Laboratories and Blood Tests

Our laboratories are equipped with the most advanced machines and devices available in order to be able to meet our patients' needs. We provide a whole array of laboratory services, with both accuracy and speed which are vital for this type of data, upon which so many treatment decisions are based.

 

Below is a list of the laboratory tests performed by our department and all of its branches. A short description of each test and its diagnostic value is provided.

 

Most of the tests are blood tests. Other tests are performed on bone marrow and some test both. In some cases, the tests are done on other body tissues (such as lymph nodes) or fluids such as spinal fluid.

 

They are divided into the following categories:


A. Routine blood tests (other than coagulation)
B. Routine tests performed on the bone marrow or spinal fluid
C. Tests related to the coagulation system
D. Tests related to thalassemia
E. Tests related to malignancies
F. Specialized tests for anemias and other conditions

 

A. Routine Blood Tests

 

  • Complete Blood Count (CBC)

A complete blood count or CBC  is a blood test that provides several pieces of information about a person's state of health based on the content of certain components within the blood. The CBC is one of the most routinely performed laboratory tests. It is a valuable screening tool for a wide variety of disorders, including:

 

  • anemia,
  • infection,
  • blood diseases,
  • excessive menstrual bleeding, internal bleeding or problems with blood clotting.

 

Adult or Adolescent

Blood is drawn from a vein (venipuncture), usually from the inside of the elbow or the back of the hand. The puncture site is cleaned with antiseptic, and a tourniquet (an elastic band) or blood pressure cuff is placed around the upper arm to apply pressure and restrict blood flow through the vein.  This causes below the tourniquet to distend (fill with blood).  A needle is inserted into the vein, and the blood is collected in an air-tight vial or a syringe.  During the procedure, the tourniquet is removed to restore circulation.  Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding. The entire procedure takes less than five minutes and produces only mild discomfort, and little, if any, bleeding after the test.

 

Infant or Child

The area is cleansed with antiseptic and punctured with a sharp needle or a lancet.  The blood may be collected in a pipette (small glass tube), on a slide, onto a test strip, or into a small container. Cotton or a bandage may be applied to the puncture site if there is any continued bleeding.

 

How to Prepare for the Test

Adults: No special preparation is necessary.

Infants and children: The physical and psychological preparation you can provide for this or any test or procedure depends on your child's age, interests, previous experience, and level of trust.  You may need to consult your pediatrician or pediatric hematologist.

 

  • Reticulocyte count

Manual methods are limited in their reproducibility by the low number of cells that can be counted. Flow cytometry allows thousands of cells to be counted rapidly, greatly improving reproducibility. The method is based on the observation that RBC newly released from the bone marrow (reticulocytes) have a higher RNA content than more mature RBC. RNA is detected by staining with a fluorescent dye, and the number of reticulocytes is determined from the number of red cells with fluorescence higher than background. This information is invaluable in the evaluation of anemia, hemolytic states, etc. as an adjunct to the CBC, complete blood count.

 

  • Peripheral Blood Smear

A small drop of the blood is placed on a slide and spread out into a thin film. After drying, it is stained with Wright's stain in an automatic stainer. The stained slide can the be viewed under a microscope, and the hematologist can assess the morphology and appearance of all the blood elements: the red blood cells, the white blood cells and the platelets. Although the automated Coulter Counter is highly sophisticated, the determining the status of the cells and the evaluation of the nature of any abnormalities is best done by human eye.

 

  • Erythrocyte Sedimentation Rate

Anticoagulated blood is placed in a thin tube to stand for two hours. The red blood cells (RBCs) settle to the bottom of the tube and a top layer of plasma is left in the tube. The result is seen through the the number of millimeters the RBCs fall in one to two hours. The speed at which the RBCs fall is determined by the concentration of a number of plasma proteins which rise during acute illnesses. A high sedimentation rate is a nonspecific, but highly sensitive, indicator for the presence of organic diseases of all sorts.

