Method for diagnosing blood clotting disorders

Assay methods for diagnosing blood clotting disorders are described. The assays use data bases for pooled normal plasma (PNP) and plasma from healthy volunteers, males and females ages 18 to 64 years. Charting on a comparative basis of patient plasma and PNP allows the results to be interpreted by reference to the data base. Simple, rapid, inexpensive and highly sensitive and specific assays devised for diagnosing blood clotting disorders are described.

BACKGROUND OF THE INVENTION 
(1) Field of the Invention 
The present invention relates to an improved method for diagnosing blood 
clotting disorders based upon clotting times for a plasma from a patient. 
In particular, the present invention relates to a method which uses a 
combination of charts, pooled normal plasma (PNP) as a control and data 
bases for normal and abnormal clotting in the presence of various clotting 
or clot inhibiting agents in diagnosing a specific clotting disorder. 
(2) Prior Art 
In the United States about 1.5 million people per year have a heart attack. 
About half survive, and most require thrombolytic (clot-dissolving) 
treatments like tPA and streptokinase. About 500,000 people are 
hospitalized each year in the U.S. because of pulmonary embolism (clot in 
blood vessels in the lungs) or deep venous thrombosis (clot in vessels of 
limbs, often in the legs). About 220,000 people per year have coronary 
by-pass surgery for treatment of heart disease resulting from clots 
blocking coronary blood vessels. 50,000 people die each year of pulmonary 
embolism. 200,000 people die each year of cerebrovascular disease. 50,000 
persons in the U.S. population have inherited clotting defects. 
Diseases caused by blood clots are among the most common life-threatening 
medical problems in the United States. Another group of important diseases 
is caused by failure of blood to clot (e.g. hemophilia); a lot of these 
are inherited defects, but some are caused by other conditions like 
cancer, Lupus, or even certain infections that affect the liver and other 
tissues involved in the production of blood proteins. Clotting diseases 
are on the increase because they are associated with life style patterns 
in the western world (obesity, lack of exercise, smoking), and with aging. 
People are living longer these days and therefore more are entering the 
high risk age groups. A lot more people are consequently put on oral 
anticoagulants to counter this trend. New types of treatment are now being 
developed that make it all the more important to be able to reach 
decisions quickly on which treatment to use and whether or not it is 
working properly. 
Accurate and rapid diagnosis of diseases caused by clots or clotting 
defects is therefore a major everyday problem for doctors and medical 
technologists. Consequently, there is a significant segment of the 
clinical diagnostic industry dedicated to providing products to meet this 
demand. 
Laboratory diagnosis depends on tests done by technicians to measure how 
long the patient's blood plasma takes to clot, as compared to normal, and 
then to find out exactly what is wrong when the plasma does not clot 
properly. To do this, laboratories buy test kits and diagnostic reagents 
that are designed to help identify the clotting problem or defect. Some 
tests are simple; others are complex, need expensive equipment and skilled 
personnel, and take a lot of technician's time. Other tests are so 
complicated or so costly in reagents that routine laboratories in 
hospitals and clinics don't even do them, so they send the blood sample 
away to a specialty laboratory where the tests are performed by experts 
for a fee. 
Several companies in the medical diagnostic field sell coagulation 
diagnostic kits. However, these kits are mostly based on technological 
principles developed in the 1950's and the procedures are frequently 
seriously flawed. Modernization in technique has come primarily in the 
form of automation by computer-controlled instruments (coagulometers) that 
provide increased accuracy and avoid human errors, and reduce technician 
time, but the test principles are still the same. These new instruments 
are expensive, particularly in their most automatic form. They handle 
multiple samples and have built-in micro-processors that perform all the 
computations of results and comparisons to normal values. For some of the 
tests needed for monitoring clot dissolving treatments (e.g. urokinase, 
tPA), additional equipment is needed. 
The trend in the industry toward automation has come about due to the high 
cost of technical time, and the demand for more, faster and better 
diagnostic testing. As pharmaceutical companies develop totally new drugs 
the need for more tests, performed more frequently and giving more 
accurate information to the doctor, will increase substantially. 
Clot-dissolving products are expected to be among the biggest growth areas 
in the medical therapy market over the next decade. For example, based on 
sale after FDA approval, tPA is the most successful new drug ever 
introduced in the United States. The diagnostic industry is going to have 
to move in the direction of speedier tests, performed as close as possible 
to the patient and his or her physician. The needs for certain kinds of 
tests that monitor, for example, tPA infusion therapy or oral 
anticoagulant prophylaxis will increase. The same is true for the 
"replacement" treatments that are now appearing for genetic and acquired 
clotting defects; these new therapies have only become possible because of 
genetic engineering technology. There are no satisfactory systematic 
diagnostic approaches available in the marketplace to meet these needs at 
the moment. There is a need for such a systematic approach. 
The prior art procedures employed in most cases are flawed; they are 
outdated and have not been optimized to meet the level of critical, 
quantitative diagnostic need in today's clinical setting. Reagents of 
uncertain value or giving unreliable results are used on the basis of 
tradition rather than on the basis of rigorous scientific evaluation. 
Consequently, it is not possible to reach a correct and accurate diagnosis 
using many of them, and they do not match up to expectations of the 
clinician for therapeutic monitoring. The more complex tests available 
today for monitoring need equipment and skills that are not routinely 
available, and therefore they are only done in reference lab settings; 
although the clinician needs these kinds of test results, the procedures 
are not run often enough to provide the best results. In some cases the 
current diagnostic reagents are not stable and their shelf lives are not 
well characterized for today's quantitative tests. The variables that 
influence the test results, like disease state and therapeutic history, 
have not been examined sufficiently in most cases to permit these kits and 
reagents to be used with confidence for quantitative purposes. 
Quantitative test data is needed when powerful therapies are being applied 
that can be disastrous in the wrong situation. 
The complications faced by the clinician in dealing with these new surgical 
and medical interventions becoming available for patients, coupled with 
the complexity of the lab technology, have resulted in a serious 
communication gap between the two. There is a need for a comprehensive 
approach to differential diagnostic logic in thrombotic and clotting 
diseases, in a modern form that makes the system available and 
understandable to both clinical and laboratory personnel. 
OBJECTS 
It is an object of the present invention to provide tests which have 
characterized reagents, improved configuration of the assays, quantitative 
reliability in a wide variety of disease states, and proven 
interrelationships of test results with clinical and pathological data. 
Further it is an object to provide tests which are relatively simple and 
economical. These and other objects will become increasingly apparent by 
reference to the following description and the drawings.

GENERAL DESCRIPTION 
The present invention relates to a method for diagnosing blood clotting 
disorders in humans which comprises: separately testing sets of a sample 
of plasma separated from the blood of a patient and sets of a sample of 
plasma from pooled normal plasma (PNP) from healthy humans for clotting 
time (CT) by addition of predetermined amounts of prothrombin time 
reagents (P) to a first set of the sample; activated partial 
thromboplastin (APT) to a second set of the sample and thrombin (T) to a 
third set of the sample, charting the results for the patient and the PNP 
together on a side-by-side basis for P, APT and T and comparing the 
results with a data base showing normal ranges of CT based upon the PNP 
for APT, T and P wherein the APT, T and P have been separately prepared in 
solution to produce a particular standardized clotting time with PNP which 
is used in all of the testing of the patient plasma; testing for 
hypercoagulation or bleeding based upon the P, APT and T tests and 
charting the results; and providing a diagnosis based upon the differences 
of CT based upon the tests. 
