Assessment of Immature Platelet Fraction in Pregnancy-Associated Thrombotic Microangiopathy
Conditions: Pre-Eclampsia, Severe, HELLP Syndrome, Microangiopathy
Sponsor: AYMAN ABDELKADER MOHAMED ABDELKADER

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Assessment of Immature Platelet 
Fraction in Pregnancy-Associated 
Thrombotic Microangiopathy 
Thesis 
Submitted for Partial Fulfillment of Master Degree 
in Clinical and Chemical Pathology 
By 
Mary Zakaria Fayek 
M.B, B.Ch.  
Misr University for Science and Technology (MUST) 
Under Supervision of 
Prof. Dr. Mohamed Ameen Mekawy 
Professor of Clinical and Chemical Pathology 
 Faculty of Medicine- Ain-Shams University 
Dr. Rasha Abd El-Rahman El-Gamal 
Lecturer of Clinical and Chemical Pathology 
Faculty of Medicine- Ain-Shams University 
Dr. Ayman Abd Elkader Mohamed 
Lecturer of Obstetrics and Gynecology 
Faculty of Medicine- Ain -Shams University 
 

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Introduction 
Thrombotic microangiopathy (TMA) syndromes are 
extraordinarily diverse. They may be hereditary or acquired. 
They occur in children and adults. The onset can be sudden 
or gradual. Despite their diversity, TMA syndromes are 
united by common, defining clinical and pathological 
features, which are microangiopathic hemolytic anemia 
(MAHA), thrombocytopenia, and organ injury (Moake, 
2002).  
Thrombotic thrombocytopenic purpura (TTP) and 
hemolytic uremic syndrome (HUS) are the two most well 
known, and are considered to be the most serious, TMA 
syndromes (Ab Rahman et al., 2013). TTP/HUS occurs 
more commonly in women and among women is commonly 
associated with pregnancy (George and Nester, 2014). 
Nevertheless, there are other pregnancy conditions that may 
manifest with TMA, including preeclampsia, eclampsia, 
HELLP syndrome (hemolysis, elevated liver enzymes, and 
low platelet count), in addition to acute fatty liver of 
pregnancy, antiphospholipid syndrome, and systemic lupus 
erythematosis (Scully et al., 2012). 
TTP may be caused by an acquired (inhibitory 
antibodies) or more rarely, constitutional deficiency in 

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ADAMTS13, a metalloprotease that cleaves the ultralarge 
multimers of von Willebrand factor (vWF). vWFs are potent 
inducers of platelets aggregation (Furlan et al., 1998). In the 
setting of ADAMTS13 deficiency, ultralarge vWF 
multimers initiate thrombi formation with ensuing 
microvascular platelet clumping, resulting in MAHA, 
fragmented 
erythrocytes 
(schistocytes), 
consumptive 
thrombocytopenia, renal dysfunction, and neurological 
symptoms. Since TTP can present without the full pentad, 
the revised diagnostic criteria state that TTP must be 
considered in the presence of thrombocytopenia and MAHA 
alone (Galbusera et al., 2006). 
HUS describes TMA that affects mainly the kidney. It 
is characterized by MAHA, thrombocytopenia and acute 
renal failure. HUS includes various subtypes: verotoxin-
associated typical (diarrheal) HUS, secondary HUS (of 
which, pregnancy-associated HUS is one type), and 
idiopathic atypical HUS (aHUS). Owing to similarity in 
clinical status & complications, diagnostic overlap with TTP 
can occur (Fakhouri et al., 2012). 
Even in patients with congenital ADAMTS13 
deficiency and recurrent acute episodes of TTP/HUS since 
infancy, pregnancy is one of the events that act as 
precipitating factors; occurring in approximately 5– 25% of 

