A British Society for Haematology Guideline: Diagnosis and management of thrombotic thrombocytopenic purpura and thrombotic microangiopathies.

The objective of this guideline is to provide healthcare professionals with clear, up-to-date and practical guidance on the management of thrombotic thrombocytopenic purpura (TTP) and related thrombotic microangiopathies (TMAs), including complement-mediated haemolytic uraemic syndrome (CM HUS); these are defined by thrombocytopenia, microangiopathic haemolytic anaemia (MAHA) and small vessel thrombosis. Within England, all TTP cases should be managed within designated regional centres as per NHSE commissioning for highly specialised services.

A British Society for haematology guideline: diagnosis and management of thrombotic thrombocytopenic purpura and thrombotic microangiopathies

The objective of this guideline is to provide healthcare professionals with clear, up-to-date and practical guidance on the management of thrombotic thrombocytopenic purpura (TTP) and related thrombotic microangiopathies (TMAs), including complement-mediated haemolytic uraemic syndrome (CM HUS); these are defined by thrombocytopenia, microangiopathic haemolytic anaemia (MAHA) and small vessel thrombosis. Within England, all TTP cases should be managed within designated regional centres as per NHSE commissioning for highly specialised services.

Changes in laboratory markers of thrombotic risk early in the first trimester of pregnancy may be linked to an increase in estradiol and progesterone.

BACKGROUND: Pregnant women are at increased risk of venous thrombosis compared to non-pregnant women. Epidemiological and laboratory data suggest that hypercoagulability begins in the first trimester but it is unknown exactly how early in pregnancy this develops. The mechanisms that result in a prothrombotic state may involve oestrogens and progestogens. METHODS: Plasma samples were taken prior to conception and five times in early pregnancy, up to Day 59 gestation, from 22 women undergoing natural cycle in vitro fertilization, who subsequently gave birth at term following a normal pregnancy. Thrombin generation, free Protein S, Ddimer, Fibrinogen, factor VIII, estradiol and progesterone were measured. To counter inter-individual variability, the change in laboratory measurements between the pre-pregnant and pregnant state were measured over time. RESULTS: Peak thrombin, Endogenous Thrombin Potential, Velocity Index and fibrinogen significantly increased, and free Protein S significantly decreased, from pre-pregnancy levels, by 32 days gestation. Ddimer and VIII significantly increased from pre-pregnancy levels by 59 days gestation. Estradiol significantly increased by Day 32 gestation with a non-significant increase of 67% by Day 24 gestation. Progesterone significantly increased by Day 32 gestation. Almost all laboratory markers of thrombosis correlated significantly with estradiol and progesterone. CONCLUSION: Our work is the first to demonstrate that the prothrombotic state develops very early in the first trimester. Laboratory markers of hypercoagulability correlate significantly with estradiol and progesterone suggesting these are linked to the prothrombotic state of pregnancy. Clinicians should consider commencing thromboprophylaxis early in the first trimester in women at high thrombotic risk.

Normal pregnancy is associated with an increase in thrombin generation from the very early stages of the first trimester.

BACKGROUND: Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis, which begins during the first trimester, but the exact time of onset is unknown. Thrombin generation, a laboratory marker of thrombosis risk, increases during normal pregnancy but it is unclear exactly how early this increase occurs. METHODS: We assessed thrombin generation by Calibrated Automated Thrombography in women undergoing natural cycle in vitro fertilization, who subsequently gave birth at term following a normal pregnancy (n=22). Blood samples were taken just prior to conception and repeated five times during very early pregnancy, up to Day 59 estimated gestation. RESULTS: Mean Endogenous Thrombin Potential (ETP), peak thrombin generation and Velocity Index (VI) increased significantly from pre-pregnancy to Day 43 gestation (p=0.024-0.0004). This change persisted to Day 59 gestation. The mean of the percentage change from baseline, accounting for inter-individual variation, in ETP, peak thrombin and VI increased significantly from pre-pregnancy to Day 32 gestation (p=0.0351-<0.0001) with the mean increase from baseline persisting to Day 59 gestation. CONCLUSION: Thrombin generation increases significantly during the very early stages of normal pregnancy when compared to the pre-pregnancy state. The increased risk of venous thrombosis therefore likely begins very early in a woman's pregnancy, suggesting that women considered clinically to be at high thrombotic risk should start thromboprophylaxis as early as possible after a positive pregnancy test.

Establishing a reference range for thrombin generation using a standard plasma significantly improves assay precision.

