antibody (35H10), recognizing residues 59-72 of human IL-2, are incubated with serial dilutions of test samples. Captured IL-2 is quantitated by adding an affinity-purified rabbit anti-rIL-2 antibody followed by an 125I-labeled goat anti-rabbit IgG. Antibodies to chemically synthesized IL-2 peptides could replace the polyspecific rabbit anti-rIL-2 antibody as the second specific reagent in the assay. This configuration was more sensitive than others tested, approaching the level of detection of the conventional IL-2 bioassay, thus allowing detection of as little as 100-200 pg IL-2. Serum or plasma fluids, however, inhibited the assay, reducing its sensitivity by approximately 5-fold. This RIA correlated well with the conventional bioassay in measuring IL-2 levels in sera from IL-2-treated patients. This, and similar, RIAs may serve as a useful adjunct or alternative to the conventional IL-2 bioassay in detecting and quantitating human IL-2 in culture supernatants and clinical samples.     

Quantitation of hemoglobin with the Vision Analyzer by use of the alkaline hematin reaction

Blood is drawn into capillary tubes containing saponin and the tubes placed into the reagent packs. Hemoglobin is denatured by mixing the hemosylate with a reagent containing lithium hydroxide and a non-ionic detergent. The absorbance is measured bichromatically at wavelengths of 577 and 633 nm. The calibration curve is stable and can be stored for at least 30 days. There are no interferences from fetal hemoglobin, glycosylated hemoglobin (20 percent), hemoglobin S, samples with hematocrits up to 0.55, paraproteins, and lipemia. Specimens with rouleau formation, nucleated and fragmented red blood cells, target cells, ovalocytes, teardrop cells, spherocytes, leukocyte counts of 29 X 10(9) per L and reticulocyte counts of 0.32; Howell-Jolly bodies did not interfere with the assay. The within run and between run precision gave average coefficient or variations of 2.3 and 1.9 percent, respectively. Comparison of the hemoglobin results obtained in 149 samples with the Vision (y) and Coulter Counter System (x) gave r = 0.987, Y = 1.01X - 1.89 g per L.     

Comparison of microchromatography and electrophoresis with elution for hemoglobin A2 (Hb A2) quantitation.

Microcolumns prepared in the authors' laboratory, two commercial microchromatography kits, and electrophoresis with elution were compared for Hb A2 quantitation. Day-to-day imprecision of microchromatographic methods was similar (CV 4.7--6.6%) and somewhat less than electrophoresis with elution (CV 8.0--9.1%). Both commercial kits showed variable imprecision in different lots; one lot of Kit B gave erratic results due to resin leakage. From 49 patient specimens, Kit A microcolumns and those of the authors identified the same 14 patients with an elevated percentage of Hb A2 and showed good correlation (P = 0.90), although Kit A showed constant bias toward higher values. Electrophoresis with elution resulted in a false-positive and a false-negative value, did not correlate well with microcolumns (P = 0.78 and 0.76), and showed proportional bias toward lower values for an elevated percentage of Hb A2. Commercial kits were convenient, relatively quick, and cost-effective. Frozen, stabilized hemolysates performed well for quality control.