Cinzia Paccapelo

Blood group genotyping for Jka/Jkb, Fya/Fyb, S/s, K/k, Kpa/Kpb, Jsa/Jsb, Coa/Cob, and Lua/Lub with microarray beads

BACKGROUND: Traditionally, blood group typing has been performed with serologic techniques, the classical method being the hemagglutination test. Serotyping, however, may present important limitations such as scarce availability of rare antisera, typing of recently transfused patients, and those with a positive direct antiglobulin test. Consequently, serologic tests are being complemented with molecular methods. The aim of this study was to develop a low‐cost, high‐throughput method for large‐scale genotyping of red blood cells (RBCs).

Outcomes of an automated procedure for the selection of effective platelets for patients refractory to random donors based on cross-matching locally available platelet products.

In 1999, we implemented an automated platelet cross‐matching (XM) programme to select compatible platelets from the local inventory for patients refractory to random donor platelets. In this study, we evaluated platelet count increments in 40 consecutive refractory patients (8·3% of 480 consecutive platelet recipients) given 569 cross‐match‐negative platelets between April 1999 and December 2001. XM was performed automatically with a commercially available immunoadherence assay. Pre‐, 1‐ and 24‐h post‐transfusion platelet counts (mean ± SD) for the 569 XM‐negative platelet transfusions containing 302 ± 71 × 109 platelets were 7·7 ± 5·5, 32·0 ± 21·0 and 16·8 ± 15·5 × 109/l respectively. Increments were significantly higher (P < 0·05, t‐test) than those observed in the same patients given 303 random platelet pools (dose = 318 ± 52 × 109 platelets) during the month before refractoriness was detected, when pre‐, 1‐ and 24‐h post‐transfusion counts were 7·0 ± 8·6, 15·9 ± 16·1 and 9·6 ± 12·8 × 109/l respectively. The cost of the platelet XM disposable kit per transfusion to produce 1‐h post‐transfusion platelet count increments >10 × 109/l was euro 447. This programme enabled the rapid selection of effective platelets for refractory patients, from the local inventory.

Immune hemolytic anemia associated with teicoplanin

BACKGROUND:  Several drugs can cause immune hemolytic anemia. Here a patient who developed hemolytic anemia after treatment with teicoplanin is described.

An acute haemolytic transfusion reaction due to anti-Jk.

The Kidd system antibodies are characteristically difficult to detect. They show variability in immunoglobulin class, subclass and serological characteristics. They are generally detected by an antiglobulin test, using a polyspecific antiglobulin or complement antiserum. Often, the antibodies are only detected using cells with a double dose (homozygous) expression of Kidd antigens, enzyme-treated cells or by using sensitive immunohaematological techniques.

New technologies in immunohaematology.

Since the discovery of the ABO system, numerous important innovations have contributed to a continuous, rapid evolution in the diagnostic methods for in vitro measurements of the antigen-antibody reaction, allowing a significant improvement in the compatibility between blood from donors and the recipients. Apart from the introduction of ABO typing, these methods include the determination of Rh type and phenotype, the direct and indirect antiglobulin tests, cross-matching and consequent identification of antigens and antibodies of clinical relevance, the use of low ionic strength additives and enzyme treatments, the development of monoclonal reagents and solid-phase and microcolumn platforms for performing the pre-transfusion tests. Since transfusion safety depends on a series of strictly inter-related processes1, among which pre-transfusion tests have a predominant role, in recent years some of the new technologies that integrate the classical techniques in immunohaematology have become valid instruments for improving the safety of transfusions. The aim of this review is to illustrate the principles and practical applications of these emerging techniques used in our laboratory to identify antigens and antibodies, in cases of red cell or platelet immunisation.