 

  • Motulsky

This test is performed on peripheral blood and is a screening test for the presence of G6PD deficiency. G6PD deficiency is an X-linked hereditary condition very common in Middle Eastern populations, particularly among Kurds. Since it is X-linked, the adverse effects of this condition are manifested more often in men. The enzyme deficiency does not allow the red blood cells to repair themselves under conditions of oxidative stress. Events, such as exposure to certain foods (fava beans, "Ful") or medicines (quinine, sulfa drugs), can cause hemolysis - destruction of the red blood cells - and can potentially be quite severe. It is important for individuals with a family history of sensitivity to fava beans to be tested for a G6PD deficiency. This test is done using a Motulsky stain on peripheral blood. If a person is found to have this condition, s/he should consult a hematologist for an accurate description of substances to avoid to prevent harmful episodes.

 

B. Routine Tests Performed on Bone Marrow or Cerebrospinal Fluid

 

  • Wright's Stain of Bone Marrow Aspiration: 

This is the routine stain allowing visualization all the cellular elements in the bone marrow.

 

  • Cytochemistry to Identify Bone Marrow Aspiration

These are special tests which use specific chemicals to determine the cellular characteristics of the bone marrow cells. These stains are used to diagnose the type of cell which may be connected in any of the leukemias, lymphomas, etc.  For example, myeloperoxidase stains myeloid cells, and esterases stain monocytic cells. (These same stains can also be performed in a peripheral blood smear, to identify the lineage and nature of immature or abnormal white blood cells).

 

  • Prussian Blue Stain to Identify Iron

A special stain using an iron compound can identify the presence of iron which is deposited in bone marrow stores. It also can also identify certain abnormal RBC precursors, such as ring sideroblasts. If present, the iron compund can denote a particular type of bone marrow abnormality indicating a specific type of myelodysplasia.

 

  • Cytospin of Cerebrospinal Fluid

For patients being treated for ALL, and for some with lymphoma, involving the central nervous system (CNS) with the malignant disease presents a difficult treatment problem. The cytospin is the tool which establishes the presence or absence of such involvement. The cerebrospinal fluid is spun down onto a glass slide, using a special type of centrifuge. The slides are stained and examined under the microscope. This cytospin test can also be done on other body fluids such as pleural fluid, which accumulates in certain disease conditions. This can enable identification of the cells as reactive or malignant.

 

 

C. Tests of the Coagulation System


There are many types of tests of the coagulation system, and these are categorized into routine tests, bleeding diathesis (tendency to bleed), hypercoagulability tests (excess blood clotting) and miscellaneous tests.


There is a whole array of indicators for tests related to the coagulation system. Some of these tests are performed routinely in a number of medical situations, such as prior to major surgical procedures. They may also be performed if there is evidence of a bleeding problem, such as easy bruising or heavy menstruation. Alternatively, other tests are performed if there is evidence of a disorder leading to excessive blood clotting (thrombosis).

 

Routine Tests

 

  • Prothrombin Time (PT)

This test is performed routinely for many patients prior to surgery, or may be done to monitor the effect of anticoagulation using Coumadin or Syntrom. The attending physician adds a substance to activates the clotting system in the plasma, and measures the time it takes to form fibrin, the primary protein of which the blood clot is made. The formation of the blood clot requires the participation of a series of proteins, and deficiency of any of these will cause an abnormal Prothrombin time. The result is usually expressed within seconds compared to the time it takes a normal plasma sample to clot.

 

Patients taking anticoagulation medications which prevent the formation of blood clots must have their dose adjusted using the prothrombin time. The usual oral drugs which inhibit anticoagulation are Coumadin and Syntrom. Both of these drugs prevent the manufacture of the normal clotting proteins which require Vitamin K for their synthesis, since these drugs are Vitamin K antagonists. By preventing the manufacture of clotting proteins, these drugs are anticoagulants.

 

The substances used to perform the prothrombin time vary widely in their strength. Therefore, the prothrombin time can vary among laboratories and even in the same laboratory from test to test. Therefore, it has become customary for laboratories to perform a correction of the prothrombin time, to "normalize" the result. This normalized test is called the "INR", for International Normalized Ratio and it is much more valid that the prothrombin time expressed in seconds or percentages of normal. A normal INR is 1.0. However, in anticoagulated patients, the INR will be higher, a level of over 1.6, indicative of anticoagulation effect. The target anticoagulation level differs for each patient. Various indications for anticoagulation will require different therapeutic INR levels. The treatment needs to be individualized and this is done with a coagulation specialist (such as Prof. David Varon of our Coagulation Clinic).

