Screening of RHD fetal genotype in RhD negative women.

OBJECTIVE: In the Czech Republic in all women within a first trimester screening a laboratory testing for RhD blood group from the peripheral blood should be performed. The aim of the screening is to diagnose RhD negative pregnant women, who may be at risk of developing RhD alloimmunization if the fetus is RhD positive. Currently, the prevention of RhD alloimmunization is carried out regardless of the knowledge of RHD fetal status. Already at the beginning of pregnancy it is possible to determine the RHD genotype of the fetus non-invasively due to cell free fetal DNA circulating in maternal peripheral blood detection. The issue of screening examination of fetal RHD genotype is solved worldwide. In some European countries, the examination is routinely established and thus contributes to the optimization of prenatal care for RhD negative pregnant women, immunoglobulin administration is targeted only in pregnancies with RhD positive fetus. The aim of our study is to evaluate clinical and laboratory effectiveness of fetal RHD genotype screening in RhD negative women by TaqMan Real-time PCR method. Designe: Prospective cohort study. SETTING: Obstetrics and Gynecology Clinic of the Faculty of Medicine University Palacky and the University Hospital Olomouc; Institute of Medical Genetics of the Faculty of Medicine UP and the University Hospital Olomouc; Transfusion Department of the University Hospital Olomouc; Institute of Biophysics of the Faculty of Medicine UP Olomouc. MATERIAL AND METHODS: In 2011-2015 at the University Hospital Olomouc 337 examinations of RHD fetal genotype were performed in pregnant women in first and second trimester and evaluated by TaqMan Real-time PCR, followed by verification of the newborn RHD genotype. RESULTS: Methodology of fetal RHD genotype examination is accurate, reliable and useful in clinical practice. The sensitivity was 97.8%. The specificity was 98.7%. When assessing the effectiveness of the introduction of non- -invasive fetal RHD genotype screening in RhD negative women, it is necessary to assess the medical, organizational and economic aspects. More consistent prevention of RhD alloimmunization in the cases actually indicated may reduce the incidence of RhD alloimmunization. CONCLUSION: From the medical point of view the RHD genotype determination in all RhD negative women at the beginning of pregnancy seems effective. It allows to diagnose about 40% of pregnancies with RhD negative fetuses that do not require administration of IgG anti-D. IgG anti-D should be administered only in indicated cases. Determination of fetal RHD genotype by using TaqMan Real-time PCR is useful in clinical practice.

The effectiveness of KEL and RHCE fetal genotype assessment in alloimmunized women by minisequencing.

OBJECTIVE: To evaluate the effectiveness of the fetal KEL and RHCE genotype assessment in alloimmunized pregnant women by minisequencing. DESIGN: Prospective cohort study. SETTING: Obstetrics and Gynecology Clinic of the Faculty of Medicine UP and the University Hospital Olomouc; Institute of Medical Genetics of the Faculty of Medicine UP and the University Hospital Olomouc; Transfusion Department of the University Hospital Olomouc; Institute of Biophysics of the Faculty of Medicine UP Olomouc. SUBJECT AND METHOD: In the years 2001-2019, 366 samples of pregnant women in the first and second trimester were assessed KEL (n = 327) or RHCE (n = 39) genotype from the free fetal DNA circulating in the peripheral blood by minisequencing. The genotype of the fetus was verified from the buccal smear of the newborn. RESULTS: The KEL genotype was assessed in 327 women (the presence of a variant of the KEL1 alele, which corresponds to the presence of the erythrocyte antigen “K“. The analysis failed in 2 cases (2/327), 16 heterozygote women (KEL1/KEL2) were excluded and in the case of 309 homozygote women (KEL2/KEL2) the fetal KEL genotype was assessed. In the case of 95.8% of the fetuses (296/309) and 95.5% of the newborns (295/309), the KEL2/KEL2 genotype was assessed. In the case of 4.2 % of the fetuses (13/309) and 4.5% of the newborns (14/309), the KEL1/KEL2 genotype was assessed. The sensitivity was 92.86%. The specificity was 100%. The RHCE genotype was assessed in 39 women. In the case of 22 women, the presence of a variant of the RHCE gene, which corresponds to the presence of the erythrocyte antigen “C“/“c“, was assessed. 5 heterozygote women (C/c) were excluded. In the case of 11 homozygote women (C/C), the RHCE genotype was assessed. In the case of 64% (7/11) of the fetuses and newborns, the C/c genotype was assessed, in the case of 36% (4/11) the C/C genotype was assessed. In the case of 6 homozygote women (c/c), the RHCE genotype was assessed. In the case of 67% (4/6) of the fetuses and newborns, the C/c genotype was assessed, in the case of 33% (2/6) the c/c genotype was assessed. The sensitivity and specificity were 100%. In the case of 17 women, the presence of the variant of the RHCE gene, which corresponds to the presence of the erythrocyte antigen “E“/“e“, was assessed. 1 heterozygote woman (E/e) was excluded. In the case of 16 homozygote women (e/e), the RHCE genotype was assessed. In the case of 75% (12/16) of the fetuses and newborns, the e/e genotype was assessed, in the case of 25% (4/16) the E/e genotype was assessed. The sensitivity and specificity were 100%. CONCLUSION: The minisequencing method using the capillary electrophoresis enabled a reliable detection of the fetal KEL and RHCE genotype from the peripheral blood of pregnant women.

