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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 45  |  Issue : 3  |  Page : 136-141

Protein Z measurement in acute myeloid leukemia patients


Department of Internal Medicine and Clinical Hematology, Ain Shams Universiy, Cairo, Egypt

Date of Submission07-Dec-2019
Date of Acceptance22-Jan-2020
Date of Web Publication23-Jun-2021

Correspondence Address:
Haydi S Mohamed
Lecturer of Internal Medicine and Clinical Hematology, Ain Shams University, Cairo, 11813
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_58_19

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  Abstract 


Background Protein Z (PZ) is a vitamin K-dependent plasma protein. The role of PZ in the pathogenesis of hemostatic disorders remains to be established. However, a significant association was found between low PZ levels and arterial vascular diseases, pregnancy complications and disseminated intravascular coagulation, stroke and antiphospholipid syndrome. On the other hand, it has been suggested that PZ deficiency is associated with hemorrhagic disorders, perhaps related to capillary fragility.
Aim Measurement of the level of PZ in newly diagnosed acute myeloid leukemia (AML) patients and correlate it with bleeding tendency or thrombosis.
Patients and methods This study was held on 90 participants divided into 60 adult (AML) patients and 30 healthy controls of matched age and sex from the Internal Medicine Department, Ain Shams University Hospital. Enzyme-linked immunosorbent assay kits are used for the estimation of PZ levels after taking informed consent.
Results A low level of PZ was detected in AML patients compared with controls (P<0.001). A statistical correlation was observed between bleeding attacks and PZ level (P=0.008). The best cutoff value for PZ as a predictor for bleeding was more than 350 ng/l by the receiver operating characteristic curve.
Conclusion Low PZ level is a cofactor of bleeding in AML patients.

Keywords: acute myeloid leukemia, bleeding, chemotherapy, protein Z


How to cite this article:
Mohamed HS, Hegab HM, El-Razzaz MK. Protein Z measurement in acute myeloid leukemia patients. Egypt J Haematol 2020;45:136-41

How to cite this URL:
Mohamed HS, Hegab HM, El-Razzaz MK. Protein Z measurement in acute myeloid leukemia patients. Egypt J Haematol [serial online] 2020 [cited 2021 Dec 8];45:136-41. Available from: http://www.ehj.eg.net/text.asp?2020/45/3/136/319161




  Introduction Top


Protein Z (PZ) is a vitamin K-dependent protein acting as a cofactor of the PZ-dependent inhibitor (ZPI), in the inhibition of activated factor Xa. Low levels of PZ were associated with increased risk of thrombotic complications, including venous thromboembolism, arterial thrombosis, and pregnancy complications. On the other hand, it has been suggested that PZ deficiency is associated with hemorrhagic disorders, perhaps related to capillary fragility [1],[2].

Studies on the potential association of PZ with cancer development have shown conflicting results and only limited research into the role of PZ in cancer-associated thrombosis was done [3].

The occurrence of various coagulation abnormalities in acute leukemia is well established, but there are no data assessing the role of PZ in hemostatic disorder in hematology cancers, including acute leukemia [1].

The aim of this study was to estimate the concentration of PZ in adult, naïve acute myeloid leukemia (AML) patients and to assess the correlation between PZ levels with the bleeding tendency or thrombosis.


  Patients and methods Top


Study design

This study included 60 adult, naïve AML patients aged above 16 years and 30 healthy controls of matched age and sex in the Clinical Hematology Department, relapsed or resistant AML patients, patients on oral anticoagulants, inherited bleeding disorder, inherited thrombophilia, and severe organ comorbidities were excluded.

Informed consents were obtained from all participants. The study was conducted in accordance with the stipulations of the local ethics and scientific committees of Ain Shams University and the procedures respected the ethical standards in Helsinki Declaration of 1964.

All the patients were subjected to:
  1. Full history and clinical examination and laboratory investigations which include: complete blood count, prothrombin time (PT), partial thromboplastin time (PTT), international normalizing ratio, serum fibrinogen level, fibrin degradation products, liver function, kidney function tests, bone marrow aspirate. Cytogenetic studies were performed on bone marrow by the fluorescent in-situ hybridization technique and karyotyping at the time of diagnosis.
  2. Radiology: echocardiography and electrocardiogram, pelvi-abdominal ultrasound.
  3. Serum PZ level by enzyme-linked immunosorbent assay before starting the chemotherapy in both patients and controls.