 

  • Partial Thromboplastin Time (PTT)

This test is performed routinely for many patients prior to surgery, or to monitor the effect of anticoagulation using heparin. The test is performed by adding a substance which activates the clotting system and measuring the time it takes to form fibrin, the primary protein of which the blood clot is made. The formation of the blood clot requires the participation of a series of proteins, and deficiency of any of these will cause an abnormal partial thromboplastin time. The result is usually expressed in seconds compared to the number of seconds it takes a control normal plasma sample to clot. A common reason for an abnormal PTT is the hereditary deficiency of Factor XI, which is found in 8% of Ashkenazim. Other reasons are Von Willebrand's Disease, lupus anticoagulants, and many more.


Although the test is used to monitor heparin, it does not become abnormal during the use of low molecular weight heparin, such as Clexane. Patients with an abnormal PTT without anticoagulation may have a bleeding disorder and they should consult with a coagulation specialist.

 

  • Thrombin Time

This measures the normal function of fibrinogen, and is a test for certain diseases (liver disease, or DIC). It can also detect the use of heparin.

 

  • Fibrinogen

This is the main protein which forms the blood clot. Its level can be measured in plasma.

 

  • Fibrin Degradation Products

This test is performed if there is evidence of a bleeding disorder caused by abnormal activity of the coagulation system which causes "DIC" - Disseminated Intravascular Coagulation.

 

Levels of Clotting Proteins

In certain diseases, there may be a hereditary or acquired deficiency of one or more of the plasma clotting proteins. Since the liver is the production site for many of the clotting proteins, liver disease is a common situation that may require measuring the precise levels of clotting factors. This is more accurate that the determination of the PT and PTT, but such testing is only required under certain circumstances.

 

Another type of situation which requires measuring the level of clotting proteins is when the routine PT and/or PTT is/are found to be abnormal. To evaluate the reason for the abnormality, factor levels are measured. The clotting proteins which can be measured in our laboratory are: Factor II, V, VII, IX, X, XI, XII, and XIII. These tests are ordered by the hematologist or other treating physician.

 

Platelet Function

In certain diseases, there is a bleeding disorder caused by poor function of the blood platelets. Platelets are small particles which help the body's blood clot. The platelets stick to each other when stimulated by various types of signals telling the body that bleeding may occur (such as injury to a blood vessel). If the platelets fail to stick to each other, then bleeding can occur.

 

  • Bleeding Time

In this test, a very superficial (less than 1 mm deep), small (1 cm long) cut is made in the top layer of the skin of the forearm using a special instrument. The time it takes for the cut to stop bleeding is a test of the function of the platelets. It is measured in minutes (the normal time is three to eight minutes). This test is delicate and is performed only by appointment with the technicians - Mrs. Luda Gelfand or Mrs. Irit Liba: 02-6776763, after consultation with a hematologist.

 

  • Platelet Aggregation with ADP, Collagen, Epinephrine and Ristocetin

These tests are performed by separating platelets from blood drawn from the patient's vein. Measurement is made of how much the platelets stick to each other after being exposed to certain substances stimulating platelet function. These tests are delicate and time consuming and are performed only by appointment with the technician, Miss Esti Hy-Am: 02-6776722.

 

Tests of Hypercoagulability

Certain clinical situations suggest that the patient has a problem leading to excess clotting (thrombosis). There are many types of diseases twhich lead to a thrombotic tendency, including problems which are hereditary or acquired. The evaluation is complex, and is expedited by consultation with Prof. David Varon of our Coagulation Clinic.

 

  • Protein C

This substance deactivates other activated coagulation proteins, slowing down the clotting process. Hereditary or acquired deficiency of this protein causes a tendency to produce excess blood clots.

 

  • Protein S

This protein deactivates other activated coagulation proteins, slowing down the clotting process. Hereditary or acquired deficiency of this protein causes a tendency to produce excess blood clots.

 

  • Activated Protein C Resistance (APCR) and Factor V Leiden

In contrast to most hereditary clotting disorders, which are very rare, APCR is quite common in Israel. The problem is due to Protein C deactivating Factor V which has previously been activated. Activated Factor V is a protein which promotes blood clotting, and by deactivating it, Protein C slows down clotting. Protein C actually cuts Factor V into 2 pieces, at a certain position on the Factor V molecule. Some individuals have a mutation (error in the genetic code) of Factor V which falls exactly at the site where Protein C is supposed to cut (deactivate) it. This mutation prevents Protein C from cutting the activated Factor V molecule. Since the activated Factor V can not be deactivated, the blood clotting process which was set in motion can not be slowed down, leading to a tendency to form excess clots. The name for this general problem is called Activated Protein C Resistance (APCR). The most common form of the mutated Factor V is called "Factor V Leiden." A screening test can be performed on peripheral blood for APC resistance. Alternately, a DNA test (performed on DNA from peripheral blood) can be done to identify the exact change in the DNA characterstic of the Factor V Leiden mutation.