IgA autoimmune haemolytic anaemia in a pregnant woman

Dear Sir, Autoimmune haemolytic anaemia (AIHA) is a clinical condition caused by IgG, IgM or IgA antibodies to red blood cells. This condition affects 1–3 per 100,000 individuals per year1. Although IgA antibodies have been reported in 14% of cases of warm AIHA, they are mostly associated with IgG and/or IgM antibodies, while warm AIHA due exclusively to IgA antibodies is rare2. Several methods have been developed for the detection of these auto-antibodies. The direct antiglobulin test (DAT) by the conventional tube technique (CTT) is the gold standard. About 12% of AIHA patients show a negative CTT-DAT, possibly caused by the level of RBC-bound immunoglobulin being below the threshold, the presence of low-affinity IgG that washes off the RBC during the CTT-DAT washing phase and RBC-bound IgA or IgM not detected by routine antiglobulin reagents. In such cases, it is important to use additional tests to characterise the autoantibody and to confirm the diagnosis. We report an unusual case of IgA-AIHA in a pregnant woman not detected by CTT-DAT. A 32-year old woman in the 21st week of gestation of her second pregnancy was admitted to our hospital because of asthenia, headache and anaemia. The day before, the patient had received two units of RBC in another hospital. The patient’s history was negative for chronic anaemia, medication use and infectious diseases. Laboratory results were as follows: haemoglobin 6.2 g/dL, reticulocytes 340×109/L, white blood cells 157×109/L, haptoglobin <6 mg/dL, lactate dehydrogenase 672 U/L, and indirect bilirubin 2.18 mg/dL. These data supported the diagnosis of haemolytic anaemia. The patient’s blood type was O Rh positive: CcDee; K−k+; Jk(a+b−); Fy(a−b+); M+N−S+s−. The CTT-DAT, performed with polyspecific anti-human globulin, monospecific anti-IgG and anti-C3 antisera from three manufacturers (Gamma Biologicals, Houston, TX, USA; Ortho-Clinical Diagnostics, Raritan, NJ, USA; Immucor Inc. Norcross, GA, USA), and anti-IgA and IgM antisera from one manufacturer (Immucor), was negative. The DAT performed by solid-phase (Capture Select, Immucor) and the mitogen-stimulated DAT3 were negative, while the DAT performed with a gel column test (BIORAD, Cressier sur Morat, Swiss) was positive (score 2+) only with anti-IgA antiglobulin. The autoantibodies eluted from the patient’s RBC (Elu-Kit II, Gamma) showed anti-e specificity. Irregular antibody screening and identification were performed by the indirect antiglobulin test in a microcolumn card (Ortho) and tube test with additive polyethylene glycol (PeG, Gamma) using anti-IgG and anti-IgA antiglobulin reagents. Free antibodies were not detected in the serum. The data led to the diagnosis of IgA-AIHA. An ultrasound of the foetus showed no abnormalities. The patient was treated with intravenous corticosteroids from day 10 to 113, with a starting daily dose of 80 mg/kg for 5 days, followed by tapering to 2.5 mg from day 83 to 113. Moreover, from day 17 to 21 the patient received 400 mg/kg/die of intravenous immunoglobulins to reduce the risk of complications due to the high steroid dosage in pregnancy (preterm premature rupture of the membranes, gestational diabetes and hypertension). The haemoglobin level began to rise after the administration of steroids and these treatments improved the patient’s condition. The laboratory data are reported in Figures 1 and ​and2.2. One month after admission the patient was discharged. At the end of gestation she delivered a healthy male neonate. A maternal DAT with anti-IgA reagent was still weakly reactive with the gel column test (1+) and free antibodies were not detected in the serum with anti-IgG and anti-IgA antiglobulin reagents. Figure 1 Haemoglobin (____), reticulocytes (...), haptoglobin (-------) and DAT (↓). Figure 2 Lactate dehydrogenase (...) and bilirubin (____). AIHA can be a very severe disease, if not promptly detected and correctly treated. However, in pregnancy the presence of maternal autoantibodies may have little relevance for the foetus1. Despite this, the correct identification of the presence of maternal autoantibodies is important for the differential diagnosis from several autoimmune conditions. For this reason, the Immunohaematology Laboratory must ensure that the techniques used for the patient’s workup include several methods with appropriate sensitivity, such as a monocyte monolayer assay, eluate concentration, the direct Polybrene test, the direct polyethylene glycol test, solid-phase, gel column test, DAT using cold washes and the mitogen-stimulated DAT. In rare cases, warm AIHA can be associated with IgA or IgM autoantibodies without IgG being present. The presence of more than one type of antibodies on RBC, even when undetected by agglutination methods, can be a major cause of haemolysis along with other factors, such as the quantity of bound IgG, IgG subclass pattern, and complement2. Recently, Chadebech et al.4 reported that trapping and sequestration of agglutinated RBC in the spleen are the principal pathogenic mechanisms of IgA-AIHA and that “elucidation of the mechanism responsible for the immune destruction of RBCs would help to guide decisions concerning the choice of first-line treatment”. Therefore, the autoantibody class, including IgA, and possibly the autoantibody specificity should be determined rapidly in order to provide AIHA patients with appropriate treatment (such as splenectomy). IgA AHIA is quite rare, with reported incidences ranging from 0.2 to 2.7%2- Its incidence is, however, likely to be underestimated, because the CTT-DAT may often be falsely negative. A threshold of 150–400 IgG molecules/RBC is required to generate agglutination sufficient for a positive result in the CTT-DAT. Therefore, in cases with <150 IgG molecules/RBC, or in cases in which a warm IgM or IgA antibody is mediating the process, the CTT-DAT is unlikely to elucidate the diagnosis. Our findings confirm that the gel column test is more suitable and more sensitive than CTT-DAT for the identification of RBC-bound IgA5. In conclusion, we recommend performing the CTT-DAT with monospecific antiglobulin anti-IgG, anti-IgM, anti-IgA, anti-C3d antisera and, before performing more complex procedures, using the gel column test, which is more sensitive, in patients in whom the suspicion of AIHA is strong and who have an apparently negative CTT-DAT.

