Pilot Study- Osmotic Fragility in Red Blood Cells of Pediatric Patients With Cholestatic Liver Disease
Conditions: Cholestasis, Red Cell Membrane and Enzyme Abnormalities
Sponsor: Children's Hospital Medical Center, Cincinnati

Page 1 of 8 
Version 05APR2018_v2                                                  
 
Cincinnati Children’s Hospital Medical Center 
Division of Gastroenterology & Hepatology 
 
Protocol Title:   Pilot Study- Osmotic fragility in Red Blood Cells of Pediatric Patients with Cholestatic 
Liver Disease  
 
Principal Investigator: Samuel Kocoshis, MD  
 
Sub investigators: Theodosia Kalfa, MD, Alexander Miethke, MD, Theodore Kocoshis 
 
1. 
ABSTRACT 
 
Background:  
Cholestasis is a condition characterized by impairment of bile flow and/or formation. The 
condition can occur from either extrahepatic obstruction or intrahepatic hepatocyte bile secretory 
defects.  
Cholestatic liver diseases are a well-recognized cause of dyslipidemia, and are closely associated 
with abnormal cholesterol and phospholipid metabolism.  Cholesterol conversion to bile acids may be 
impaired, and apo-lipoprotein B binding to cholesterol may be impaired, resulting in the formation of 
lipoprotein X which contains apolipoprotein C.  The end result of dyslipidemia in cholestatic patients is 
hypercholesterolemia and an increased membrane cholesterol to phospholipid ratio.   
Elevated red cell membrane cholesterol to phospholipid ratios result in decreased osmotic 
fragility and decreased deformability of the red blood cell (RBC).  When red cell membranes are 
compromised, and especially when membrane peroxidation is increased (due to exposure to an 
excessive oxidant load or due to reduction in antioxidants such as vitamin E), spur cells will form (1,2).   
We hypothesize that even in the absence of spur cell formation, subclinical changes in osmotic 
fragility will appear and these can be measured by ektacytometry.    
Ektacytometry is a tool to measure the deformability and osmotic fragility of red blood cells 
subjected to shear stress while exposed to an osmotic gradient (Figure 1). Ektacytometry can be used as 
a diagnostic tool to determine the presence of abnormalities in cell membrane structure. It does this by 
analyzing the RBC rheological behavior in a variety of physiologic and non-physiologic conditions. 
 
 
 
 
 
 
 
 
 
 

Page 2 of 8 
Version 05APR2018_v2                                                  
 
 
 
 
 
 
Objective:  
 
 
We propose to perform ektacytometry on 20 pediatric patients over age one with cholestatic 
liver diseases and a direct bilirubin level of greater than 2 gm/dl.  The most common diagnoses will be 
extrahepatic biliary atresia, progressive familial intrahepatic cholestasis, Alagille syndrome, autoimmune 
hepatitis, primary sclerosing cholangitis, and parenteral nutrition-associated cholestasis.   We will 
correlate the osmotic fragility and deformability with their direct bilirubin levels, their serum cholesterol 
levels, their serum bile acid levels, and their vitamin E levels.  
 
Design/Methods:  
 
This pilot study will be a single center, prospective cross-sectional investigation of red blood cell 
ektacytometry in pediatric patients with extrahepatic cholestasis who are followed at Cincinnati Children’s 
Hospital Medical Center. We will include all subjects with cholestasis regardless of the etiology in order 
to maximize the number of subjects.  While the population will be heterogeneous, we will stratify patients 
according to diagnosis, recognizing that only a few patients may fall into each diagnostic category.    
Ektacytometry will be the method utilized to measure osmotic fragility and deformability of the RBC 
membrane.   The ektacytometry of red cells from cholestatic patients will be compared to that of red cells 
obtained from contemporaneous age-matched controls recruited among patients without liver disease or 
red cell membrane defects undergoing blood sampling for evaluation of other entities including but not 
confined to functional abdominal pain.  
 