Assay of protein Z level

Blood sampling

Under aseptic conditions 20 ml of blood sample was collected from the patients in tubes containing ethylene diamine tetra acetic acid as an anticoagulant. Samples were centrifuged for 15 min at 2000–3000 rpm at 2–8°C within 30 min of collection. PZ was measured by an enzyme-linked immunosorbent assay as follows:
  1. After the samples are collected, the reagent and the standard solution are diluted and prepared at room temperature.
  2. Add 50 ml standard solution to a standard well.
  3. Add 40 ml sample to sample wells and then add 10 ml PZ antibody to sample wells, then 50 ml streptavidin–HRP to samples wells and standard wells (not blank control well). Mix well. Cover the plate with a sealer. Incubate 60 min at 37°C.
  4. Remove the sealer and wash the plate five times with a wash buffer. Soak wells with at least 0.35 ml wash buffer for 30 s–1 min for each wash buffer, overfilling wells with a wash buffer. Blot the plate onto paper towels or other absorbent material.
  5. Add 50 ml substrate solution A to each well and then add 50 ml substrate solution B to each well. Incubate the plate covered with a new sealer for 10 min at 37°C in the dark.
  6. Add 50 ml stop solution to each well, the blue color will change into yellow immediately, determine the optical density value of each well immediately using a microplate reader set to 450 nm within 30 min after adding the stop solution. The manufacturer gave a measuring range: 2–600 ng/l, sensitivity: 1.05 ng/l.


Statistical analysis

Data were collected, revised, coded, and entered to the Statistical Package for Social Sciences (IBM SPSS),version 23 (IBM Corp, released 2015, IBM SPSS statics for windows version 23 Armonk, NY: IBM Corp.). The quantitative data were presented as mean, SDs, and ranges when parametric and median with interquartile range when nonparametric. Comparison between groups regarding qualitative data was made using the χ2 test.

The comparison between two independent groups regarding quantitative data with parametric distribution was done by using independent t test, while with nonparametric distribution was done by using the Mann–Whitney test. Also, the comparison between more than two groups with quantitative data and nonparametric distribution was done using Kruskal–Wallis test.

Receiver operating characteristic curve was used to assess the best cutoff point for PZ with its sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve.

The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the P value was considered nonsignificant if P value more than 0.05, significant if P value less than 0.05, and highly significant if P value less than 0.01.


  Results Top


Demographic and laboratory characteristics of the patients are summarized in [Table 1].
Table 1 Demographic data of the studied patient groups

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A total of 60 AML patients were recruited in the study between January 2018 and October 2018. Approximately 53.3% (32 patients) were aged from 16 to 55 years and 46.7% (28 patients) were above 55 years and 60% were men.

Thrombocytopenia was reported in 90% (54 patients) and normal PT, PTT was found in about 96.7% of the patients. Upon measurement of fibrin degradation products it was negative in 80% of the studied group and fibrinogen level was normal in 40%, low in 56.7%, and high in only 3.3% of the patients.

According to FAB classification of AML, the patients were divided into 12 (20% M0) patients, 22 (36.7% M1,2) patients, 24 (40% M4,5) patients, and two (3.3% M6) patients; 56 (93.3%) patients received 3 days anthracycline and 7 days cytarabine and four (6.7%) patients received 2 days anthracycline and 5 days cytarabine as an induction chemotherapy protocol.

Bleeding episodes were observed in 20 (33.3%) patients that varied from mucocutaneous, gastrointestinal tract (GIT), and vaginal bleeding (23.3, 26.7, 3.3%, respectively).

No thrombotic events were reported in the studied group.

Complete remission was detected in 46 (76.7%) patients while six (10%) patients were resistant and eight (13.3%) patients died from neutropenic fever and septic shock, bleeding episodes, hypovolemic shock, and intestinal obstruction (37.5, 25, 25, 12.55, respectively) ([Table 1]).

The median (interquartile range) PZ level in the studied patients was 205 ng/l (range, 100–800 ng/l), which was significantly lower in comparison to its level in the control group (349.5 ng/l; range, 200–490 ng/l) (P<0.001) ([Table 2]).
Table 2 Comparison between patients group and control group regarding protein Z level (ng/l)

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Although PZ level was higher in thrombocytopenic patients, no statistically significant correlation was detected between PZ level and initial platelet count (P=0.323).

