The UK National External Quality Assessment Scheme in Blood Group Serology. Compatibility testing 1981–1982: performance and practice

Summary. The design of exercises of compatibility testing was modified in 1981 in order better to accommodate participants' serological practices. Ten reference laboratories were also enrolled in order to determine the ‘correct’ results for each exercise. In 1981–1982 3.5 to 36% of participants missed incompatibilities in exercises in which undiluted antibodies were issued and this did not represent an improvement over performance obtained in 1979–1980. Surveys were undertaken of antiglobulin test procedures and revealed that serological practices continue to change, old techniques are being modified, new techniques are being employed but standardization shows little overall improvement. Surveys of quality control procedures and of cross-match procedures for agglutination in albumin and for agglutination of enzyme treated cells show equal lack of standardization.     

Hemolytic Anemia with Cold Detectable IgG Antibodies

Erythrocytes obtained from patients whomanifest autoimmune hemolytic anemiacan be divided into at least three categories by the nature of their proteincoats as determined by direct antiglobulin (Coombs) test: IgG alone, IgG andcomplement (C), C alone. IgG antibodieswere detected by direct Coombs Test at4°C but not at 37°C in patients of type3 A.H.A. Experiments at 4, 10, 15, 20,30 and 37°C demonstrated that the IgGantibody was not eluted from the redcells at 37°C but apparently underwenta configurational change above 10°Csuch that agglutination no longer occurred with the Coombs reagent. Thischange was reversible. The presence ofcold detectable IgG antibodies providesa mechanism for C deposition on erythrocytes in some cases of A.H.A., ostensibly due to complement alone.

Submitted on May 5, 1970 Revised on June 12, 1970 Accepted on June 15, 1970     

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

Summary When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group 0-test red cell panels at 37°C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.     

Hemolytic anemia associated with multiple autoantibodies and low serum complement

A 37 year old woman with extravascular hemolytic anemia had a positive Monospot test associated with positive antiglobulin and anticomplement Coombs' tests, cold agglutinins and warm autoantibodies. IgG-kappa (κ) antibodies, which reacted with all panel red cells at 37 °C, were eluted from her circulating red cells. However, neither immunoglobulins nor C3 was detected after her serum was adsorbed with heterologous red cell stroma at 37 °C and eluted at the same temperature in glycine buffer. In contrast, IgM-κ and IgM-Iambda (λ), IgG3-κ, IgG4-λ, IgA-λ and C3 were eluted at 37 °C from heterologous red cell stroma after adsorption with her serum at 0 °C. Thus, antibodies of several types, which were present in the patient's serum, reacted optimally with red cell antigens at low temperature. Cold-reactive IgG3-κ antibodies, which were also capable of interacting with red cells at 37 °C, probably accounted for the IgG-κ antibodies eluted from the patient's circulating red cells.The patient's serum C4 titers were decreased, with low normal to moderately depressed C3 and low normal C5, indicating that the anti-red cell IgM and/or IgG3-κ antibodies probably fixed complement.A localized cold stress test resulted in a transient increase in plasma hemoglobin and a decrease in serum C3 titer. These findings, and the beneficial clinical response obtained with small doses of prednisone, suggest that both the cold-reactive antibodies and the IgG-κ on circulating red cells were pathophysiologically significant.This is the first report of a patient with multiple red cell autoantibodies in whom serum complement component titers were determined in conjunction with characterization of the anti-red cell immunoglobulins. Subclinical infectious mononucleosis may have preceded the prolonged hemolytic episode. Clinical evidence of systemic lupus erythematosus has not appeared.     