2. 
PURPOSE OF STUDY   
 
Primary Aim:  
The primary aim is to determine if ektacytometry of red blood cells is abnormal in a population of pediatric 
patients over age one with cholestasis.    
 
 
 
 
Figure 1.  Normal RBC ektacytometry.  The curve provides 
the following characteristics: Elongation Index (Elmax): 
Elmax corresponds to the maximum deformability of the 
erythrocyte.  Its value depends mostly on the cytoskeleton 
mechanics. Omin: Omin corresponds to the value of the 
hypotonic osmolality where 50% of the cells hemolyze in an 
osmotic fragility assay and provides information on the 
initial surface to volume ratio of the cell sample. A shift to 
the right reflects a decrease in the surface area/volume 
ratio. Ohyp: The declining portion of the curve is 
represented by the Ohyp value. This is the osmolality value 
where the cell’s maximum diameter is half of Elmax and 
correlates with the initial intracellular viscosity of the cell 
sample. A shift to the left reflects increased intracellular 
viscosity of the erythrocyte caused by increased 
intracellular concentration of hemoglobin and/or a 
dehydration state. 

Page 3 of 8 
Version 05APR2018_v2                                                  
 
Secondary Aims:   
 
The secondary aim is to ascertain the extent at which abnormalities in bilirubin levels, bile acid levels, 
vitamin E levels or cholesterol levels in cholestatic patients are related to changes in red blood cell 
ektacytometry in this patient population.  
 
 
3. 
BACKGROUND 
 
Cholestatic liver disease is an umbrella term for reduced bile flow from either extrahepatic 
obstruction of bile ducts or faulty hepatocyte bile secretion. Cholestasis is a well-recognized cause of 
dyslipidemia, and are closely associated with abnormal cholesterol and phospholipid metabolism (3).  
Cholesterol conversion to bile acids may be impaired, and apo-lipoprotein B binding to cholesterol may 
be impaired, resulting in the formation of lipoprotein X which contains apo-lipoprotein C (3).  The end 
result of dyslipidemia in cholestatic patients is hypercholesterolemia and an increased membrane 
cholesterol to phospholipid ratio (3). 
The lack of bile entering the digestive tract can lead to increased serum cholesterol, and 
abnormally high red cell membrane cholesterol: phospholipid ratios, resulting in altered red cell 
membrane structural integrity (4,5). To test for this phenomenon in patients with extrahepatic biliary 
atresia, 20 pediatric patients will be tested using ektacytometry to test the deformability and osmotic 
fragility of red blood cells. 
Ektacytometry is a diagnostic tool used to measure the deformability of red blood cells on an 
osmotic gradient (6). More specifically it measures deformability due to shear stress, while the RBC are 
suspended in an osmotic medium (6,7). It can differentiate among several blood maladies such as 
hereditary spherocytosis, hereditary elliptocytosis, Southeast Asian ovalocytosis, and pyropoikilocytosis 
that are otherwise difficult to diagnose (8). It does this by analyzing their rheological behavior under 
constant shear stress in vitro and using photometric analysis of deformability. This is then quantified and 
provides a specific ektacytometry curve (Figure 1).  
The ektacytometry curve will have several unique features depending on the red blood cell 
being analyzed. The Omin portion of the curve represents the value of the osmolality in which 50 
percent of the red blood cells lyse. The Elmax is the value of the elongation index that reaches its 
maximum near physiologic osmolality (300 mOsm/Kg). And lastly the Ohyp, which represents the 
declining portion of the curve and correlates to the initial intracellular viscosity of the cell sample. 
To date, ektacytometry has not been employed systematically to characterize potential red cell 
abnormalities in cholestatic patients.  In our analysis the ektacytometry profiles of cholestatic patients 
will be compared to those of historical non-cholestatic age-matched controls.  The degree of fragility will 
be correlated with serum direct bilirubin levels, serum cholesterol levels, and vitamin E levels.   
 