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TTP cases (Scully et al., 2008). This is because pregnancy 
may provide multiple risk factors that can provoke an acute 
episode of TTP/HUS in a susceptible woman. These risk 
factors include increasing concentrations of procoagulant 
factors, decreasing fibrinolytic activity, loss of endothelial 
cell 
thrombomodulin, 
and 
decreasing 
activity 
of 
ADAMTS13 (George, 2003). These abnormalities become 
progressively more severe through the course of pregnancy, 
with the maximum abnormalities occurring at delivery and 
immediately postpartum. This is also the time of greatest risk 
for the occurrence of the more common pregnancy-related 
syndromes (pre-eclampsia, eclampsia, HELLP syndrome, 
and HUS). Differentiating these other syndromes from TTP 
may difficult or impossible because of similar clinical 
presentation in some aspects (Stirling et al., 1984). In many 
women, the distinction can only be determined by the course 
of illness following delivery (McMinn and George, 2001). 
Reliable laboratory criteria specific for TTP are 
largely lacking. The diagnostic value of ADAMTS13 
deficiency remains uncertain since not all TTP patients have 
severe deficiency (Kremer Hovinga et al., 2010), and not 
all documented severe ADAMTS13 deficiency patients meet 
TTP diagnostic criteria (Scully et al., 2012). Moreover, it is 
not feasible to constantly monitor ADAMTS13 activity 

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during therapeutic plasma exchange (TPE) in order to adjust 
therapy and monitor disease progress. Conjointly, relying on 
the therapeutic response to TPE harbors its own hazards and, 
besides, it has not been demonstrated that TPE is beneficial 
in treating non-idiopathic TTP. Thus, additional tests that 
could aid in the diagnosis and management of TTP are 
needed (Sadler et al., 2008). 
  
Assessment of immature platelets was introduced as a 
non-invasive test of real time thrombopoeisis. They are 
newly released in the circulation with a larger size & greater 
RNA content than mature platelets, and can be measured by 
automated hematology analyzer equipped with reticulocyte 
detection channel and described as immature platelet 
fraction (IPF-%) and absolute immature platelet count (A-
IPC). A high IPF-% has been suggested as an indicator of 
thrombocytopenia due to rapid platelet consumption, while 
a low IPF-% is characteristic of bone marrow suppression 
states. IPF-%/A-IPC has the competency to be performed 
routinely and, therefore, can provide therapeutic and 
diagnostic feedback in the compatible life threatening 
conditions (Hong et al., 2013). 

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Aim of work 
The aim of this work is to show the utility of estimating IPF-% and A-IPC 
using a reticulocyte detection channel CBC auto analyzer as a simple 
reproducible blood analysis to be employed in the differential diagnosis of 
pregnancy-associated thrombotic microangiopathic conditions. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Patients and Methods 
This study aiming to show the utility of estimating IPF-% and A-IPC using 
a reticulocyte detection channel CBC auto analyzer as a simple reproducible 
blood analysis to be employed in the differential diagnosis of pregnancy-
associated thrombotic microangiopathic conditions. Patients were recruited from 
Ain Shams University Maternity Hospital.  
Study sample was 57 women and were classified into three groups: 
Group 1: SPE/HELLP group. This group will include 24 pregnant women 
(gestational age of >20 weeks) who are diagnosed as having TMA with 
provisional diagnosis of pre-eclampsia, HELLP syndrome. 
Group 2: TTP/HUS group. This group will include 13 pregnant women 
(gestational age of >20 weeks) who are diagnosed as having TMA with 
provisional diagnosis of TTP/HUS. 
Group 3: Control group. This group will include 20 pregnant women (gestational 
age of >20 weeks) having normal pregnancy with normal blood 
pressure and platelet count. 
Inclusion criteria  
Women will be included if they are: 
- Older than 20 years of age  
- Pregnant with singleton intrauterine pregnancy  
- More than 20 weeks of gestation 
Exclusion criteria 
Patients will be excluded from the study if there was any of the following: 
- Congenital malformation and fetuses with chromosomal or genetic 
syndrome. 

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- Recent blood transfusion. 
- Refusal to participate in the study. 
- BMI <18. 
- Placental abnormalities like velamentous insertion. 
- Multiple pregnancies. 
- Known kidney disease.  
- History of auto immune disease.  
Ethical Considerations 
An informed consent will be taken from each patient. The aim of the 
research was explained to patients. No harm was caused to patients as blood 
samples were routinely collected from patients. 
Consent form: 
Patient name: 
Hospital number: 
Information to the participant: 
The procedure 
• The results of your blood results and hospital data will be used in this 
clinical trial. 
• An extra blood sample will be withdrawn at the time of diagnosis which 
is a simple procedure with no known complications. 
• All your data and blood results, will saved confidentially and it will 
appear as a number in the results. 
• The results of these investigation will not affect your management plan. 
Methodology  
An informed consent will be taken from each participant. 

