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#1
15.08.2011, 20:14
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Псевдотромбоцитопения - способы коррекции в трудных случаях
Замена ЭДТА на цитрат или гепарин - наиболее простое решение, но что можно сделать, если и это не помогает?: добавление аминогликозидного антибиотика (канамицин, гентамицин) в пробирку перед взятием крови может помочь:
Clin Chem Lab Med. 1999 Nov-Dec;37(11-12):1063-6. Accurate platelet counting in an insidious case of pseudothrombocytopenia. Lombarts AJ, Zijlstra JJ, Peters RH, Thomasson CG, Franck PF. Department of Clinical Chemistry, Leyenburg Hospital, The Hague, The Netherlands. Anticoagulant-induced aggregation of platelets leads to pseudothrombocytopenia. Blood cell counters generally trigger alarms to alert the user. We describe an insidious case of pseudothrombocytopenia, where the complete absence of Coulter counter alarms both in ethylenediaminetetraacetic acid blood and in citrate or acid citrate dextrose blood samples was compounded by the fact that the massive aggregates were exclusively found at the edges of the blood smear. Non-recognition of pseudothrombocytopenia can have serious diagnostic and therapeutic consequences. While the anti-aggregant mixture citrate-theophylline-adenosine-dipyridamole completely failed in preventing pseudothrombocytopenia, addition of iloprost to anticoagulants only partially prevented the aggregation. Only the prior addition of gentamicin to any anticoagulant used resulted in a complete prevention of pseudothrombocytopenia and enabled to count accurately the platelets. --- EDTA-dependent pseudothrombocytopenia confirmed by supplementation of kanamycin; a case report. Ahn HL, Jo YI, Choi YS, Lee JY, Lee HW, Kim SR, Sim J, Lee W, Jin CJ. Korean J Intern Med. 2002 Mar;17(1):65-8. [Ссылки доступны только зарегистрированным пользователям ] --- Br J Haematol. 1997 Dec;99(4):817-23. Aminoglycosides prevent and dissociate the aggregation of platelets in patients with EDTA-dependent pseudothrombocytopenia. Sakurai S, Shiojima I, Tanigawa T, Nakahara K. Department of Central Laboratory, Tokyo University Hospital, Japan. Although EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is of practical importance because failure to recognize this clinical entity may result in misdiagnosis and subsequent mismanagement of the patients, the pathophysiological nature of EDTA-PTCP remains unknown. To develop an effective way to evaluate the platelet counts in patients with EDTA-PTCP, we introduced aminoglycosides-supplemented anticoagulating agents. When kanamycin was pre-supplemented with EDTA for anticoagulating blood samples from EDTA-PTCP patients there was no significant change in the platelet counts and the morphology of blood cells after 150 min of incubation at room temperature. Furthermore, when kanamycin was added to EDTA-anticoagulated blood samples from EDTA-PTCP patients within 30 min after blood withdrawal, rapid dissociation of platelets without apparent morphological changes of blood cells was observed, and complete blood cell counts as well as the histogram patterns were almost the same as those examined immediately after blood sampling. The dissociation of aggregated platelets was also detected when other antibiotics were used, although it was associated with some extent of morphological changes of blood cells. These findings indicate that the supplementation of aminoglycosides either before or after blood sampling is a useful method for the diagnosis EDTA-PTCP and for the evaluation of platelet counts in patients with EDTA-PTCP. 
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#2
12.12.2012, 00:35
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еще некоторые недавние публикации по теме:
Am J Clin Pathol. 2011 Oct;136(4):646-52. Amikacin can be added to blood to reduce the fall in platelet count. Zhou X, Wu X, Deng W, Li J, Luo W. [Ссылки доступны только зарегистрированным пользователям ] ---- Clin Chem Lab Med. 2012 Mar 3;50(8):1387-91. Novel method to dissociate platelet clumps in EDTA-dependent pseudothrombocytopenia based on the pathophysiological mechanism. Chae H, Kim M, Lim J, Oh EJ, Kim Y, Han K. Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, South Korea. BACKGROUND: EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is an in vitro phenomenon of platelet clumping that leads to spuriously low platelet counts by automatic hematology analyzers. The mechanism is not clearly defined, but is known as an immunologically mediated phenomenon due to the presence of EDTA-dependent antiplatelet auto-antibodies that induce platelet clumping. The purpose of this study was to identify antiplatelet antibodies in EDTA-PTCP samples and to design a method to dissociate platelet clumps based on the pathophysiological mechanism. METHODS: The antiplatelet antibody was investigated using direct and indirect immunofluorescent flow cytometric methods in 23 EDTA-anticoagulated whole blood (WB) samples and 12 serum samples of EDTA-PTCP patients, respectively. A novel mixture containing 9 mmol/L CaCl(2) and 0.1 unit/L sodium heparin, that provides calcium replacement while curbing coagulation, was designed to dissociate platelet clumps. The effect on dissociation was demonstrated in 26 samples of EDTA-PTCP and compared with the established method of kanamycin supplementation. RESULTS: The direct test was positive for IgM and IgG