4. STUDY DESIGN 
 
This pilot study will be a single center, prospective cross-sectional investigation into the 
ektacytometry of red blood cells taken from 20 cholestatic pediatric patients followed at Cincinnati 
Children’s Hospital.  They are seen regularly for continued follow up in the hepatology clinic. Parents of 
all of these patients will be asked to participate in this investigation.  We will also contemporaneously 
analyze ektacytometry of 20 age-matched controls recruited from the general GI clinics with neither 
cholestatic liver disease nor know red cell membrane defects. 
 
 

Page 4 of 8 
Version 05APR2018_v2                                                  
 
 
5. DURATION 
 
The study is anticipated to take approximately 6-12 months to complete. This time table includes 
3-6 months to recruit patients and perform the assays and another 3-6 months to analyze the data and 
complete a manuscript.  
 
6. 
SELECTION & RECRUITMENT OF PARTICIPANTS 
• 
Inclusion Criteria 
 
               Disease Cohort  
Age greater than one year old 
Direct bilirubin level of >2 mg/dl.  
No known congenital red cell membrane defect  
 
Controls 
Age matched to disease cohort 
No evidence for cholestatic liver disease 
No known congenital red cell membrane defect 
 
• 
Exclusion Criteria.  
 
Disease Cohort  
Parental refusal 
Age <1 year old.  
Known congenital red cell membrane defect 
 
Controls 
Parental refusal 
Age <1 year old.  
Known congenital red cell membrane defect 
Cholestatic liver disease 
 
The study Principal Investigator (PI), PI designate, or research coordinator (research team) will have the 
responsibility for case finding and subject recruitment.  Potential study participants will be identified by 
the PI and a further member of the research team will enroll the participants. 
 
7. 
PROCESS OF OBTAINING CONSENT 
 
The Research Team will discuss appropriateness for study inclusion with the clinical team, and contact 
the family, most likely during their hepatology outpatient clinic visit or an inpatient hospitalization. The 
study will enroll children, a vulnerable population, therefore patients and parents will be explicitly 
informed that choosing not to participate, will in no way affect the quality of the medical care that they 
will receive.  
 
Details of the study will be reviewed with one or both parents prior to obtaining consent. All patients 
will be under the age of 18 years old and therefore only parental consent will be necessary along with 
the participant’s assent if older than 11 years of age.  

Page 5 of 8 
Version 05APR2018_v2                                                  
 
 
8. 
STUDY PROCEDURES 
 
All patients are routinely seen in outpatient clinic by one of the hepatologists from Cincinnati Children’s 
Hospital Medical Center.   Control patients will be gastroenterology patients seen by one of the general 
gastroenterologists in the general gastroenterology clinics. The research team will approach the family in 
clinic or in the hospital and after informed consent will obtain a 500-1500 mcl aliquot left-over from blood 
that has been drawn into a lavender top (EDTA) tube for measurement of a complete blood count. or a 
yellow top tube for measurement of serum chemistries.  To avoid research-only blood draws, blood will 
only be obtained if the patient is undergoing clinical laboratory testing.  The team’s PI, Dr. Samuel 
Kocoshis, and coinvestigators, Dr. Theodosia Kalfa and Mr. Theodore Kocoshis, will have access to the 
individual results of the assay.   
 
All blood samples will be de-identified with the assigned research number. 
 
In addition to providing a blood sample, we will record in a secure database the patient’s demographics 
such as current age at time of blood draw, race and ethnicity, birth history, past medical and surgical 
history as well as contemporaneous direct bilirubin levels, hemoglobin levels, hematocrit levels, and red 
cell morphologic changes noted in the hemogram. Cholesterol levels, bile acid levels, and vitamin E levels 
which were obtained as close as possible to the ektacytometry analysis will also be recorded.   Only the 
investigators at Cincinnati Children’s will have access to this database. 
 
Study Completion 
 
Once the blood sample has been obtained, no additional study procedures will be performed and the 
remaining blood will be properly disposed once the assay is complete.  
 