Direct antiglobulin (“Coombs”) test-negative autoimmune hemolytic anemia: A review

We have reviewed the literature to identify and characterize reports of warm-antibody type, autoimmune hemolytic anemia in which the standard direct antiglobulin reaction was negative but a confirmatory test indicated that the red cells were opsonized with antibody. Three principal reasons account for the absence of a positive direct antiglobulin test in these cases: a) IgG sensitization below the threshold of detection by the commercial antiglobulin reagent, b) low affinity IgG, removed by preparatory washes not conducted at 4 °C or at low ionic strength, and c) red cell sensitization by IgA alone, or rarely (monomeric) IgM alone, but not accompanied by complement fixation, and thus not detectable by a commercial antiglobulin reagent that contains anti-IgG and anti-C3. In cases in which the phenotype is compatible with warm-antibody type, autoimmune hemolytic anemia and the direct antiglobulin test is negative, an alternative method to detect low levels of IgG sensitization, use of 4 °C, low ionic strength washes to prepare the cells for the direct antiglobulin test reaction to permit retention and identification of low affinity IgG antibodies, and, if the latter are uninformative, testing for sensitization with an anti-IgA, and, if necessary, an anti-IgM reagent identifies cases of warm-antibody type, immune hemolysis not verified by a commercial reagent.     

The role of macromolecular components in anti-gamma, alpha, mu, kappa, lambda, C3 and C4 antiglobulin sera

Summary . Studies are described of the early and late responses of rabbits immunized with human IgG protein. Early macroglobulins (19S) antiglobulin antibody is compared with 7S antibody in tests against red cells strongly and weakly coated by IgG anti-Rh(D) antibody. The 19S antibody, when reacted on a glass plate, is slower to produce maximum agglutination than 7S antibody, is enhanced by 4% human serum albumin, and is of at least equal sensitivity in detecting very weakly sensitized erythrocytes. The 19S antibody shows no loss of potency during 1 yr storage at - 20°C, but is unstable beyond 6 mth storage at 4°C. The 19S antibody is non-precipitating against its specific antigen, which complicates assessment of its specificity. It is shown that admixture of 19S with 7S antiglobulins alters the manifestation of the prozone characteristic of the 7S antibody alone. Macroglobulin antiglobulin reagents raised against human γ, α, μ, K, λ, C₃ and C₄ antigens are compared with 7S reagents of similar specificities in tests against coated cells from 14 patients with acquired autoimmune haemolytic anaemia (AAHA), three patients receiving methyldopa, and against normal cells sensitized with anti-D, anti-Fy^a, anti-Kell, anti-Le^a and anti-Jk^a isoantibodies. The results put macroglobulin antiglobulin reagents in perspective in relation to the broad field of antiglobulin testing.     

Preparation and evaluation of a glycerol-preserved direct agglutination antigen for long-term preservation: a comparative study of the detection of anti-Leishmania infantum antibodies in human and dog

ObjectiveTo prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum (L. infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination tests (FD-DAT) that use freeze-dried antigen.MethodsGlycerol-preserved DAT antigen was prepared and stored at different temperatures. We tested antigen stored at 4 °C, 22-37 °C and 50 °C over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009, and 80 of these samples were pooled to produce sera. Each pooled serum contained 10 sera. All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT, FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens; tests were performed on both human and dog sera over a period of 12 months.ResultsThere was strong agreement between the results obtained using GP-DAT and FD-DAT antigens stored at 22–37 °C for 12 months for both human (100%) and dog (100%) pooled sera. The direct agglutination test results were highly reproducible (weighted kappa: GP=0.833, FD=0.979 and FF=0.917).ConclusionsBecause GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field, this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.     

Mild and moderate hypothermia increases platelet aggregation induced by various agonists: a whole blood in vitro study

The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30–34°C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37°C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate® at five different test temperatures between 30°C and 34°C. Aggregation responses at 37°C served as controls. At temperatures of mild and moderate hypothermia (30–34°C), overall platelet aggregation was increased compared to 37°C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31°C and 34°C and in response to adenosine diphosphate between 30°C and 34°C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30°C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30°C and 34°C requires further detailed study.     

