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Platelets play an important role in haemostasis. The interaction of the platelet glycoproteins with the extracellular proteins is very crucial. Clinical observations of pathological conditions involving antibodies against platelet-specific antigens resulted in investigating alloantigens which are produced by Human Platelet Antigens (HPA). Since then dramatic progress has been made in unravelling the biochemical nature, function and molecular biology of platelet membrane GPs. These advances have resulted in detection and identification of platelet antibodies and antigens in clinical diagnosis. Although a variety of techniques are involved in the detection of HPA only four techniques or modifications have become the most common and they are PIFT, MAIPA, red-cell adherence assay and ELISA based techniques. In the following report a previously known rare disease known as Neonatal alloimmune thrombocytopenia (NAITP) was detected by the flow cytometer through PIFT. This disease is caused by immunization of the mother against antigens present on the platelets of her child. These antibodies belong to the IgG class and seem to have anti-HLA specificity. (Metcalfe, 2004). This report focuses on NAITP, the pathology of this disease, the mode of diagnosing the disease along with the mode of treatment that is required.
Six samples were utilised for this protocol- 6 negative controls (AB serum), a positive sample which was Anti HLA and test sample which is the mother's serum. The father's platelets were centrifuged at 15000g. The pellet was re-suspended in 10ml PBS/EDTA/BSA. A 1:100 dilution of the platelet suspension was added to a final volume of 100μl. The platelets were counted using improved Neubauer chamber and adjusted to a concentration of 1x10 platelets per ml in PBS/ETDA/BSA. 8 1.5 ml eppendorf tubes were used where 6 contained negative controls (AB serum), one for a positive control (Anti HLA) and the other for the test sample (mother's serum). 100μl of the father's pellets were mixed and dispensed in all the tubes and incubated at room temperature in the dark for 30 minutes. 10μl anti-human IgG FITC was added to all tubes and then incubated in the dark for 10 minutes. The IgG-fluorophone conjugated antibodies are binding to the platelets in the sample. After each step the cells were washed and centrifuged at 6000 rpm for 1 minute with 1.5 ml PBS/EDTA/BSA. This washing process removes any non-specific immunoglobulins and any platelet-bound antibodies are detected with fluorescent labelled polyclonal or monoclonal antibody specific for IgG. The cell pellet were washed and re-suspended in 0.5 ml of sheath fluid and transferred to appropriate flow cytometry tubes. The platelets were gated and analysed for fluorescence in the flow cytometer. The graphs obtained were analysed and the two standard deviations were obtained from the mean fluorescence of all AB serum. This standard deviation acted as a negative cut off. Values within this range were not considered as they were considered to be statistically insignificant and vice-versa.
Table 1: Table showing the mean florescence of the negative control (AB serum), test sample (mother's serum) and positive control (anti HLA).
Mean of AB serum is 1.72833
One standard deviation of AB serum: 0.066
1.72+ 0.06 = 1.78.
1.72 - 0.06 = 1.66 (both values correspond to 66%)
Two standard deviation 1.72 - 0.06 = 1.66, 1.66-0.06 = 1.6
1.72 + 0.06 + 0.06 = 1.84 (both values correspond to 95%)
Values between this range are statistically in- significant i.e. negative result, not suffering from NAITP.
The mean of the sample is 12.79
1.60 1.66 1.72 1.78 1.84
This time line was generated, values beyond 1.84 are statistically significant i.e. suffering from NAITP.
From the calculations the mean sample value is 12.79. From the two standard deviations of the AB serum i.e. 1.6 and 1.8, both these values correspond to 95% of platelets in the negative control sample. Any value above these limit are statistically significant. Also our fluorescence value of the test sample is above this limit i.e. 2.31. Therefore 95% of IgG-fluorophone conjugated antibodies are binding to the platelets in the sample. Therefore this patient is suffering from NAITP.
Neonatal alloimmune thrombocytopenia (NAITP) is a devastating bleeding disorder. It often presents unexpectedly and ranges in severity from mild thrombocytopenia at birth to intracerebral haemorrhage (ICH) in utero, at delivery, or in the first days of life. NAITP is caused by maternal IgG alloantibodies stimulated by foetal platelet antigens inherited from the father. Foetal platelet antigens are expressed as early as 16 weeks of gestation and, soon enter the maternal circulation by mechanisms that are not understood yet. The IgG alloantibodies cross the placenta and target foetal platelets, causing rapid Fc-mediated clearance by the foetal reticule-endothelial system. The passage of blood through the lungs immediately after birth increases the exposure of IgG-sensitized platelets to reticule-endothelial cells and explains the postnatal drop in platelet count that is often observed. Anti-platelet antibodies may persist in maternal circulation for many years and can be further stimulated by additional exposures through subsequent pregnancies or transfusions. (Arnold, et al; 2008) This disease was previously thought to be a rare disease but after clinical investigations carried out on 25,000 pregnancies, it was found that 1/2000 pregnancies suffer from this disease. The leading cause of this disease is HPA-1a in 80% of the cases. These HPA antigens are a class of antigens found on the surface of platelets i.e. antibodies directed against these HPA antigens. NAITP is caused by antibodies to platelet-specific antigens, which present epitopes of the platelet surface glycoproteins (GPs). One of the most severe symptoms of this disease is ICH (Intracranial Haemorrhage) which in severe can results to death of the infant in utero.