Further the present invention relates to a method for diagnosing blood 
clotting disorders in humans which comprises: separately testing a first 
set of samples of plasma from a patient and pooled plasma from normal 
healthy humans (PNP) for the time to coagulate by prothrombin time 
reagents (P), activated partial thromboplastin (APT) and thrombin (TCT), 
charting the results together on a side-by-side basis, and comparing the 
results from the samples with a data base showing abnormal and normal 
ranges of coagulation times (CT) for healthy humans, wherein the APT, T 
and P have been prepared in solution to produce particular standardized 
clotting time with PNP which is used in all of the testing of patient 
plasma; optionally testing second sets of a sample of the plasma from a 
patient and PNP by mixing the patient plasma suspected to be genetically 
deficient in a blood factor selected from the group consisting of Factors 
V, VII, VIII, IX, X, XI, XII, F1.F and HMWK (HMWK is HMW-kininogen and 
F1.F is Factor 1 fibrinogin) with a volume factor deficient of a genetic 
plasma in an amount between about 40 to 60 percent by volume of GFPD to 
PNP and patient plasma for coagulation by an appropriate one of P, APT or 
both separately, charting the results together on a side-by-side basis and 
comparing the results with a second data base showing normal ranges of CT 
based upon PNP wherein the CT of GFPD is corrected by the PNP and normal 
patient plasma; optionally testing the samples of the plasma for 
anti-thrombin III by determining CT for the patient and PNP, charting the 
results together on a side-by-side basis and comparing the results with an 
antithrombin III data base showing normal ranges for PNP; optionally 
testing the samples of the plasma for protein C by determining CT and 
charting the results and comparing the results with a protein C data base 
showing normal ranges of CT based upon the PNP; optionally testing the 
samples of plasma for fibrinogen from the patient plasma and PNP and 
determining CT for coagulation by T at known dilutions of the plasma, 
charting the results as CT on a side-by-side basis and comparing the 
results with a fibrinogen data base showing normal ranges of CT for the 
PNP; and providing a diagnosis based upon the differences in coagulation 
times in the tests. 
In the present invention the PNP is used to rigidly standardize the CT 
reagents P, APT and T and are formulated to always produce the same CT 
with PNP. For T the CT is preferably 8 to 9 seconds. This is 1.2 units per 
100 ul of solution. For AP the CT is preferably 26.4 seconds .+-.2 
seconds. For P the CT is preferably 11.6 seconds .+-.1 second. Generally 
the tests are conducted in 200 ul of PNP. If this procedure is not 
followed, the results of the assays will be variable from lot to lot of P, 
T, and APT and from day to day and the results in the manner of the 
present invention can not be achieved which relies upon producing the same 
CT with normal plasma. 
It has been found that when PNP is mixed with a Factor deficient plasma, in 
a volume between about 40 and 60% of the volume of the Factor deficient 
plasma (GFDF) in a PT, APTT or TCT/TT assay that the PNP will correct the 
factor deficiency and if it is normal, the patient plasma will correct the 
factor deficiency of a factor deficient plasma. If there is a particular 
factor deficiency in the patient plasma it will not correct the GFDF thus 
showing that the patient plasma is also deficient in this factor. This 
mixing technique is a basic concept of the present invention. Histograms 
for FVII, FX and FV by the PT assay and FVIII by the APTT assay are shown 
in FIGS. 24 to 28. 
Diseases associated with hemostatic disorders are broadly categorized into 
bleeding and thrombosis. Bleeding can be external or internal. Internal 
bleeding includes the types that manifest under the skin such as 
hematomas, bruises, purpura and petichiae. Thrombosis can be acute or 
chronic. The acute form of thrombosis if untreated, will result in severe 
uncontrollable bleeding. Chronic thrombosis can be caused by deranged 
protein coding or by environmental factors. 
To investigate the pathogenesis of bleeding and thrombotic diseases a 
multitude of standardized procedures were developed. Some are complicated, 
with a high degree of sophistication and can be carried out only in 
research laboratories. Others are relatively simple and suitable for 
clinical testing. 
FIGS. 1 to 8 show the diagnosis of various diseases and the CT for normal 
and abnormal patient plasma by the APT, PT and T tests. These tests are 
performed and charted as shown in FIGS. 10 to 18. The individual assays 
are for factor deficiency, inhibitors, contact phase antithrombin III, 
protein C, and fibrinogen as well as other standard assays as shown in the 
charts. 
SPECIFIC DESCRIPTION 
Routine Assays 
There are several well established screening procedures of a very general 
nature which are ordered routinely on individuals who present for the 
first time with a bleeding disorder. 
(1) The Tourniquet Test (Rumple Leede Test). This is a non-invasive, easy 
to perform procedure to identify capillary fragility. It is performed by 
applying a blood pressure cuff for 20 to 40 minutes and applying a 40 mm 
Hg pressure. In fact, it is the only available testing procedure for the 
blood vessel component of the hemostatic system. Blood vessel disease is 
the least commonly encountered hemostatic abnormality. It is frequently 
seen associated with viral infections, drug induced vasculitis, and 
collagen diseases such as lupus erythematosus and others. It is also 
frequently seen in the elderly. 
(2) Platelet Count. A platelet count of 50,000 per cubic milliliter of 
blood ensures adequate hemostasis unless challenged by stressful 
conditions such as trauma, surgery or childbirth. Platelet counts of 
10,000 per cubic milliliter of blood are dangerously low and may lead to 
spontaneous hemorrhage. Thrombocytosis is a platelet count above 500,000 
per cubic milliliter. Thrombocytosis predisposes to thrombosis in most 
cases. In myeloproliferative disorders platelet counts may reach 1 million 
per cubic milliliter. In such conditions platelet functions are defective 
and thrombocytosis is associated with excessive bleeding rather than 
thrombosis. 
(3) Bleeding Time Tests. The Duke and Ivy time bleeding procedures are 
technically difficult to perform. Adequate standardization is essential 
for the interpretation of the results. In expert hands a normal bleeding 
time of greater than 15 minutes is frankly abnormal and indicates: 
Severe impairment of platelet functions of genetic or acquired origin. 
Very low blood levels of von Willebrand Factor. 
Afibrinogenemia and severe Factor V deficiency. 
A bleeding time greater than 8 minutes and less than 15 minutes is more 
difficult to interpret. A moderately low plasma level of vWF is by far the 
most common underlying pathogenesis. Antiplatelet drugs, lupus-like 
inhibitors and Factor XI deficiency should be considered in the 
differential diagnosis. 
(4) Prothrombin Time (PT). The PT is a screening test to identify 
coagulation abnormalities of the extrinsic and common pathways and 
fibrinogen. This test is performed by mixing tissue thromboplastin/calcium 
chloride solution (TTP/CaCl.sub.2) with the plasma. Oral anticoagulant 
medication is the most frequent cause of prolonged PT. The only 
abnormality that causes a prolonged PT and no changes in the other 
screening tests is a Factor VII deficiency. The PT is sensitive to slight 
decreases in plasma levels of Factor V. 
(5) Activated Partial Thrombo-Plastin Time (APTT). The APTT is a screening 
test to identify coagulation abnormalities of the contact phase and the 
intrinsic and common pathways of plasma activation. This test is performed 
by mixing activated partial thrombin reagent (APTT) with the plasma. The 
APTT is not sensitive to fluctuation in plasma fibrinogen levels. Of 
conditions that cause prolongation of the APTT, the most frequent is 
contamination of plasma with heparin. Other conditions are Factors VIII 
and IX deficiencies as well as lupus-like anticoagulants. A factor XII 
deficiency of less than 1% gives an APTT of 260 to 300 seconds, while 
deficiencies of Factor VIII or IX of less than 1% will give a more modest 
increase of 78 to 82 seconds in the APTT. 
(6) Thrombin Clotting Time/Thrombin Time Assay (TCT/TT). TCT or TT is the 
same assay given two slightly different names. TCT/TT measures the time 
taken by exogenously added thrombin to proteolyze plasma fibrinogen and to 
form a clot. TCT/TT assays are not standardized by the prior art. Each 
laboratory determines the activity (strength) of thrombin to be used in 
the assay. It is customary to adjust the thrombin activity to give a 
clotting time of 8 to 9 seconds with 0.2 ml citrated pooled normal plasma 
(PNP). This is equivalent to 1.2 NIH units of thrombin activity. 
The usefulness of TCT/TT as a screening assay is underestimated since it is 
not specific for any disease condition. However, routine use of TCT/TT 
alongside the PT and APTT is invaluable to differentiate efficiencies of 
the intrinsic pathway from heparin, and separate lupus-like anticoagulants 
from afibrinogemia and paraproteinemia. 
(7) Euglobulin Lysis Time Test. This is a crude but effective screening 
test for accelerated fibrinolysis. If the clot dissolves much faster than 
the control plasma, it is correct to attribute the patient's condition to 
a deficiency of the major inhibitor of plasmin, alpha-2-AP or to a 
deficiency of the major inhibitor of plasmin, alpha-2-AP or to a 
deficiency of activated Factor XIII, the fibrin cross linking 
transglutaminase. The concentration of alpha-2-AP in plasma is half the 
capacity of plasma for forming plasmin. Therefore, conditions of 
accelerated fibrinolysis do not necessarily imply a genetic deficiency of 
alpha-2-AP, but could mean a transient depletion of the inhibitor. 