An Investigation into the Effect of Reagent Volume on the Spin Indirect Antiglobulin Test

Three polyspecific antiglobulin reagents were tested in a spin antiglobulin technique with 51 antibodies of varying blood group specificities in which the volume of antiglobulin reagent used ranged from 1 volume (35 microliters) to 3 volumes (105 microliters). Statistical analysis of results showed that a significant decrease in avidity and agglutination scores occurred as the volume of antiglobulin reagent used was increased ('volume effect'). The volume effect was shown by 33/51 (65%) of antibodies with all three antiglobulin reagents and by 48/51 (94%) of antibodies with at least one antiglobulin reagent. Only 3/51 (6%) failed to show the volume effect with all three antiglobulin reagents.     

Red cell alloimmunization in multitransfused patients and multiparous women

Purpose  To determine the incidence of alloimmunization against red cell antigens and the thermal amplitude and specificity of antibodies in multitransfused patients and multiparous women. Methods  Antibody screening was performed in 30 nontransfused orthopedic surgery cases (control), 504 multitransfused patients and 325 multiparous women. Antibody screening at 4°C, 22°C and 37°C was carried out in a saline phase, by indirect antiglobulin technique (IAT), using papain cystein, low ionic strength solution (LISS) and polyethylene glycol (PEG). Results  In multitransfused patients IgM antibodies were more frequently detected at 4°C and the IgG antibody incidence was 7.1% by enzyme method, 7.7% IAT, 9.4% LISS, 10.2% using PEG & 10.2% multiparous women. Bad obstetric history cases had significantly higher incidence of alloimmunization. The antibody specificity of antibodies was mainly in Lewis, Rh, Kidd and MN systems. Conclusion  Antibody screening before transfusion, at set time intervals after transfusion and during antenatal period is recommended.     

Evaluation of A newly developed DOT-ELISA kit for the diagnosis of human brucellosis

A new dot-ELISA kit for detection of Brucella antibodies in human sera was developed and compared with that of serum agglutination test, Rose Bengal plate test, rapid slide agglutination and Coomb's antiglobulin test. Following testing of 120 human sera from suspected patients of occupational risk, 25 gave positive reaction in Rose Bengal plate test, 25 in rapid slide agglutination test, 26 in serum agglutination test, 27 in Coomb's antiglobulin test and 28 in dot-ELISA kit. Dot-ELISA kit picked up more positive than any other Serological test, indicating its superiority over the other laboratory tests for the diagnosis of brucellosis.     

EDTA‐induced pseudo‐neutropenia resolved with kanamycin

This report describes a case of spurious neutropenia caused by EDTA‐dependent in vitro agglutination of neutrophils. After raising the temperature of the sample to 37°C the agglutination was irreversible, but it resolved completely after addition of kanamycin. Previously this method has been shown to be effective in EDTA‐dependent pseudo‐thrombocytopenia, but this is the first report demonstrating successful application in EDTA‐dependent pseudo‐neutropenia.     

Quantitation of IgG on Erythrocytes: Correlation of Number of IgG Molecules per Cell with the Strength of the Direct and Indirect Antiglobulin Tests

Abstract. The number of IgG molecules bound to the erythrocyte surface for a given agglutination score in the antiglobulin test was studied with several different examples of anti-D, anti-E, anti-c, anti-Kell, anti-Fy^a, anti-Jk^a and immune anti-A antisera. The serological scores show a significant correlation with the mean values for bound IgG molecules within a restricted range, although the number bound for a given score may vary by up to 20%. The limit of detection was 100–120 IgG molecules per cell and when over 1,000 were bound, the cells were completely agglutinated. Anti-Kell bound under low ionic strength saline conditions required a greater number of molecules for a given agglutination strength. The relatively low levels of bound IgG necessary to give strong agglutination make the direct antiglobulin test (DAT) less valuable for following the progress of auto-immune haemolytic anaemia (AIHA) than a quantitative test. The latter test does not, however, provide any additional information in AIHA cases with a negative DAT as in these the anaemia does not appear to be due simply to the number of bound IgG molecules. Detection of certain antibodies may not be achieved simply by increasing the sensitivity of the antiglobulin test when correctly performed.     