 

  • Prothrombin Mutation

Another mutation in the genetic code associated with excess blood clotting is a mutation near the gene for the clotting protein - prothrombin. This mutation can be identified directly, using DNA from peripheral blood. The prothrombin mutation is a fairly common cause of excess blood clotting in the Israeli population. Patients with a history of clotting or a family history of such disorders can be tested for this mutation, as suggested by the treating physician, preferably a hematologist.

 

  • Antithrombin III level

This is a very rare cause of excess blood clotting, and can be tested on peripheral blood.

 

Miscellaneous Tests

  • Factor Xa

This test is used to monitor the proper dose of an anticoagulant, low molecular weight heparin. This test is performed with advance consultation with the technician, Miss Esti Hy-Am, 02-6776722.

 

  • Circulating Anticoagulants

In some diseases, the body makes antibodies to the coagulation proteins, creating a tendency to bleed excessively. However, in some situations, these antibodies may actually cause excess clotting by activating the clotting cascade. This test is performed with advance consultation with the technician, Miss Esti Hy-Am, 02-6776722.

 

D. Tests Related to Thalassemia

 

  • Hemoglobin Electrophoresis (Including Hemoglobin A2 and Hemoglobin F)

These tests are performed on peripheral blood. The red blood cells are opened and the hemoglobin inside them is dissolved in solution. The hemoglobin is loaded into a special apparatus and is electrophoresed (run in an electrical field) onto a special membrane. Adult hemoglobin is the main hemoglobin type identified. It is possible that this is the only Hb present however, there are some minor hemoglobins which may be present, such as fetal hemoglobin (HbF), and hemoglobin A2. These two minor hemoglobins generally are present in 1-2% of normal hemoglobin. However, they increase in production when an individual is a carrier of beta-thalassemia trait. The levels are not elevated in alpha thalassemia trait. Other conditions can raise hemoglobin F (but not HbA2), such as recovery from anemic states or pregnancy, therefore, HbA2 elevation is quite specific for beta thalassemia trait. If an individual of childbearing age is found to be a carrier of thalassemia, his spouse must be tested to determine if they require genetic counseling. Such individuals or couples should see a hematologist, such as Prof. Deborah Rund or Dr. Ada Goldfarb . They may need molecular diagnosis of thalassemia, see below.

 

  • Heinz Bodies

These are clumps of proteins (mostly hemoglobin) which have lost their proper form (denatured) and which stick to the RBC membrane. They can be visualized under the microscope using a special stain. They are found in a number of different states associated with hemolysis, such as G6PD deficiency, various congenital hemolytic anemias, or thalassemia

 

  • Molecular Diagnosis of Thalassemia

Thalassemia is caused by mutations in the genes for alpha or beta globin of the hemoglobin molecule. These genetic errors cause either alpha or beta thalassemia, respectively. Our laboratory is the central reference laboratory in Israel for the identification of these mutations. In order to enable precise diagnosis of the patient and prenatal diagnosis of couples at risk for the birth of a thalassemic child, the knowledge of the precise mutation(s) carried by the individuals is crucial. This enables assessment of the severity of the illness and of the prognosis for the future. Recommendations for therapy, such as the need for bone marrow transplantation, can be made based on the analysis of mutations. The test is performed on DNA which is prepared from blood samples.


Using the most modern technology, mutation(s) is/are identified in either the beta globin or the alpha globin genes which encode the hemoglobin molecule. There are several hundred point mutations (errors in a single letter of the DNA code) which cause beta thalassemia, of which over 30 have been found in Israeli patients. In some individuals, there are mutations of both the alpha and the beta globin genes. Therefore, identifying the mutations causing beta thalassemia is quite complex and needs to be individualized for each family. In Israel, 18 genetic lesions have been found to cause alpha thalassemia. The analysis of alpha thalassemia is even more complex, since not only are there point mutations (involving a single letter substitution in the DNA code) but there also deletions (missing segments) of large sections of DNA. Identification of alpha globin deletions require specialized tests, some of which are highly sophisticated. The laboratory can be contacted for advice. Prof. Ariella Oppenheim is the responsible scientist in the laboratory and Dr. Dvora Filon is responsible for coordinating the prenatal and patient diagnosis programs. Prof. Deborah Rund and Dr. Ada Goldfarb are happy to advise couples or individuals who have a family history of thalassemia or suspect they are carriers.