9. 
DATA ANALYSIS/METHODS 
 
Sample size:  
The sample size of 20 cholestatic patients and 20 controls is estimated based on the number of patients 
followed at CCHMC.  We estimate a 75 % participation rate for the study.  Knowing that we have a small 
patient pool within each diagnostic category of cholestatic patients, we will stratify patients according to 
diagnosis, but suspect that we will have only 3-5 patients within each diagnostic grouping, so it may be 
difficult to draw conclusions regarding relative differences in osmotic fragility and deformability within 
the overall cholestatic population. Because this is a pilot study we do not know what the difference in 
Omin or deformability between patients and controls will be. Hence we cannot conduct power analysis. 
However, we can utilize our preliminary data to facilitate power analysis for future studies. 
 
Laboratory Methods: 
 
Within 24 hours of blood sampling, we will conduct our ektacytometric analysis. Samples will be drawn 
into EDTA tubes. In the clinical hematology lab, hemoglobin, hematocrit, red cell indices, and RBC number 
will be measured by standard methods.  A blood smear will be stained with a Giemsa stain and reviewed 
by clinical cytologists. Ektacytometry will be performed in the CCHMC Erythrocyte Diagnostic Lab per 
clinical testing SOP. Specifically, a 300 microliter sample will be injected into the Osmoscan LoRRca MaxSis 
(Mechatronics Instruments BV, Zwaag, The Netherlands) ektacytometer. Patients' RBCs deformation will 
be monitored while exposed to a constant shear stress of 30 Pa and an increasing osmotic gradient in 

Page 6 of 8 
Version 05APR2018_v2                                                  
 
order to generate the ektacytometry curves. A positive result is seen as a divergence of the Omin, Elmax, 
and Ohyp of the sample compared to a concurrently-run normal control and the normal control range for 
the assay. Deformability will also be tested under an increasing shear stress for further investigation of 
the RBC cytoskeleton mechanics.  The Erythrocyte Diagnostic Lab will then discard the remaining sample 
once a result is obtained.  
 
Bilirubin levels, bile acid levels, lipid profiles, and vitamin E levels will be measured by standard laboratory 
methods employed by the CCHMC clinical laboratory.  Bile acids will be measured by radioimmunoassay.  
 
Statistical analysis 
We will compare the Omin and EI max values between patients and controls by Student’s t test of the 
means.  The ektacytometry characteristics (mainly values of Omin and Elmax) will be correlated with direct 
bilirubin levels, serum cholesterol levels and vitamin E levels by linear regression analysis.  
 
10. 
FACILITIES AND PERFORMANCE SITES 
 
The research study, all patient recruiting and blood draws will be performed in either the inpatient 
hepatology unit, the hepatology clinic, the general GI unit, or one of the general GI clinics at CCHMC.  
Ektacytometery will be performed in the CCHMC Erythrocyte Diagnostic Laboratory and analyzed by Dr. 
Theodosia Kalfa. Bile acids will be analyzed within Dr. Miethke’s laboratory.  
 
11. 
POTENTIAL BENEFITS 
 
There is minimal risk associated with participation in this study. The potential benefits of this study 
pertain to the advancement of scientific knowledge in the area red cell membrane changes among 
cholestatic patients.  
 
There is unknown direct medical benefit for patients.  
 
12. 
POTENTIAL RISKS, DISCOMFORTS, INCONVENIENCES AND PRECAUTIONS 
 
There are no increased risks to participate in this study.  The analyses will be conducted only upon blood 
that has been drawn for clinical purposes so there are no additional risks beyond what the patient would 
encounter for sampling conducted for clinical monitoring even if patients did not participate in the study. 
Blood will not be drawn specifically for this study, and a minimal amount of whole blood will be required. 
 
All unanticipated problems and adverse events will be reported consistent with institutional policy. 
 