FIG. 1 shows the possible diagnosis based upon the AP, T and P tests. All 
of these tests are performed if there is any abnormal bleeding or a 
thrombosis. 
Other Assays: 
(8) Fibrinogen Assays 
The unique features of this assay are: 
1) it is a qualitative as well as a quantitative thrombin time clotting 
assay; 
2) serial dilutions of patient and control plasma are performed 
simultaneously and clotted with thrombin; 
3) fibrinogen in PNP has been quantified by Lowry's assay; and 
4) Slopes of curves for control PNP and patient plasma are compared. 
Calculations for patient fibrinogen levels are read off a regression line. 
The prior art assays: 
Prior Art Assays: 
1) are based on Clauss method (citation). 
2) evaluated patient fibrinogen duplicate clotting times of one plasma 
dilution. Calculations are made from a semi-logarithmic plot of the 
regression line of a standard curve performed with purified fibrinogen. 
Clinically, the most significant fibrinogen disorders result from:(a) 
impairment of the velocity with which fibrinogen converts to fibrin and 
coagulates in plasma; (b) increases or decreases of levels of circulating 
fibrinogen. 
Determination of fibrinogen has, throughout the years, been performed with 
a variety of methods, each in itself burdened with its own inherent 
limitations. A brief listing of various methods includes the turbidimetric 
estimation of fibrinogen after salting out procedures, estimation of 
fibrinogen by zone electrophoresis, spectrophotometric assays of the 
purified protein, assays of fibrinogen as fibrin after coagulation, and 
gravimetric and heat precipitation. 
For clinical testing in the present invention, two procedures are 
performed: 
A. Thrombin Clottable Fibrinogen (TCF) Assay, and 
B. Heat Precipitatable Plasma Fibrinogen (HPPF) determination by Lowry's 
Assay. 
A. Thrombin clottable fibrinogen assay (TCF) 
In the TCF Assay, a thrombin solution (1.2-1.5 NIH units; CT 7-8 secs) is 
added to serial dilutions of patient plasma and pooled normal plasma 
(PNP). The dilutions are done with defribilated PNP. Enzyme concentration 
is always constant; substrate is serially diluted. The defibrilated PNP 
will be partially corrected by the normal PNP on a reproducible basis. The 
velocity of fibrinogen conversion and fibrin polymerization is recorded on 
a coagulation analyzer. Two graphs are sketched by plotting the points for 
the clotting times of the patient and PNP as shown in FIGS. 19 to 21. The 
plasma dilutions are preferably on the x-axis and the clotting times in 
seconds on the y-axis. By finding the slope and y-intercept of both lines 
of the graph the following information can be derived: 
(1) If the slopes of the line of the patient and the control are parallel; 
and the y-coordinates (y-intercept mean clotting time) are at the same 
place on the axis, the diagnosis is that levels of biologically functional 
fibrinogen are within the normal range. 
(2) If the slopes of the line of the patient and the control are parallel 
but the y-coordinate of the patient is translated upward (more prolonged 
clotting time) the diagnosis is that levels of biologically functional 
fibrinogen are decreased. 
(3) If the slopes of the line of the patient and the control are parallel 
but the y-coordinate of the patient is translated downward (shorter 
clotting times) the diagnosis is that levels of biologically active 
fibrinogen are increased. 
(4) If the graph of the patient has a different slope from that of the 
control the only possibilities are: heparin or an abornmal fibrinogen. 
Heparinized plasma will give a false positive result for 
dysfibrinogenemia. 
To rule out the interference by heparin, 1 ml of the patient's plasma is 
reacted with a heparin absorbent chemical (Hepasorb.TM.) (need source) for 
10-20 minutes. The absorbant approach is less costly and just as effective 
as the better known reptilase test (citation). 
Following treatment of patient plasma with absorbant, the TCF assay is 
repeated. If the slopes of the graph continue to be different, an abnormal 
fibrinogen is verified by HPPF determination. The diagnostic findings for 
dysfibrinogenemia are: discrepancy in levels of fibrinogen obtained by the 
HPPF assay (higher) and the TCF assay (lower). 
Determination of Fibrinogen Levels 
The points for the fibrinogen levels and clotting times of control plasma 
are entered on the keyboard of a scientific calculator. The slope, 
y-intercept and the correlation coefficient of the linear regression line 
are obtained. To calculate the level of fibrinogen in the patient's plasma 
enter a y'-coordinate (a clotting time for one of the patient plasma 
dilutions) into the keyboard. The calculator will find and print the 
corresponding x-coordinate (control plasma (PNP) dilution) equivalent to 
the fibrinogen level for the patient. 
Materials and Methods 
PREATION OF PLASMA 
Pooled Normal Plasma (PNP) 
PNP is prepared by pooling human citrated plasma obtained from 40 healthy 
volunteers. The PNP can be stored in 0.5 ml aliquots in small conical 
plastic tubes at -70.degree. C. and has a stability of 5 years. 
Defibrinated Pooled Normal Plasma (Defibr PNP) 
Pooled Normal Plasma (PNP) for defibrination is prepared by pooling ten 
units of outdated citrated human plasma. The pooled plasma is aliquoted 
into 25 ml conical plastic centrifuge tubes. Plasma is defibrinated by 
placing the plastic centrifuge tubes containing the plasma in a water bath 
heated to 56.degree. C. The temperature of the plasma is brought to 
56.degree. C. and maintained at that temperature for 5 minutes. This 
process denatures fibrinogen. Centrifugation at 2,000 rpm for 15 to 20 
minutes then precipitates the denatured fibrinogen to the bottom of the 
tubes. 
The supernatant is the defibrilated PNP that is used as the diluent in the 
TCt Assay. A thrombin clotting time is performed to verify the 
completeness of removal of fibrinogen. Packaging is by placing 1.5-2 ml of 
defibrilated PNP into plastic tubes. Storage is at -70.degree. C. and the 
stability is 10 years. 
Chemical estimation of fibrinogen: 
The denatured fibrinogen pellet is reconstituted in 25 ml distilled water. 
A Lowry's Assay is performed as described hereinafter. The concentration 
of fibrinogen in plasma is expressed in milligrams per deciliter. 
Patient Plasma 
Blood is collected in sodium citrate. Quantity: 5 ml blood to yield about 2 
ml plasma. Patient's plasma is not debrinated, and is used in the assay as 
whole decalcified plasma. 
REAGENTS 
Tissue Thromboplastin: prepared from rabbit brain. 
Calcium Chloride: 0.02M. 
Activated Phospholipid Reagents: activating agents can be ellagic acid, 
kaolin, silica or soybean extract, etc. 
Thrombin Reagent: the powdered thrombin is reconstituted in a solution of 
CaCl.sub.2 0.1M. Clotting activity of thrombin is adjusted to 1.2 unit/per 
100 ul which will give when added to 200 ul PNP a clotting time of 8-9 
seconds. 
EQUIPMENT 
Fibrometers or photo optical systems. 
Assay Procedures 
EXPERIMENTAL PROCEDURES 
Plasma Dilutions 
Two duplicate sets of fibrocups or cuvettes are labeled 1 to 6. Pipette in 
all the cuvettes or fibrocups 200 ul defibrilated PNP. Label one duplicate 
set PNP and the other duplicate set Patient. 
In #1 PNP pipette 200 ul PNP perform a serial dilution of PNP in Defibr 
PNP. Discard 200 ul from #6. 
In #1 Patient pipette 200 ul patient plasma. Perform a serial dilution of 
patient plasma in Defibr PNP. Discard 200 ul from #6. 
Measurement of Clot Formation 
A. A prothrombin time (PT) assay or a thrombin clotting time (TCT) assay 
can be performed. The clotting times are recorded on the report chart 
(FIG. 7). 
Analysis of the Properties of Plasma Fibrinogen 
This can be performed on a scientific calculator, on a personal computer 
with graphic capabilities, or on a photo optical coagulation analyzer with 
graphics capabilities. 
Enter on the keyboard two separate sets of results: 
y: clotting time of plasma dilutions of PNP. 
y': clotting time of plasma dilutions of patient. 
x: plasma dilution of PNP 
x': plasma dilution of patient 
FIG. 20 shows the results of the assay with thrombin. FIG. 21 shows the 
results with prothrombin. 