 

 

E. Tests Related to Malignancies

 

  • Molecular Diagnosis of Malignancies

Genetic mutations are a common feature of many hematological malignancies. The identification of these mutations is of great impact, both from a diagnostic and a prognostic standpoint. In addition, if a genetic mutation is identified, it provides an excellent tool for the followup of minimal residual disease which may remain after therapy. Tissues which are generally analyzed for these abnormalities include blood, bone marrow and lymph nodes, although any substance can be analyzed under specific circumstances - body fluids such as pleural fluid (from around the lungs) and spinal fluid. These tests are performed in the laboratory of Prof. Dina Ben Yehuda, using the most modern of molecular techniques of PCR technology. Recently our department has acquired sophisticated equipment for the most exquisite precision of these PCR reactions. This state-of-the-art equipment is a Real-Time PCR apparatus. This machine performs PCR with fluorescent probes, and provides the most sensitive method of measuring PCR products, for quantitative measurements which are crucial for making decision for our patients.

 

The following tests are done by the Department of Hematology:

 

  • Tests for Myeloid Leukemias: PCR-based tests for the 8;21 and 15;17 translocations (for M2 and M3 AML), analysis of MLL gene rearrangment, PCR-based tests for the Philadelphia chromosome (for CML).

  • Tests for Lymphoid Malignancies (Leukemia and Lymphoma): B-cell and T-cell gene rearrangements, Philadelphia chromosome (for ALL), BCL1 and BCL2 rearrangements, myc oncogene analysis.


These are the most frequently used tests, though an infinite selection of other molecular tests are available, depending on the individual patient's needs. Most importantly, all of these tests can be utilized to provide the utmost in diagnostic information, and for accurate follow-up during and after therapy.

 

  • Multi-Drug Resistance Gene (MDR1)

This test is performed on blood or bone marrow using a fluorescent dye with FACS  analysis. It is jointly performed by the laboratories of Prof. Deborah Rund and Prof Eitan Fibach. Malignant cells can possess de novo or acquire the ability to resist chemotherapy due to the ability to "pump" out cytotoxic drugs. There are a number of "pump" mechanisms, the most well-known being the P-Glycoprotein pump, a transmembrane molecule which can eject many substances from the cell. Malignant cells which possess this molecule on their surface are more likely to be resistant to specific chemotherapy drugs. In addition, the presence of the molecule connotes a bad prognostic parameter, even if drugs are used which are not ejected by the P-Glycoprotein system. The test is performed at diagnosis on all leukemias, and some lymphomas and on follow up bone marrow tests, during and after continued chemotherapy administration.

 

  • FLT3 Mutations

Very recently, it was discovered that abnormalities of this gene are highly indicative of a poor prognosis in AML. This test is performed in the laboratory of Prof. Deborah Rund. It can be used to plan postremission therapy for these patients.

 

F. Specialized Tests Related to Anemia and Other Conditions

 

  • Schilling Test

A Schilling test investigates the cause of vitamin B12 deficiency. There are several diseases known to affect the body's uptake of vitamin B12.  The Schilling Test checks to see if the B12 deficiency is caused by low levels of a compound called intrinsic factor, which must combine with B12 before the vitamin can be absorbed.

 

For the first part of the Schilling Test you will take a capsule that contains a small amount of radioactive vitamin B12. One hour later we will give you an injection of nonradioactive vitamin B12.  We will ask you to collect your urine for the next 24 hours.  We will provide you with a container for this collection, which you must return to us the next day.  It is very important to collect all the urine excreted in a 24-hour period. In the nuclear medicine laboratory we will check your urine for the radioactive B12. If it was absorbed, it will be detected in the urine. If the first part of the Schilling Test shows that the B12 was not absorbed, your doctor may ask you to return for the second part of the test some days later.  The second part is exactly like the first, except that you will also take capsule containing intrinsic factor. If there is now radioactive B12 in the urine and there was none in the first study, it will mean that intrinsic factor deficiency is likely to be the cause of the B12 deficiency.

