Variant Creutzfeldt-Jakob disease: implications for the health care system.

The recognition of the first cases of variant Creutzfeldt-Jakob Disease in the United Kingdom (UK) in 1996 and the realisation that this new disease represented the human form of the cattle disease BSE has prompted a considerable investment in research, particularly in the UK, Europe and the United States (US). Much has been learnt about this disease but much is still unknown. Infectivity is not destroyed by conventional sterilisation and disinfection treatment methods. This, combined with the widespread distribution throughout the lymphoid system as well as the central nervous system, raises the spectre of transmission through both surgical and ophthalmological procedures. Reports in 2004 of two likely transfusion-transmitted cases of vCJD suggest the probability of infection through blood transfusion and tissue transplantation. The risk of hospital-based and community-based transmission has not been quantified. To complicate matters even further, there is no suitable ante-mortem screening test or effective treatment for this fatal disease. The incubation period prior to onset of clinical disease is many years and there is good evidence for the existence of subclinical infection and infectivity during this stage. The extent of under-diagnosis and misdiagnosis is probably significant, adding to the risk of human-to-human transmission.

Evaluation of high performance liquid chromatography for routine estimation of haemoglobins A2 and F.

AIMS: To compare high performance liquid chromatography (HPLC) with conventional methods for the estimation of blood haemoglobin A2 (HbA2) and haemoglobin F (HbF) concentrations in routine thalassemia screening. METHODS: An HPLC system (the VARIANT Hemoglobin Testing System) was tested for precision and reproducibility in the measurement of HbA2 and HbF, and reference ranges were obtained for a local healthy adult population. HPLC was compared with column anion exchange chromatography for HbA2 measurement, and radial immunodiffusion, or alkaline denaturation for HbF measurement. The reliability of HbA2 measurement by HPLC for the detection of beta thalassaemia and HbE was assessed in 200 consecutive samples for routine thalassaemia screening. RESULTS: HPLC was rapid, technically easy, and gave good precision and reproducibility. In all comparisons linear regression analysis showed good correlation between HbA2 or HbF concentrations determined by HPLC and by the respective conventional methods. All beta thalassaemia or haemoglobin E carriers presumptively identified by conventional methods in 200 consecutive samples were detected by HbA2 measurement using HPLC, without any false positive or false negative results. CONCLUSIONS: The measurement of HbA2 and HbF by HPLC is rapid, reproducible, and precise. It is as reliable as column chromatography for the measurement of HbA2 and radial immunodiffusion or alkaline denaturation for the measurement of HbF. HPLC may be an appropriate method for rapid screening in population surveys for beta thalassaemia and HbE carriers.

PCR detection of cytomegalovirus: a review.

CMV is of major concern in immunocompromised and immunosuppressed patients. Since CMV can be transmitted by leucocyte transfusions from healthy seropositive donors (Hirsch, 1984) it has been stated that leucocytes are the natural reservoir of latent CMV (Jiwa et al, 1989). Although tissue culture is the method currently used for the diagnosis of CMV infection, this technique is time consuming, expensive and does not detect latent virus. As 80% of normal Australian blood donors are seropositive for CMV (Ho, 1990) the amount of blood available for high risk patients is greatly reduced. The dramatic gains in sensitivity of viral detection made possible by the PCR technique offers new hope for the detection of otherwise elusive latent genomes as well as more routine application in the detection of viraemia or other active infection. However, for this technique to be adopted by clinical laboratories it must be shown to be easily reproducible and cost-effective. Thus, the PCR may have an important role in the development of CMV-negative blood products, as well as being a powerful test in diagnostic virology. It is expected that it will reduce the morbidity and mortality rate in susceptible patients at risk of CMV when given transfusions of blood from subjects who may carry the virus.

Detection of cytomegalovirus in blood donors by the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed and optimized to detect cytomegalovirus (CMV) DNA in the blood of 86 normal donors who had originally tested seropositive for CMV. Evidence of previous or current infection with CMV was determined by rescreening of the blood for CMV antibodies and by detecting the presence of infectious virus in the white cells by cell culture. DNA was extracted from the blood of donors by a manual or an automated method and amplified by PCR using primers from the major immediate early gene of CMV DNA. The amplified product was detected by visualization of a fluorescent 435-base pair DNA band in an electrophoretic agarose gel after ethidium bromide staining and confirmed by slot-blot DNA hybridization using an oligonucleotide probe with complementarity for the major immediate early gene. Seven (8%) of the 86 donors were positive for CMV DNA in both fluorescence and hybridization studies. These donors were also antibody positive. While 74 (86%) of the 86 donors were positive for the presence of CMV antibodies in enzyme-linked immunosorbent assay, none was positive for virus in cell culture. PCR has the potential to be an effective and reliable procedure for the detection of CMV DNA in donor blood, but further study is required for this technique to be used for diagnostic or routine screening purposes.

