Laboratory recognition of a rare hemoglobinopathy: hemoglobins SS and SG(Philadelphia) associated with alpha-thalassemia-2.

This article describes the laboratory investigation of an unusual hemoglobinopathy involving hemoglobin (Hb) S, HbSG(Philadelphia), and alpha-thalassemia-2 in a patient whose phenotype was HbSC by alkaline electrophoresis. Findings of a mean corpuscular volume of 62 fL and microcytes on the blood smear were inconsistent with HbSC disease. The patient's clinical course over several years had been mildly symptomatic. Testing in our hospital laboratory using isoelectric focusing and cation-exchange high-performance liquid chromatography to separate hemoglobins showed an unknown variant. Additional studies, including globin chain electrophoresis, reverse-phase high-performance liquid chromatography, and polymerase chain reaction-based DNA analysis were performed at reference laboratories, which reported the following findings: HbG(Philadelphia) associated with alpha-thalassemia-2, HbS and HbG(Philadelphia), and the alpha-globin deletions defining the -alpha3.7/-alpha3.7 genotype. The hemoglobin molecular defects, alpha-thalassemia-2, and the pattern of inheritance are discussed.

Two sensitive immunometric assays for serum thyroid stimulating hormone evaluated.

This study was conducted to evaluate the analytical performance (functional sensitivity, reproducibility, parallelism, and accuracy) of two recent commercial kits marketed as third generation immunometric assays for measuring serum thyroid stimulating hormone (TSH). One assay is automated; the other is manual. Accuracy by method comparisons was evaluated using 86 patient samples assayed by an established third generation immunometric assay as the comparative method. The new assays met the third generation criterion for functional sensitivity (CV < or = 20 percent at TSH < or = 0.02 mIU/L), were reproducible (CVs < 11 percent), and measured serum TSH in parallel with the calibrator curves. Linear regression analysis of the intermethod comparison data showed highly correlated (R > .095) results; however, the regression slopes were non-unity, indicating patient sample results were not transferable between methods. Clinical laboratories choosing a third generation TSH assay should validate the performance characteristics of the selected method to ensure reliable results for patient care.

Identification and quantification of hemoglobins A, F, S, and C by automated chromatography.

The Bio-Rad Variant Hemoglobin Testing System is an automated HPLC analyzer marketed with a Beta-thalassemia Short Program to quantify hemoglobins (Hbs) F and A2 and assist in detecting Hbs A, S, C, D, and E. We evaluated this system to replace several traditional methods for Hb in our hospital laboratory. Analytical performance relevant to quantifying Hbs A, S, C, and F was assessed with blood samples obtained from our local patient population. Studies of precision (CVs < 3%) and analytical limits (% of total Hb) of Hbs A (2-86%), F (1-89%), S (5-90%), and C (3-92%) demonstrated results comparable with or exceeding those of traditional methods. Results for patients' samples (n) for Hbs A (107), F (157), S (128), and C (27) correlated well (r > 0.93) with results by traditional methods. The satisfactory performance and efficiency led us to implement this system for routine quantification of clinically significant Hbs.

Comparison of liquid and dried blood for neonatal hemoglobinopathy screening: laboratory and programmatic issues.

OBJECTIVES: To compare laboratory and programmatic issues in neonatal hemoglobin screening in two systems using either liquid cord blood or heel puncture blood dried on filter paper (DB) to determine the accuracy of hemoglobin phenotype; the collection rate of cord blood versus DB; and the types of and reasons for errors. METHODS: Cord blood samples for 6904 newborns were analyzed by electrophoresis in a large hospital laboratory. DB samples on the same cohort were analyzed by isoelectric focusing in a state public health laboratory. RESULTS: Interlaboratory concordance was 99.4% for 6904 matched specimen pairs, which included 27 disease and 596 carrier phenotypes. In 42 pairs, discordances for potential disease phenotypes occurred with cord blood samples. Errors involving carrier phenotypes occurred in 15 cord blood samples and 14 DB samples. Inconclusive results requiring repeat testing occurred more often in the cord blood testing system (92) than in the DB system (23). Clerical transcriptions and limitations of the techniques accounted for most laboratory errors. Noncompliance in sample collection occurred more frequently with cord blood (181) than DB (86). CONCLUSIONS: Both systems are subject to errors, but are equally reliable for neonatal hemoglobinopathy screening.

Analytical evaluation of methods for serum creatine kinase-MB. Electrophoresis, immunoinhibition and solid phase separation.

The purpose of this study was to evaluate immunoassay methods for the measurement of serum cardiac creatine kinase isoenzyme (CK-MB) with respect to sensitivity and specificity. The CK-MB electrophoretic assay (Helena Laboratories) was used as the reference. Two principles of immunoassay were included in the evaluation,--immunoinhibition and solid phase separation. The direct immunoinhibition techniques were from Beckman Instruments (CKMB reagent) and DuPont Medical Products (CKMB). Three solid phase separation techniques were from Abbott Laboratories (IMx CKMB), DuPont (acaPlus MCKMB), and Tosoh Medics Inc. (AIA-Pack CKMB). The electrophoretic method for separation of the CK isoenzymes has good specificity but lacks sensitivity for CK-MB in low concentrations. The immunoinhibition methods lack specificity and correlate poorly with the electrophoresis method and with the solid phase methods. The solid phase separation techniques are highly sensitive and show an excellent correlation with electrophoresis when based on specificity. The solid phase separation methods correlate well with each other.