B. Heat precipitatable plasma fibrinogen determination by modified Lowry's 
Assay. 
Test Procedure: 
1. Percent plasma fibrinogen dilutions are made in the following way: 
100% PNP is pooled normal plasma. 
50% PNP is 1 ml 100% PNP and 1 ml distilled water. 
25% PNP is 1 ml 50% PNP and 1 ml distilled water. 
12.5% PNP is 1 ml 25% PNP and 1 ml distilled water. 
6.2% PNP is 1 ml 12.5% PNP and 1 ml distilled water. 
3.1% PNP is 1 ml 6.2% PNP and 1 ml distilled water. 
2. Dilution sets are made up of 1 ml of each plasma fibrinogen dilution. 
Dilution sets are then incubated separately in a 56.degree. C. water bath. 
Two dilution sets are incubated for 5 minutes; another two dilution sets 
are incubated for 30 minutes. Centrifugation of each dilution set at 2,000 
rpm for 15 minutes immediately after incubation yields the fibrinogen 
precipitate. 
3. Each precipitate is then reconstituted with 1 ml distilled water and 
determined by Lowry's Assay which is described hereinafter. 
In the Lowry's assay, proteins including fibrinogen (5-100 micrograms 
sensitivity range) are measured with the Folin Phenol Reagent after 
alkaline copper treatment. Following alkaline treatment, free tyrosine and 
tryptophan residues react with copper, resulting in a 3- to 15-fold 
increase in color. This reaction is complete in 5-10 minutes at room 
temperature. The copper-treated protein will reduce the 
phosphomolybdicphosphotungstic (Folein) reagent. The final color is 
greatly enhanced. This reaction is complete in 30 minutes at room 
temperature. 
Reagents 
Reagent A: 2% Na.sub.2 CO.sub.3 (10 g/500 ml) in 0.10M NaOH (2 g/500 ml). 
Reagent B: 2% Na tartrate and 1% CuSO.sub.4 (1:1 volume). 
Reagent C: Alkaline Copper solution: 100 ml A and 2 ml B. (Discard after 1 
day). 
Reagent E: Folin reagent 2N diluted 1:2 in distilled water to make 1N 
solution. 
Procedure: 
The sample is 0.2 ml to which 1 ml of Reagent C is added and vortexed 
immediately. 
The sample with Reagent C sits at room temperature for 10 minutes and then 
0.1 ml of Reagent E is added and vortexed immediately. 
The sample then sits at room temperature for 30 minutes and is then read on 
Spectrophotometer at 750 nm. 
Calibration Curve: 
The curve prepared by dissolving 1 mg albumin in 1 ml distilled water (1 
microgram/1 microliter). 
Blanks: 
The blanks are: 
Distilled water 0.2 ml 
Reagent C: 1 ml 
Reagent E: 0.1 ml 
PROCEDURE FOR CALIBRATION CURVE 
Albumin 1 mg/1 ml (1 ug/1 ul) 
5 ul 
10 ul 
20 ul 
50 ul 
100 ul 
The albumin solution is q.d. to 0.1 or 0.2 ml in distilled water or buffer. 
(Disposable glass tubes and duplicate samples). 
Blanks: 0.1 to 0.2 ml distilled water or buffer. 
Protein sample: 5-10 ug sensitivity range. 
Add 1 ml of Reagent D and vortex immediately. The sample then sits at room 
temperature for ten minutes. 
PROCEDURE 
Prepare Folin reagent. 
Pipette 3 ml Folin Reagent and 3 ml distilled water to produce 6 ml. 
Combine in small beaker. Add 0.1 ml Folin Reagent to: 
(1) Standard curve; (2) Blank; and (3) Protein samples and vortex 
immediately. 
The sample then sits for 30 minutes at room temperature and the optical 
density of samples is read on a spectrophotometer (which can be single or 
double beam). 
SPECTROPHOTOMETER 
Set at 750 nm (visible) 
1. Warm instrument for 30 minutes prior to use. 
2. Calibrate with BLANK 
3. Use quartz cuvettes (washed in chromerge acid(?)) 
4. Read optical density (O.D.) of calibration curve) 
5. Read O.D. of protein samples 
6. Construct on calculator linear regression for calibration curve. 
Estimation of slope, intercept and protein concentration of unknown samples 
can be obtained by entering the: 
Concentration (x values) of albumin used in the calibration curve, followed 
by entry of O.D. (y values) obtained on spectrophotometer. 
Procedure: 
______________________________________ 
Press: 2nd Pgm 1 SBR CLR to clear the calculator. 
Then 
Press: Value of x (5 mg) 
Press: x = tg Then 
Press: value of y (O.D. reading) 
Press: 2nd + Then 
______________________________________ 
Repeat process for values of x: 10 micrograms, 30 micrograms, 50 
micrograms, 100 micrograms. 
To calculate: 
The y intercept of the line fitted to the data points, PRESS 2nd OP 12. The 
slope of the line, PRESS 2nd OP 12 x.congruent.t. The correlation 
coefficient, PRESS 2nd OP 13. The linear estimate of x on the regression 
line, enter y value on the keyboard, followed by 2nd OP 15. 
The amount of fibrinogen is related to an equivalent amount of the albumin 
and is expressed in mg/ml. The modified Lowry's assay produces results 
which are compared to the TCF assay. 
The results are charted as set forth in FIG. 16. 
(9) Inhibitor Assays 
Until about five years ago the best known of the coagulation inhibitors 
were those that developed in hemophiliacs in response to Factor VIII 
replacement therapy. Nowadays the lupus-like anticoagulant or lupus-like 
inhibitor has taken in the scientific literature a place of considerable 
importance. There are several types of lupus-like inhibitors. 
Some are purely in vitro phenomena. They are easily identified in 
phospholipid-dependent coagulation tests. They cause significant 
prolongation of the clotting times of normal plasma when equal amounts of 
lupus inhibitor plasma are added to the reaction mixture. Also, plasma 
with lupus inhibitor does not correct the prolonged clotting times of 
assorted single factor deficient plasma reagents. Significantly in vitro 
type lupus inhibitors are neutralized by Platelet Factor III activity. 
Other lupus inhibitors have been found to interfere with the activation of 
coagulation factors and impair the in vivo biological function of these 
factors. With this type of lupus inhibitor a bleeding diathesis may 
develop under challenge to the hemostatic system. More often however, they 
tend to cause an increase in the susceptibility to thrombosis. Lupus 
inhibitors directed against the biological activity of Factors V, VIII and 
prothrombin have been identified. These were exclusively present in 
elderly females with connective tissue disorders. Inhibitors have been 
identified in other categories of individuals and in association with a 
variety of diseases. This type of lupus inhibitor is best identified in 
mixing studies with PNP and single factor deficient plasma reagents as 
indicated in the chart (FIG. 3) labeled "Inhibitor Screen". The method for 
the Inhibitor Screen is very simple. Mix 50 ul PNP or single deficient 
plasma reagent and perform a PT or an APTT assay. The activator substance 
in the APTT assay should be kaolin, soybean extract, silica, never ellagic 
acid. If evidence points to a specific coagulation factor inhibition, an 
inhibitor assay is performed. 
The only difference between in vivo lupus-like anticoagulants directed 
specifically against the activity of a coagulation factor and the 
inhibitors that develop in hemophiliacs is our lack of our understanding 
of the site of action of lupus-like anticoagulants. The inhibitor Assay 
can accurately measure the antibody titer of inhibitors to Factors VIII or 
Factor IX that develop in hemophiliacs as well as the in vivo lupus-like 
anticoagulants. By definition an antibody titer is the amount of antibody 
that can bind to one unit of antigen. Unit can be in measures of weight or 
activity. In our assay we measure the inhibition 1 unit of activity of a 
single coagulation factor. 
INHIBITOR ASSAY 
Specificity of the inhibitor assay is for: 
1) Inhibitors that develop in hemophiliacs and other individuals. These 
types of inhibitors are antibodies directed specifically against a 
procoagulant factor. 
2) Lupus like inhibitors. These are inhibitors directed specifically 
against phospholipids. These antibodies are recognized by their effect on 
the clotting times of the APTT assay. Two types of lupus like inhibitors 
can develop: 
i) a non-specific type that reacts in vitro with phospholipid reagents to 
inactivate them. These inhibitors do not cause bleeding problems. 
ii) a specific type that reacts with platelet phospholipids and prevents 
complex formation and assembly. These inhibitors can cause serious 
bleeding problems. 