[Postpancreatectomy haemorrhage (PPH), prevalence, diagnosis and management]. / Krvácení po pankreatektomii (PPH), prevalence, diagnostika a resení.

INTRODUCTION: Postpancreatectomy haemorrhage (PPH) is considered to be the most severe specific postoperative complication following pancreatic resections and its treatment is difficult and requires coordinated interdisciplinary collaboration. PPH causes 11-38% of all post-pancreatectomy deaths. The aim of this study was to determine the prevalence of PPH in a set of patients operated on within the last 10 years, and to analyze the diagnostic methods, treatment modalities and the outcomes. METHODS: A retrospective analysis of patients undergoing pancreatic resections between 2006 and 2015. Clinically relevant PPH (types B and C) were the subject of interest. The onset, location and severity of PPH were analysed. Other factors analysed included operation diagnosis of PPH, diagnostic methods along with signs of sentinel bleeding, treatment options undertaken including the number of transfusions. 30-day, 90-day and in-hospital mortality, as well as the length of hospital stay and readmission rate were calculated. A descriptive statistical method was used. RESULTS: A total of 449 patients were operated on. Pancreatoduodenectomy (DPE) or pylorus-preserving pancreatoduodenectomy (PPPD) was done in 76.4%, left sided pancreatectomy (LPE) in 19.8% and total pancreatectomy (TPE) in 3.8%. 190 of the patients (42.3%) were women and 259 (57.7%) men, with the mean age of 61.5±11.1 years. A total of 23 (5.1%) PPH cases were identified, 21 (4.7%) were clinically relevant. Eight patients (35%) developed early PPH with direct reoperation, late PPH was seen in 14 patients after DPE and in one after LPE. Sentinel bleeding was present in 53.3% of late PPH cases. CT/CTA was performed in four patients with subsequent DSA performed in three. DSA identified a gastroduodenal artery stump pseudoaneurysm in one patient, which was resolved using a stent. Surgical intervention for late PPH was required in 10 patients in total, six of whom needed direct surgery due to the rapid development of circulatory instability and 3 due to inconclusive radiological management. One patient needed surgical drainage of both an abscess and haematoma. In two patients the origin of bleeding was due to a gastric ulcer, which was proven and solved endoscopically and 2 patients required conservative treatment only. The specific mortality for PPH was 17.4%. In the group of patients that suffered with any PPH following DPE and PPDPE the mortality rate was 22.2%, and 28.6% for late PPH. If late PPH developed coincidentally with postoperative pancreatic fistula (POPF), the mortality was 44%. In the early PPH group, an average of 10.1±2.5 transfusion units (TUs) were used with an average length of hospital stay 17.5±4.8 days and zero mortality in comparison to an average of 11.7±10 TUs and 29.9±14.6 days in hospital and 26.6% mortality in the late PPH group. CONCLUSION: PPH is a severe complication, which has a high mortality rate. It also often coincidentally develops with POPFs. Early clinical diagnosis with identification of its cause plays a key role in management. The use of interventional radiology in the treatment of PPH has begun to dominate other treatment modalities due to a very high success rate, and close collaboration with interventional radiologists is necessary in order to reduce the rate of surgical intervention required in PPH. KEY WORDS: haemorrhage - pancreas - resection - complications - mortality.

[Spontaneous antepartal RhD alloimmunization]. / Spontánní antepartální RhD aloimunizace.