The main goal which needs to be followed when diagnosing this disease is to determine if a maternal-foetal antigen incompatibility is present and to detect platelet alloantibodies (IgG) in maternal serum. The risk of the foetus is based on the results of these two criteria. The only possible way to control disease is immunising the mothers against HLA antigens but this connection has not been proven yet. (Metcalfe, 2004) Diagnostic tests for NAITP usually include detection of maternal antibody together with finding out whether the mother and the father have the respective platelet antigens. Maternal platelet antibodies are usually detectable against paternal platelets. (Rayment, et al; 2003). For women at risk of NAITP, antenatal treatment should be performed which involves several ultrasounds to monitor ICH or foetal distress. (Arnold, et al; 2008). The diagnosing procedure involves the following: 1. Antigen testing. This involves DNA based serological testing of both the frequently and infrequently encountered HPA antigens. Platelet antigen typing can also be carried out with the help of oligonucleotide probes. Various ELISA based kits and fluorescence-based tests can be performed for screening of positive antigens. 2. Alloantibody testing- i.e. the MAIPA assay which is utilised during pregnancy to monitor antibody levels. MAIPA and the radioimmunoprecipitation (RIP) assay allowed for the detection of alloantibodies with specificity for platelet antigens even at low levels of expression. The best method for detection is Flow Cytometry using fluorescent dyes through PIFT. This method has proven to be the best method to detect platelet-reactive antibodies in patient sera that binds to intact platelets. The platelets are constantly washed and finally detected using fluorescent labelled polyclonal or monoclonal antibodies which are specific for human immunoglobulins. The platelets are then analysed in the flow cytometer and interpreted as a ratio of the mean or peak channel fluorescence of normal platelets sensitised with patient serum over that of normal platelets incubated in normal serum. This technique has proven to be a very sensitive method for the detection of alloantibodies. This assay is capable of detecting very small numbers of antibody bound to platelets. It has been advocated as a platelet cross-matching method. (McFarland, 2003). To confirm diagnosis of NAITP caused by maternal IgG antibodies against neonatal platelets, both the mother and father have to be typed for the important platelet-specific antigens. The maternal serum is cross-matched with paternal serum and with a panel of typed donors (negative controls). In this panel the platelets are homozygous for the clinically relevant platelet-specific antigens because some antibodies do not react with heterozygous platelets. (Porceljin, et al; 1998). Along with PIFT another method is used as a coupled method in the diagnosis of such diseases and one such technique is the MPHA (mixed passive hemagglutination assay). Apart from these the other techniques used for the diagnosis of this disease are nonspecific platelet-associated IgG (PAIgG), anti-ABH, and anti-HLA antibodies. The use of flow cytometry allowed for the detection of antibodies at the individual platelet level; however, like PIFT, flow cytometry also detects nonspecific antibodies and reactivity to HLA.
Management of this disease requires a multidisciplinary treatment team that includes hematologists, obstetricians, and neonatologists, with support from transfusion medicine services and a specialized platelet testing laboratory. The other important point to consider is ICH. This may occur during or soon after delivery, or antenatally. Hence management of NAITP must begin prenatally and include considerations of delivery. (Johnson, et al; 1997) The therapeutic approaches involve 1. Monitoring the efficacy of the therapeutic interventions in foetuses using FBS. This approach has reportedly proven to drop foetal loss rate of 0.2%-7.2%. 2. Blind treatment-maternally administrating the foetus with IVIG and IVIG with a combination of dexamethasone which helped manage the ICH levels. Other corticosteroids also are used like prednisone. 3. Intrauterine platelet Transfusions- this treatment involves using washed maternal platelets which are then injected into the umbilical vein of the infant, also known as PUBS precutaneous umbilical blood sampling. Through this technique a fine gauge spinal needle is inserted and pure foetal blood can be obtained and analysis can be carried and the platelet count checked. (Johnson, et al; 1997)Unfortunately these platelets have a short half-life and therefore repeated transfusions are required. Current study has also stated that transfusing blood from random donors also helps reduce the risk of the infant. (Bussel, et al; 2008). The mode of delivery for this disease is usually caesarean although vaginal delivery is also considered depending on the platelet count of the foetus i.e. greater than 50x10. All women with a history of NAITP must be counselled on the risk of NAITP with subsequent pregnancies. For antigen-incompatible couples where maternal antibody is detected, all future pregnancies should be closely monitored. After birth the infant is at a high risk and therefore its platelet count should be monitored daily. To control this, the infant is administered with IVIG. (Johson, et al; 1997) In conclusion for better safety of the mother and the child, while still in utero an IVIG dose of 1g/kg/week should be administered with or without prednisone. PUBS should be an essential protocol which needs to be carried frequently. PUBS should also be carried out just before delivery. Intrauterine platelet transfusion should be carried out to avoid haemorrhage and to raise the foetal platelet count. After birth frequent ultrasounds and transfusions should be carried out to assess and monitor the well being of the infant.
The treatments for NAITP are still evolving. Several fundamental features of this disease such as the mechanism of placental passage of platelets, the identification of reliable clinical and laboratory predictors of disease severity still need to be fully understood. Better and more accurate therapies are required to improve the outcome of this disease and further better strategies are required for screening first pregnancies.