Removal of cytomegalovirus DNA from donor blood by filtration.

Blood from five donors, previously shown to be positive for cytomegalovirus (CMV) DNA following polymerase chain reaction (PCR) amplification, was filtered through commercially available leucocyte filters. Analysis of pre- and post-filtration samples by PCR with ethidium bromide staining has shown that filtration was successful in removing CMV DNA from all samples. This is evidence that leucocyte filtration of red cell concentrates may greatly decrease the risk of CMV disease following transfusion to susceptible patients.

Status of the MNSs antigens on human platelets.

A sensitive and specific radioimmunoassay was used to determine whether human platelets possess antigens of the MNSs blood group. Mouse monoclonal IgG anti-M and anti-N were purified by Staphylococcus protein A chromatography, labeled with 125I, and incubated with platelet pellets from donors of various MN phenotypes. Human IgG anti-M, -S, and -s were purified by absorption-elution, incubated with platelet pellets from donors of different MNSs phenotypes, washed, and incubated with 125I-labeled mouse monoclonal anti-human IgG. In both assays, the platelet pellets were centrifuged through phthalate ester oils and the radioactivity in the pellets was counted. Dose-response curves and ligand bound per cell indicated no significant difference in the binding of mouse or human anti-M and anti-N to platelets from donors of the MM, MN, or NN phenotype or of human anti-S and anti-s to platelets from donors of the Ss or ss phenotype. Contrary to many previous studies, our data indicate that the MNSs antigens are not expressed on the circulating human platelet. Therefore, antibodies to these antigens probably do not play a role in refractoriness to platelet transfusion.

The expression of ABH antigens during in vitro megakaryocyte maturation: origin of heterogeneity of antigen density.

An indirect immunofluorescence technique with single and double labelling has been used to examine cultured human megakaryocytes for ABH antigens. This technique demonstrated the presence of these antigens on megakaryocytes and a population of small mononuclear cells that probably represent the differentiated precursors of megakaryocytes. In contrast to the intense homogeneous labelling with human anti-PlA1, the labelling with human anti-A, mouse monoclonal anti-A and anti-type 2H is light and heterogeneous, with many cells staining weakly or not at all. This variability in blood group ABH surface antigen expression appears to occur at the level of cell proliferation within colonies. While cells within an individual colony are homogeneous in fluorescence intensity, there is considerable variation between colonies. The progeny of individual megakaryocytes also appear uniform in ABH antigen expression.

Status of major red cell blood group antigens on neutrophils, lymphocytes and monocytes.

Previous investigators have reported that a number of red cell (RBC) antigens are found on neutrophils (PMN) and lymphocytes. However, there is a lack of consensus in the literature. Furthermore, few data are available concerning the occurrence of RBC antigens on monocytes. To address this problem, leucocyte fractions prepared from donors of known RBC antigen phenotype were analysed by fluorescence flow cytometry. Based on recent reports that PMN do not express ABH antigens and on the data presented here, the occurrence of major RBC antigens on leucocytes may be defined as follows: (1) lymphocytes express A, B. Lea and Leb antigens, depending on the ABH secretor (Se) status of the donor; (2) ABH and Lewis antigens cannot be detected on monocytes and PMN regardless of Se status; (3) D, E, e, C, c, Fya, Fyb, Fy5, Jka, Jkb, Jk, K, k, M, N, S, s, U, Vel, Coa, Lan, Jk3Yta, Dib, Ge, Sc:1 or Lub antigens were not detected on lymphocytes, monocytes and PMN; (4) lymphocytes, monocytes and PMN all express I, i, P and P1 antigens. The absence of selected Rhesus, Duffy, Kell, Kidd and other antigens on PMN is at variance with some previous reports. Furthermore, the distribution of RBC antigens on lymphocytes and monocytes has not been previously characterized using immunofluorescence flow cytometry.

Heterogeneous distribution of antigens on human platelets demonstrated by fluorescence flow cytometry.