AIM OF THE STUDY: Assess the incidence of spontaneous antepartal RhD alloimmunization in RhD negative pregnant women with an RhD positive fetus. DESIGN: Clinical study. SETTING: Department of Obstetrics and Gynecology, Medical School and University Hospital Olomouc. METHODS: A total of 906 RhD negative women with an RhD positive fetus and without the presence of anti-Dalloantibodies at the beginning of pregnancy were examined. Always it was a singleton pregnancy, RhD blood group of the pregnant women was assessed in the 1st trimester of pregnancy, RhD status of the fetus was determined after delivery. Screening for irregular antierythrocyte antibodies was performed in all women in the 1st trimester of pregnancy, at 28-32 weeks gestation and immediately prior to delivery at 38-42 weeks gestation. Screening for irregular antierythrocyte antibodies was performed also at 6 months following delivery in all cases of positive antibodies before delivery. Antibody screening was performed using the indirect antiglobulin (LISS/NAT) and enzyme (papain) test with their subsequent identification using a panel of reference erythrocytes by column agglutination method Dia-Med. After delivery, the volume of fetomaternal hemorrhage was assesed in all RhD negative women and RhD alloimmunization prophylaxis was performed by administering the necessary IgG anti-D dose; none of the women were administered IgG anti-D antepartally. RESULTS: During screening for irregular antierythrocyte antibodies at 28-32 weeks gestation, anti-D alloantibodies were diagnosed in 0.2% of the women (2/906); immediately prior to the delivery at 38-42 weeks gestation, anti-D alloantibodies were diagnosed in 2.3% of the women (21/906) and repeatedly even at 6 months following delivery (21/157). In 82.7% of the women (749/906), examination at 6 months following delivery was not performed, therefore in these women spontaneous antepartal RhD alloimmunization cannot reliably be ruled out. Alloimmunization may not be diagnosed yet at term of delivery. If anti-D alloantibodies were not present prior to the delivery, these women were all administered IgG anti-D in a dose of at least 125 µg after delivery. CONCLUSION: In RhD negative women with an RhD positive fetus, the incidence of spontaneous antepartal RhD alloimmunization was at least 2.3%. Most cases may theoretically be prevented by prophylactic administration of 250 µg of IgG anti-D to all RhD negative women at 28 weeks gestation.

[Guideline for prevention of RhD alloimmunizationin RhD negative women]. / Doporucení k provádení prevence RhD aloimunizace u RhD negativních zen.

Events following which immunoglobulin (Ig) G anti-D should be given to all RhD negative women with no anti-D alloantibodies: First trimester indications (IgG anti-D sufficient dose of 50 µg*) - termination of pregnancy, spontaneous abortion followed by instrumentation, ectopic pregnancy, chorionic villus sampling, partial molar pregnancy; Second and third trimester indications (IgG anti-D sufficient dose of 100 µg*) - amniocentesis, cordocentesis, other invasive prenatal diagnostic or therapeutic procedures, spontaneous or induced abortion, intrauterine fetal death, attempt at external cephalic version of a breech presentation, abdominal trauma, obstetric hemorrhage; Antenatal prophylaxis at 28th weeks of gestation (IgG anti-D sufficient dose of 250 µg*); Delivery of an RhD positive infant** (IgG anti-D sufficient dose of 100 µg*); Minimal dose*: before 20 weeks gestation - 50 µg (250 IU), after 20 weeks gestation*** - 100 µg (500 IU); Timing: as soon as possible, but no later than 72 hours after the event. In cases where prevention of RhD alloimmunization is not performed within 72 hours of a potentially sensitising event, it is still reasonable to administer IgG anti-D within 13 days, and in special cases, administration is still recommended up to a maximum interval of 28 days postpartum; Legend: *administration of a higher dose of IgG anti-D is not a mistake, ** also if the D type is not known, *** simultaneous assessment of the volume of fetomaternal hemorrhage (FMH) to specify the dose is suitable; The FMH volume assessment - If the volume of fetal erythrocytes (red bood cells, RBCs) which entered maternal circulation is assessed, intramuscular administration of IgG anti-D in a dose of 10 µg per 0.5 mL of fetal RBCs or 1 mL of whole fetal blood is indicated. IgG anti-D in a dose of 10 µg administered intramuscularly should cover 0.5 mL of fetal RhD positive RBCs or 1mL of whole fetal blood. FMH is the fetal RBC volume; fetal blood volume is double (expected fetal hematocrit is 50%).

[Incidence of erythrocyte alloimmunization in pregnant women in olomouc region]. / Incidence erytrocytární aloimunizace u tehotných zen v olomouckém regionu.