 

Preparation for the Test - Please do not eat or drink anything after midnight the night before the test.  You must continue to fast for two hours after swallowing the radioactive vitamin B12 capsule in the hospital. Also, you should not have received any vitamin B12 injections for at least one week prior to the test.

 

Safety - Nuclear medicine procedures are very safe.  The radiation dose from this test is extremely small (less than 5 mrem). It is equivalent to the radiation exposure you would receive from naturally occurring cosmic rays during a round trip flight from Boston to Hawaii.

 

Results - Your doctor will discuss the results of the test with you after reviewing them with the nuclear medicine physician.

 

  • Schilling Test with Intrinsic Factor

If the Schilling Test is abnormal, the Schilling is repeated with the addition of a capsule of "intrinsic factor," which is a protein made by the stomach. If this improves the absorption measured by the Schilling, then this proves the diagnosis of "pernicious anemia." The term pernicious anemia refers to anemia characterized by large red blood cells which results from the body's inability to absorb vitamin B12 due to the lack of "intrinsic factor." The treatment for this problem is lifelong replacement of B12 by a route OTHER than pills, which can not be absorbed. The vitamin can be give by injection, intranasal, or under the tongue. Results of therapy must be monitored to prevent recurrent anemia or neurological problems.

 

  • Vitamin E Levels

In certain diseases, the levels of vitamin E may be low, and they can be measured by this blood test.

 

  • Osmotic Fragility

In certain diseases, the red blood cells are susceptible to being broken up when put in a diluted salt water solution. The most common condition reason for this condition is hereditary spherocytosis, which is a common cause of mild to moderate anemia in Ashkenazic Jews. This is a specialized blood test which requires an appointment with the laboratory technician in advance. Please call Mrs. Olga Fradkin at 02-6776758.

 

  • Serum Haptoglobin

In certain types of anemia, the body destroys its own red blood cells. The haptoglobin level is a test which can help determine if this is occurring.

 

  • Plasma Hemoglobin

In certain types of anemia, the body destroys its own red blood cells. The plasma hemoglobin level is a test which can help determine if this is occurring.

 

  • Red Blood Cell Survival

In certain cases, it appears that the body is destroying its own red blood cells, and quantification is needed. The red blood cell survival test can determine the lifespan of the patient's red blood cells.

 

  • Erythropoietin Level

Erythropoietin is a substance which is made by the body to stimulate the production of red blood cells. Its level can be measured in the blood. Erythropoietin is synthesized by the kidney. If the bone marrow is unable to make the normal amount of red blood cells, the kidney responds by producing more erythropoietin meant to stimulate the bone marrow to work harder. This blood test can be used to determine if anemia is due to a bone marrow problem or a kidney problem.

 

  • Erythroid Progenitor Cultures

In certain diseases, the body produces an excess of red blood cells in the absence of the erythropoietin signal. This can be proven using cultures of erythroid precursor cells from peripheral blood, in the presence and absence of erythropoietin. The autonomous function of the bone marrow proves the diagnosis of polycythemia vera. This is a specialized and labor intensive procedure and requires notification in advance to Mrs. Aliza Treves at 02-6776749.

 

  • Myeloid Progenitor Cultures

In certain diseases, the production of white blood cells by the bone marrow is abnormal and their development can be studied using cultures in the laboratory. This is a specialized and labor intensive procedure and requires notification in advance to Mrs. Aliza Treves at 02-6776749.

 

  • Leukocyte Alkaline Phosphatase (LAP Score)

This special stain of the peripheral blood is used to determine the amount of a particular enzyme in the patient's white blood cells. It is used to diagnose certain blood diseases characterized by overactivity of the bone marrow (myeloproliferative diseases), which are manifested by an elevated level of the enzyme. If the levels are abnormally low, this suggests that the patient may have chronic myeloid leukemia, and further testing may be required.

 

  • Ham Test

This test is used to diagnose a rare bone marrow disorder called paroxysmal nocturnal hemoglobinuria (PNH). Another method of diagnosis is a special test using FACS analysis, performed by Dr. Zelig Orly, of our General Hematology Clinic and the Blood Bank.