Hypothermia slows sequential and parallel steps initiated during caerulein pancreatitis

Background and objectivesMultiple deleterious signaling cascades are simultaneously activated in acute pancreatitis (AP), which may limit the success of pharmacologic approaches targeting a single step. We explored whether cooling acinar cells slows distinct steps initiated from a stimulus causing pancreatitis simultaneously, and the temperature range over which inhibition of such deleterious signaling occurs.MethodsCaerulein (100 nM) induced trypsinogen activation (TGA), CXCL1, CXCL2 mRNA levels, cell injury were studied at 37 °C, 34 °C, 31 °C, 29 °C and 25 °C in acinar cells. Trypsin, cathepsin B activities and cathepsin B mediated TGA were studied at 37 °C, 23 °C and 4 °C.ResultsThere was >80% reduction in TGA, CXCL1 and CXCL2 mRNA levels at 29 °C, and in cell injury at 34 °C, compared to those at 37 °C. Trypsin activity, cathepsin B activity and cathepsin B mediated TGA at 23 °C were respectively, 53%, 64% and 26% of that at 37 °C. Acinar cooling to 31 °C reduced LDH leakage even when cooling was initiated an hour after caerulein stimulation at 37 °C.ConclusionsHypothermia synergistically and simultaneously slows parallel and distinct signaling steps initiated by caerulein, thereby reducing TGA, upregulation of inflammatory mediators and acinar injury.     

Oxidative stress in hepatocytes and stimulatory state of Kupffer cells after reperfusion differ between warm and cold ischemia in rats

Rat liver was kept at 4°C or 37°C in MEM, and reperfused through a closed circulation from the hepatic vein to the portal vein at 37°C with the same solution. Although purine nucleoside phosphorylase and ALT activities were increased in the perfusate, depending on the duration of ischemia at both 4°C and 37°C, the ratio of the latter to the former was significantly higher after 37°C-ischemia than after 4°C-ischemia. The stimulation stage of Kupffer cells evaluated in situ by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was elevated after 4°C-ischemia longer than 1 h, but not after 37°C-ischemia. In contrast, the degree of oxidative stress in hepatocytes assessed by formazan deposition after liver perfusion with nitro blue tetrazolium alone was greater after 37°C-ischemia than after 4°C-ischemia. These results suggest that oxidative stress in hepatocytes and the stimulatory state of Kupffer cells after ischemia-reperfusion may differ between 4°C-ischemia and 37°C-ischemia, probably leading to different development of liver damage.     

Thermotolerance of Histoplasma capsulatum at 40 °C predominates among clinical isolates from different Latin American regions

The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America.     

A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test

In vivo coating of red cells by antibody and/or complement is detected using various sensitive techniques, however most hospitals even today rely on the conventional tube technique (CTT). We compared the performance of the CTT and recently introduced gel test (GT) in the evaluation of direct antiglobulin test (DAT).

The CTT and GT were first compared using in-house prepared control cells. The polyspecific DATs were performed simultaneously by CTT and GT on 170 consecutive blood samples. Positive samples were further tested for monospecific IgG and C3d by both techniques.

GT demonstrated stronger agglutination scores (60 vs. 43) compared to CTT using control cells. The sensitivity and specificity of the GT was 98.4 and 95.2%, respectively as compared to CTT for polyspecific DAT. Discordance between the two test systems was seen in 6/170 patients. Of these, 5 were missed by CTT while GT failed to detect in vivo coating in only 1 case. The agreement between two methods of DAT was 96.4% (κ = 0.926) using polyspecific AHG and 95.7% (κ = 0.379) with mono specific anti-IgG. We conclude that GT is a better alternative to CTT for detecting red cell bound antibodies in various clinical conditions.     