OBJECTIVE: To determine the incidence of clinically significant anti-erythrocyte alloantibodies in pregnant women, which can cause severe hemolytic disease in the fetus and newborn. DESIGN: Retrospective-prospecitive clinical study. SETTING: Transfusion Department, University Hospital Olomouc, Department of Obstetrics and Gynecology, University Hospital Olomouc. SUBJECT AND METHOD: Between the years 2000-2011, a total of 45 435 pregnant women were examined at the Department of Transfusion Medicine at the University Hospital Olomouc. Screening for irregular anti-erythrocyte antibodies followed by identification of the alloantibody was performed in all women at the beginning of the pregnancy. RESULTS: Clinically significant anti-erythrocyte antibodies were diagnosed in 1.5% pregnant women (683/45435). The most common cause of maternal alloimmunization was antigen E with an incidence of 5.7 (258/45435), followed by antigen D 4.0 (181/45435), M 1.5 (70/45435), C 1.2 (54/45435), K 1.2 (55/45435), c 0.6 (26/45435), S 0.4 (20/45435), Jka 0.2 (9/45435), PP1pk (Tja) 0.1 (3/45435) and antigen Fya 0.0 (2/45435). CONCLUSION: Despite performing prophylaxis for RhD alloimmunization by administering anti-D immunoglobulin to RhD negative women during pregnancy and after the birth of an RhD positive child, antigen RhD still represents the 2nd most frequent cause of maternal erythrocyte alloimmunization. The remaining clinically significant alloimmunizations are caused by non-D antigens of the Rh system, antigens of the Kell system, and rarely observed antigens of the MNS and Kidd blood systems.

[Erythrocyte alloimmunization in pregnant women, clinical importance and laboratory diagnostics]. / Erytrocytární aloimunizace tehotných zen, klinický význam a laboratorní diagnostika.

OBJECTIVE: The aim of this review is to give comprehensive summary of erythrocyte alloimunization of pregnant women, laboratory dignostics and clinical importance. DESIGN: Review. SETTING: University Hospital Olomouc, Transfusion Department, Department of Obstetrics and Gynecology. SUBJECT AND METHOD: Based on literature analysis using database search engines PubMed, Google Scholar, Ovid in field of erythrocyte antibodies, laboratory diagnostics and clinical importance up-to-date knowledge. CONCLUSION: Erythrocyte alloimunization anti-D antibodies decreases in connection with the introduction of immunoprofylaxis. Immunization of non RhD antibodies with impossibility using of immunoprofylaxis remains still clinical problem.

[Epidemiology and management of thyroid disorders in pregnancy]. / Epidemiologie a management poruch stítné zlázy v tehotenství

OBJECTIVE: Determine the incidence of asymptomatic decrease of thyroid function at pregnant women in the first trimester of pregnancy in the Olomouc region. MATERIAL AND METHODS: The authors examined 461 women in the first trimester of pregnancy. There was measured serum concentrations of thyrotropin (TSH), free thyroxine (FT4) and thyroidperoxidaseantibody (TPOAb). RESULTS: TSH concentration > 2.5 mIU/l was in 21% women (96/461). Reduction of FT4 < 9.8 pmol/l was found in 15 of the 96 women screened with a TSH greater than 2.5 mU/l. For women with TSH 2.5 mU/l, however, was found reduction of the FT4 < 9.8 pmol/l in 11 cases. TSH concentration > 3 mIU/l was found in 14% women (64/461). Reduction of FT4 < 9.8 pmol/l was found in 13 of the 64 women screened with a TSH greater than 3 mU/l. TSH concentration> 3.5 mIU/l in 10% of women (45/461). Reduction of FT4 <9.8 pmol/l was found in 11 of 45 examined women with TSH greater than 3.5 mU/l. TSH concentration > 4 mIU/l in 8% of women (35/461). Reduction of FT4 < 9.8 pmol/l was found in 10 of35 examined women with TSH greater than 4 mU/l. TPOAb was raised over 20 kU/l in 17% of women (78/461). CONCLUSIONS: Asymptomatic decrease of thyroid function was during direct examination in the first trimester of pregnancy diagnosed at more than 8% of pregnant women. Untreated decreased of thyroid function at mothers may have a negative impact on the course of pregnancy, as well as on fetal development, especially in the psychomotoric area. The presence of TPOAb indicate an increased risk of developing thyroid fictional disorders in pregnancy or postpartum thyroiditis. TSH in pregnancy may be affected by elevated levels of hCG and may slightly decrease because hCG and TSH have a common alpha subunit. Discussed the specific standards for TSH in pregnant and implementation of systematic screening for women in early pregnancy or before planned conception. Perspective, however, we see in tests of thyroid function already in preconception period.