No significant difference was detected between PZ level and initial PT, PTT levels (P=0.057) ([Table 3]).
Table 3 Comparison between protein Z level as regards initial platelet count and initial prothrombin time, partial thromboplastin time of the studied cases

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PZ level was significantly higher in patients with bleeding episodes during induction chemotherapy (P=0.008).

Statistically significant difference was detected as regards mucocutaneous, GIT and vaginal bleeding with a P value of 0.001, 0.005, and 0.026, of respectively ([Table 4]).
Table 4 Correlation between protein Z levels with bleeding episodes among the studied cases

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Receiver operating characteristic curve showed that the best cutoff value for PZ as a predictor for bleeding episodes during induction was found to be more than 350 ng/l, with 52.38% sensitivity, 97.44% specificity, positive predictive value of 91.7%, negative predictive value of 79.2%, and area under the curve of 70.9% ([Figure 1]).
Figure 1 Receiver operating characteristic (ROC) curve for protein Z level as a predictor for bleeding episode during induction.

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Patients with PZ level more than 350 ng/l are more liable for bleeding episodes.

Median PZ was higher in resistant AML patients (340 ng/l; range, 340–340 ng/l) than in remitted patients (200 ng/l; range, 100–800 ng/l) without statistically significant difference which was found between PZ and remission status (P=0.272) ([Table 5]).
Table 5 Correlation between protein Z levels with remission status among the studied cases

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  Discussion Top


Thrombohemorrhagic complications are frequently seen in patients with hematologic malignancies as in those with solid tumors and significantly affect morbidity and mortality. In acute leukemia, thrombosis and bleeding manifestations may occur concomitantly as a part of the same thrombohemorrhagic syndrome. Furthermore, hypercoagulable state is present in virtually all of these patients, even without clinical manifestations [4].

Acute leukemia is often complicated with disseminated intravascular coagulopathy due to abnormal expression of coagulation-related factors in leukemic cells and external factors, such as infection and vascular endothelial damage, caused by chemotherapy. Hence, it can be improved by treatment with the reduction of leukemic cells [5].

AML-associated bone marrow failure produces marked thrombocytopenia with small, dysfunctional platelets, increasing the risk of hemorrhage. Chemotherapy is also a risk factor for thrombocytopenia and bleeding complications [6].

The incidence of venous thromboembolism in AML varies markedly among studies, ranging from 2 to 13%. Acute promyelocytic leukemia represents about 10% of all AML cases and its course can be characterized by either hemorrhagic or thrombotic events [7].

The role of PZ in the pathogenesis of hemostatic disorders in humans remains to be established. From investigations performed so far, no clear link was evidenced between PZ concentrations and bleeding or thrombosis tendency [7].

As regards PZ level in our study, PZ level was lower in AML patients in comparison to the control group with high statistical significance with (P<0.001),which matched with the study done by Galar et al. [1] that was held on AML and acute lymphoblastic leukemia patients, and observed a statistically significant lower concentration of PZ in groups of patients in comparison to healthy individuals.

Similar results were reported in the study by Gutwein et al. [3] that was performed on plasma cell neoplasm patients and found that PZ level was lower in patients than healthy controls with statistical significance.

The Shang et al. [8] study reported that PZ levels in patients with various malignancies were lower than in healthy controls which agree with our results.

Contrary to our study results, PZ levels were not significantly lower in acute lymphoblastic leukemia children and control group of matched age and sex in the study performed by Çankal et al. [9].

In our study, PZ level was higher in participants above 55 years old than those younger among the studied patients and control group and it was detected to be higher in male than female patients without statistical significance observed neither with age nor with sex (P=0.312 and 0.204, respectively). This matched with a previous study held by Gutwein et al. [3] in which there was no correlation between PZ level and age.

No statistically significant difference was detected by Çankal et al. between patients and controls as regards the correlation between PZ with age and sex which was in line with our results which matched with our study results [9].

As regards the correlation between PZ level with initial platelet count and initial PT, PTT of the studied patients’ group, it was found that PZ level was higher in thrombocytopenic patients without statistical significance (P=0.323). And it was lower in patients with elevated PT and PTT without significant difference with a P value of 0.057. This is in agreement with the study by Gutwein et al. [3].