BACKGROUND: Thrombin generation is a global coagulation assay which appears to be an effective method for assessing the potential for an individual's plasma to coagulate. However for this assay to become accepted in routine clinical practice it requires standardization. OBJECTIVES: To establish a reference range for the NIBSC reference plasma (TGT-RP) which can then become an internal quality control (IQC) for thrombin generation assays. PATIENTS/METHODS: Thrombin generation was measured in TGT-RP in 153 independent experiments using 4 assay conditions; 1 pM tissue factor (TF) or 5 pM TF +/- thrombomodulin (TM). A target value +/- 2 SD was calculated to provide an acceptance range under the 4 conditions. A plasma sample from a healthy volunteer was subsequently tested in 11 separate experiments using the TGT-RP for (i) normalisation and (ii) exclusion of experimental results when the TGT-RP results did not fall within the established acceptance range. RESULTS: An acceptance range was established for TGT-RP for the 4 assay conditions. Normalisation of all results from a healthy volunteer reduced inter-assay variability significantly (ETP: p=0.0003; Peak: p=0.001). Exclusion of results from the volunteer when concurrently run TGT-RP results fell outside the acceptance range reduced inter-assay variability significantly when reporting raw data (ETP: p=0.001; Peak: p=0.004). However normalisation of this data had no beneficial effect (ETP: p=0.126; Peak: p=0.232). CONCLUSIONS: Our work represents further progress in the standardization of thrombin generation techniques with the establishment of an IQC reference range. Using an IQC reduces inter-assay variability, whilst allowing reporting of raw data and ensures production of accurate and reproducible data.

The effect of estrone on thrombin generation may explain the different thrombotic risk between oral and transdermal hormone replacement therapy.

BACKGROUND: The metabolism of estrogen contained within hormone replacement therapy (HRT) is influenced by the route of administration, and this may affect the risk of venous thromboembolism. Thrombin generation, a global coagulation assay, is a marker of hypercoagulability and is of potential use in determining the thrombotic risk associated with particular HRT administration routes. OBJECTIVES: To determine whether any effect of oral and transdermal HRT on thrombin generation is related to the plasma estrogen profile. METHODS: We investigated the effects of oral, transdermal and no HRT (controls) in 52, 39 and 52 postmenopausal women, respectively, on thrombin generation, standard markers of thrombophilia, estradiol level and estrone level. RESULTS: All parameters of thrombin generation were altered in women using oral HRT as compared with controls (P<0.001 for all comparisons). No such differences were found in women using transdermal HRT. Estrone levels correlated with peak thrombin generation (R=0.451, P<0.001) in women using oral HRT, but there was no correlation in women using the transdermal route. CONCLUSIONS: Thrombin generation is significantly increased in women who use HRT administered by the oral route. This is probably mediated by the hepatic first-pass metabolism of estrone, the main metabolite of oral estradiol, which is avoided by the transdermal route. The effect of estrone on thrombin generation may provide the explanation for the higher thrombotic risk seen in women using oral rather than transdermal HRT.

Maternal Hoxa10 is required for pinopod formation in the development of mouse uterine receptivity to embryo implantation.

Hoxa10 is a homeobox gene that is expressed both during the embryogenesis of the genitourinary tract and in the adult reproductive tract. Maternal Hoxa10 expression is necessary for endometrial receptivity to blastocyst implantation. The mechanism by which Hoxa10 induces endometrial development to a state of receptivity is unknown as HOXA10-deficient endometrium appears histologically normal. We altered the expression of Hoxa10 in the uterus of cycling adult female mice and examined the uterus at the time of implantation by transmission electron microscopy for alterations in epithelial morphology. Pinopods are projections on the surface of the uterine endometrial epithelial cells that develop transiently at the time of endometrial receptivity. Blocking Hoxa10 expression by transfection of Hoxa10 antisense into the cycling mouse uterus before implantation dramatically decreased pinopod number. Constitutively expressing Hoxa10 in the uterus just before the normal time of pinopod formation resulted in increased pinopod number. Therefore, Hoxa10 is necessary for pinopod development. Hox genes have been implicated in both the regulation of cellular proliferation and the determination of developmental fate. Hoxa10 exemplifies this dual role in the uterus by regulating both endometrial stromal cell proliferation and epithelial cell morphogenesis. Taken together, these results demonstrate that maternal Hoxa10 has an essential role in pinopod development and this function of Hoxa10 likely contributes to endometrial receptivity for the purpose of blastocyst implantation.

Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation.

Mice with a targeted mutation of the Hoxa10 gene demonstrate uterine factor infertility. It is unclear if the defect in the uterine environment arises due to the absence of Hoxa10 expression during embryonic development or in the adult. We have recently demonstrated that HOXA10 expression in human endometrium rises dramatically at the time of implantation, suggesting maternal expression of Hoxa10/HOXA10 may be essential to the process. To assess the importance of maternal Hoxa 10 expression, the uteri of day 2 pregnant mice were injected with a DNA/liposome complex containing constructs designed to alter maternal Hoxa10 expression before implantation. Transfection with a Hoxa10 antisense oligodeoxyribonucleotide significantly decreased the number of implantation sites. Transfection with a plasmid which constitutively expresses Hoxa10 optimized survival of implanted embryos resulting in increased litter size. These results demonstrate that maternal Hoxa10 expression is essential for implantation and is the first report of the maternal alteration of a gene known to affect implantation specifically. We also demonstrate that DNA/liposome complexes containing the same Hoxa10 constructs that alter fertility in mice, can affect Hoxa10 expression in a human endometrial cell line. Alteration of human endometrial HOXA10 via liposome-mediated gene transfection is a potential contraceptive agent or fertility treatment.