We have used fluorescence flow cytometry to analyse cell-to-cell variability in the density of platelet ABH, Ii, Lewis, P, P1A1, Bak,a and HLA class I antigens. Human IgG and IgM antibodies were used in a two-stage assay with goat FITC-conjugated antihuman IgG (H&L) antibody as the label, followed by single cell analysis of 10 000 platelets per sample using a 256-channel fluorescence flow cytometer (Becton-Dickinson FACS Analyser). Computer analysis of fluorescence intensity histograms for mean and peak channel and coefficient of variation shows that the degree of heterogeneity in platelet antigen density varies with each particular blood group. The broad fluorescence distribution curves with oligosaccharide antigens (CVs: A = 53, B = 40, I = 44, Lea = 40, P = 40) indicate that these antigens possess a greater variability in the number of sites per cell compared to the more homogeneous distribution of P1,A1 BaK,a and HLA (CVs: P1A1 = 24, HLA = 30). These findings may partly account for the mechanism by which transfusion of ABO-incompatible platelets results in a biphasic survival curve, with a period of early rapid removal of those platelets with a high density of antigen sites, followed by a relatively normal survival curve for those platelets that possess only a few or no antigen sites. In contrast, P1A1 and HLA sites are less variable in number from one platelet to another in a given donor, and immune-mediated removal would be more likely to approximate a single exponential curve.

Use of fluorescence flow cytometry to study the binding of various ligands to platelets.

Fluorescence flow cytometry can be used to analyze the binding of different ligands to platelets. However careful choice of volume gates is essential in selecting the population of platelets for analysis. The use of fluorescein isothiocyanate conjugated to staphylococcal protein A or F(ab')2 fragments of immunoglobulin G anti-immunoglobulin offers no advantage in sensitivity or specificity in fluorescence studies of platelets and prefixation of washed platelets with paraformaldehyde has no effect on nonspecific fluorescence. The application of this technology to platelets facilitates quantitation of fluorescence intensity and may yield additional useful information.

Stability of platelet surface antigens during storage.

We measured changes in A, B, 2H, PlA1, and HLA Class I antigens on human platelets stored as routine platelet concentrates (PCs) in 50 to 60 ml of citrate-phosphate-dextrose-adenine (CPDA-1) plasma in polyolefin (PL 732) bags at 22 degrees C with continuous cartwheel rotation. Samples were obtained at 1, 3, 5, and 10 days of storage; incubated with human IgG anti-A, -B, -HLA and -PlA1; incubated with mouse monoclonal 125I-labeled anti-human IgG; centrifuged through phthalate ester oils; and assayed in a gamma scintillation counter. Additionally, group O platelets were analyzed using 125I-labeled IgM mouse monoclonal anti-Type 2H. Mean values for molecules of Ig bound per platelet showed that platelet surface antigens A, B, 2H, PlA1, and HLA Class I showed no significant change during 10-day storage as routine PCs in CPD-A1 in PL 732 bags. Identical radioassays were performed with platelets incubated at 22 degrees C in plastic test tubes for 24 hours in homologous plasma from donors negative for the respective antigens and in a variety of artificial media with albumin and lipids. No significant changes occurred in any of the surface antigens, except for the loss of approximately 50 percent of the blood group A antigen from platelets stored in O plasma or in albumin media. These data indicate that HLA, PlA1, and type 2H structures do not readily dissociate from the platelet membrane during storage, while some blood group A antigens, presumably acquired passively from the plasma, will elute from the platelet under certain conditions. Routine storage conditions are unlikely to alter the immunogenicity of platelets due to a loss of antigen expression.

Absence of ABH antigens on neutrophils.

Many investigators have concluded that polymorphonuclear leucocytes (PMN) express ABH antigens in parallel to red cells (RBC). We have examined human PMN for ABH antigens using human isoantibodies and mouse monoclonal antibodies with three highly sensitive and specific two-stage assay systems: fluorescence flow cytometry, immunofluorescence microscopy, and avidin-biotin immunoperoxidase microscopy. In all three assays the ABH antigens could not be detected on the surface of PMN. Previous reports alleging that ABH antigens occur on PMN probably represent false positive reactions due to inherent technical problems.

Presence of P blood group antigens on human platelets.

Except for ABH antigens, the presence of red cell (RBC) antigens on human platelets has been a source of disagreement among investigators. Because ABH antigens share precursor sequences with P-system saccharides, the authors examined human platelets for evidence of P blood group antigens. Anti-P was directly labeled with 125I and incubated with normal platelets in a one-stage radioimmunoassay (RIA). Alternatively, platelets from donors of known RBC phenotype were incubated with anti-P, anti-P1, or anti-Tja, washed, incubated with FITC-labeled goat anti-human immunoglobulin and evaluated by fluorescence flow cytometry. The results of these assays demonstrate that platelets express P blood group antigens in parallel to the donor's RBCs. The role of these antigens in platelet transfusion is not known.

Posttransfusion purpura. Report of a case with anti-P1A1 masked by HLA antibodies.