 
13. 
PRIVACY AND CONFIDENTIALITY 
 
All study material will be stored in a locked storage cabinet in the CCHMC Gastroenterology office that 
will remain locked when not in use by study personnel. Access to original data and study materials will be 
limited to the investigative team. Informed consent/assent documents will be stored in a locked storage 
cabinet separate from study data. All raw data and computer files containing data will have confidentiality 
protected with the use of unique assigned study identifier codes; computer files will also be password 
protected. As mentioned previously, only a few selected study team members will have access to the 

Page 7 of 8 
Version 05APR2018_v2                                                  
 
database, completed forms and results of the assay. This dataset will only be used for research purposes. 
Each subject will be assigned a study identification code so that the study information will be confidential.  
All blood samples will be collected and processed in blood tubes containing the subject’s unique 
identification code.  All blood samples will be destroyed at the earliest opportunity as consistent with the 
conduct of the research, after all data on that patient has been collected and processed. 
 
Information from this research study may be published. However, participants will not be identified in any 
publication. All subject identifiers (e.g. medical record number, date of birth) as defined by HIPAA will be 
removed, so that identities are not traceable within or outside the study. The privacy and confidentiality 
of patient information will be maintained in accordance with HIPAA regulations.  
 
14. 
COST OF PARTICIPATION  
 
There is no cost to the patient to participate in this study. The cost of performing ektacytometry will be 
borne by the Kalfa laboratory. The cost of performing bile acids will be borne by Dr. Miethke’s laboratory.  
Other, clinically indicated testing, will be covered by the patients’ insurers.  
 
15. 
PAYMENT FOR PARTICIPATION 
 
There is no payment to patients for participation in this study. 
 
16. 
FUTURE USE 
 
All data collected will be stored in a data repository for future analysis regarding similar research interests.  
All future projects will undergo separate IRB approval.  The repository will be stored on a password 
protected file. There will be no linking information to patient identifier numbers and it will not be used as 
a means for future recruitment. 
 
17. 
References 
 
1. Cooper RA. Anemia with spur cells: a red cell defect acquired in serum and modified in the circulation. 
J Clin Invest. 1969 Oct; 48(10):1820-31. 
 
2. Kohno M1, Murakawa K, Yasunari K, Yokokawa K, Horio T, Kano H, Minami M, Yoshikawa J.  
Improvement of erythrocyte deformability by cholesterol-lowering therapy with pravastatin in 
hypercholesterolemic patients. Metabolism. 1997 Mar; 46(3):287-91. 
 
3. Corey KE, Cohen DE. Lipid and Lipoprotein Metabolism in Liver Disease. [Updated 2015 Jun 27]. In: De 
Groot LJ, Chrousos G, Dungan K, et al., editors. Endotext [Internet]. South Dartmouth (MA): 
MDText.com, Inc.; 2000-.Available from: https://www.ncbi.nlm.nih.gov/books/NBK326742/ 
 
4. Cynamon HA, Isenberg JN, Gustavs LP, Gourley WK. Erythrocyte lipid alterations in pediatric 
cholestatic liver disease: spur cell anemia of infancy. J Pediatr Gastroenterol Nutr 1985;4:542-9 
 
5. Allen DW, Manning N. Cholesterol Loading of Membranes of Normal Erythrocytes Inhibits 
Phospholipid Repair and Arachidonoyl-CoA: 1-Palmitoyl-sn-Glycero-3-Phosphocholine Acyl Transferase. 
A Model of Spur Cell Anemia. Blood 1996; 87:3489-3493  

Page 8 of 8 
Version 05APR2018_v2                                                  
 
 
6. Bessis M, Mohandas N, Feo C. Automated Ektacytometry: a new method of measuring red cell 
deformability and red cell indices. Blood Cells 1980; 6(3):315-27 
 
7. Mohandas N, Clark MR, Jacobs MS, Shohet SB. Analysis of factors regulating erythrocyte 
deformability. J Clin Invest. 1980; 66(3):563-573. 
 
8. Da Costa L, Suner L, Galimand J, et al. Diagnostic tool for red blood cell membrane disorders: 
Assessment of a new generation ektacytometer. Blood Cells Mol Dis. 2016; 56(1):9-22.