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BACKGROUND Recent studies suggest that HPA-1a–specific, low-avidity maternal antibodies not detectable by conventional methods can cause neonatal alloimmune thrombocytopenia (NAIT). We performed studies to further define the incidence and clinical significance of this type of antibody. STUDY DESIGN AND METHODS Surface plasmon resonance analysis was used to detect low-avidity antibodies in HPA-1a–negative, “antibody-negative” mothers of suspected NAIT cases. The ability of antibodies detected to promote immune destruction of human platelets (PLTs) was examined in a newly developed NOD/SCID mouse model. RESULTS Among 3478 suspected cases of NAIT, 677 HPA-1a–negative mothers were identified. HPA-1a–specific antibodies were detected by conventional antibody testing in 616 cases (91%). Low-avidity HPA-1a–specific antibodies were identified in 18 of the remaining 61 cases (9%). Clinical follow-up on 13 cases showed that eight were referred because of suspected NAIT and five because the mother’s sister had previously had an infant with NAIT. Only six infants born to the 13 sensitized mothers had clinically significant thrombocytopenia at birth. Three of four low-avidity antibodies tested in the mouse caused accelerated clearance of HPA-1a/a but not HPA-1b/b PLTs. Only 3 of 12 mothers with low-avidity HPA-1a antibodies were positive for HLA-DRB3*0101. CONCLUSIONS The findings confirm previous reports that low-avidity HPA-1a antibodies can cause NAIT but show that the presence of such an antibody does not predict that an infant will be affected. The low incidence of HLA-DRB3*0101 in this cohort (p < 0.0001) suggests that women negative for DRB3*0101 may be predisposed to produce low-avidity HPA-1a antibodies.

Drug-induced immune thrombocytopenia (DITP) is a relatively common and sometimes life-threatening condition caused by antibodies that bind avidly to platelets only when drug is present. How drugdependent antibodies (DDAbs) are induced and how drugs promote their interaction with platelets are poorly understood, and methods for detecting DDAbs are suboptimal. A small animal model of DITP could provide a new tool for addressing these and other questions concerning pathogenesis and diagnosis. We examined whether the nonobese diabetic/ severe combined immunodeficient (NOD/ scid) mouse, which lacks xenoantibodies and therefore allows infused human platelets to circulate, can be used to study drug-dependent clearance of platelets by DDAbs in vivo. In this report, we show that the NOD/scid model is suitable for this purpose and describe studies to optimize its sensitivity for drug-dependent human antibody detection. We further show that the mouse can produce metabolites of acetaminophen and naproxen for which certain drug-dependent antibodies are specific in quantities sufficient to enable these antibodies to cause platelet destruction. The findings indicate that the NOD/ scid mouse can provide a unique tool for studying DITP pathogenesis and may be particularly valuable for identifying metabolite-specific antibodies capable of causing immune thrombocytopenia or hemolytic anemia. (Blood. 2010;116(16): 3033-3038) IntroductionDrug-induced immune thrombocytopenia (DITP) can be triggered by various medications through several distinct mechanisms. 1,2 In a comprehensive survey of DITP cases reported since 1998, George and coworkers identified 17 drugs that were considered to be "probable" causes of DITP and 51 thought to be "definite" causes on the basis of having met, respectively, 3 or 4 well-defined clinical criteria. 3,4 This analysis has helped greatly to define which drugs are capable of causing DITP but does not provide proof in an individual patient that a particular drug was responsible. Evidence supporting a cause-and-effect relationship between drug exposure and thrombocytopenia can be obtained by identifying a drugdependent antibody (DDAb) that reacts with platelets only when the implicated drug is present. 1,2,5 However, relatively few laboratories are experienced in DDAb detection, and it is not rare for antibody testing to be negative in a patient with a clinical history strongly suggestive of DITP. 1,2 Moreover, there is not uniform agreement as to whether detection of a DDAb provides conclusive evidence that the antibody caused platelet destruction. Partly for these reasons, identification of a DDAb is not included among the George criteria. The most stringent of these criteria calls for thrombocytopenia to recur when a patient is exposed a second time to the implicated drug. 3 While a rechallenge can provide convincing evidence that thrombocytopenia was drug-induced, it is often impractical and can be difficult to justify for reasons of patient safety.A surrogate small animal model for direct demonstration...