A case of posttransfusion purpura is reported in which the laboratory determination of the specificity of the causative antibody was initially confused by the presence in the patient's serum of HLA-directed antibodies. The patient was a multiparous 65-year-old woman with a previous history of blood transfusion. She developed the typical clinical features of posttransfusion purpura 8 days following the transfusion of 6 units of packed red cells. The patient was P1A1 negative, and her serum reacted strongly in a radiolabeled monoclonal antibody assay with both P1A1-positive and -negative platelets. Radioimmunoprecipitation demonstrated that the patient's serum contained antibodies both of anti-P1A1 and anti-HLA specificity. Western blotting of normal platelets incubated with the patient's serum verified that the anti-P1A1 antibody was directed against platelet membrane glycoprotein III.

The origin of ABH antigens on human platelets.

ABH antigens are present on platelets from individuals of the corresponding red cell phenotype, but the extent to which these antigens are intrinsic or adsorbed remains undefined. To evaluate platelets for intrinsic H substance, an IgM mouse monoclonal antibody against type 2H chain (the intrinsic H structure found on erythrocytes) was labeled with 125I and incubated with platelets from donors of different ABO type. The antibody showed dose-response saturation curves, and binding to platelets paralleled that of the red cell ABO type, with O greater than B greater than A1 greater than A1B greater Oh cells, giving a single factor variance F of 190 (P less than .0005). Passive adsorption of A antigens by platelets has been previously reported. To verify this phenomenon for A and B antigens and to quantitate the elution of A and B antigens from platelets, the following assay system was used. Platelets from group A1 and B donors were incubated in plasma from group O donors, and platelets from group O donors were incubated in plasma from different ABO, Lewis, and presumed secretor-type donors. Human IgG anti-A or anti-B was added to the platelets. The amount of antibody bound was determined with a 125I-labeled mouse monoclonal anti-human IgG. When incubated for 96 hours in group O plasma, group A1 platelets showed a 45% to 50% decrease in binding of anti-A. There was no significant change in the level of type 2H antigen on these platelets during the same incubation period. Group O platelets incubated in A or B plasmas rapidly acquired the antigens, but if returned to their original plasma, 95% of this passively adsorbed antigen eluted off within 18 hours. The maximum uptake of A and B substances was influenced by the Lewis and secretor type of donor plasma. Our present study demonstrates that ABH antigens on platelets consist of type 2H chains, which are presumably intrinsic as when found on red cells, and of passively adsorbed ABH structures, which are presumably type 1H chains.

Lea blood group antigen on human platelets.

One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined.

Biochemical characterisation of A blood group activity on human platelets.

A butanol extraction procedure has been used to isolate the blood group A antigen determinants from platelet membranes and to determine their biochemical structure-glycolipid and/or glycoprotein. The blood group A serological activity was found to reside entirely in the butanol (organic) fraction. When this fraction was analyzed by thin-layer chromatography, the A-inhibitory activity was found to comigrate with extracts of erythrocyte type Aa, Ab and Ac variant structures. The results of this study indicate that the ABO blood group A determinants on human platelets are glycolipid, similar in structure to glycolipid A determinants on erythrocytes.

The presence of the Ii blood group system on human platelets.

To examine platelets for the Ii antigens, high-titer human IgM anti-I and anti-i antibodies were affinity purified, radiolabeled with 125I, and incubated with adult and cord platelets. Saturation binding curves were performed by incubating adult platelets with serial dilutions of concentrated 125I-anti-I. Inhibition binding curves were performed by incubating adult platelets with doubling dilutions of concentrated unlabeled anti-I mixed with 125I-labeled anti-I. Adult platelets bound significantly more anti-I than anti-i, while cord platelets bound more anti-i than anti-I (P less than 0.025). Both anti-I and anti-i show a temperature-dependent dose-response curve of maximum binding at 4 degrees C. Binding of 125I-labeled anti-I was inhibited by preincubation with 100-fold concentration of unlabeled anti-I. The authors conclude that platelets express I/i antigens in parallel with that of red blood cells.

Erythrocyte antigens on human platelets. Absence of Rh, Duffy, Kell, Kidd, and Lutheran antigens.

There is considerable controversy as to whether antigens of the Rh, Duffy, Kidd, Kell, or Lutheran red cell systems are present on human platelets. The majority of previous investigators of this topic have reported them to be present. We have used a sensitive two-stage radioimmunoassay to examine human platelets for the presence of antigens of these five red cell systems. Platelets from donors of appropriate red cell phenotype were incubated with monospecific anti-erythrocyte IgG, followed by a second-stage incubation with 125I-labeled mouse IgG monoclonal anti-human IgG (Fc). Analysis of ligand bound per cell demonstrated no significant difference in binding of erythrocyte antibodies to platelets from donors homozygous, heterozygous, or negative for D, C, c, E, e, Fya, Fyb, Jka, Jkb , K, k, and Lub antigens. These findings indicate that major antigens of the Rh, Duffy, Kidd, Kell, and Lutheran systems are not expressed on the surface of human platelets.