 

  • LE Cells

This test is used to diagnose lupus, an autoimmune disease.

 

Bone Marrow Examination

Marrow examination complements clinical and laboratory information in determining the cause of anemia, leukopenia, leukocytosis, thrombocytopenia and thrombocytosis. It also contributes to the staging of lymphoproliferative disease and various solid tumors.

 

Bone marrow is the tissue found within your bones where blood cells are made. In infants, all the bone marrow is capable of manufacturing blood cells. However, in adults, blood cells are made only in the flat bones of the pelvis, sternum (breast bone) and skull. The bone marrow is like a blood cell factory.  It contains stem cells that have the capacity to evolve into all three types of blood cells. A stem cell can develop into a red cell and carry oxygen, or it can evolve into a white cell and fight infection. The bone marrow is responsible for maintaing the normal number of the three types of cells by replacing old cells as they naturally die off. It also increases production of any type of blood cell if there is a special demand for it, such as more white blood cells if you are fighting an infection. The bone marrow is a place where cells divide very quickly in order to keep up with your body's constant demand for blood cells. Since chemotherapy affects the cells that divide quickly (like cancer cells), it temporarily affect your bone marrow. Unlike cancer cells, your bone marrow will recover and resume its normal production of blood cells.

 

Chemotherapy doesn't affect the blood cells that are already in circulation, since these cells are not dividing themselves. Only the production of new cells in the bone marrow is slowed down. Chemotherapy's effect on your bone marrow usually shows up in your blood cell count about a week to ten days after your treatment, which it is noticeable that blood cells have not been replaced at the normal rate. But in another week or so, the number of blood cells in circulation will return to normal. Your chemotherapy treatments are timed to allow your bone marrow to recover. Your doctor will always check your blood cell count before each treatment to be sure that your bone marrow is back on the job of producing blood cells.

 

Marrow Aspiration

This is the preferred method for examining the cytogenetics or bone marrow morphology. Examination of the marrow aspirate is a useful method to review cellular morphology and focal lesions. The technique lends itself to special stains which cannot be done on decalcified material. These stains are essential for properly diagnosing acute leukemia. In patients with anemia, iron stains can determine the etiology of the anemia.

 

Marrow Biopsy
This is the preparation of choice for assessment of cellularity and marrow architecture.  The technique lends itself to the evaluation of cellular distribution, as well as the pattern and extent of fibrosis or of infiltrative processes. Granulomata can be examined for microorganisms with special stains. Biopsies evaluate some forms of anemia, but it is a routine procedure for staging of all the lymphomas.


Ancillary Tests

Flow Cytometry (FACS analysis)


Immunophenotyping is used as an adjunct study for diagnosis and classification of most lymphoproliferative diseases and acute leukemias. The technique complements peripheral blood analysis and may replace marrow examination in some disorders. The flow cytometry laboratory utilizes highly advanced equipment which is applied toward patient care needs as well as scientific research goals. The laboratory offers its services to other departments upon request with remuneration on a fee for service basis.

 

Flow cytometry analyzes cells in liquid suspension (i.e. blood, bone marrow, body fluids or tissue cell suspensions) that have been incubated with antibodies tagged with a fluorescent label. The antibodies are directed against specific cellular antigens. A number of different antibody panels are available, depending on the clinical question. The instrument is capable of examining up to four different antigens simultaneously. Several thousands cells are counted with each antibody combination, with the percentage of positive cells calculated. The relative fluorescence intensity of the positive cells indicates the amount of antigen studied.

 

The specific panel of antibodies used is selected based on the patient's clinical history, referring physician information, and the morphologic appearance of the cells present in the specimen. Antibodies of particular interest may be requested by the referring physician. A written report and interpretation for each case are issued. In addition, printouts of flow histograms are provided upon request. Specimen requirements: Generally, 5 ml heparin-anticoagulated (green top tube) blood or bone marrow.

 

Immunophenotyping of Acute Leukemias

The acute leukemia panel is designed to determine whether leukemic blasts are of myeloid or lymphoid origin, and to further classify the cells as B or T cell, monocytic, megakaryocytic, etc. Each antibody combination includes three antibodies directed against lineage-specific antigens and a third antibody against CD45, which is present on all hematopoietic cells. The inclusion of this third antibody allows the identification of small numbers of blasts in the presence of large numbers of nonmalignant cells, and is based on the blasts' lower expression of CD45. If the routine panel is insufficient to adequately characterize the leukemic cells, additional antibodies may be used to determine the expression of other marker combinations. The multidrug resistance (MDR)  phenotype of the malignant cells can be also evaluated with anti-P-glycoprotein antibodies or by a functional assay using rhodamine-123.