The Immunohaematology Reference Laboratory: the experience of the Policlinico Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan

A careful evaluation of the presence of irregular red cell antibodies must be carried out in patients undergoing transfusion therapy1–3. For immunised patients, the availability of donors of rare blood groups and access to banks and international registries enables compatible blood components to be found and assigned correctly even in the most difficult cases. The accreditation of laboratories that provide these services indicates not only fulfilment of the accreditation bodys requisites regarding the quality system, structure and organisation, but also those related to the technical and professional capacities of the laboratory and its staff. The requisites on which accreditation is based are technical (warranting satisfactory levels of performance), economic (controlling the appropriateness of the services) and educational (promoting continuous training and exchange of knowledge and experience). Accreditation also facilitates the evaluation and comparison of structures, since the services are delivered and controlled according to internationally recognised models.

Four novel silenced RHCE : RHCE NULL ALLELES

T he ISBT (International Society of Blood Transfusion) documents more than 100 RHCE variant alleles. More than 20 encode RHCE null phenotypes with loss of expression of Cc/Ee antigens, most often recognized in samples homozygous for the mutant allele and presenting with a Dred blood cell (RBC) phenotype. RHCE null alleles result from diverse molecular mechanisms including insertions or deletions, nonsense mutations, splice site changes, and replacement of RHCE with the corresponding regions of RHD. We report here four samples referred for investigation—three with discrepant Rh serological and RH molecular results and one for complete RH genotyping with D+ RBC phenotype and anti-D in the plasma.

Implementation and Assessment of High-Throughput Donor Typing at the Milan, Italy, Immunohematology Reference Laboratory

In 2005, the Centro Transfusionale e di Immunomatologia, Dipartimento di Medicina Rigenerativa, an Immunohematology Reference Laboratory in Milan, Italy, instituted a rare donor program to address the transfusion needs of patients with complex immunization to red cell antigens with a rare phenotype. From June 2005 to December 2008, the laboratory used a high-productivity system (Galileo, Immucor, Norcross, GA) for mass-scale antigen screening with profile 1 and 2 antigens for select donors, where 48,715 blood donors were typed with the identification of 6,634 rare blood donors. In April 2009, the laboratory adopted the BeadChip™ platform (BioArray Solutions, Ltd., Warren, NJ) for large-scale DNA typing. The decision to implement was to expand the panel of red blood cell and platelet antigens using the human erythrocyte antigen (HEA) and human platelet antigen (HPA) BeadChip™ formats. As recommended by international guidelines, a validation plan was used to evaluate the sensitivity and specificity of the method. The results of our testing are described in this chapter.