The principle of the inhibitor assay for inhibitors that develop in 
hemophiliacs and other individuals is a direct inhibitor assay similar to 
the passive hemaglutination assay. The end point of this assay determines 
the amount of factor activity inhibited by a known quantity of patient 
plasma. Thereby calculations are made as to how much factor concentrate is 
needed to overcome the inhibitor. 
The principle of the assay for lupus like inhibitors is to mix in equal 
proportions of the patient's plasma and a control plasma. If the prolonged 
clotting times by the APTT assay are corrected, a lupus like inhibitor can 
be ruled out with certainty. To provide proof for a lupus like inhibitor, 
the patient's plasma is mixed in equal proportions with Factor VIII 
deficient plasma and an APTT assay is performed. If the prolonged clotting 
time by the APTT is not corrected, evidence is very strong of a lupus like 
inhibitor. 
Reagents: 
Citrated pooled normal plasma (about 1 ml). 
Factor VIII deficient plasma (about 2 ml) 
Activated Partial Thromboplastin Reagent ("Actin.TM."), purchased from 
American Dade (ellagic acid), or Helena.TM. APTT Reagent (kaolin) 
CaCl.sub.2 (0.02M) 
MLA pipettes and pipette tips: 200 microliters, 50 microliters, 30 
microliters, 20 microliters and 10 microliters. 
Method 
Step 1: 
Prepare 5 tubes, label 1-5 
Tube # Plasma Mixture (Antigen dilution, FVIII deficient) 
1 200 microliters PNP 
2 200 microliters PNP+200 microliters FVIII deficient. Mix. 
3 Add 200 microliters from tube 2+200 microliters FVIII deficient. Mix. 
4 Add 200 microliters from tube 3+200 microliters FVIII deficient. Mix. 
5 Add 200 microliters from tube 4+200 microliters FVIII deficient. Mix and 
discard 200 microliters. 
6 200 microliters patient's plasma. 
Step 2: Add inhibitor to antigen dilution. 
Tube 1 0.16 unit FVIII/200 microliters, add 50 microliters patient plasma. 
Tube 2 0.08 unit FVIII/200 microliters, add 30 microliters patient plasma. 
Tube 3 0.04 unit FVIII/200 microliters, add 20 microliters patient plasma. 
Tube 4 0.02 unit FVIII/200 microliters, add 10 microliters patient plasma. 
Tube 5 0.01 unit FVIII/200 microliters, add 10 microliters patient plasma. 
Control: Standard curve of PNP in Factor VIII deficient plasma. 
Label tubes 1-10. 
80% 1. 200 microliters PNP 
40% 2. 200 microliters PNP+200 microliters FVIII deficient. Mix. 
20% 3. Add 200 microliters from tube 2 to 200 microliters FVIII deficient. 
Mix. 
10% 4. Add 200 microliters from tube 3 to 200 microliters FVIII deficient. 
Mix. 
5. Add 200 microliters from tube 4 to 200 microliters FVIII deficient. Mix. 
2.5% 6. Add 200 microliters from tube 5 to 200 microliters FVIII deficient. 
Mix. 
1.2% 7. Add 200 microliters from tube 6 to 200 microliters FVIII 
deficient. Mix. 
0.6% 8. Add 200 microliters from tube 7 to 200 microliters FVIII deficient. 
Mix. 
0.3% 9. Add 200 microliters from tube 8 to 200 microliters FVIII deficient. 
Mix. 
0.1% 10. Add 200 microliters from tube 9 to 200 microliters FVIII 
deficient, mix and discard 200 microliters. 
Step III: 
Incubation: Plasma mixtures are incubated for 1 hour at 37.degree. C. 
At the end of the incubation period, 100 microliters of plasma mixture from 
each tube is added to 100 microliters prewarmed activated partial 
thromboplastin reagent in fibrocups. After 3 minutes, the mixture is 
clotted with 100 microliters CaCl.sub.2 0.02M. 
Calculations 
Follow the instructions outlined on the report chart FIG. 12. Results are 
interpreted as follows: 
1. A powerful inhibitor will overcome and neutralize all of the activity 
present in all plasma dilutions, and consequently one is unable to 
determine the antibody titer. In this instance the test should be repeated 
using 20 ul, 10 ul, 5 ul and 1 ul of patient's plasma while preserving the 
same initial factor activity in the plasma mixture. 
2. In another case, 1 ml of patient's plasma inhibits 3 units of Factor 
VIII activity. Since an adult human of average weight and height will have 
3 liters of circulating plasma, it is probable that 9,000 units Factor 
VIII concentrate will neutralize the inhibitor in vivo. 
3. A third example is that of a lupus-like anticoagulant. When tested in 
this system it will neutralize any of the plasma activity in the mixture. 
(10) Antithrombin III Assays 
The unique features of the Antithrombin III assay are: 1) it is a clotting 
assay; 2) the plasma is not diluted and is defibrinated; 3) the thrombin 
used in the assay is standardized; 4) heparin added to the assay mixture 
is standardized; 5) fibrinogen used in the assay is ATIII deficient plasma 
fibrinogen (200 micrograms per 100 microliters); 6) calculations are based 
on molecular interaction of ATIII and thrombin in the inactivation 
process. 
In all other ATIII clinical clotting assays: 1) plasma is variably diluted; 
2) thrombin activity is not recorded or has not been determined; 3) 
heparin is standardized; 4) purified fibrinogen is used for the end point; 
5) calculations are based on a standard curve of thrombin fibrinogen 
interaction. 
ATIII levels are decreased in hereditary ATIII deficiency, intravascular 
coagulation, hepatitis and hepatic cirrhosis and chronic nephritis. 
Significant laboratory findings in acute disseminated intravascular 
coagulation are low levels of ATIII activity measured by clotting assays, 
low platelet count, and decreased levels of fibrinogen. Hereditary ATIII 
deficiency is characterized by reduced ATIII activity and normal or 
reduced concentrations of ATIII measured by immunological assays. Other 
hemostatic parameters are normal. 
In clinical laboratories, both clotting and amidolytic assays are used to 
evaluate the heparin cofactor activity of ATIII. It has been found that 
ATIII selectively inhibits the clotting activity of thrombin. In other 
words, when ATIII first forms a complex with thrombin the inhibitor binds 
the enzyme at the fibrinogen binding site so that even though the clotting 
activity of thrombin is inhibited, complexed thrombin has the ability to 
hydrolyze chromogenic substrates. It has been found that the non-clotting 
forms of thrombin or "autolyzed thrombins" (beta and gamma thrombin) also 
form complexes with ATIII. In commercial thrombin preparations, the 
autolyzed thrombins are present in higher proportion than the clotting 
thrombin "autolyzed thrombins" have the ability to hydrolyze chromogenic 
substrates as well as to bind ATIII but do not hydrolyze fibrinogen. 
Therefore, chromogenic substrate assays measure clotting thrombin forms, 
thrombin complexed with ATIII as well as autolyzed thrombins. The use of 
such assays in clinical testing is questionable. The clinical significance 
of ATIII lies in its ability to inhibit the clotting form of thrombin from 
hydrolyzing fibrinogen to fibrin. 
ATIII levels are measured in the present invention by a clotting assay and 
an immunologic assay. Chromogenic substrate assays are never used. 
To devise an assay to measure the heparin cofactor activity of ATIII in 
plasma, several very important facts about the effect of heparin on 
coagulation proteins were taken into account. 
Heparin sodium is a mixture of active principles having the property of 
prolonging blood clotting. The route of administration is intravenous or 
subcutaneous. Its mode of action is to bind to ATIII. The greater impact 
of heparin is its in vivo effect on ATIII. This effect is known as the 
anticoagulant effect of heparin. The discovery of heparin-ATIII 
interaction provided an explanation for the long-known action of heparin. 
There is evidence that heparin interacts with ATIII by electrostatic 
binding to lysine residues of the ATIII molecule. In the presence of 
heparin, the preferential target of ATIII appears to be thrombin. Some 
subspecies of heparin may enhance the interaction of ATIII with Factor Xa 
more than with thrombin or vice versa. When formation of the 
ATIII-thrombin complex has occurred, heparin readily dissociates from the 
complex. Thus, it acts as a catalyst of the neutralizing process. 