PZ level was correlated with fibrinogen levels among the studied cases. It was higher in patients with high serum fibrinogen level than in patients with normal or low serum fibrinogen level without statistical significance (P=0.296).

There was correlation between PZ levels with initial bleeding among the studied cases. We detected that PZ level was higher in patients with bleeding episodes during induction than those without bleeding with statistically significant difference with a P value of 0.008.

Statistical significance was detected also between PZ level and (mucocutaneous, GIT, vaginal bleeding) in studied patients with a P value of 0.001, 0.005, and 0.026, respectively, which matched with a previous study performed by Galar et al. [1], who observed a statistically significant correlation between PZ level and bleeding episodes in the AML patient group.Thrombosis is one of the most frequent complications in cancer and the second leading cause of death among patients with malignant diseases. The risk of thrombosis is up to seven times increased in cancer patients as compared with the general population. However, the risk of thrombosis in patients with hematologic malignancies was considered lower than that in patients with solid tumors, and much of the attention was directed toward bleeding and infectious complications due to associated thrombocytopenia and neutropenia [10].

Our studied naïve AML patients group did not experience any thrombosis during the period of induction which might be due to the short follow-up period [3].

As regards PZ level correlation with remission status among the studied cases, PZ was higher in the resistant AML patients than in remitted patients without statistical significance which matched with the study by Galar et al. [1], who did not find any significant correlation between PZ concentration and stages of disease after the treatment.

Acknowledgements

Hany M. Hegab: participated in the research design, in research performance, data analysis, and in the revision of the paper for submission. Mostafa K. El-Razzaz: participated in the performance of the research and revision of the paper for submission. Haydi S. Mohamed: participated in the research design and idea of research, in the writing of the paper, in the performance of the research, and participated in data analysis.

Financial support and sponsorship

Nil.

Conflict of interest

There are no conflicts of interest.



 
  References Top

1.
Galar M, Piszcz J, Bolkun L, Szumowska A, Kloczko J. Protein Z concentrations in patients with acute leukemia. Clin Appl Thromb Hemost 2012; 18:542–545.  Back to cited text no. 1
    
2.
Sofi F, Cesari F, Abbate R, Genisini G, Broze G. Ameta-analysis of potential risks of low levels of protein Z for diseases related to vascular thrombosis. Thromb Haemost 2010; 103:749–756.  Back to cited text no. 2
    
3.
Gutwein O, Rahimi-Levene N, Herzog-Tzarfati K, Garach-Jehoshau O, Nagler A, Izak M, Koren-Michowitz M. Low protein Z levels in patients with plasma cell neoplasms are inversely correlated with IL-6 levels. Leuk Res 2017; 62:104–107.  Back to cited text no. 3
    
4.
Anna F, Laura R, Carmen T. Pathogenesis and treatment of thrombo-hemorrhagic diathesis in acute promyelocytic leukemia. Mediterr J Hematol Infect Dis 2011; 3:e2011068.  Back to cited text no. 4
    
5.
Rickles FR, Falanga A, Montesinos P, Sanz M, Brenner B, Barbui T. Bleeding and thrombosis in acute leukemia: what does the future of therapy look like? Thromb Res 2007; 120:S99–S106.  Back to cited text no. 5
    
6.
Wada H, Matsumoto T, Yamashita Y. Diagnosis and treatment of disseminated intravascular coagulation (DIC) according to four DIC guidelines. J Intensive Care 2014; 2:15.  Back to cited text no. 6
    
7.
Fang Y, Garnier D, Lee TH, D’Asti E, Montermini L, Meehan B, Rak J. RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells. Angiogenes J 2016; 19:25–38.  Back to cited text no. 7
    
8.
Shang Y, Pan XY, Ding CP, Yang XM, Cai XY, Ding Y, Zhang RL. Clinical significance of protein Z detection in patients with malignant tumors. Ai Zheng 2005; 24:1144–1147.  Back to cited text no. 8
    
9.
Çankal A, Tüfekçi Ö, Gözmen S et al. The evaluation of protein Z levels of children with acute lymphoblastic leukaemia during induction therapy. Blood Coagul Fibrinolysis 2013; 24:375–380.  Back to cited text no. 9
    
10.
Timp J, Braekkan S, Versteeg H, Cannegieter S. Epidemiology of cancer-associated venous thrombosis. Blood 2013; 122:1712–1723.  Back to cited text no. 10
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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