266 Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal antibodies specific for fetal platelet antigens and is the most common cause of intracranial hemorrhage in full term infants. The antigen HPA-1a, carried on beta 3 integrin (GPIIIa), is the most common trigger for NAIT and the 2% of women who are HPA-1a-negative are at risk to produce such antibodies. Most HPA-1a antibodies causing NAIT can be easily detected but it is not rare for an HPA-1a negative mother who lacks detectable antibodies to give birth to an infant with thrombocytopenia. Several recent reports suggest that low avidity HPA-1a antibodies not detected by conventional serologic methods are responsible for some of these cases (Socher 2009, Bakchoul 2011). To examine this question, we retroactively analyzed a cohort of 3478 suspected NAIT cases referred for laboratory diagnosis. Among 677 HPA-1a-negative mothers, we identified 61 in whom HPA-1a-specific antibodies were not detected by conventional methods. Surface plasmon resonance (SPR) analysis enables ligand-receptor interaction to be studied in real time without washing the target. Using this approach, we studied reactions of IgG from the 61 archived serum samples against purified GPIIb/IIIa isolated from group O platelets and identified 18 samples that reacted preferentially with the HPA-1a-positive version of the integrin in comparison with HPA-1a-negative integrin. Information defining clinical status was obtained on 13 of these cases by follow-up communication. Seven cases had been referred because of neonatal thrombocytopenia. Platelet nadirs ranged from 8,000/ul to 141,000/ul (median 38,000/ul). Five of the 7 infants had bleeding and were given maternal platelet transfusions and/or IVIgG. Normal platelet counts were achieved after 4 to 70 days (median 7 days). One infant had a normal platelet count, however IVIgG had been given to its mother throughout pregnancy. The remaining 5 cases were referred in mid-pregnancy because an HPA-1a-negative sister previously gave birth to an infant with NAIT. Four of these infants had normal platelets at birth; one had mild TP (platelets 125,000/ul). Only 3 of 12 mothers typed for HLA were positive for HLA-DRB3*0101, a marker found in >95% of women who make “conventional” HPA-1a antibodies during pregnancy (p < 0.001). The ability of human antibodies to cause destruction of human platelets in vivo can be studied in the NOD/SCID mouse, which lacks xenoantibodies normally found in other species (Newman 2007). Serum from 4 mothers was available in quantities sufficient for mouse studies; three of the four maternal sera caused accelerated destruction of HPA-1a-positive, but not HPA-1a-negative platelets. These findings indicate that low-avidity HPA-1a antibodies not detectable by conventional serologic methods are made by a subset of women exposed to the HPA-1a antigen during pregnancy and that some, but not all of these antibodies are capable of causing NAIT, which is usually mild, but can be severe. Women negative for HLA-DRB3*0101 may be especially prone to produce antibodies of this type. Maternal-fetal incompatibility for platelet antigens other than HPA-1a is very common and many apparent NAIT cases not involving HPA-1a go unresolved. The possibility that low avidity antibodies specific for antigens other than HPA-1a are responsible for some of these cases deserves study. Disclosures: No relevant conflicts of interest to declare.

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Strong and healthy saplings are a prerequisite to establish a successful forest. Therefore, an attempt has been made to develop the best package for nutrient supplementation to raise healthy Acacia mangium saplings, especially in acidic soil. The seeds were sown in pots, receiving different combinations of Arbuscularmycorrhizal (AM), Rhizobium inoculation with application of lime, and mustard oil cake (MOC). The highest spore count and infection percentage (3220 kg−1 soil and 69) were recorded in the AM + MOC + R treated pot, whereas the lowest (2553 kg−1 soil and 37) were recorded in the AM + L treated pot. Nitrogen concentration and uptake in the sapling were higher in the Rhizobium-inoculated treatments than the uninoculated ones. The sulfur concentration and uptake were higher in the MOC-supplemented treatment. Similarly, the P, K, Ca, and Mg concentrations and uptakes were higher in the limed treatments than the unlimed ones. The micronutrient concentration and uptake were higher in the unlimed treatments compared to the lime practice. The concentration of N in Rhizobium-treated pots, P and K in lime-treated pots, and S in MOC-treated pots were increased, whereas the soil pH decreased in all treatments except in the integrated package (AM + MOC + R + L) after 120 days. The Ca and Mg were reduced in all treatments, whereas micronutrients were reduced in all packages except the control. Under different nutrient management practices, plant height and stem girth continuously increased by 9.5 to 12 cm and 3 to 4 times, respectively. The production of robust saplings required integrated application of lime, MOC, AM, and Rhizobium in an acid soil that facilitated better root growth with availability of adequate nutrients for saplings.

Biochemical changes in black gram varieties inoculated with root-knot nematode, Meloidogyne incognita were investigated. Observations were recorded in the biochemical modifications relating to various parameters like total chlorophyll , total sugar contents, protein, and proline content during post infection periods. The variation in total chlorophyll, total protein, proline and total sugar content in six cultivars i.e PU 09-36(S), MU-44(S) ,VBG 11-031(R) ,VBG 11-016(R) ,KUG-715 (R)and NUL-205(R) were studied 45 days after inoculation . Reduced percentage of total chlorophyll contents were observed in inoculated samples than the healthy counterparts. However, an increase in amount of total protein ,proline and total sugar contents was observed in the diseased tissues.