However, the following facts though less well known are equally important. 
1. In vitro heparin has not one but two actions on blood coagulation. The 
less known one is a direct inhibitory effect. Heparin inhibits thrombin 
and Factor Xa by a direct effect that is due to electrostatic attraction. 
The inhibition involves the formation of reversible complexes that 
interfere with the procoagulant effect of both enzymes. Thus heparin has 
an anticoagulant and antithrombotic effect. The antithrombotic effect of 
heparin is one of the side effects of heparin therapy. 
2. At low heparin concentration, fibrinogen acts as an antithrombin, 
apparently due to an induced change in the charge of fibrinogen by 
heparin. This effect is absent in defibrinated plasma and in serum and is 
significant when the ATIII/heparin cofactor activity of plasma is 
evaluated. 
For clinical testing two procedures are performed: 
ATIII/heparin cofactor activity assay. 
Counter electrophoresis or cross over. 
Immunoelectrophoresis (cross over IEP) immuno assay. 
In the ATIII/Heparin Assay: 
(1) Test plasma (patient and PNP) is heat defibrinated. This is done to 
eliminate the effect of fibrinogen on heparin. 
(2) Low concentrations of heparin are used: 0.3-0.4 unit/ml. The purpose is 
to eliminate the direct effect of heparin on thrombin. 
(3) Patient and control PNP are never diluted. Again, here the purpose is 
to eliminate the direct effect of heparin on thrombin. When plasma ATIII 
is greatly diluted thrombin inhibition will occur as a result of 
inactivation by ATIII but also as a result of direct inactivation by 
heparin. Electrostatic binding of the negatively charged heparin to the 
active site of the thrombin will prevent the enzyme from hydrolyzing 
fibrinogen. Thus falsely high values for ATIII levels can be obtained. 
(4) Thrombin concentration is calculated to be less than plasma ATIII level 
in the test sample. 
PREATION OF REAGENTS 
1. Patient plasma and control PNP are heat defibrinated by the method 
described in the fibrinogen assay. 
A thrombin solution (1.2 to 1.5 NIH units/100 ul) to give a clotting time 
of 7-8 seconds when added to 200 ul PNP is prepared in 0.1M CaCl.sub.2 in 
one single 15 ml batch. Thrombin is not frozen and is stored in a plastic 
tube at 4.degree. C. in the refrigerator. The activity lasts for over 3 to 
4 months or even longer if properly handled. Small aliquots for the assay 
are used at room temperature. 
3. A heparin solution is prepared in distilled water as follows: take from 
the stock solution one unit of heparin. Add this one unit to 10 ml 
distilled water, mix gently. Heparin concentration is 0.1 unit/100 ul. To 
titrate the heparin activity suitable for the assay the following should 
be done. 
a. take 5-6 fibrocups or cuvettes and place 200 ul PNP. 
b. take MLA pipettes calibrated 20, 30, 40, 50, 60 ul. 
c. add 20 ul heparin solution to plasma in fibrocup and clot with thrombin. 
Record clotting time. Repeat with 30 ul, 40 ul, etc. 
d. ideal clotting time for the assay is 30-45 seconds. 
ASSAY PROCEDURE 
The results are reported in the chart (FIG. 14). 
1. 100 ul defibrinated patient plasma are placed in 4 fibrocups. 
2. 100 ul defibrinated PNP plasma are placed in 4 fibrocups. 
3. Add heparin to 2 fibrocups. Add thrombin then clot with PNP (100 ul). 
Record clotting time. 
4. Other 2 fibrocups, add thrombin then clot with PNP. 
CALCULATIONS 
The stoichiometry of the inactivation of thrombin by ATIII is 1:1. The 
clotting assay for measurement of ATIII is an indirect assay that reflects 
the loss of thrombin activity. Therefore, the ATIII levels are calculated 
indirectly by calculating the loss of thrombin activity. From FIG. 23, the 
ATIII levels can be obtained from the clotting times. 
(11) 
IMMUNOLOGICAL ASSAY 
HEIN ASSAYS 
The unique features of the heparin assay are that: 
(1) the sensitivity and specificity of the PT, TCT, and APTT for heparin at 
therapeutic ranges; (2) the specificity and sensitivity of heparin assays 
are not conferred by the heparin source (beef or porcine), or the purity 
of the thrombin reagent; (3) powdered heparin is more stable than liquid 
reagents; (4) computerized standard curves for heparin assays can be 
obtained. 
Heparin assays in other laboratories: 
(1) The APTT assay is the most widely used. APTT is sensitive to heparin 
but cannot measure heparin levels in plasma;(2) There is widespread 
confusion as to the sensitivity of heparin assays using different animal 
sources and curves are prepared fresh each day. 
In the present invention, test sensitivity to heparin was measured for 
three tests: the PT, APTT, and TCT. Their sensitivity to both liquid and 
powdered heparin was measured. The experimental condition and procedures 
were the same for both types of heparin. 
Historically, therapeutic heparin ranges were described as the amount of 
heparin that will prolong the whole blood clotting time to twice baseline 
value by the Lee and White assay (citation). Later, clinical trials were 
conducted with heparin on patients with thrombotic disease. It was then 
determined that plasma levels of 0.2 to 0.4 unit/ml heparin were 
sufficient to keep the patients anticoagulated. When heparin was 
administered at higher doses it was found that plasma heparin levels of 
0.5 unit/ml or higher resulted in increased bleeding tendency. Heparin 
plasma levels of 0.2 unit/ml were not adequate to check the thrombotic 
process. 
At the present, the Lee and White clotting time assay is rarely performed. 
The most widely used laboratory procedure for monitoring heparin therapy 
is the APTT. The TCT is considered by some to be a more reliable test. 
However, there are no reports on the use of the PT assay for monitoring 
heparin. 
Powdered heparin: 
2 mg of 162 units/mg powdered heparin (sodium), obtained through Sigma 
Chemical Co., St. Louis, Mo., was added to 32.4 ml of distilled water. 
This stock solution was reconstituted form each test to a concentration of 
0.1 unit/ml. 
Liquid heparin: 
10 ul of liquid heparin (sodium), obtained through Elkins-Sinn, Inc., 
Cherry Hill, N.J., was added to 10 ml of distilled water. This solution 
was then reconstituted to a concentration of 0.1 u/ml. 
Test Procedures: 
All tests were performed on Helena Laboratory's Dataclot-2.TM. fibrometer 
using pooled normal plasma obtained from 40 healthy donors age 18 to 64 
stored at a temperature of -70.degree. C. All plasma and reagents were 
transferred using MLA pipettes. 
Thromboplastin-C (Rabbit brain) obtained from American Dade AHS del Caribe, 
Inc., Aguada, Puerto Rico, 00602, was used as the reagent for the PT 
tests. 
For the TCT test, thrombin was prepared to give an activity of 1.2 NIH 
unit. 
The PT and TCT were performed 10 times on pooled normal plasma with no 
heparin added to obtain baseline clotting times. Then, 10 ul of 0.1 u/ml 
heparin was added to the plasma and four clotting times were obtained. 
Then, 20 ul of 0.1 u/ml heparin was added to the plasma and four clotting 
times were obtained. Each new concentration of heparin was increased by 
0.1 u/kml increments in this manner until the clotting times became so 
long that accurate data collection was no longer possible. 
The entire procedure was repeated three times, each time with newly 
reconstituted heparin and other reagents. 
The APTT reagents were comprised of Thrombosil-I (Rabbit brain cephalin and 
silica activator) Lot 3 1TH111 and Calcium Chloride Solution (0.02M aq.) 
Lot #CAL 300. Both were obtained from Ortho Diagnostic Systems, Inc., 
Raritan, N.J. 08869. 
The APTT test was performed in the same manner with the exception that the 
initial heparin concentration was 0.05 u/ml and each new heparin 
concentration value was increased by 0.05 u/ml, rather than by 0.1 u/ml as 
in the PT and TCT test procedures. 
A representative graph is shown in FIG. 23. FIG. 14 shows the chart for 
recording antithrombin III. 