Increased whole blood viscosity during coronary artery bypass surgery. Studies to evaluate the effects of soluble fibrin and poloxamer 188.

This study was designed to test the hypothesis that soluble fibrin complexes resulting from the trauma of surgery could produce elevated blood viscosity, to characterize the soluble fibrin polymers, and to evaluate in vitro the effect of a new hemorheologic agent, poloxamer 188, on viscosity in these abnormal situations. Ten patients undergoing aortocoronary bypass surgery were studied before and at various times after surgery. By 6 h after surgery, the mean hematocrit decreased by 23%, fibrinogen decreased 48%, and erythrocyte sedimentation rate decreased 33%, whole blood viscosity at a low shear rate rose on average of 69% and soluble fibrin rose 118%. Over the 6-day observation period, the concentrations of soluble fibrin paralleled the changes in viscosity, whereas the concentrations of fibrinogen varied nearly inversely with viscosity. The effects of various forms of fibrinogen and fibrin were tested by additions to normal blood. Soluble fibrin polymers, but not fibrin monomers, increased blood viscosity two to three fold. Poloxamer 188 reduced the viscosity of all patient samples to the normal range. These data support the hypothesis that increased whole blood viscosity at low shear rates is caused by hydrophobic adhesion of fibrin polymers to red cells and that poloxamer 188 normalizes viscosity by effectively disrupting the weak hydrophobic bonds.

Precision and reliability of paraprotein determinations by high-resolution agarose gel electrophoresis.

Agarose gel electrophoresis has recently replaced cellulose acetate electrophoresis as the preferred technique for monitoring paraprotein levels in patients with plasma cell dyscrasias. The authors studied the accuracy and precision of this method for paraprotein determination. Twenty-seven serum samples with paraprotein concentrations ranging from 5 to 73 g/L were aliquotted and assayed on 20 separate occasions, and the mean and standard deviation for the paraprotein concentration in each serum was established. Linear regression analysis showed that the standard deviation of paraprotein concentration (SD) increased as a function of paraprotein concentration (PC). For IgG paraproteins, the regression equation was SD = 0.041 (PC) + 1.06; R = 0.942; standard error = 0.32. For non-IgG paraproteins the equation was SD = 0.101 (PC) - 0.04; R = 0.851; standard error = 0.5. The accuracy of paraprotein determinations by the agarose gel electrophoretic technique was assessed by comparison with values obtained with the use of a previously validated enzyme-linked immunosorbent assay (ELISA) method for quantitation of IgG subclasses. Results obtained by the two methods were similar and highly correlated: (concentration by electrophoresis) = 0.921 (concentration by ELISA) + 0.46; R = 0.988; standard error = 0.34. The laser densitometric scanning procedure showed a loss of linearity above 60 g/L, indicating the need to dilute sera with very high paraprotein concentrations in order to obtain accurate results. A table is presented that should help pathologists who interpret such scans to determine whether small changes in paraprotein measurements occurring over time represent true changes in paraprotein concentration or merely reflect the analytic variability inherent in the technique.

IgG subclass distribution in patients with multiple myeloma or with monoclonal gammopathy of undetermined significance.

Multiple myeloma provides a unique model for studying factors affecting IgG isotype distribution in humans. Evaluations were made as to whether monoclonal immunoglobulins (M-proteins) of different IgG isotypes are associated with different extents of hypogammaglobulinemia and whether all residual subclasses are decreased comparably. The isotype patterns were analyzed in the context of the gene order of the constant regions of gamma (gamma) heavy chains on chromosome 14. Using monoclonal antibody-based immunoenzymometric assays, IgG subclasses were quantitated in the sera of 50 patients having IgG M-proteins, 38 with multiple myeloma and 12 with monoclonal gammopathy of undetermined significance. Thirty-three (66 percent) patients had IgG1, nine (18 percent) had IgG2, four (8 percent) had IgG3, and four had IgG4 M-proteins, paralleling the normal IgG subclass distribution. The concentration of residual IgG (sum of the evaluatable polyclonal IgG subclasses) was significantly decreased in patient sera (p less than 0.05). However, in only seven (14 percent) of the patients were all three subclasses below the reference range, suggesting some selectivity of immunosuppression. Patients with M-proteins of different IgG subclasses had markedly different patterns of suppression. Patients with IgG2 M-proteins (78 percent) were more likely to have depressed residual IgG than patients with IgG3 (50 percent), IgG1 (27 percent) or IgG4 (0 percent) M-proteins. Some patients had deficits of only one or two IgG subclasses. When considering all sera together, residual IgG1 was disproportionately reduced, followed by residual IgG2, IgG3, and IgG4. Next it was determined whether or not patterns of suppression were predicted by the gamma heavy-chain gene order (5' to 3'): gamma 3, gamma 1, gamma 2, gamma 4, as seen in some other immunologic disorders. Interestingly, the normal isotypes encoded by genes in juxtaposition to that of the M-protein were most often decreased (p less than 0.05). Thus, the patterns of hypogammaglobulinemia in multiple myeloma are heterogeneous. They may be influenced by the M-protein itself, possibly through interactions with regulatory cells. In addition, factors at the gene rearrangement level may contribute.