 

Immunophenotyping of Lymphomas/Lymphoproliferative Disorders

The lymphoma panel is designed to characterize lymphoproliferative disorders, which usually are comprised of mature B or T cells. For B cell malignancies, demonstration of the presence of a monoclonal population by restricted kappa or lambda immunoglobulin light chain expression is diagnostic. For T cell malignancies, in many cases the finding of an abnormal T cell phenotype is strong evidence for a T cell malignancy. To prove clonality for T cell malignancies, gene rearrangement studies need to be done. The routine lymphoma panel is performed using the B cell markers CD19, CD20, CD23, kappa and lambda, etc. and the T cell markers CD3, CD4, CD8, CD7, CD5 etc. Based on the results of this initial panel, additional B or T cell markers are added to help further classify B cell processes or to try to demonstrate an abnormal phenotype in T cell processes.

 

T Cell Subsets

To follow the immunologic status of patients our panel includes the T cell markers CD3, CD4, and CD8, and the pan-hematopoietic marker CD45 which is used for gating and quality control. The percentage of cells bearing each marker is determined by this method. In addition, the absolute number of CD4+ and CD8+ T cells is determined by including a calibrated number of fluorescent beads with the patient specimen, and comparing the number of lymphocytes to the number of beads that pass through the flow cytometer. This method eliminates the need for a separate lymphocyte count and allows determinations on up to 3 day old specimens.

 

Post-Transplant Monitoring

The immunologic status of post-transplant (renal, cardiac, liver, pancreas, heart-lung) patients receiving immunosuppressive treatment is routinely followed by lymphocyte counts. The transplant antibody panel includes CD2, CD3, CD4, CD8, and CD19. The rejection antibody panel determines only the total number of T cells based on CD3 staining, and is generally used to follow the therapeutic efficacy of the therapy.

 

Pre-Transplant Evaluation of Hematopoietic Stem Cell Harvests

Bone marrow, mobilized peripheral blood and neonatal cord blood harvests are evaluated by staining with antibodies to CD34, CD45, CD38 surface antigens, as well as other antigens as requested.

 

Measurement of Fetal Hemoglobin (HbF)-Containing RBC and Reticulocytes

Measurement of RBC with high content of HbF (RBC of fetal origin) in the blood of pregnant women helps evaluate the severity of fetal-maternal hemorrhaging. Measurement of HbF-containing cells of the patient's origin is important for diagnosis of chronic hereditary anemias, e.g., beta-thalassemia and sickle-cell anemia. HbF-containing reticulocytes are specifically informative in transfusion-dependent patients, and patients undergoing treatment with agents, e.g., hydroxyurea, that stimulate HbF production.
In these assays, peripheral blood cells are fixed and permeabilized, followed by staining with HbF-specific antibodies.

 

Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay
In PNH, a clonal marrow stem cell population gives rise to mature blood cells lacking the expression of a variety of different cell surface proteins. The feature common among these proteins is their link to the cell membrane via a glycosyl-phosphatidyl-inositol (GPI) link. Flow cytometry can be used to detect the loss of GPI-linked proteins (e.g., CD56, CD59) on each cell type. For the erythroid cell line, preferential analysis of reticulocytes increases the sensitivity of the assay. The loss of GPI-linked proteins in some cases of aplastic anemia having features of PNH may be useful for precise diagnosis.

 

Custom Flow Cytometry

Customized antibody panels for research or clinical use can be designed. Issues such as cell activation, expression of adhesion molecules etc, can be addressed with special panels. In addition, cell cycle, hypo/hyper ploidy, apoptosis etc. can be studied. Sorting (including aseptic sorting) of specific subpopulation can be accomplished using the sorter module. Please inquire by calling 02-6777458.

 

Cytogenetic Studies

This adjunctive method provides diagnostic and prognostic information in a wide range of malignant hematologic disorders. It reveals chromosomal abnormalities which can be crucial in determining the diagnosis and prognosis of many hematological malignancies.

 

Location: Sharett Institute of Oncology, floor -3

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