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For all patients included in the study the following were done  
1- Thorough history taking with special emphasis on: 
- The gestational age will be estimated according to the last menstrual period 
and confirmed by a first trimesteric ultrasound. 
- Past history to ensure the absence of any medical disorder included in 
exclusion criteria. 
2- General, abdominal and local examination with particular emphasis on vital 
sign monitoring records. 
Laboratory Investigations: 
▪ Haematological tests 
o Sampling: 
Five (5.0) ml of venous blood will be collected from each patient, it will be 
divided as follows: 
- Two (2.0) ml added to an EDTA containing sterile tube for assessment of: 
a) Complete blood count (CBC), for estimation of hemoglobin concentration 
and platelet count using XE-2100® (Sysmex Corporation, Kobe, Japan) 
automated hematology system. 
b) Morphological assessment of different blood cells with special emphasis 
on estimation of schistocyte counts (if present), achieved by microscopic 
examination of Romanwsky-stained PB smears prepared from an EDTA-
anticoagulated PB sample. Schistocytes counting will be performed in at 
least 1000 RBCs in optimal areas of the film, with interpretation of results 
following the ICSH recommendations for identification, diagnostic value, 
and quantitation of schistocytes (Zini et al., 2011).  
c) Reticulocyte count: by microscopic examination of supravitally stained PB 
smear.  

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d) Estimation of IPF-% and A-IPC: All samples will be analyzed within 4 h 
of collection, using the XE-2100® (Sysmex Corporation, Kobe, Japan) 
automated hematology system. Using a carefully designed gating system in 
the XE-2100 optical (fluorescence) reticulocyte/platelet channel it is now 
possible to reliably quantify the IPF, employing upgraded software (XE-Pro 
Series; Sysmex). The flow cytometric IPF determination uses a proprietary 
fluorescent dye containing polymethine and oxazine. These two dyes 
penetrate the cell membrane, staining the RNA in the red cell and platelet 
reticulocytes. The stained cells are passed through a semiconductor diode laser 
beam and the resulting forward scatter light (cell volume) and fluorescence 
intensity (RNA content) measured. A computer algorithm discriminates the 
mature and IPF by the intensity of forward scattered light and fluorescence. 
The XE-2100 instrument used in the study was equipped with upgraded 
software for the data analysis of the IPF, which applies a preset gate to separate 
the two platelet populations. Mature platelets appear as blue dots and the 
immature platelets are displayed as green dots, the latter constituting the IPF 
parameter. Immature platelet fraction is usually expressed as a proportional 
value of the total optical platelet count to indicate the rate of platelet 
production, although an absolute count can also be obtained (Briggs et al., 
2004). 
- Three (3.0) ml are to be added to a sterile plain tube for serum assessment of: 
lactate dehydrogenase (LDH), serum bilirubin, liver enzymes (ALT & AST), 
serum uric acid, kidney function tests, using HITACHI 912 automatic analyzer 
(Roche Diagnostics GmbH. Sandhofer Str. Mannheim, D-68298 Germany). 
 
 
 

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Statistical methods: 
Data will be analyzed using using IBM© SPSS© Statistics version 22 
(IBM© Corp., Armonk, NY, USA) and MedCalc© version 14 (MedCalc© 
Software bvba, Ostend, Belgium).  
The Shapiro-Wilk test to examine the normality of numerical data 
distribution. Normally distributed numerical variables will be presented as mean 
± SD and inter-group differences were compared using one-way analysis of 
variance (ANOVA) for multiple group comparison with application of the 
Schéffé test for post hoc pair-wise comparison, whenever the ANOVA test 
revealed a statistically significant difference among the groups.  
Skewed numerical will be presented presented as median (interquartile 
range) and inter-group differences were compared non-parametrically using the 
Kruskal-Wallis test.  
Categorical variables were presented as number (%) and differences will 
be compared using Fisher’s exact test.  
Correlations among numerical variables will be tested non-parametrically 
using the Spearman rank correlation.