Inspired gas temperature in ventilated neonates

The warming and humidification of inspired gases for ventilated neonates are routine. There are no data on the temperature of the gas at the airway opening in ventilated neonates. Is the inspired gas temperature at the airway opening, as expected and set on the humidifier, around 37°C? We aimed to measure temperature at the airway opening and compare this with the circuit temperature. This was an observational study in a neonatal intensive care unit. Twenty‐five mechanically ventilated infants were studied. All had humidifiers with chamber temperature set at 36°C and the circuit temperature set at 37°C. Two temperature probes were inserted and rested at the circuit‐exit and at the airway opening, and temperatures were measured for 2 min in each infant. At this time, the circuit temperature was also noted. The mean (SD) temperature at the airway opening in infants nursed in incubators was 34.9 (1.2)°C, compared with radiant warmers where the mean (SD) was 33.1 (0.5)°C. The mean (SD) difference in temperature from the circuit temperature probe to the airway opening was greater under radiant warmers, with a mean (SD) drop of 3.9 (0.6)°C compared with a mean (SD) drop of 2.0 (1.3)°C in the incubators. In conclusion, the temperature at the circuit temperature probe does not reflect the temperature at the airway opening. Inspired gas temperatures are lower than the expected 37°C with the normal circuits and usual humidifier settings. Pediatr Pulmonol. 2004; 38:50–54. © 2004 Wiley‐Liss, Inc.     

The effect of storage on adhesion and aggregation of platelets

Abstract. Aggregation and adhesion of stored platelets were studied by a modified Wright technique. Comparisons of platelet rich plasma collected in ACD solution were made with and without aggregating agents, and at storage temperatures of 5°C, 24°C and 37°C. Platelets showed a loss of response to 0.5 μg ADP/ml, collagen and agar after storage for 4–6 h at the three temperatures with the greatest loss at 37°C and the least change at 5°C. The response to 5μg ADP/ml also was best maintained at 5°C. After 6–8 h of storage at 5°C. there was some decrease in free (single) platelets after rotation in the absence of aggregating agents, with a marked decrease after 48 hours storage. With storage at 24°C and 37°C, decrease in single platelets after rotation was not demonstrated during the first 24 h. A response to 5 μg ADP/ml was maintained for more than a week with the 5°C preparations. Acidification diminished the response to ADP for the first few days of storage and also depressed platelet aggregation and adhesion in the absence of aggregating agents on prolonged storage. The results of this in vitro study would appear to correlate with the loss of hemostatic effect of stored platelets on transfusion. The sustained response to 5 μg ADP/ml at 5°C suggests that this may be the best temperature for short term storage, although there is less spontaneous aggregation of platelets at the end of 24 h with 24°C storage. Also the ‘storage lesion’ appears to include loss of ADP or inability to release this substance from platelets.     

A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test

In vivo coating of red cells by antibody and/or complement is detected using various sensitive techniques, however most hospitals even today rely on the conventional tube technique (CTT). We compared the performance of the CTT and recently introduced gel test (GT) in the evaluation of direct antiglobulin test (DAT). The CTT and GT were first compared using in-house prepared control cells. The polyspecific DATs were performed simultaneously by CTT and GT on 170 consecutive blood samples. Positive samples were further tested for monospecific IgG and C3d by both techniques. GT demonstrated stronger agglutination scores (60 vs. 43) compared to CTT using control cells. The sensitivity and specificity of the GT was 98.4 and 95.2%, respectively as compared to CTT for polyspecific DAT. Discordance between the two test systems was seen in 6/170 patients. Of these, 5 were missed by CTT while GT failed to detect in vivo coating in only 1 case. The agreement between two methods of DAT was 96.4% (kappa = 0.926) using polyspecific AHG and 95.7% (kappa = 0.379) with monospecific anti-IgG. We conclude that GT is a better alternative to CTT for detecting red cell bound antibodies in various clinical conditions.