antiplatelet antibody in 60.9% and 4.4% of patients, respectively [mean median fluorescence intensity (MFI) of 223.9 and 128.4, respectively]. The indirect test was positive for IgM antiplatelet antibody in 58.3% of patients (mean MFI of 123.4). The novel method dissociated the platelet clumps with a mean increased platelet count of 242.3% and was equivalent in efficiency to kanamycin supplementation. CONCLUSIONS: The novel method is an easily applicable and efficient measure that allows dissociation of platelet clumps, based on the pathophysiological mechanism of EDTA-PTCP. --- Clin Chem Lab Med. 2012 Aug;50(8):1281-5. EDTA-dependent pseudothrombocytopenia: further insights and recommendations for prevention of a clinically threatening artifact. Lippi G, Plebani M. Ethylenediaminetetra-acetic acid (EDTA) is widely used as anticoagulant in laboratory medicine. EDTA-dependent pseudothrombocytopenia is a rare phenomenon (i.e., around 0.1% in the general population), which is mostly due to the presence of EDTA-dependent antiplatelet antibodies that react optimally between 0°C and 4°C, recognize the cytoadhesive receptors gpIIb-IIIa, stimulate the expression of activation antigens, trigger activation of tyrosine kinase, platelet agglutination and clumping in vitro, which finally lead to a spuriously decreased platelet count. The reliable and timely identification of this artifact is essential, since there a high chance that it may be confused with other life-threatening platelet disorders, or otherwise lead to inappropriate clinical and therapeutic decision-making. Five basic criteria should be fulfilled to raise the clinical suspicion of EDTA-dependent pseudothrombocytopenia, i.e., (i) abnormal platelet count, typically <100×10(9)/L; (ii) occurrence of thrombocytopenia in EDTA-anticoagulated samples at room temperature, but to a much lesser extent in samples collected with other anticoagulants and/or kept warmed at ~37°C; (iii) time-dependent fall of platelet count in the EDTA specimen; (iv) evidence of platelet aggregates and clumps in EDTA-anticoagulated samples with either automated cell counting or microscopic analysis; (v) lack of signs or symptoms of platelet disorders. Several remedies have been proposed, such as warming the sample to 37°C or using additives or specific formulations of anticoagulants including buffered sodium citrate, heparin, ammonium oxalate, β-hydroxyethyltheophylline, sodium fluoride, CPT (trisodium citrate, pyridoxal 5'-phosphate and Tris), antiplatelet agents, potassium azide, amikacin, kanamycin or other aminoglycosides, and calcium replacement with the simultaneous addition of calcium chloride/heparin. According to available evidences, the most suitable and practical approach so far for most clinical laboratories seems, however, the recollection of blood samples using sodium citrate, CPT or calcium chloride/heparin as additives, maintaining the specimen at 37°C until the platelet count has been completed.
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#3
16.12.2012, 19:19
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Почему-то сообщение, набранное с большим усилием и содержащее "многобукв", не отправилось
 Видимо что-то делаю не так...постараюсь исправиться, т.к. вопрос мой актуален.*** Попытка №2: Как молодому специалисту пришлось столкнуться с проблемой ЭДТА-индуцированной псевдотромбоцитопении (при обучении на кафедре об этом конечно же не упоминали). Поэтому в целях повышения качества лабораторной диагностики и единообразного подхода к выполнению ОАК на автоматических гем.анализаторах позвольте описать ситуацию и задать несколько вопросов. Ситуация: пациент неоднократно сдаёт кровь на общий анализ, из раза в раз - низкое количество тромбоцитов+флаг "PLT Clumps" (анализатор Sysmex 2000i-XT), пересчёт тромбоцитов "глазом" в мазке с выдачей результата в формате "количество тромбоцитов 10^9/л + комментарий: на щётке наличие большого количества агрегатов тромбоцитов". Врач и пациент требуют точную цифру. Задал вопрос более опытным коллегам: а можно ли взять кровь с другим а/коагулянтом и выполнить анализ из неё? Получаю категоричный ответ - нет, нельзя. Вопросы: 1. можно ли для подсчёта тромбоцитов на гем.анализаторе использовать кровь с другим а/коагулянтом? если можно, то с каким (цитрат 3,2%, 3,8%; гепарин)?. 2. при положительном ответе на 1й вопрос: в таком случае для исключения влияния цитрата/гепарина/др. на морфологию клеток следует ли брать кровь в 2 пробирки: с солью ЭДТА - для определения всех показателей, исключая тромбоциты и с другим а/коагулянтом для подсчёта только лишь тромбоцитов? Не будет ли это "вредным" для анализатора? Благодарю!
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#4
16.12.2012, 23:09
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Цитата:
2. Вредным не будет, но ради 0,5% проб брать все в 2 пробирки немного чересчур. Кроме того не забудьте о разведении - нужен пересчет.
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#5
17.12.2012, 01:32
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можно и с гепарином; или в ЕТДА пробу добавить гепарин-кальциевую смесь...
кстати, частота ИТП у взрослых 9.5-23.6 на 100 тыс. (0.01-0.024%) гораздо ниже... In a systematic review of previously published literature, incidence of ITP among adults ranged from 1.6 to 2.68 per 100 000 persons per year; prevalence ranged from 9.5 to 23.6 per 100 000 persons.//Eur J Haematol. 2009 Aug;83(2):83-9.