(12) Protein C 
Protein C is the zymogen of a serine protease, Protein C.sub.A. Protein 
C.sub.A exerts an anticoagulant effect in plasma by the selective 
inactivation of non-enzymic activated cofactors FVa and FVIIIa. It has 
been shown by several investigators that native Factors V and VIII are 
poor substrates for Protein C.sub.A. It has also been shown that on 
endothelial cell surfaces in blood, Protein C is activated to a protease 
by thrombin complexed with thrombomodulin. Thrombomodulin is an integral 
endothelial cell surface protein. 
In vitro Protein C is slowly activated by thrombin alone or by 
thrombin/thrombomodulin at a much faster rate. Also Protein C is activated 
by purified Factor X.sub.a and by Akistrodon Akistrodon Contortrix venom 
of the Southern Copperhead snake. The component in the Akistrodon venom 
that is selective for the activation of Protein C has been purified and is 
given the trade name "PROTAC".RTM. (available from Sigma Chemical Company, 
St. Louis, Mo.). In addition to activating Protein C, the purified 
component of Akistrodon Akistrodon Contortrix has been found to decrease, 
by direct proteolysis, the procoagulant activities of purified Factors II, 
VII, IX, X, and to cleave the A alpha chain of fibrinogen (Thrombomodulin 
Activityin Commercial Thromboplastin Preparations. Thrombos Res. 43, 
265-274 (1986)). Thus, in vitro, the Protein C activator from the Southern 
Copperhead snake venom exerts a broad substrate specificity. 
Several assays to measure the biological activity of Protein C in plasma 
have been published. Some utilize lengthy and rather complicated 
experimental procedures that preclude their use in clinical diagnostic 
laboratories. Others use the purified Protein C activator from Akistrodon 
venom to measure Protein C activity as a function of the prologation of 
the APTT clotting times. 
A. Principle of the Protein C Assay 
Plasma is activated by thrombomodulin/tissue Factor and calcium chloride. 
Amount of tissue Factor and calcium chloride that activate plasma was 
carefully calculated. As little as 20 ul of commercial 
ThromboplAstin/CaCl.sub.2 0.02M solution activates PNP. Evidence for 
generation of thrombin in the activated plasma is obtained by an increase 
in Factor V and Factor VIII activity without detectable fibrin formation. 
The maximum amount of Thromboplastin/CaCl.sub.2 0.02M solution that fully 
activates PNP without detectable fibrin formation is 50 ul. 
To prepare a suitable commercial Thromboplastin/CaCl.sub.2 0.02M solution 
that will activate plasma at the recommended 20 ul to 50 ul range, 
distilled water is added to the dried powder to give in a fibrometer a 
clotting time by the PT assay for PNP of 11.6 seconds (0.5). The reagent 
recommended is Ortho Thromboplastin for its high thrombomodulin activity. 
Measurement of Activated Factors V and VII by the APTT assay. 
30 ul plasma before activation and 30 ul plasma after activation are each 
added to 70 ul Factor V and Factor VIII deficient plasma. Measurement of 
change in activity is a change in the clotting time by the APTT assay. 
Activity is derived from the standard curves presented in the Table shown 
in FIG. 29. The Table of FIG. 29 shows standard curves for descending 
ranges of Factors V, VIII, IX, X and XI activities and the mean clotting 
times obtained by the APTT assay. A least squares linear regression of the 
actual data points from the straight lines of the best fit are shown in 
FIG. 29. Factor activities and the corresponding clotting that represent 
the critical threshold procoagulant Factor V, VIII, IX, X or XI activities 
are shown. 
Protein C is activated in Activated Plasma by Akistrodon Contortrix 
Protein C in Activated Plasma is activated by 80 to 100 ng Akistrodon 
Akistrodon Contortrix (Southern Copperhead Venom). The proteolytic 
activity of snake venom used, 80 to 100 ng, is selective for Protein C. 
The activation of Protein C with Akistrodon is used to speed the process 
that is started by thrombin/thrombomodulin CaCl.sub.2 as described above. 
If the snake venom step is omitted, a time interval of at least 4 hours is 
necessary for the inactivation of Factors V.sub.a and Factors VIII.sub.a 
by "thrombin activated Protein Ca". 
B. Biological Activity of Protein C.sub.A is measured as a function of 
percent change in Factor V and Factor VIII activity. 
At the end of one hour incubation, 30 ul of plasma mixture are added to 70 
ul of Factor V and Factor VIII deficient plasma. Factor activity is 
obtained from clotting times by means of standard curves in FIG. 29. 
MATERIALS AND REAGENTS 
Preparation of Pooled Normal Plasma (PNP) 
Human pooled normal plasma (PNP) was prepared from forty healthy blood 
donors ages 18 to 64 years. Blood (4.5 ml) was drawn from each donor into 
vacutainer tubes each containing 0.5 ml of 3.85% acidified sodium citrate 
solution. Blood was spun at 2,000 r.p.m. in a refrigerated Beckman table 
top centrifuge at 2.degree. C. for 10 to 15 minutes. Platelet poor plasma 
was pooled into a polystyrene beaker placed on ice. The pooled plasma was 
assayed for procoagulant factor levels by the PT and APTT assays. 
Fibrinogen levels were determined by clotting and chemical assays. PNP 
aliquots (1 ml) were pipetted into 4 ml polystyrene capped tubes and 
stored at -80.degree. C. for use in the Protein C experiments. 
Akistrodon Contortrix Venom (Southern Copperhead venom) 
One gram freeze dried venom powder was purchased from Sigma Chemical 
Company, St. Louis, Mo. Twenty samples of dried powder were weighed 0.1 mg 
each and stored in 15 ml graduated capped plastic centrifuge tubes at 
4.degree. C. until further use. The dried venom was dissolved in distilled 
water (0.1 mg/10 ml) and assayed for stability by adding 500 ng/50 ul to 1 
ml PNP. The proteolytic anticoagulant activity was tested by the APTT 
assay. Proteolytic anticoagulant activity was markedly decreased within 24 
hours after reconstitution in distilled water. Proteolytic anticoagulant 
activity was retained in the dried powder. Fresh solutions therefore were 
prepared daily by adding 10 ml distilled water to the graduated plastic 
centrifuge tubes containing 0.1 mg of dried powder. Venom solutions were 
kept on ice for the duration of the experiments. 
The venom was also tested at two concentrations of 80 ng and 500 ng added 
to 1 ml PNP for substrate selectivity by PT, APTT and thrombin Clotting 
Time (TCT) assays. 
Tissue Thromboplastin/Calcium Chloride Powder (TTP/CaCl.sub.2) 
This was purchased from Ortho Diagnostic Systems Inc., Raritan, N.J. Ortho 
Brain thromboplastin ISI Standard lot 871007 was obtained from the same 
source. The commercially prepared tissue thromboplastin/calcium chloride 
powder was reconstituted to give a clotting time of 11.6.+-.0.5 seconds on 
100 ul PNP. This reagent has high thrombomodulin activity (Thrombomodulin 
Activity in Commercial Thromboplastin Preparations. Thrombos Res. 43, 
265-274 (1986)). 
Activated Partial Thromboplastin Reagent 
Thrombosil I, a commercially prepared brain cephalin with silica activator, 
was purchased from Ortho Diagnostic Systems. 
Calcium Chloride Reagent 
0.02 molar solution and Thrombofax Reagent. A bovine brain cephalin 
solution were also purchased from Ortho Diagnostic Systems. 
Human Alpha Thrombin with a specific activity of 3,000 units/ug was 
prepared. Clotting activity of the thrombin in 0.1M CaCl.sub.2 solutions 
is retained for several years. A preservative, Thimerosal purchased from 
Sigma Chemical Company is added to the thrombin solutions at 1/100,000 
(weight in mg/volume). Thrombin solutions (1.5 to 1.2 unit per 100 ul 0.1M 
CaCl.sub.2) were prepared to give a clotting time of 8-10 seconds with 200 
ul PNP. 
Equipment 
A Dataclot 2 fibrometer, Helena Laboratories, Beaumont, Tex. was used for 
the clotting experiments. A Macintosh Apple Computer and an IBM PC were 
used for the analysis and graphing of the data. 
EXPERIMENTAL PROCEDURES 
Prothrombin Time (PT) Assay 
PNP or plasma mixture (100 ul) were clotted with 200 ul TTP/CaCl.sub.2 
solution. The clotting times were recorded on a fibrometer. 
Activated Partial Thromboplastin Time (APTT) Assay 
PNP or plasma mixture (100 ul) were incubated with APTT reagent for 3 to 5 
minutes then clotted with 100 ul CaCl.sub.2 0.02 M. 