Human immunoglobulin G and immunoglobulin G subclasses: biochemical, genetic, and clinical aspects.

Human IgG consists of two identical heavy (H) chains and two identical light (L) chains joined by interchain disulfide bridges. Heterogeneity in the amino acid sequences of the H and L polypeptides results in at least three types of IgG variants at the structural and genetic levels. The four isotypic forms are IgG1, IgG2, IgG3, and IgG4, which share extensive homologies in the primary structure of their H chains. As a result, the subclasses cross-react antigenically, but they can be differentiated on the basis of subtle architectural dissimilarities. The biological and effector properties of the IgG isotypes have been associated, in part, with their structural differences. Genes determining the synthesis of human IgG heavy chains are located on chromosome 14. In several clinical situations the isotypes appear to be regulated or expressed in patterns reflecting the gene arrangement. The numeric designations of the subclasses correspond to the order of their proportional amounts in healthy adult serum: IgG1 greater than IgG2 greater than IgG3 greater than IgG4. Awareness of the importance of the roles of the four IgG isotypes in human health has steadily increased since they were first described in the 1960s. The recognition that deficits or increases in selected IgG subclasses may have clinical consequences has prompted considerable interest in quantifying the four isotypes in clinical specimens. In particular, deficiencies of IgG2, IgG3, and IgG4, singly or combined, are associated with chronic infections which may not be readily recognized in otherwise healthy people with normal serum total IgG concentrations. Different assay methods using polyclonal or monoclonal antisera with various calibrants have been applied; however, no standardized method exists at the present. IgG deficits are associated with gene defects and are acquired in secondary immunodeficiencies in conjunction with other disorders. IgG isotype selectivity has been recognized in autoimmune diseases and in response to carbohydrate and protein antigens derived from pathogenic microorganisms and common allergens.

Monoclonal antibody-based solid-phase immunoenzymometric assays for quantifying human immunoglobulin G and its subclasses in serum.

We developed quantitative immunoenzymometric assays for human IgG and its subclasses by using monoclonal antibodies, an avidin-biotin detection system and, as the calibrant, the U.S. National Reference Preparation for Specific Human Proteins. The assays are sensitive (detecting as little as 6 micrograms/L), precise (average inter-assay CV less than 11%), and vary linearly with concentrations over a five- to 10-fold range, depending on the monoclonal antibody. We evaluated 22 different monoclonal antibodies, many of which remained highly reactive when immobilized in wells of microtiter plates coated with bovine serum albumin-glutaraldehyde to "capture" total IgG or subclasses of IgG in the sample. We demonstrated the specificity of the most reactive antibodies by using a panel of 20 purified myeloma proteins. The sum of IgG subclass concentrations correlated well (r = 0.84, p less than 0.001) with the total IgG measured in sera from 63 apparently healthy adults (26 men, 37 women). We estimated 95 percentile reference intervals for the immunoglobulins in these subjects and determined the following mean percentage distributions of IgG subclasses: IgG1 49, IgG2 33, IgG3 9, and IgG4 7. The availability of these assays should facilitate studies of the clinical significance of the subclasses.

The effect of storage conditions on ion exchange and affinity chromatographic assays for glycated hemoglobin.

Experiments were performed to evaluate the effect on glycated hemoglobin determinations when erythrocytes were stored under various conditions. We studied the influence of time, temperature, glucose concentration, and pH on results obtained by three commercially available column chromatographic procedures designed to quantify glycated hemoglobin. An affinity chromatographic procedure which measures total glycated hemoglobins (GHb) was compared with two ion-exchange column methods. One ion-exchange method measures total hemoglobin A1 and the other is purported to measure hemoglobin A1c. Stability studies were performed using specimens from nondiabetics and diabetics, supplemented in vitro with glucose at pH 7.4 and pH 5.0. Results obtained from the A1c and the GHb tests were unchanged over time for samples stored under the described conditions. Erythrocytes were incubated with glucose to produce labile glycated hemoglobin and assayed before and after overnight incubations in saline. These experiments showed that the A1c and GHb assays were unaffected by the presence of labile glycated hemoglobin. Column fractions were analyzed by isoelectric focusing and scanned with a laser densitometer for quantitative assessment of hemoglobin species found in each fraction. These experiments showed that the three column methods measure very different hemoglobin species. Focusing of eluates from the A1 and GHb tests revealed carryover of several hemoglobin species into the measured fractions, while the A1c test was found to be much more specific for HbA1c. Our data suggest that it may be possible to analyze glycated hemoglobins in samples transported to a reference laboratory without special treatment.