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#6
17.12.2012, 21:43
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Цитата:
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#7
18.12.2012, 13:48
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Цитата:
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#9
06.02.2013, 13:46
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Случай из практики
Проиллюстрирую тему псевдотромбоцитопении (с картинками интересней
 ).Венозная кровь пациента, система Моновет с ЭДТА. Первое измерение через 20 минут после взятия крови, второе - через час. На втором бланке: стрелкой указан артефакт на гистограмме лейкоцитов (агрегаты). Галочкой отмечена погрешность при подсчете лейкоцитов и лимфоцитов. При ручном подсчете тромбоцитов 250. [Ссылки доступны только зарегистрированным пользователям ] Мазок крови приготовлен непосредственно перед первым измерением. [Ссылки доступны только зарегистрированным пользователям ]
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#10
05.05.2014, 22:56
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Br J Haematol. 2013 Sep;162(5):684-92. Effective estimation of correct platelet counts in pseudothrombocytopenia using an alternative anticoagulant based on magnesium salt.
Schuff-Werner P et al. [Ссылки доступны только зарегистрированным пользователям ]
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#11
06.05.2014, 17:31
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еще один кейс в полной версии по теме: псевдотромбоцитопения была частично обратима после инкубации при 37С и добавлении канамицина
Pseudothrombocytopenia observed with ethylene diamine tetra acetate and citrate anticoagulants, resolved using 37°C incubation and Kanamycin. Kamath V, Sarda P, Chacko MP, Sitaram U. Indian J Pathol Microbiol. 2013 Jul-Sep;56(3):306-8. [Ссылки доступны только зарегистрированным пользователям ] гистограммы с псевдотромбоцитопенией [Ссылки доступны только зарегистрированным пользователям ]
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#12
10.05.2014, 19:41
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Цитата:
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#13
10.05.2014, 21:17
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да, конечная концентрация магния сульфата 33,8 микромоль, к сожалению не указано, что такая концентрация в крови или только в плазме? Если Ваши расчеты верны, то судя по обьему шприца 2.7 мл, он уже презаполнен 0.3 мл 0.2% магния сульфата.
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#14
19.08.2014, 19:17
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еще кейс с иллюстрациями и типичными отклонениями в гистограмме - исключение псевдотромбоцитопении было сделано при помощи забора крови с CPT (citrate 17 mmol/l, pyridoxal 5′-phosphate 11.3 mmol/l and Tris 24.76 mmol/l):
A case of EDTA-dependent pseudothrombocytopenia: simple recognition of an underdiagnosed and misleading phenomenon [Ссылки доступны только зарегистрированным пользователям ]
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#15
21.10.2014, 20:32
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Еще две публикации: с образцах крови с псевдотромбоцитопенией почти всегда ложноповышенное число лейкоцитов, правда в большинстве случаев (74%) их число не превышает норму:
Platelets. 2014 Oct 2:1-5. Concomitant spuriously elevated white blood cell count, a previously underestimated phenomenon in EDTA-dependent pseudothrombocytopenia. Xiao Y1, Xu Y. The proportion and potential risk of concomitant spuriously elevated white blood cell count (SEWC) are underestimated in ethylenediaminetetraacetic acid (EDTA)-dependent pseudothrombocytopenia (PTCP). The proportion, kinetics and prevention of SEWC remain poorly understood. A total of 25 patients with EDTA-dependent PTCP were enrolled in this study. With the hematology analyzer Coulter LH 750, we determined the time courses of WBC count, WBC differential and platelet count in EDTA- and sodium citrate-anticoagulated blood, respectively. Blood smears were prepared to inspect the presence of platelet clumps using light microscopy. The effect of automatic instrumental correction on the extent of SEWC was evaluated. The proportion of SEWC was 92% in EDTA-dependent PTCP and 73.9% of SEWCs were within the normal range. The development of SEWC was time-dependent, and neutrophils and lymphocytes were the main subpopulations involved in SEWC. A strong and significant correlation (r = 0.9937, p < 0.001) was found between the increased WBC count and the decreased platelet count. Both corrected and uncorrected WBC counts at 15 minutes or later after blood collection in EDTA were significantly higher than their basal counts, respectively, p < 0.05. Interestingly, in citrated blood, WBC counts after blood collection were not significantly different from its basal counts, p > 0.05. A high proportion of concomitant SEWCs, which are mainly within normal range, are present in patients with EDTA-dependent PTCP. Proper interpretation of SEWC is crucial to avoid clinic errors. SEWC develops in a time-dependent pattern, although the Coulter LH 750 only partly mitigates the extent of SEWC, sodium citrate is able to effectively prevent SEWC. --- у пациентов с псевдотромбоцитопенией функция тромбоцитов тоже снижена (тромбоцитарная агрегация при PRP-турбидиметрии): Platelet Aggregation Tests Are Affected in Pseudothrombocytopenia [Ссылки доступны только зарегистрированным пользователям ]
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Линейный вид