Thrombin Clotting Time (TCT) Assay 
200 ul PNP or plasma mixture was clotted with 100 ul thrombin solution (1.5 
to 1.2 unit). 
Single Factor Genetically Deficient Plasma Reagents (less than 1% activity) 
Factor XI deficient reagent was purchased from George King, Biomedical, 
Inc., Overland Park, Kans. All other factor deficient plasmas were 
obtained by plasmapherisis from patients at Michigan State University, 
East Lansing, Mich. 
Standard Curves for Factors V, VII, VIII, IX, X, XI were constructed using 
single factor genetically deficient plasma and PNP. The clotting times by 
PT and APTT assays for about forty estimates per point were analyzed. 
Standard deviation, linear regression, Pearson's correlation coefficient, 
as well as mean and median were calculated for each curve. 
In FIG. 28 the clotting times by PT for Factors V, VII and X activities 
ranging from 80% to less than 1% are presented. 
In FIG. 29 the data presented are the clotting times by the APTT assay for 
Factors V, VIII, IX, X, and XI activities ranging from 80% to less than 
1%. 
Factor Assays: 
In the Protein C assay experiments, most of the Factor assays were 
performed by PT or APTT assay after adding 30 ul plasma mixtures to 70 ul 
single factor deficient plasma (Factors V, VII, VIII, IX, X, XI or XII) 
and recording the mean clotting times. The mean clotting time was never 
less than four estimates with an average of ten estimates per point. 
Factor activities were then derived from the corresponding clotting times 
on the linear regression of the standard curves. 
Activation of Plasma 
One milliliter of PNP or patient plasma was activated by adding 50 ul, 30 
ul, 20 ul or 10 ul solutions of TTP/CaCl.sub.2. Tube was gently shaken and 
incubated at 37.degree. C. for times ranging from zero to one hour. PT, 
APTT, TCT, and Factor assays were performed on activated plasma and on 
plasma prior to activation. 
Standard Curves for Protein C 
Three standard curves for Protein C were contructed in: 
1) Protein C freeze dried deficient plasma reagent purchased from 
Diagnostica Stago, Asniere, France. 
2) Plasma obtained froma 16 year-old patient who tried to commit suicide by 
ingestion of three packages of a long lasting coumarin derivative prepared 
commercially and used as a rat poison (trade name: Enforcer.TM.). PT was 
72 secs (control 11.2 secs), APTT 132.4 secs (control 26.4 secs), and TCT 
9.9 secs (control 9.2 secs). Factor VII activity in this patient's plasma 
was less than 1%, Factor X activity 2%, and FIX activity 2.5%. 
3) PNP immunodepleted of Protein C by anti-Protein C insolubilized rabbit 
immunoglobulins. 
Human anti Protein C antibodies were purchased from Diagnostica Stago 
Asniere, France. The commercial antibodies were not charcterized in my 
laboratory for antigen specificity and cross-reactivity. Coupling of the 
anti Protein C antibodies to sepharose beads and immuno depletion of PNP 
by insolubilized antibodies was performed exactly as described (H. I. 
Hassouna and J. A. Penner. Sem. Thromb. Haemost. Vol. 7, No. 2, pp. 61-111 
(1981)). 
Patient Tests 
Following surgery for cancer of the pancreas, a 61 year old patient 
suffered a pulmonary embolism. He was placed on coumadin, 5 mg/day. Two 
months later, while still on coumadin, he was hospitalized for spontaneous 
bleeding, bruising and a hematoma on the left thigh. He had lost eight 
pounds because he was not eating. 
At the time of admission, his PT was 60 seconds, APTT greater than 100 
seconds, platelet count 140,000/cc, fibrinogen 425 mg/dl, and fibrin split 
products moderately elevated. Liver function tests were unchanged from 
previous records. 
He was diagnosed as disseminated intravascular coagulation (DIC), was taken 
off coumadin and given fresh frozen plasma. His PT and APTT corrected for 
six hours. For the next three days, his PT and APTT were still prolonged; 
PT 20-22 seconds (control 11.6 secs.) and his APTT 38 seconds (control 
26.4 secs.). Fibrinogen remained unchanged, at 425 mg/dl. Fibrin split 
products were not ordered. 
Discussion 
The diagnosis of DIC was made on the basis of a prolonged PT and APTT and a 
mild elevation of fibrin split products. The fact that fibrinogen levels 
and platelet counts were within normal range was attributed to a possible 
decline in initially higher than normal values. Other possible diagnoses 
were disregarded, owing to the myth that an APTT is never prolonged with 
coumadin therapy and that a PT of 60 seconds is possible even with 
fibrinogen levels of 425 mg/dl. Also the specificity of immunoassays for 
the determination of fibrin split products was never questioned. 
In this case, access to the present invention data would have given the 
clinician the ability to make the correct diagnosis. 
1) The Prothrombin Time (PT) test measures Factor VII, as well as 
Fibrinogen, Factors V, X, and prothrombin. This can be seen in FIG. 1 
(Coagulation Screening Tests). Also, maximal prolongation of the clotting 
times by the PT test for a single factor deficiency is less than 50 
seconds, provided fibrinogen levels are normal (see FIG. 2 top section and 
FIG. 3). Similarly, maximal prolongation of the clotting times by the 
Activated Partial Thromboplastin Time (APTT) test for Factor VIII 
deficiency is 77-80 seconds (see FIG. 2--lower section and FIG. 5). Drugs, 
such as coumadin, that interfere with the synthesis of biologically active 
vitamin K dependent factors have an effect on the PT as well as the APTT 
(see FIG. 5--4th section from bottom of page). PT or APTT tests are 
considered prolonged if the clotting times are outside the normal 
distribution. Normal distributions for both the PT and APTT are indicated 
in the top section of Table V and detailed in the histograms marked FIGS. 
6 and 7. In DIC, Factors V and VIII are the major procoagulant factors 
consumed. The half lives of Factors V and VIII are 12-36 hours and 2.9 
days respectively (see FIG. 8). 
2) When the patient was given fresh frozen plasma, the PT and APTT 
corrected for six hours then became prolonged again. The prolonged 
coagulation times, though modest, were significant. 
3) We can therefore contemplate one of two conditions: 
a) a low grade ongoing disseminated intravascular coagulation 
or 
(b) a multiple deficiency involving the vitamin K dependent factors due to 
malnutrition and possible malabsorption associated with coumadin therapy. 
4) Confirmatory tests are indicated in table IX (Diagnosis of Acute 
Thrombosis). Testing for Antithrombin III, Protein C, and plasminogen will 
provide proof for or refute DIC. 
Another approach would be to test for Factor V. If Factor V levels are 
within the normal range, a diagnosis for vitamin K deficiency can be made. 
Diagnosis 
Vitamin K deficiency due to malnutrition and possible malabsorption 
associated with coumadin therapy. 
Better diagnostic tests for diseases caused by abnormal blood clot 
formation (heart disease, strokes, deep clots in lungs, legs) are in 
demand. These conditions are an enormous health problem and public 
awareness of them has been increasing steadily with growth in health 
consciousness. They are life threatening diseases associated with aging, 
lack of exercise, poor dietary habits, smoking and oral contraception so 
they have a lot of visibility. Thrombotic (clotting) disorders are being 
handled now with new treatments, like tPA and oral anticoagulants, that 
complicate the interpretation of laboratory tests needed for diagnosis and 
for monitoring therapy. 
There is already an established market for coagulation disease diagnostic 
kit sales to the medical-technology community. The fact is though that 
this traditional technology, based on concepts prevalent in the 1950's, 
does not provide the quantitative precision that is essential for this 
expanding market and for the complexities that result from modern medical 
and surgical management of coagulation diseases. The present invention is 
a validated, proven system of original assays, founded on a very extensive 
data-base of thousands of test results on samples from diseased and normal 
subjects. It is compiled in a comprehensive, differential-diagnostic 
format and relies on some preferred reagents with highly desirable 
characteristics. The system makes high quality information available to 
the diagnostician conveniently and quickly, improving the decision-making 
process for the clinician. It can even identify those "at risk" of clot 
formation and therefore has the potential for incorporation into screening 
panels for routine use. 
The preceding description is only illustrative of the present invention and 
it is intended that the present invention be limited only to the 
hereinafter appended claims.