[Disorders of the thyroid gland in pregnancy]. / Poruchy funkce stítné zlázy v tehotenství

Functional disorder of the thyroid gland affects individuals of all ages. Incidence and nature of the disease is influenced by supplying iodine, genetic and immunological factors and other external effects. Increased function occurs in 1-2% of the adult population, reduced function in the younger population diagnosed in about 5%, in women aged over 50 years we have been at 10-15%. The incidence of thyroid diseases is higher in women than in men (5-8:1) and significantly increases with age. Approximately as common as overt forms of thyroid disease are their subclinical forms. Thyroid disease, especially asymptomatic reduction of function for direct examination is diagnosed up to 5% of pregnant women. Untreated decreased thyroid function of mothers may have significant negative impact on the course of pregnancy, as well as on fetal development, especially in the psychomotoric area. Women with the presence of antithyroid antibodies are at risk of pregnancy deepening malfunctions and/or postpartum thyroiditis. Czech Endocrinological Society currently pursuing a systematic screening of other risk population groups, including notably the pregnant and nursing women, women in old age and ill with some other disease or treatment with certain drugs.

[RHD genotyping from cell-free fetal DNA circulating in pregnant women peripheral blood and sensitivity assessment of innovated diagnostic approaches for introduction into the clinical practice]. / Stanovení RHD genotypu plodu z plazmy periferní krve tehotné zeny a posouzení citlivosti nových diagnostických postupu pro zavedení do klinické praxe.

OBJECTIVE: Introduction of fetal RHD genotyping from cell-free fetal DNA circulating in the peripheral blood of pregnant women to clinical practice. Sensitivity assessment of innovated method using range of dilution series and internal control of amplification. DESIGN: Procedure creating of noninvasive determination of fetal RHD genotyping from blood plasma of pregnant women. Detection of limit of minority representation RHD+/- sample in the RHD-/- sample. SETTING: University Hospital Olomouc, Institute of Medical Genetics and Fetal Medicine, Clinic of Obstetrics and Gynecology, Transfusion Department. METHODS: TaqMan Real-Time PCR without an internal amplification controls. Optimization and calibration of RHD genotyping using RHD multiplex by TaqMan Real-Time PCR with an internal amplification control and by minisequencing (Snapshot - multiplex) with an internal amplification controls. RESULTS: RHD positive or negative fetuses were determined by amplification curves from Real-Time PCR system that matches the parameters for the evaluation of the output data using series of amplification and contamination parallel controls. TaqMan based Real-Time PCR and minisequencing (SNaPshot) based quantification were able to detect 0.22% of artificial RHD+/- sample diluted in RHD-/- sample. In addition, SNaPshot assay is suitable for heterozygozity and homozygozity recognition. CONCLUSION: Current established and routinely used procedure is based on the detection of exon 7 of the RHD gene and on the series of parallel amplification and contamination controls. Both newly developed methods could be, after validation of the larger set of control samples, introduced into clinical practice.

[Guideline for prevention of RhD alloimmunization in RhD negative women]. / Doporucení k provádení prevence RhD aloimunizace u RhD negativních zen.

Events following which anti-D immunoglobulin should be given to all RhD negative women with no anti-D antibodies: First trimester indications (50 microg)--termination of pregnancy, spontaneous abortion followed by instrumentation, ectopic pregnancy, chorionic villus sampling, partial molar pregnancy; Second and third trimester indications (100 microg)--amniocentesis, cordocentesis, other invasive prenatal diagnostic or therapeutic procedures, spontaneous or induced abortion, intrauterine fetal death, attempt at external cephalic version of a breech presentation, abdominal trauma, obstetric haemorrhage; Antenatal prophylaxis at 28th weeks of gestation (250 microg); Delivery of an RhD positive infant * (100 microg); Minimal dose: before 20 weeks gestation--50 microg (250 IU), after 20 weeks gestation **--100 microg (500 IU); Timing: as soon as possible, but no later than 72 hours after the event. In cases where prevention of RhD alloimmunization is not performed within 72 hours of a potentially sensitising event, it is still reasonable to administer anti-D immunoglobulin (IgG anti-D) within 13 days, and in special cases, administration is still recommended up to a maximum interval of 28 days postpartum.; FMH (fetomaternal haemorrhage)--If the amount of fetal erythrocytes which entered the maternal circulation is quantitatively determined, administration of 10 microg IgG anti-D per 0.5 ml of fetal erythrocytes or 1 ml of whole blood is indicated. *also if the RhD type of the infant has not been determined or is in doubt, **in conjunction with a test to assess the volume of any fetomaternal hemorrhage.